Lactobacilli and human dental caries: more than mechanical retention.

Lactobacilli have been considered as major contributors to human dental caries for over a century. Recent in vitro model studies have shown that when compared to Streptococcus mutans, a keystone pathogen of human dental caries, the ability of lactobacilli to form biofilms is poor, although differences exist between the different major species. Further studies using molecular and bioinformatics approaches provide evidence that multiple mechanisms, including adhesin-receptor mediated physical contact with S. mutans, facilitate the adherence and establishment of lactobacilli on the tooth surface. There is also evidence that under conditions like continuous sugar consumption, weak acids and other antimicrobials such as bacteriocins from lactobacilli can become detrimental to the microbial community, especially those in the proximity. Details on the underlying mechanisms of how different Lactobacillus sp. establish and persist in the highly complex microbiota on the tooth surface await further investigation.

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.  However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.  Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.  It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Deficiency of BrpA in Streptococcus mutans reduces virulence in rat caries model.

Our recent studies have shown that BrpA in Streptococcus mutans plays a critical role in cell envelope biogenesis, stress responses, and biofilm formation. In this study, a 10-species consortium was used to assess how BrpA deficiency influences the establishment, persistence, and competitiveness of S. mutans during growth in a community under conditions typical of the oral cavity. Results showed that, like the wild-type, the brpA mutant was able to colonize and establish on the surfaces tested. Relative to the wild-type, however, the brpA mutant had a reduced ability to persist and grow in the 10-species consortium (P < .001). A rat caries model was also used to examine the effect of BrpA, as well as Psr, a BrpA paralog, on S. mutans cariogenicity. The results showed no major differences in infectivity between the wild-type and the brpA and psr mutants. Unlike the wild-type, however, infection with the brpA mutant, but not the psr mutant, showed no significant differences in both total numbers of carious lesions and caries severity, compared with the control group that received bacterial growth medium (P > .05). Metagenomic and quantitative polymerase chain reaction analysis showed that S. mutans infection caused major alterations in the composition of the rats' plaque microbiota and that significantly less S. mutans was identified in the rats infected with the brpA mutant compared with those infected with the wild-type and the psr mutant. These results further suggest that BrpA plays a critical role in S. mutans pathophysiology and that BrpA has potential as a therapeutic target in the modulation of S. mutans virulence.

Synthesis, antibacterial activity, and biocompatibility of new antibacterial dental monomers.

PURPOSE: To synthesize a small library of antibacterial dental monomers based on quaternary ammonium salts and to test their antibacterial activity against cariogenic bacteria. METHODS: Five new antibacterial monomers were synthesized and characterized by NMR, IR and HRMS. RESULTS: Cytotoxicity assays using human gingival fibroblast cells showed that these new antibacterial monomers were biocompatible at concentrations of 10⁻5 M and displayed less cytotoxicity than BisGMA, a common dental monomer. When analyzed in vitro, all new monomers demonstrated strong inhibitory activity against biofilm formation by cariogenic Streptococcus mutans and Lactobacillus casei. Results indicated that antibacterial monomers containing a long alkyl (i.e. hexadecyl) chain are superior to their shorter-chain counterparts. The cross-linking monomers based on glycerol dimethacrylate also consistently outperformed their monomethacrylate analogs. Finally, the ammonium salts containing the dimethylbenzyl moiety were superior to the similar structures containing 1,4-diazabicyclo[2.2.2]octane (DABCO) in some cases. CLINICAL SIGNIFICANCE: All five new monomers were deemed biocompatible at concentrations of 10⁻5 M or less, and most had better biocompatibility than BisGMA. Dimethacrylate monomers 5 and 6 generally demonstrated high antibacterial activities, with the highest activity shown for the most lipophilic monomer 6, and these new antibacterial monomers have potential future application in dental composites and bonding agents.

Deficiency of RgpG Causes Major Defects in Cell Division and Biofilm Formation, and Deficiency of LytR-CpsA-Psr Family Proteins Leads to Accumulation of Cell Wall Antigens in Culture Medium by Streptococcus mutans.

Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG, encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, "squarish" dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation (P < 0.01). Double and triple mutants with deficiency in brpA and/or psr, genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically (P < 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope.IMPORTANCEStreptococcus mutans, a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans, indicative of a significant role of RGP in cell division and biofilm formation in S. mutans These results are novel not only in S. mutans, but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans This study reveals new potential targets to develop anticaries therapeutics.

Photo-cross-linked Antibacterial Zein Nanofibers Fabricated by Reactive Electrospinning and its Effects against Streptococcus mutans.

Native zein electrospun nanofibers have shown poor solvent resistance and low mechanical strength. Compared to other toxic cross-linkers, a safer method of stabilizing zein based fibers while retaining or with improved mechanical strength is needed to convert these materials for biomedical applications where culture media or body fluids may be present. We report here a method of fabricating non-toxic zein nanofibers using reactive electrospinning coupled with in situ photo-cross-linking. The cross-linked zein nanofibers exhibited significantly improved mechanical strength and sustained morphology against water and aqueous ethanol solution. This process doesn't require additional conventional cross-linking agents to form cross-linking network, which is advantageous for biomedical applications. Antimicrobial monomer with photo-reactive moiety was coupled with methacrylate zein nanofibers and showed strong inhibitory activity against cariogenic Streptococcus mutans. Cytotoxicity test with human gingival fibroblasts revealed high biocompatibility.

Streptococcus mutans Displays Altered Stress Responses While Enhancing Biofilm Formation by Lactobacillus casei in Mixed-Species Consortium.

Like Streptococcus mutans, lactobacilli are commonly isolated from carious sites, although their exact role in caries development remains unclear. This study used mixed-species models to analyze biofilm formation by major groups of oral lactobacilli, including L. casei, L. fermentum, L. rhamnosus, L. salivarius ssp. salivarius, and L. gasseri. The results showed that lactobacilli did not form good biofilms when grown alone, although differences existed between different species. When grown together with S. mutans, biofilm formation by L. gasseri and L. rhamnosus was increased by 2-log (P < 0.001), while biofilms by L. fermentum reduced by >1-log (P < 0.001). L. casei enhanced biofilm formation by ~2-log when grown with S. mutans wild-type, but no such effects were observed with S. mutans deficient of glucosyltransferase GtfB and adhesin P1. Both S. mutans and L. casei in dual-species enhanced resistance to acid killing with increases of survival rate by >1-log (P < 0.001), but drastically reduced the survival rates following exposure to hydrogen peroxide (P < 0.001), as compared to the respective mono-species cultures. When analyzed by RNA-seq, more than 134 genes were identified in S. mutans in dual-species with L. casei as either up- or down-regulated when compared to those grown alone. The up-regulated genes include those for superoxide dismutase, NADH oxidase, and members of the mutanobactin biosynthesis cluster. Among the down-regulated genes were those for GtfB and alternative sigma factor SigX. These results further suggest that interactions between S. mutans and oral lactobacilli are species-specific and may have significant impact on cariogenic potential of the community.

Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6.

BACKGROUND: Cross-linking of IgE antibody by specific epitopes on the surface of mast cells is a prerequisite for triggering symptoms of peanut allergy. IgE epitopes are frequently categorized as linear or conformational epitopes. Although linear IgE-binding epitopes of peanut allergens have been defined, little is known about conformational IgE-binding epitopes. OBJECTIVE: To identify clinically relevant conformational IgE epitopes of the two most important peanut allergens, Ara h 2 and Ara h 6, using phage peptide library. METHODS: A phage 12mer peptide library was screened with allergen-specific IgE from 4 peanut-allergic patients. Binding of the mimotopes to IgE from a total of 29 peanut-allergic subjects was measured by ELISA. The mimotope sequences were mapped on the surface areas of Ara h 2 and Ara h 6 using EpiSearch. RESULTS: Forty-one individual mimotopes were identified that specifically bind anti- Ara h 2/Ara h 6 IgE as well as rabbit anti-Ara h 2 and anti-Ara h 6 IgG. Sequence alignment showed that none of the mimotope sequences match a linear segment of the Ara h 2 or Ara h 6 sequences. EpiSearch analysis showed that all the mimotopes mapped to surface patches of Ara h 2 and Ara h 6. Eight of the mimotopes were recognized by more than 90% of the patients, suggesting immunodominance. Each patient had distinct IgE recognition patterns but the recognition frequency was not correlated to the concentration of peanut specific IgE or to clinical history. CONCLUSIONS: The mimotopes identified in this study represent conformational epitopes. Identification of similar surface patches on Ara h 2 and Ara h 6 further underscores the similarities between these two potent allergens.

PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a)-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v) after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans.

Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans.

Streptococcus mutans, the primary aetiological agent of dental caries, possesses an YjeE-like protein that is encoded by locus SMU.409, herein designated brpB. In this study, a BrpB-deficient mutant, JB409, and a double mutant deficient of BrpB and BrpA (a paralogue of the LytR-CpsA-Psr family of cell wall-associated proteins), JB819, were constructed and characterized using function assays and microscopy analysis. Both JB409 and JB819 displayed extended lag phases and drastically slowed growth rates during growth in brain heart infusion medium as compared to the wild-type, UA159. Relative to UA159, JB409 and JB819 were more than 60- and 10-fold more susceptible to acid killing at pH 2.8, and more than 1 and 2 logs more susceptible to hydrogen peroxide, respectively. Complementation of the deficient mutants with a wild-type copy of the respective gene(s) partly restored the acid and oxidative stress responses to a level similar to the wild-type. As compared to UA159, biofilm formation by JB409 and JB819 was drastically reduced (P<0.001), especially during growth in medium containing sucrose. Under a scanning electron microscope, JB409 had significantly more giant cells with an elongated, rod-like morphology, and JB819 formed marble-like super cells with apparent defects in cell division. As revealed by transmission electron microscopy analysis, BrpB deficiency in both JB409 and JB819 resulted in the development of low electron density patches and formation of a loose nucleoid structure. Taken together, these results suggest that BrpB likely functions together with BrpA in regulating cell envelope biogenesis/homeostasis in Strep. mutans. Further studies are under way to elucidate the mechanism that underlies the BrpA- and BrpB-mediated regulation.

Psr is involved in regulation of glucan production, and double deficiency of BrpA and Psr is lethal in Streptococcus mutans.

Streptococcus mutans, the primary causative agent of dental caries, contains two paralogues of the LytR-CpsA-Psr family proteins encoded by brpA and psr, respectively. Previous studies have shown that BrpA plays an important role in cell envelope biogenesis/homeostasis and affects stress responses and biofilm formation by Strep. mutans, traits critical to cariogenicity of this bacterium. In this study, a Psr-deficient mutant, TW251, was constructed. Characterization of TW251 showed that deficiency of Psr did not have any major impact on growth rate. However, when subjected to acid killing at pH 2.8, the survival rate of TW251 was decreased dramatically compared with the parent strain UA159. In addition, TW251 also displayed major defects in biofilm formation, especially during growth with sucrose. When compared to UA159, the biofilms of TW251 were mainly planar and devoid of extracellular glucans. Real-time-PCR and Western blot analyses revealed that deficiency of Psr significantly decreased the expression of glucosyltransferase C, a protein known to play a major role in biofilm formation by Strep. mutans. Transmission electron microscopy analysis showed that deficiency of BrpA caused alterations in cell envelope and cell division, and the most significant defects were observed in TW314, a Psr-deficient and BrpA-down mutant. No such effects were observed with Psr mutant TW251 under similar conditions. These results suggest that while there are similarities in functions between BrpA and Psr, distinctive differences also exist between these two paralogues. Like Bacillus subtilis but different from Staphylococcus aureus, a functional BrpA or Psr is required for viability in Strep. mutans.

The redox-sensing regulator Rex modulates central carbon metabolism, stress tolerance response and biofilm formation by Streptococcus mutans.

The Rex repressor has been implicated in regulation of central carbon and energy metabolism in gram-positive bacteria. We have previously shown that Streptococcus mutans, the primary causative agent of dental caries, alters its transcriptome upon Rex-deficiency and renders S. mutans to have increased susceptibility to oxidative stress, aberrations in glucan production, and poor biofilm formation. In this study, we showed that rex in S. mutans is co-transcribed as an operon with downstream guaA, encoding a putative glutamine amidotransferase. Electrophoretic mobility shift assays showed that recombinant Rex bound promoters of target genes avidly and specifically, including those down-regulated in response to Rex-deficiency, and that the ability of recombinant Rex to bind to selected promoters was modulated by NADH and NAD(+). Results suggest that Rex in S. mutans can function as an activator in response to intracellular NADH/NAD(+) level, although the exact binding site for activator Rex remains unclear. Consistent with a role in oxidative stress tolerance, hydrogen peroxide challenge assays showed that the Rex-deficient mutant, TW239, and the Rex/GuaA double mutant, JB314, were more susceptible to hydrogen peroxide killing than the wildtype, UA159. Relative to UA159, JB314 displayed major defects in biofilm formation, with a decrease of more than 50-fold in biomass after 48-hours. Collectively, these results further suggest that Rex in S. mutans regulates fermentation pathways, oxidative stress tolerance, and biofilm formation in response to intracellular NADH/NAD(+) level. Current effort is being directed to further investigation of the role of GuaA in S. mutans cellular physiology.

Synthesis and characterization of antibacterial dental monomers and composites.

The objective of this study is to synthesize antibacterial methacrylate and methacrylamide monomers and formulate antibacterial fluoride-releasing dental composites. Three antibacterial methacrylate or methacrylamide monomers containing long-chain quaternary ammonium fluoride, 1,2-methacrylamido-N,N,N-trimethyldodecan-1-aminium fluoride (monomer I), N-benzyl-11-(methacryloyloxy)-N,N-dimethylundecan-1-aminium fluoride (monomer II), and methacryloxyldecylpyridinium fluoride (monomer III) have been synthesized and analyzed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The cytotoxicity test and bactericidal test against Streptococcus mutans indicate that antibacterial monomer II is superior to monomers I and III. A series of dental composites containing 0-6% of antibacterial monomer II have been formulated and tested for degree of conversion (DC), flexure strength, water sorption, solubility, and inhibition of S. mutans biofilms. An antibacterial fluoride-releasing dental composite has also been formulated and tested for flexure strength and fluoride release. The dental composite containing 3% of monomer II has a significant effect against S. mutans biofilm formation without major adverse effects on its physical and mechanical properties. The new antibacterial monomers can be used together with the fluoride-releasing monomers containing a ternary zirconiun-fluoride chelate to formulate a new antibacterial fluoride-releasing dental composite. Such a new dental composite is expected to have higher anticaries efficacy and longer service life.

Novel amelogenin-releasing hydrogel for remineralization of enamel artificial caries.

Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels.

Replication or death: distinct fates of pathogenic Leptospira strain Lai within macrophages of human or mouse origin.

Pathogenic leptospires evoke severe diseases in humans but only cause mild chronic or asymptomatic infection in many host animals. The reasons for this diversity of infection remain unclear. Here, we demonstrated that Leptospira interrogans serovar Lai strain Lai had a similar ability to adhere to and enter primary and immortal (THP-1 and J774A.1) macrophages from human and mouse, but its intracellular fate in human macrophages differed markedly from that in mouse. The leptospires resided within membrane-bound vacuoles in the murine macrophages, but occurred free in the cytosol of human macrophages, with no surrounding vesicular membrane. Most leptospires in murine macrophages co-localized with the late-endosomal/lysosomal marker LAMP-1 and then were killed by lysosomal hydrolases, while most leptospires in human macrophages did not co-localize with this marker and survived. Enumeration of colony-forming units plus quantitative fluorimetry showed that in human, but not in murine, macrophages, the amounts of leptospires increased with incubation time. The infected human macrophages differed from mouse macrophages by displaying gradually enhanced apoptosis, in parallel with the increase in number of leptospires. These data strongly suggest that the outcome for intracellular leptospires depends on differences among host macrophages, which may account for some of the differences in the severity of leptospirosis in humans and animals.

Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai.

BACKGROUND: Pathogenic Leptospira species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in Leptospira, and paricularly in pathogenicity remains unknown. RESULTS: A suicide plasmid for the fliY gene of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (bla) was constructed, and the inactivation of fliY gene in a mutant (fliY-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of fliP and fliQ genes which are located downstream of the fliY gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased. CONCLUSION: Inactivation of fliY, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of fliY inactivation, but also a consequence of the polar effects.

[Analysis on the volatile oil of Xinhui Pericarpium Citri Reticulatae in different years by GC/MS].

OBJECTIVE: To analyze the difference of the volatile oil of Xinhui Pericarpium Citri Reticulatae in different years. METHODS: The components and contents of the volatile oil of Xinhui Pericarpium Citri Reticulatae were analyzed in different years by GC/MS. RESULTS: The components and contents of the volatile oil of Xinhui Pericarpium Citri Reticulatae changed along with the years. CONCLUSION: With the years' extension, the components of the volatile oil changed slowly during 3 years but quickly after 3 years, a few new components appeared evenly, So there's relevance between using old Xinhui Pericarpium Citri Reticulatae and its component changing.