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Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents

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Polygonum multiflorum is an important medicinal plant. Hairy roots systems obtained by transforming plant tissues with the natural genetic engineer Agrobacterium rhizogenes can produce valuable biological active substances, which have immense potential in the pharmaceutical industry. To optimize the inductive and cultural conditions of P. multiflorum hairy roots and to identify the major active secondary metabolites in hairy roots. P. multiflorum hairy root were mediated with A. rhizogenes R1601 to induce hairy roots. Four combinations, including Murashige-Skoog (MS), 1/2 MS, B5, and White, were investigated to optimize the culture medium. MS medium was selected for the growth measurement. The qualitative and quantitative determinations of free anthraquinone in hairy roots were compared with the calli and aseptic plantlets using high-performance liquid chromatography. The inductive rates of hairy roots by leaves were higher than for any other explants. The presence of agropine in the P. multiflorum hairy roots confirmed that they were indeed transgenic. MS medium was the most suitable of the four media for hairy root growth. Meanwhile, the growth kinetics and nutrient consumption results showed that the hairy roots displayed a sigmoidal growth curve and that their optimal inoculation time was 18-21 days. The determination of the anthraquinone constituents indicated that the rhein content of the hairy roots reached 2.495 μg g(-1) and was 2.55-fold higher than that of natural plants. Transgenic hairy roots of P. multiflorum could be one of the most potent materials for industrial-scale production of bioactive anthraquinone constituents.
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High-speed counter-current chromatography methods, combined with solvent partition, were applied to the systematic separation and purification of chemical components from Chinese medicinal herb Polygonum multiflorum extract. The aim of this paper is summing up the rules of solvent system selection for diverse fractions of herbal extract, and establishing the systematic pattern to screen the bioactive constituents rapidly. Nine compounds including emodin, chrysophanol, rhein, 6-OH-emodin, emodin-8-beta-D-glucoside, polygonimitin B, 2,3,5,4'-tetrahydroxystilbene-2-beta-D-glucoside, gallic acid and an unknown glycoside, which differed in quantity and polarity remarkably, were obtained. The purities of them were all above 97% as determined by high-performance liquid chromatography (HPLC), and their structures were identified by 1H NMR and electrospray ionization mass spectrometry (ESI-MS). The results demonstrated that HSCCC is a speedy and efficient technique for systematic isolation of bioactive components from traditional medicinal herbs.
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Withania somnifera (L.) Dunal. (Indian ginseng) is an important medicinal plant which yields pharmaceutically active compounds called withanolides. The present work deals with optimization of parameters of hairy root culture of W. somnifera for the production of biomass and withanolide A. We also investigated the effects of carbon source [sucrose, glucose, fructose, maltose, glucose + fructose (1:1), fructose + sucrose (1:1) and sucrose + glucose (1:1)], sucrose concentration (1%, 2%, 3%, 4%, 6% and 8%) and the initial medium pH (4.0, 4.5, 5.0, 5.5, 5.8, 6.0 and 6.5) on growth and production of withanolide A in hairy root cultures of W. somnifera. We found that biomass accumulation and production of withanolide A was highest when sucrose was used as the carbon source (11.92 g l−1 DW and 11.96 mg g−1 DW of withanolide A). Further 3% sucrose concentration was found to be optimal for biomass accumulation (11.92 g l−1 DW) and 4% sucrose favoured the production of withanolide A (13.28 mg g−1 DW) in the tested range of concentrations (1–8%). The biomass of hairy roots was optimal when the initial medium pH was 5.8 (12.1 g l−1 DW) and the withanolide A production was highest in the medium pH set at 6.0 (13.84 mg g−1 DW).
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The neuroprotective effects of Polygonum multiflorum extract (PME) and its two fractions, ethanol-soluble PME (PME-I) and -insoluble PME (PME-II), on the degeneration of nigrostriatal dopaminergic neurons induced by a combination of paraquat and maneb (PQMB) were investigated in male C57BL/6 mice. The mice were treated twice a week for 6 weeks with intraperitoneal injections of PQMB. This combination caused a reduction of spontaneous locomotor activity, motor incoordination, and declines of dopamine level in the striatum and tyrosine hydroxylase-positive neurons in the substantia nigra. Administration of PME and PME-I once daily for 47 days during 6 weeks of PQMB treatment and last 8 days after PQMB significantly attenuated the impairment of behavioral performance and the decrease in striatal dopamine level and substantia nigral tyrosine hydroxylase-positive neurons in the PQMB-treated animals, whereas the administration of PME-II had no effect on these behavioral, neurochemical and histological indices. The present findings suggest that PME has a beneficial influence on parkinsonism induced by PQMB and that the effects of PME are attributable to some substance(s) included in the ethanol-soluble fraction of PME (PME-I).
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The leaf, stem and root of fresh Polygonum multiflorum plant were investigated. The contents of bioactive compounds and their antioxidant potential were studied by several radicals scavenging assay, and the antioxidative substances measured. The total phenolic contents were 179.0, 70.3 and 118.0 mg/g, whereas flavonoids contents were 7.3, 8.0 and 37.5 mg/g for water extracts from root (R), stem (S) and leaf (L), respectively. Further HPLC analysis revealed that the leaf and stem of this functional plant contained quercetin at 13,469 and 1,095 mg/kg, respectively. Furthermore, the root and stem contained rich emodin-related compounds that were 6,620 and 1,245 mg/kg, respectively. And different concentrations of emodin-related compounds and quercetin in root, stem and leaf showed different antioxidative ability. We observed that the DPPH scavenging activity, total antioxidant activity (TEAC), reducing power and NO scavenging ability of the three parts of the plant samples and found that the activities were R > L > S except that was S > L > R on superoxide anion scavenging effect. Our results indicated that the radical scavenging abilities of the three sample parts were more potent than that of emodin and quercetin. P. multiflorum contains rich emodin-related compounds and quer-cetin, indicating that these compounds played important roles in antioxidative effect. We suggested that the antioxidative effect of the fresh P. multiflorum plant correlate-well with emodin-related compounds and quercetin.
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Prolonged application of ammonium as a source of nitrogen leads to serious physiological and morphological disorders in many plants, including mustard (Sinapis alba L,) seedlings (ammonium toxicity syndrome). Ammonium tolerance was previously observed in mustard seedlings in the presence of considerable amounts of nitrate in the medium. In the present study, the question was addressed as to what extent accumulation of nitrate and ammonium occurs in the mustard seedling and how this relates to ammonium toxicity and tolerance. Emphasis was on light control of accumulation in the attached cotyledons. Both NQ3 and NH4 became strongly accumulated in the mustard cotyledons once the concentration in the medium exceeded 1 mM, In the cotyledons, we measured concentrations > 30 mM in the case of nitrate and > 50 mM in the case of ammonium 4 days after sowing. Accumulation of inorganic nitrogen in the mustard cotyledons did not depend on photosynthesis nor on intact chloroplasts. However, the rate of nitrate accumulation was strongly stimulated by light, operating through phytochrome, while ammonium accumulation was not affected by light in short-term experiments, i,e, within 24 h and only weakly (and probably indirectly) in long-term light.
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Primary hairy root tissues as well as aseptic hairy root culture lines contain specific compounds that have been biologically characterized as opines. These substances are agropine, mannopine, mannopinic acid, and agropinic acid; they have been synthesized and their electrophoretic behavior has been studied. Hairy root tissues also contain agrocinopines. According to the opine content of hairy root tissues, two types of Agrobacterium rhizogenes strains have been identified. Agropine-type strains (A4, 15834, HRI) elicit roots containing agropine, mannopine, mannopinic acid, and agropinic acid, whereas mannopine-type strains (8196, TR7, TR101) elicit roots containing only mannopine, mannopinic acid and agropinic acid. A. rhizogenes strains catabolize the opines whose synthesis they induce in the hairy root tissues. However, strain HRI only catabolizes agropine. Except for strain HRI, all A. rhizogenes strains studied contain three plasmids, of which the largest appears to be a cointegrate of the two others. Transconjugants of A. rhizogenes plasmids in A. tumefaciens have been obtained by selection on opines. Their properties have been studied and related to their plasmid content. In the mannopine strain C58C1(pRi8196), the virulence functions and the opine-related functions are located on the same plasmid (pRi8196). In agropine strains the catabolic functions are dissociated: agropine degradation is specified by the virulence plasmid, which also specifies opine synthesis in hairy root tissue, however, mannopine, mannopinic acid and agropinic acid degradation are specified by the smaller plasmid. Strain HRI contains only the virulence plasmid, which explains its inability to degrade mannopine, mannopinic acid, and agropinic acid.
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Centella asiatica is a herbaceous plant used in medicine for its wound-healing and anti-inflammatory properties. Its bioactive compounds are ursane-type triterpene saponins known as centellosides. With the aim of increasing the biotechnological production of these compounds, C. asiatica cell suspensions were established and treated with two concentrations (100 and 200μM) of methyl jasmonate (MeJA). The maximum centelloside production was observed in the stationary growth phase, reaching 0.16mgg−1 dry weight (DW) at day 25 of the culture in the control and 1.11mg−1 g DW at day 15 in the MeJA-elicited cultures. The elicitor did not change the centelloside pattern, with madecassoside being the main compound, followed by asiaticoside. Reverse transcription polymerase chain reaction (RT-PCR) analysis of the β-amyrin synthase gene (CabAS, the specific oxidosqualene cyclase that leads to centelloside formation) showed higher levels of expression in the elicited cultures than in the control. The maximum content of centellosides was obtained at day 15, with a time lag between gene activation and centelloside biosynthesis. In the cultures elicited with 200μM MeJA, the centelloside production did not increase compared to the control. Both elicitor concentrations decreased the content of phytosterols. Thus, MeJa elicitation in this type of culture was dose-dependent and its inducing role was apparent at low concentrations. KeywordsAsiaticoside-Cell suspension cultures- Centella asiatica -Centellosides-Madecassoside-Methyl jasmonate
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Rheum ribes L. (Polygonaceae) is the source of one of the most important crude drugs in Asiatic regions .The medicinal character of rhubarb is due to its anthraquinone content . Different parts of sterile seedlings were cultured on MS medium to study the generation of callus. The explants were cultured with different ranges of plant growth regulators and the best range of plant growth regulators for generation of callus was IBA (1 mg l–1) and BA (1 mg l–1). The content of anthraquinones were determined by HPLC . The concentration of sucrose, vitamins and Myo-inositol and ratio of NO3 to NH4 in the medium was changed and growth rate and content of 2 anthraquinones was determined . The growth rate of callus declined with increased rate of secondary metabolites production. Myo-inositol 100 mg l–1 in the medium increased anthraquinone content and in medium that had NO3/NH4:1/1 the maximum content of anthraquinone was obtained.
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In this study, methyl jasmonate (MJ)-induced changes of triterpene saponins in ginseng (Panax ginseng C.A. Meyer) hairy roots and expression profiling of relevant responsive genes were analyzed. The transcription of PgSS (squalene synthase), PgSE (squalene epoxidase), and PNA (dammarenediol synthase-II) genes in hairy root cultures elicited by MJ treatment increased as compared with the controls, whereas that of PNX (cycloartenol synthase) decreased slightly. In order to select candidate genes encoding for cytochrome P450-dependent hydroxylase or glucosyltransferase associated with triterpene biosynthesis, RT-PCR analysis was conducted following MJ elicitation. No differences were observed in any expression among the five genes associated with the cytochrome P450 family, when compared to that of control. For candidates of the glucosyltransferase gene,expression of EST IDs PG07020C06, PG07025D04, and PG07029G02 was upregulated. In an effort to assess the effects of MJ elicitation on the biosynthesis of triterpene saponin, protopanaxadiol saponin (Rb group) and protopanaxatriol saponin (Rg group) contents in hairy roots were evaluated by HPLC analysis. With 7 days of MJ elicitation, levels of all ginseonsides of the two-groups increased much higher than that observed in the control. In particular, protopanaxadiol-type saponin contents increased by 5.5–9.7 times that of the control, whereas protopanaxatriol-type saponin contents were increased by 1.85–3.82-fold. In the case of Rg1 ginsenoside after MJ elicitation, the content was affected negatively in ginseng hairy root cultures.
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To study the production of secondary metabolites of Maesalanceolata and Medicagotruncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1M sucrose. The encapsulated hairy roots were precultured for 3days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35–40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.
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Cotyledon explants of two Oriental melons produced hairy roots when cultured on Murashige and Skoog basal medium after infection by the Agrobacterium rhizogenes. Hairy roots were produced from the wounded surface of the cotyledon explants of Cucumis melo L. cv. Geumssaragi-euncheon on Murashige and Skoog selective medium and 86% of the GUS stained hairy roots were positive for the expression of beta-glucuronidase. The insertion of the gfp-gus fusion gene in the genomic DNA and the presence of the gfp-gus-specific transcript in the total RNAs of transgenic hairy roots were confirmed by PCR and RT-PCR, respectively. An immunoblot analysis of the transgenic hairy root extract revealed 97kDa single bands coincident with the molecular weight of the GFP-GUS fusion proteins. ELISA demonstrated that the highest level of GFP-GUS fusion protein expression was 0.47% of the total soluble protein in a transgenic hairy root. The MS medium showed the fastest growth among three media types tested. Infection of the hairy roots with a root-knot nematode resulted in the development of a mature egg mass about 4–5weeks after inoculation. The highest number of egg mass was obtained on the hairy roots cultured in SH medium containing 0.3% agar.
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An acidic polysaccharide, designated AEPS-1, was fractionated from the exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1 in mycelial culture. The molecular structure of AEPS-1 was characterised and elucidated by spectral and chromatographic analyses, and through derivatization by periodate oxidation, Smith degradation and methylation. AEPS-1 was composed of glucopyranose (Glcp) and pyrano-glucuronic acid (GlcUp) in an 8:1 M ratio plus a trace amount of mannose, having an average molecular weight of about 36 kDa. AEPS-1 had a linear backbone of (1→3)-linked α-d-Glcp residues with two branches, α-d-Glcp and α-d-GlcUp, attached to the main chain by (1→6) glycosidic bonds at every seventh α-d-Glcp unit. Atomic force microscopy revealed that AEPS-1 formed large networks in water that are connected primarily with triple helical strands. In Raw264.7 macrophage cell cultures, AEPS-1, at suitable doses between 25 and 250 μg/ml, significantly stimulated the release of several major cytokines, demonstrating an immunomodulatory property.
Article
In the past three decades, hairy roots research for the production of valuable biological active substances has received a lot of attention. The addition of knowledge to enhance the yields of desired substances and the development of novel tools for biomass engineering offer new possibilities for large-scale cultivation of the plant hairy root. Hairy roots can also produce recombinant proteins through the transfer of Agrobacterium T-DNA into the plant genome, and thereby hold immense potential for the pharmaceutical industry. This review highlights some of the significant progress made in the past few years and outlines future prospects for exploiting the potential utility of hairy root cultures as "chemical factories" for producing bioactive substances.
Article
Polygonum multiflorum has traditionally been used for treating patients suffering from baldness and hair loss in East Asia. The present study sought to investigate the hair growth promoting activities of Polygonum multiflorum and its mechanism of action. The Polygonum multiflorum extract was topically applied to the shaved dorsal skin of telogenic C57BL6/N mice. To determine the effect of Polygonum multiflorum extract in telogen to anagen transition, the expression of β-catenin and Sonic hedgehog (Shh) was determined by immunohistochemistry analysis. Polygonum multiflorum extract promoted hair growth by inducing anagen phase in telogenic C57BL6/N mice. In Polygonum multiflorum extract treated group, we observed increase in the number and the size of hair follicles that are considered as evidence for anagen phase induction. Immunohistochemical analysis revealed that earlier induction of β-catenin and Shh were observed in Polygonum multiflorum extract treated group compared to that in control group. These results suggest that Polygonum multiflorum extract promotes hair growth by inducing anagen phase in resting hair follicles.
Article
Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.
Article
To investigate the protective effects of preconditioning human umbilical vein endothelial cells (HUVECs) with Polygonum multiflorum stilbeneglycoside (PMS) under anoxia/reoxygenation (A/R), and the mechanism of protection. Prior to A/R, HUVECs were incubated with PMS (0.6 x 10(-11), 1.2 x 10(-11), or 2.4 x 10(-11) mol/L) for 3 h. Cell injury was subsequently evaluated by measuring cell viability with an MTT assay and lactate dehydrogenase (LDH) release, whereas lipid peroxidation was assayed by measuring malondialdehyde (MDA) content. Antioxidant capacity was quantified by superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Nitric oxide (NO) production was determined by nitrite accumulation. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein expression was detected by Western blotting. Guanylate cyclase activity and cyclic GMP (cGMP) activity were assessed by an enzyme immunoassay kit. PMS incubation attenuated A/R-induced injury in a concentration-dependent manner, as evidenced by a decrease in LDH activity and an increase in cell viability. PMS exerted its protective effect by inhibiting the A/R-mediated elevation of MDA content, as well as by promoting the recovery of SOD and GSH-Px activities. Additionally, PMS incubation enhanced NO and cGMP formation by increasing iNOS expression and guanylate cyclase activity. The protective effects of PMS were markedly attenuated by NOS inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ or PKG inhibitor KT5823. PMS preincubation resulted in the enhancement of antioxidant activity and anti-lipid peroxidation. The NO/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway was involved in the effect of PMS on HUVECs.
Article
Due to their fast growth rates and biochemical stability, 'hairy root' cultures remain unsurpassed as the choice for model root systems and have promise as a bioprocessing system. Applications are wide-ranging, from the production of natural products and foreign proteins to a model for phytoremediation of organic and metal contaminants. Hairy roots will have a continuing role as an experimental model in plant metabolic engineering.
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Dihydroartemisinic acid hydroperoxide (2) was isolated for the first time as a natural product from the plant Artemisia annua in a 29% yield. Its structure was identified by (1)H and (13)C NMR spectroscopy. Compound 2 is known as an intermediate of the photochemical oxidation of dihydroartemisinic acid (1) leading to artemisinin (3). The presence of 1 and 2 in the plant and the conditions under which 1 can be converted into 2, which can very easily oxidize to 3, provide evidence for a nonenzymatic, photochemical conversion of 1 into 3, in vivo, in the plant.
Article
The in vivo antioxidant action of a lignan-enriched extract of the fruit of Schisandra chinensis (FS) and an anthraquinone-containing extract of the root of Polygonum multiflorum (PME) was compared with their respective active constituents schisandrin B (Sch B) and emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and carbon tetrachloride (CCl4)-intoxicated mice. FS and PME pretreatments produced a dose-dependent protection against CCl4 hepatotoxicity, with the effect of FS being more potent. Pretreatment with Sch B, emodin or α-tocopherol (α-Toc) also protected against CCl4 hepatotoxicity, with the effect of Sch B being more potent. The extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl4 toxicity was found to correlate well with the degree of enhancement in hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in reduced glutathione level and activities of glutathione reductase, glutathione peroxidase as well as glutathione S-transferases, in both control and CCl4-intoxicated mice. α-Toc, which did not enhance mitochondrial glutathione antioxidant status, seemed to be less potent in protecting against CCl4 hepatotoxicity. The ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than α-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin. Abbreviations ALT:alanine aminotransferases CCl4:carbon tetrachloride FS:lignan-enriched extract of Schisandra fruit GRD:glutathione reductase GSH:reduced glutathione GSH-Px:Se-glutathione peroxidase GST:glutathione S-transferases mt:mitochondrial MDA:malondialdehyde PME:anthraquinone-containing fraction of Polygonum root Sch B:schisandrin B SDH:sorbitol dehydrogenase α-Toc:α-tocopherol
Article
Polygonum multiflorum stilbeneglycoside (PMS) is a water-soluble fraction of Polygonum multiflorum Thunb., one of the most famous tonic traditional Chinese medicines, that has protective effects on the cardiovascular system. The purpose of the present study is to elucidate the effects of PMS on macrophage-derived foam cell functions and the reduction of severity of atherosclerosis in hypercholesterolemic New Zealand White (NZW) rabbits. NZW rabbits were fed for 12 weeks with a normal diet, a high cholesterol diet, or a high cholesterol diet associated with irrigation with different doses of PMS (25, 50, or 100 mg/kg). Treatment of NZW rabbits fed with high cholesterol diet with 100 mg/kg PMS attenuated the increase in plasma cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, and plasma triglyceride. Treatment with 50 and 100 mg/kg PMS caused 43% and 60% decrease in atherosclerotic lesioned area ratio to total surface area, respectively. In U937 foam cells, PMS could decrease the high expression of intercellular adhesion molecule (ICAM)-1 protein and the vascular endothelial growth factor (VEGF) protein levels in the medium induced by oxidized lipoprotein when analyzed by flow cytometry. The results proved that PMS is a powerful agent against atherosclerosis and that PMS action could possibly be through the inhibition of the expression of ICAM-1 and VEGF in foam cells.
Callus culture of Polygonum multiforum and the production of anthraquinones
  • RM Yu
  • H Zhang
  • JQ Chen
  • LP Dong
A revised medium for rapid growth and bioassays with tobacco tissue cultures
  • T Murashige
  • F Skoog