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Objective: To study the anti-lipopolysaccharide (LPS) effect of the ultrafine granular powder of Houttuynia cordata and the effects in comparison with traditional decoction pieces. Methods: BALB/c mice were injected with LPS through nose to establish lung inflammation model. The number of leukocytes in mice whole blood was examined, and the degree of the inflammation of lung tissue by pathology was observed. Rat inflammatory model was induced by injection of LPS into tail vein. The number of leukocytes in mice whole blood, content of LPS in plasma were examined, the content of IL-1β, IL-6, and TNF-α in serum were detected by enzyme-linked immunoadsordent assay (ELISA). The limulus test method was used to detect the anti- lipopolysaccharide effect of the ultrafine granular powder of H. cordata in vitro. Results: The ultrafine granular powder of H. cordata and traditional decoction pieces can reduce the level of leukocytes in mice whole blood in various degrees, and alleviated the infiltration of inflammatory cells in pathological lung tissue, and there was a positive dosage dependent relation. For the two decoction pieces, the number of leukocytes in rat whole blood, the content of LPS in plasma, the levels of IL-1β, IL-6, and TNF-α in serum decreased in different degrees were found. Compared with traditional decoction pieces group, the content of IL-1β and TNF-α of serum in ultrafine granular powder group were significant decreased. The ultrafine granular powder of H. cordata showed better anti-inflammatory activity than traditional decoction pieces in vitro at the same concentration and same dilution ratio. Conclusion: The ultrafine granular powder of H. cordata has satisfactory anti-lipopolysaccharide effect, and the effect is better than traditional decoction pieces in some extent.
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AIM To prepare the thermosensitive intestinal gels of Houttuynia cordata Thunb volatile oils hydroxypropyl-β-cyclodextrin (HPCD) inclusion compound.METHODS For the gels prepared by cold dissolving method,poloxamer 407 consumption and poloxamer 188 consumption were taken as influencing factors,together with phase transition temperature as an evaluation index,central composite design-response surface method was applied to optimizing the formulation.With 2-undecanone as an index component,the gels' dissolution rate and in vitro release rate were investigated by non-membrane dissolution method and dialysis bag method respectively,whose stability was then evaluated by high temperature (40,60 ℃),low temperature (4 ℃),strong light [(4 500 ±500) 1x] and acceleration (three months) tests.RESULTS The optimal conditions were determined to be 20.61％ for P407 consumption and 3.03％ for P188 consumption,the phase transition temperature was 36.5 ℃.Within the time range of 30-150 min,the HPCD inclusion compound gels exhibited higher accumulative dissolution rate than the volatile oils gels,which tended to be consistent in 150-210 min,but the former exhibited higher accmulative release rate (0-50 h) than the latter all the time.The obtained gels showed good stability at low temperature,whose appearance,characteristic (except for high temperature) and pH were stable at high temperature,strong light and acceleration with obviously decreased 2-undecanone content.CONCLUSION The thermosensitive intestinal gels of Houttuynia cordata Thunb volatile oils HPCD inclusion compound should be stored at low temperature (4 ℃).
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Objective To clone and analyze the expression difference sequence of 3-hydroxy-3-methylglutaryl coenzyme A redutase (HMGR) gene from Houttuynia cordata. Methods The sequence of HMGR was cloned from H. cordata by RT-PCR strategy. The physical and chemical properties, secondary structure, and three-dimensional structure of the HMGR protein were forecasted and analyzed, and its structure and function were predicted. And the different expression of HMGR gene in rhizome, stems, leaves, and flowers were analyzed by fluorescent quantitative PCR. Results The cDNA contained a 1 626 bp open reading frame and encoded a predicted protein of 541 amino acids. Two transmembrane regions and no signal peptide were present in HMGR. Relative real-time PCR analysis indicated that HMGR showed the highest transcript abundance in the flowers, and the lowest levels in the rhizomes. Conclusion This study cloned and expression analyzed HMGR gene from H. cordata for the first time. The result will provide a foundation for exploring the mechanism of terpenoid biosynthesis in H. cordata.
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Abstract The aim of this paper is to identify and investigate an endophytic fungus (strain 28) that was isolated from Houttuynia cordata Thunb, a famous and widely-used Traditional Chinese Medicine. Based on morphological methods and a phylogenetic analysis of ITS sequences, this strain was identified as Chaetomium globosum. An antifungal activity bioassay demonstrated that the crude ethyl acetate (EtOAc) extracts of strain 28 had a wide antifungal spectrum and strong antimicrobial activity, particularly against Exserohilum turcicum (Pass.) Leonard et Suggs, Botrytis cinerea persoon and Botrytis cinerea Pers. ex Fr. Furthermore, the fermentation conditions, extraction method and the heat stability of antifungal substances from strain 28 were also studied. The results showed that optimal antifungal activity can be obtained with the following parameters: using potato dextrose broth (PDB) as the base culture medium, fermentation for 4–8 d (initial pH: 7.5), followed by extraction with EtOAc. The extract was stable at temperatures up to 80 °C. This is the first report on the isolation of endophytic C. globosum from H. cordata to identify potential alternative biocontrol agents that could provide new opportunities for practical applications involving H. cordata.
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Chaetomium/isolation & purification , Chaetomium/metabolism , Houttuynia/microbiology , Endophytes/metabolism , Antifungal Agents/metabolism , Phylogeny , Chaetomium/classification , Chaetomium/genetics , Endophytes/isolation & purification , Endophytes/classification , Endophytes/genetics , Fungi/growth & development , Fungi/drug effects , Antifungal Agents/pharmacologyABSTRACT
Objective: Simple sequence repeats (SSR) loci information in the transcriptome of Houttuynia cordata was analyzed in this study and the more powerful tools were provided for molecular marker-assisted breeding in this plant. Methods: SSR loci were searched in all 63 954 unigenes by using MISA. SSR loci information was analyzed and SSR primers were designed by Primer 3. Furthermore, 50 pairs of primers were randomly selected for the polymorphic analysis on 16 H. cordata plants collected from different habitats. Results: A total of 4 800 SSRs were found in the transcriptome of H. cordata, which distributed in 4 413 unigenes with the distribution frequency of 7.51%. Tri-nucleotide repeat was the main type, accounted for as much as 41.54% of all SSRs, followed by mono-nucleotide repeat motif (27.35%). The mononucleotide repeat motifs of A/T were the predominant repeat type (27.0%). A total of 3068 pairs of SSR primers were designed by using Primer 3. For validating the availability of those SSR primers, 50 pairs of primers were randomly selected for PCR amplification. Among them, 43 pairs of primers (86.0%) produced clear and reproductive bands, which showed polymorphism, and 16 H. cordata plants collected from different places were divided into two groups by UPGMA. Conclusion: There are numerous SSRs in H. cordata transcriptome with high frequency and various types, this will provide the basis for study on genetic diversity and genetic map for H. cordata.
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Objective: For rapid identification of Houttuynia cordata and Gymnotheca chinensis, the specific PCR for mutual authentication of them was established based on the SNPs in matK sequence. Methods: H. cordata and G. chinensis samples from different origins were collected, total DNA of all samples was extracted, and the matK gene was seqenced. SNPs in the matK sequences of all the samples were found by ClustulX 2.1 program. Primers for identifying H. cordata and G. chinens were designed according to the SNP site, and specific PCR method was established to identify them, for rapid detection by addition of SYBR Green I dye. In addition, constructing a multi-PCR reaction system, and then the PCR reaction system was optimized. Results: The band special for H. cordata (185 bp) and band special for G. chinensis (389 bp) were found using specific PCR reaction and multi-PCR reaction, and SYBR Green I dye can be used for rapid detection. Conclusion: The multi-PCR reaction system could be used to identify H. cordata and G. chinensis.
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Objective: Acetyl-CoA C-acetyltransferase (AACT) is the initial enzyme in the terpenoid biosynthesis pathway of mevalonate (MVA), two units of acetyl-CoA were catalyzed to acetoacetyl-CoA. To clone the full length cDNA of AACT gene and carry out the bioinformatics analysis and expression analysis in order to provide the basis on resolving the mechanism of biosynthesis for terpenoid secondary metabolites from Houttuynia cordata. Methods: The cDNA sequence of AACT gene was obtained from H. cordata by using RT-PCR strategy. And the different expression of AACT gene in the rhizomes, stems, leaves, and flowers of H. cordata was analyzed by fluorescent quantitative PCR. Results: The cDNA contains a 1 218 bp open reading frame and encodes a predicted protein of 405 amino acids. No transmembrane region and signal peptide were present in AACT protein by bioinformatics prediction. Relative real-time PCR analysis indicated that AACT gene showed the highest transcript abundance in the stems and rhizomes of H. cordata lower levels in the flowers and leaves, the values of them were 1.49, 0.96, 0.20, and 0.10, respectively. Conclusion: This AACT gene is cloned from H. cordata for the first time. The results will provide a foundation for exploring the mechanism of the gene in terpenoid biosynthesis and metabolism in H. cordata.
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Objective: To clone the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene from Houttuynia cordata and analyze the differential expression. Methods: The cDNA sequence of DXR was cloned from H. cordata by using RT-PCR strategy. The physical and chemical properties, secondary structure, and three-dimensional structure of the DXR protein were forecasted and analyzed, and its function was predicted. The differential expression of DXR gene in rhizome, stems, leaves, and flowers was analyzed by fluorescent quantitative PCR. Results: The cDNA contained a 1 416 bp open reading frame and encoded a predicted protein of 471 amino acids. Bioinformatics predicted that no transmembrane region and signal peptide were present in DXR. Relative real-time PCR analysis indicated that DXR showed the highest transcript abundance in leaves, moderate level in rhizomes, lower level in stems, and the lowest level in flowers. Conclusion: This study clones DXR gene from H. cordata for the first time, and provides a foundation for exploring the mechanism of this gene for the terpenoid biosynthesis in H. cordata.
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Objective: To clone the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) gene from Houttuynia cordata and to analyze the expression difference. Methods: The cloning primers were designed based on the transcriptome dataset of H. cordata, one unique sequence encoding DXS1 was discovered. The sequence of DXS1 was cloned from H. cordata by RT-PCR. The physicochemical properties, secondary structure, and three-dimensional structure of the DXS1 protein were forecasted and analyzed, and its structure and function were predicted. And the different expression levels of DXS1 gene in rhizome, stems, leaves, and flowers were analyzed by fluorescent quantitative PCR. Results: The cDNA (named as DXS1) contains a 2 172 bp open reading frame and encodes a predicted protein of 723 amino acids. No transmembrane region and signal peptide were present in DXS1. The conserved domain of DXS was present in DXS1. Relative real-time PCR analysis indicated that DXS1 showed the highest transcript abundance in the flowers, moderate level in the leaves, lower level in the rhizomes, and the lowest level in the stems. Conclusion: This study cloned the DXS1 gene from H. cordata for the first time. The results will lay a foundation for exploring the mechanism of terpenoid biosynthesis in H. cordata plants.
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Objective To explore the effects of volatile oil and 2-undecanone from Houttuynia Cordata Thunb. (H. cordata) on LPS-TLR4 / MD-2-TNF-α signaling pathway. Methods TLR4 / MD-2 blocking agent was used to mask the TLR4 /MD-2 site,then protein expression levels of TLR4 in cells treated with volatile oil and 2-undecanone were analyzed by western blot. ELISA was used to detect the secretion of the inflammatory cytokines such as TNF-α,IL-1β and IL-10. Comparison analysis was then performed from the results of cell experiments in vitro and anti-inflammatory effects through xylene-induced ear edema test in vivo. Results In concentrations between 1 to 10 μg · mL-1 ,Houttuynia volatile oil showed better effect than 2-undecanone on inhibition of TLR4 protein in LPS-induced RAW264. 7 cells, and had some differences in the effects on inflammatory factors. Compared with the LPS+TLR4 / MD-2 group,the LPS+TLR4 / MD-2 + volatile oil group had no significant difference in the expression of TLR4 protein (P>0. 05),but the LPS+TLR4 / MD-2 +2-undecanone group reduced the expression of TLR4 protein obviously. It appeared that volatile oil exerts its anti-inflammatory effect through LPS-TLR4 / MD-2-TNF-αpathway,but 2-undecanone may exert its anti-inflammatory effect by other means. Houttuynia volatile oil showed better anti-inflammatory activity than 2-undecanone in vivo at the same dose. Conclusion There are some differences in anti-inflammatory effects and related mechanisms between volatile oil and 2-undecanone, probably owing to the synergistic effects of multi-ingredients in the volatile oil.
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Objective: To clone and sequence the chalcone synthase1 (CHS1) gene from the leaves of Houttuynia cordata. Methods: The cloning primers were designed on the basis of the conserved sequences of the cloned CHS gene in other plants. The total RNA was extracted from the leaves of H. cordata. The sequence of CHS gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) and SON-PCR techiniques, and then the gene was ligated with pMD18-T Simple vector. The positive clone was sequenced after the identification of clone by PCR. Results: We cloned a fragment of 1 188 bp. The analysis of sequencing indicated that the fragment encoded 395 amino acids and shared the sequence homology of more than 62.3% with CHS gene sequences from other higher plants. Bioinformatic analysis indicated that the CHS amino acids had no signal peptide sequences, but possessed the CHS family's characteristic sequences, RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Relative real-time PCR analysis indicated that CHS1 showed the highest transcript abundance in the flowers, moderate levels in the stems, lower levels in the rhizomes, and the lowest levels in the leaves. Conclusion: It is the first report that a novel CHS gene is cloned from H. cordata, which lays a foundation for the effective use of CHS1 gene.
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Development of a therapy providing protection from, or reversing gentamicin-sulfate (GS)-induced oxidative stress and nephrotoxicity would be of great clinical significance. The present study was designed to investigate the protective effects of Houttuynia cordata Thunb. (HC) against gentamicin sulfate-induced renal damage in rats. Twenty-eight Sprague-Dawley rats were divided into 4 equal groups as follows: group 1, control; group 2, GS 100 mg/kg/d, intraperitoneal (i.p.) injection; group 3, GS 100 mg/kg/d, i.p. + HC 500 mg/kg/d, oral; and group 4, GS 100 mg/kg/d i.p. + HC 1000 mg/kg/d, oral administration). Treatments were administered once daily for 12 d. After 12 d, biochemical and histopathological analyses were conducted to evaluate oxidative stress and renal nephrotoxicity. Serum levels of creatinine, malondialdehyde (MDA), and blood urea nitrogen (BUN), together with renal levels of MDA, glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were quantified to evaluate antioxidant activity. Animals treated with GS alone showed a significant increase in serum levels of creatinine, BUN, and MDA, with decreased renal levels of GSH, SOD, and CAT. Treatment of rats with HC showed significant improvement in renal function, presumably as a result of decreased biochemical indices and oxidative stress parameters associated with GS-induced nephrotoxicity. Histopathological examination of the rat kidneys confirmed these observations. Therefore, the novel natural antioxidant HC may protect against GS-induced nephrotoxicity and oxidative stress in rats.
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Animals , Cats , Rats , Blood Urea Nitrogen , Catalase , Creatinine , Drug Combinations , Gentamicins , Glutathione , Glycerides , Houttuynia , Kidney , Malondialdehyde , Monoterpenes , Oxidative Stress , Rats, Sprague-Dawley , Superoxide DismutaseABSTRACT
Objective To study the genetic diversity of different geographical populations of Houttuynia cordata in China.Methods The genetic diversity of 15 geographical populations of H.cordata from 13 provinces in China was estimated using amplified fragment length polymorphism(AFLP) markers.The data of amplified bands were analyzed by the software POPGENE and MEGA.Results The ten AFLP primers employed produced a total of 110 discernable and reproduceable amplified fragments.The percentage of polymorphic bands within different populations was 70.51%.Genetic diversity analysis showed that effective number of alleles(Ne) was 1.210,Nei′s gene diversity(H) was 0.119,and Shannon′s genetic diversity index(I) was 0.186.The coefficient of genetic distance was 0.008 9—0.181 8 among populations.A UPGMA dendrogram based on Nei′s(1972) genetic distance visualized that the 15 populations were grouped into three different clusters,Emei population was one individual cluster group and the other populations were grouped into two different clusters corresponding to the different geographical areas.Conclusion The genetic diversity within different geographical populations of H.cordata in China is plentiful.
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Objective Taking methyl nonyl ketone as index to establish pharmacokinetic compartment model for study volatile oil from the whole herb of Houttuynia cordata by pharmacokinetic parameters in rabbits in vivo. Methods Using GC to determine the blood concentrations of methyl nonyl ketone in the volatile oil from whole herbs of H. cordata in rabbit plasma. The GC system used a capillary SPB-5 column and helium as carrier gas; the initial temperature was at 90 ℃, then raised to 190 ℃ by 10 ℃/min and kept for 5 min; the injector temperature was 250 ℃ and the FID detector temperature was 300 ℃; the injection volume was 5.0 ?L with splitless injection mode; n-hexane was used as solvent and n-pentadecane as interior standard substance. The blood concentration value of methyl nonyl ketone was fitted with 3P97 procedure. Results A capillary GC method was established for the detemination of methyl nonyl ketone in the volatile oil from whole herbs of H. cordata in rabbit plasma. Methyl nonyl ketone linearity was good (r=0.987 0) within 0.04—60.0 ?g/mL.The blood concentration-time course of precision in rabbit plasma conformed to two compartment model. Conclusion The experiment provides pharmacokinetic evidence for the rational administration and the further development of H. cordata and some methods for pharmacokinetic study of volatile oils from traditional Chinese medicinal materials.
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Object To study the relationship between the essential oil constituents and the chromosome numbers of Houttuynia cordata and compare the difference of the essential oil constituents from the fresh and the dried ones, aerial and underground parts of the fresh plant. Methods The essential oil constituents of H. cordata with different chromosome numbers from the aerial and underground parts of the fresh, and the aerial parts dried in shade as well were analyzed by TLC. Results According to the difference of the spot number and definition of the essential oil TLC pattern, the essential oil constituents in the aerial parts of 20 fresh plants were classified into five types, those in their underground parts into three types, and those in 19 dried plants into four types. Conclusion There is obvious difference in the essential oil constituents between the fresh aerial and underground parts, and between the fresh and dried plants. The difference among the fresh plants is more significant than that of the dried ones. And the difference among the aerial parts of the fresh plants is more significant than that of the underground parts. There is a tendency that the spot numbers increase with the chromosome numbers. The difference of essential oil constituents in H. emeiensis and H. cordata is insignificant.
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Object To explore the bacteriostatic and anti inflammation activities of ultrasonically nebulized Houttuynai cordata Thunb in patients after pneumonectomy Methods The patients were randomized into 2 groups Patients of the experimental group, inhaled ultrasonically nebulized H cordata, while patients in the control group inhaled ultrasonically nebulized vapors without the addition of H cordata Results Total leucocyte counts on the third and fourth day in the experimental group were significantly lower than that in the control group Neutrophil ratio in experimental group was also lower in all the 5 days of treatment although nonsignificantly Meanwhile, occurrence of Gram negative bacilli colonization in experimental group occurred less than that in the control group But incidence of fungi colonization appeared higher Conclusion Inhalation of ultrasonically nebulized H cordata can ease the inflammation and inhibit the colonization of Gram negative bacilli in respiratory tract