Apigenin-7-O-ß-D-glycoside isolation from the highly copper-tolerant plant Elsholtzia splendens.

Elsholtzia splendens (Lamiaceae) is a copper-tolerant plant species growing on copper deposits in the south of China. Chromatographic separation of n-BuOH extracts from the flowering aerial biomass afforded apigenin-7-O-ß-D-glycoside, using macroporous resin, Sephadex™ LH-20 gel, polyamide resin as well as preparative high-performance liquid chromatography (P-HPLC) columns. Chemical structure was elucidated using HPLC/ESI-MS (electrospray ionization-mass spectrometry), Fourier transform infrared (FTIR), and (1)D- and (2)D-nuclear magnetic resonance (NMR). Apigenin-7-O-ß-D-glycoside could be the post-harvesting product from E. splendens biomass.

Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas.

Elsholtzia splendens (ES) is, rich in flavonoids, used to repair copper contaminated soil in China, which has been reported to benefit cardiovascular systems as folk medicine. However, few direct evidences have been found to clarify the vasorelaxation effect of total flavonoids of ES (TFES). The vasoactive effect of TFES and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFES (5-200mg/L) caused a concentration-dependent vasorelaxation in endothelium-intact rings, which was not abolished but significantly reduced by the removal of endothelium. The nitric oxide synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (100µM) and the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (30µM) significantly blocked the endothelium-dependent vasorelaxation of TFES. Meanwhile, NOS activity in endothelium-intact aortas was concentration-dependently elevated by TFES. However, indomethacin (10µM) did not affect TFES-induced vasorelaxation. Endothelium-independent vasorelaxation of TFES was significantly attenuated by KATP channel blocker glibenclamide. The accumulative Ca(2+)-induced contraction in endothelium-denuded aortic rings primed with KCl or phenylephrine was markedly weakened by TFES. These results revealed that the NOS/NO/cGMP pathway is likely involved in the endothelium-dependent vasorelaxation induced by TFES, while activating KATP channel, inhibiting intracellular Ca(2+) release, blocking Ca(2+) channels and decreasing Ca(2+) influx into vascular smooth muscle cells might contribute to the endothelium-independent vasorelaxation conferred by TFES.

Improved cadmium uptake and accumulation in the hyperaccumulator Sedum alfredii: the impact of citric acid and tartaric acid.

The elucidation of a natural strategy for metal hyperaccumulation enables the rational design of technologies for the clean-up of metal-contaminated soils. Organic acid has been suggested to be involved in toxic metallic element tolerance, translocation, and accumulation in plants. The impact of exogenous organic acids on cadmium (Cd) uptake and translocation in the zinc (Zn)/Cd co-hyperaccumulator Sedum alfredii was investigated in the present study. By the addition of organic acids, short-term (2 h) root uptake of (109)Cd increased significantly, and higher (109)Cd contents in roots and shoots were noted 24 h after uptake, when compared to controls. About 85% of the (109)Cd taken up was distributed to the shoots in plants with citric acid (CA) treatments, as compared with 75% within controls. No such effect was observed for tartaric acid (TA). Reduced growth under Cd stress was significantly alleviated by low CA. Long-term application of the two organic acids both resulted in elevated Cd in plants, but the effects varied with exposure time and levels. The results imply that CA may be involved in the processes of Cd uptake, translocation and tolerance in S. alfredii, whereas the impact of TA is mainly on the root uptake of Cd.

Extraction and isolation of the salidroside-type metabolite from zinc (Zn) and cadmium (Cd) hyperaccumulator Sedum alfredii Hance.

The active metabolite in the post-harvested biomass of zinc (Zn) and cadmium (Cd) hyperaccumulator Sedum alfredii Hance from phytoextraction is of great interest in China. The current study demonstrates that a salidroside-type metabolite can be yielded from the Zn/Cd hyperaccumulator S. alfredii biomass by means of sonication/ethanol extraction and macroporous resin column (AB-8 type) isolation. The concentrations of Zn and Cd in the salidroside-type metabolite were below the limitation of the national standards.

Caffeic acid product from the highly copper-tolerant plant Elsholtzia splendens post-phytoremediation: its extraction, purification, and identification.

In the current study, caffeic acid was an important metabolite in the highly copper-tolerant plant Elsholtzia splendens. Preparation and purification of caffeic acid were performed on the dried biomass of the plants by means of sonication/ethanol extraction, followed by purification using a macroporous resin (D101 type) column and silica gel chromatography. The faint-yellow caffeic acid product was yielded with a purity of 98.46%, and it was chemically identified from spectra of Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance ((1)H NMR)/carbon nuclear magnetic resonance ((13)C NMR), and electrospray ionization mass spectrometry (ESI-MS). Caffeic acid is a possible product from the post-harvest processing of Elsholtzia splendens biomass.

Colonic exclusion and combined therapy for refractory constipation.

AIM: To investigate the therapeutic effectiveness of colonic exclusion and combined therapy for refractory constipation. METHODS: Thirty-two patients with refractory constipation were randomly divided into treatment group (n = 14) and control group (n = 18). Fourteen patients in treatment group underwent colonic exclusion and end-to-side colorectal anastomosis. Eighteen patients in control group received subtotal colectomy and end-to-end colorectal anastomosis. The therapeutic effects of the operations were assessed by comparing the surgical time, incision length, volume of blood losses, hospital stay, recovery rate and complication incidence. All patients received long-term follow-up. RESULTS: All operations were successful and patients recovered fully after the operations. In comparison of treatment group and control group, the surgical time (h), incision length (cm), volume of blood losses (mL), hospital stay (d) were 87 +/- 16 min vs 194 +/- 23 min (t = 9.85), 10.4 +/- 0.5 cm vs 21.2 +/- 1.8 cm (t = 14.26), 79.5 +/- 31.3 mL vs 286.3 +/- 49.2 mL (t = 17.24), and 11.8 +/- 2.4 d vs 18.6 +/- 2.6 d (t = 6.91), respectively (P < 0.001 for all). The recovery rate and complication incidence were 85.7% vs 88.9% (P = 0.14 > 0.05), 21.4% vs 33.3% (P = 0.73 > 0.05), respectively. CONCLUSION: Colonic exclusion has better therapeutic efficacy on refractory constipation. It has many advantages such as shorter surgical time, smaller incision, fewer blood losses and shorter hospital stay.

Phytoextraction of copper from contaminated soil by Elsholtzia splendens as affected by EDTA, citric acid, and compost.

Phytoextraction of copper (Cu) from contaminated soils greatly depends on the metal bioavailability in the soils and metal uptake ability of the plant. In this study, the effects of chelators [ethylenediamine tetraacetic acid (EDTA), citric acid (CA)] and compost amendments on Cu phytoextraction potential by a tolerant and accumulating plant species (E. splendens) were examined in two types of contaminated soils, ie., the mined soil from Cu-mined area (MS) and a paddy soil polluted by Cu refining (PS). The results showed that EDTA application at 2.5-5.0 mmol kg(-1) increased phytoextraction of Cu by four- and eight-fold from both MS and PS, respectively, which is mainly attributed to increased H2O extractable Cu in the soil. The Cu amount extracted by the shoots of E. splendens reached 800-1000 microg Cu plant(-1) from the MS and 400-700 microg Cu plant(-1) from the PS at EDTA application rates of 2.5-5.0 mmol kg(-1). The application of CA at 5.0 mmol kg(-1) had minimal effects on Cu extractability in both soils and slightly decreased Cu extraction efficiency by E. splendens. Plant biomass production was enhanced by CA at 0.25 mmol L(-1) in nutrient solution, but inhibited by CA at 5.0 mmol kg(-1) in both MS and PS. Increasing the compost rate significantly decreased H2O extractable Cu in the MS, but raised H2O-extractable Cu in the PS, which resulted mainly front the reduced exchangeable Cu in the MS and the increased exchangeable and organic fractions of Cu in the PS by compost. At high compost rate (5%), the shoots of E. splendens extracted 3.6-fold higher Cu from the PS than from the MS. These results indicate that, among the soil amendments, efficiency of Cu phytoextraction is enhanced mostly by 2.5-5.0 mmol kg(-1) EDTA, followed by 5% (w:w) compost, whereas < 5.0 mmol kg(-1) CA has minimal effects on Cu phytoextraction by E. splendens in the PS. As for the MS, only 2.5-5.0 mmol kg(-1) EDTA can elevate the efficiency of Cu, while 5% compost amendment and < 5.0 mmol kg(-1) CA application have no marked effects on Cu phytoextraction by E. splendens.

Changes of root morphology and Pb uptake by two species of Elsholtzia under Pb toxicity.

Elsholtzia argyi and Elsholtzia splendens, which are Chinese endemic Pb/Zn mined and Cu mined ecotype respectively, were investigated in the aspect of their response to Pb toxicity in the presence or absence of EDTA addition. After 8 d's Pb treatment, root length, root surface area and root volume of E. splendens decreased much more than those of E. argyi, and reduced considerably with increase of Pb, while no marked change was noted for root average diameter. Compared to E. argyi, length of root with diameter (D)<0.2 mm was significantly reduced for E. splendens as Pb increasing. Root with cross-sectional area of D<0.1 mm for E. splendens was at Pb> or =10 mg/L, while for E. argyi, it was at Pb> or =25 mg/L. DW of E. splendens decreased much more than that of E. argyi with increase of Pb. E. argyi exhibited much more tolerance to Pb toxicity than E. splendens. Treatment with 100 mg/L Pb plus 50 mmol/L EDTA significantly decreased the length and surface area of D< or =0.2 mm root, increased the length and surface area of 0.2< or =D< or =0.8 mm root for the case of E. argyi, while for E. splendens, length and surface area of D<0.6 mm root reduced, as compared to 100 mg/L Pb treatment, alone. At 100 mg/L Pb, shoot Pb accumulation in E. splendens and E. argyi were 27.9 and 89.0 microg/plant DW respectively, and much more Pb was uptaken by the root and translocated to the stem of E. argyi as compared to E. splendens. Treatment of the plant with 100 mg/L Pb plus 50 mmol/L EDTA increase leaf Pb accumulation from 16.8 to 84.9 g/plant for E.splendens and from 18.8 to 52.5 g/plant for E. argyi, while both root and stem Pb pronouncedly reduced for both Elsholtzia species. The increased translocation of Pb to the leaf of E. splendens than that of E. argyi at the treatment of 100 mg/L Pb plus 50 mmol/L EDTA should be further investigated.

Accumulation and ultrastructural distribution of copper in Elsholtzia splendens.

Copper accumulation and intracellular distribution in Elsholtzia splendens, a native Chinese Cu-tolerant and accumulating plant species, was investigated by transmission electron microscope (TEM) and gradient centrifugation techniques. Copper concentrations in roots, stems and leaves of E. splendens increased with increasing Cu levels in solution. After exposure to 500 micromol/L Cu for 8 d, about 1000 mg/kg Cu were accumulated in the stem and 250 mg/kg Cu in the leaf of E. splendens. At 50 micromol/L Cu, no significant toxicity was observed in the chloroplast and mitochondrion within its leaf cells, but separation appeared at the cytoplasm and the cell wall within the root cells. At >250 micromol/L Cu, both root and leaf cell organelles in E. splendens were damaged heavily by excessive Cu in vivo. Copper subcellular localization in the plant leaf after 8 days' exposure to 500 micromol/L Cu using gradient centrifugation techniques was found to be decreased in the order: chloroplast>cell wall>soluble fraction>other organelles. The plant root cell wall was found to be the site of highest Cu localization. Increase of Cu exposure time from 8 d to 16 d, increased slightly Cu concentration in cell wall fraction in roots and leaves, while that in the chloroplast fraction decreased in leaves of the plants grown in both 0.25 micromol/L and 500 micromol/L Cu. TEM confirmed that much more Cu localized in cell walls of E. splendens roots and leaves, but also more Cu localized in E. splendens' chloroplast when the plant is exposed to Cu levels>250 micromol/L, as compared to those in the plant grown in 0.25 micromol/L Cu. Copper treatment at levels>250 micromol/L caused pronounced damage in the leaf chloroplast and root organelles. Copper localization in cell walls and chloroplasts could mainly account for the high detoxification of Cu in E. splendens.

Copper phytoavailability and uptake by Elsholtzia splendens from contaminated soil as affected by soil amendments.

Pot and field experiments were conducted to evaluate bioavailability of Cu in contaminated paddy soil (PS) and phytoremediation potential by Elsholtzia splendens as affected by soil amendments. The results from pot experiment showed that organic manure (M) applied to the PS not only remarkably raised the H2O exchangeable Cu, which were mainly due to the increased exchangeable and organic fractions of Cu in the PS by M, but also stimulated plant growth and Cu accumulation in E. splendens. At M application rate of 5.0%, shoot Cu concentration in the plant increased by four times grown on the PS, so as to the elevated shoot Cu accumulation by three times as compared to the control. In the field trial, soil amendments by M and furnace slag (F), and soil preparations like soil capping (S) and soil discing (D) were performed in the PS. Soil capping and discing considerably declined total Cu in the PS. Application of M solely or together with F enhanced plant growth and increased H2O exchangeable Cu levels in the soil. The increased extractability of Cu in the rhizosphere of E. splendens was noted, which may have mainly attributed to the rhizospheric acidification and chelation by dissolved organic matter (DOM), thus resulting in elevating Cu uptake and accumulation by E. splendens. Amendments with organic manure plus furnace slag (MF) to the PS caused the highest exactable Cu with saturated H2O in the rhizospheric soil of E. splendens after they were grown for 170 days in the PS, thus achieving 1.74 kg Cu ha(-1) removal from the contaminated soil by the whole plant of E. splendens at one season, which is higher than those of the other soil treatments. The results indicated that application of organic manure at a proper rate could enhance Cu bioavailability and increase effectiveness of Cu phytoextraction from the contaminated soil by the metal-tolerant and accumulating plant species (E. splendens).

Volatile constituents in the flowers of Elsholtzia argyi and their variation: a possible utilization of plant resources after phytoremediation.

Phytoremediation effectiveness and remediation costs are driving factors of this project. Full utilization of plant resources after their being used for phytoremediation is an unsolved problem. GC/MS technique was used to investigate the volatiles of the flowers from Elsholtzia argyi (PFE1: Purple Flower Elsholtzia) and their variation (WFE: White Flower Elsholtzia), naturally growing in Pb/Zn mined area, and Elsholtzia argyi (PFE2: Purple Flower Elsholtzia), naturally growing in Jiuxi uncontaminated agriculture soil. Seventeen compounds constituting 86.88% of total essential oils were identified in PFE1, with 2,6-octadienoic acid,3,7-dimethyl-methyl ester being the main constituent (63.30%). Sixteen compounds accounting for 95.32% of total essential oils were identified in WFE, with caryophyllene being the main component (55.02%). Compared to PFE1, PFE2 contains lower level of 2,6-octadienoic acid,3,7-dimethyl-methyl ester (31.76%), which is the main constituent in the total essential oils of PFE2. Caryophyllene is the main ingredient of flavor. Elsholtzia ketone was identified in all the three Elsholtzia plants. It can be concluded that the selected Elsholtzia argyi plants can be exploited on their versatile uses as fragrances and antiseptics due to the perfume ingredient and antibacterial components existing in their essential oils.

Gama-aminobutyric acid accumulation in Elsholtzia splendens in response to copper toxicity.

A solution with different Cu supply levels was cultured to investigate gama-aminobutyric acid (GABA) accumulation in Elsholtzia splendens, a native Chinese Cu-tolerant and accumulating plant species. Increasing Cu from 0.25 to 500 micromol/L significantly enhanced levels of GABA and histidine (His), but considerably decreased levels of aspartate (Asp) and glutamate (Glu) in the leaves. The leaf Asp level negatively correlated with leaf Cu level, while leaf GABA level positively correlated with leaf Cu level. The leaf Glu level negatively correlated with leaf GABA level in Elsholtzia splendens. The depletion of leaf Glu may be related to the enhanced synthesis of leaf GABA under Cu stress.