Two Chloranthus The Chemical Composition Of Species And Biological Activity

Chloranthus japonicus Sieb. and Chloranthus multistachys Pei, which belong to the genus Chloranthus in Chloranthaceae family are two perennial herbaceous plants. As folklore medicines native to the Qinling Mountains of China, the whole plants and their roots have been traditionally used for hundreds of years to treat boils, dermatopathia, enteric fever, detumescence, snake bite, bone fractures, cough, and rheumatic pain. Their various bioactivities have now gained attention from researchers because of their antifungal, cytotoxic, hepatoprotective, and tyrosinase-inhibitory properties. However, the active constituents and the mechanism for their function still remain unclear. In this study, we used anatomical, histochemical, phytochemical, pharmacological and toxicological methods were used to investigate structure, chemical composition and biological activities of C. japonicus and C. multistachys. The research will be beneficial to the further exploitation and utilization of the Chloranthus plants. The main results are as follows:1. The morphological structure of vegetative organs of C. japonicus and C. multistachys were analyzed and compared. The main similarities were as follows:cortexs in the root and the rhizome were broad; stems got large hollow pith cavities; leaves had the bifacial structure, palisade tissue and sponge tissue were clearly differentiated, and leaf veins had a narrow phloem and a developed xylem; there were no hairs on the upper and lower epidermis of the leaf, the stomatal apparatuses fell into anomocytic type and occurs only on the lower epidermis. In addition, there were some obvious differences in the structures of two species. In C. japonicus, the root cortex cells were arranged in order and had smaller intercellular spaces, and the root phloem was narrow; the stem cortex cells were also arranged in order, and the stem phloem was surrounded by strands of fiber; the leaf sponge tissue of were composed of cells with various shapes, which had a small intercellular space, there were parenchyma cells with irregular shapes, which have large intercellular spaces inside collenchyma of main leaf vein, and the epidermis cells of the main vein had no papilla. In C. multistachys, however, the root cortex cells got arranged irregularly and had large intercellular spaces, the root phloem was broad as well; the stem cortex cells were misaligned, with its phloem surrounded by a few layers of fiber; the leaf sponge tissue was composed of round-like cells which had large intercellular space; there were nearly round parenchyma cells which had small intercellular space inside the collenchyma of main leaf vein, and the epidermis cells of the main vein had obvious papilla.2. The histochemistry results of terpenoids and flavonoids localization were similar in different vegetative organs in C. japonicus and C. multistachys, both of which were mainly distributed in the cortex of root, the cortex of stem and phloem cell. Otherwise, the only difference just lied in their contents in two species. Terpenoids were dyed reddish in the cortex parenchyma stem cells and phloem cells in C. japonicus, and was dyed reddish brown in C. multistachys, which indicated that C. multistachys. contained more terpenoids than those in C. japonicus. Flavonoids showed strong yellow-green fluorescence in the cortex cells of root and displayed yellow-green fluorescence in stem cortex and phloem cells in C. japonicus. On the contrary, in C. multistachys, strong green fluorescence was shown in the cortex cells and blue-green fluorescence in stem cortex and phloem cells. From the above, the conclusion could be drawn that the contents of flavonoids in C. multistachys were more than those in C. japonicus, which was consistent with the phytochemical results.3. The essential oils of C.japonicus and C. multistachys were obtained by hydrodistillation and analyzed by GC and GC-MS. In all of them,48 and 39 compounds were identified, accounting for 95.56% and 94.58% of all constituents in the oil from C.japonicus and C. multistachys, respectively. Up to 28 compounds were found in common between the two oils, which featured a high relatively content of monoterpenes and their oxygenated monoterpenes (77.04%,46.64%). Bornyl acetate was the most abundant compound in these oils, which ranged from 30.98% to 35.99%. In addition, the telling distinguishing feature was that there was a high proportion of myristicin in C. multistachys.4. The fat-soluble components in C. japonicus and C. multistachys were extracted by soxlet extraction, and then esterified and analyzed by GC-MS. There were twenty-four fat-soluble components detected and identified in C. japonicus, amounting to 99.03% of the total contents, among which the saturated fatty acid and unsaturated fatty acid were 22.32% and 70.02%, respectively. There are fifteen fat-soluble components detected and identified in C. multistachys, amounting to 92.59% of the total contents, among which the saturated fatty acid and unsaturated fatty acid were 15.73% and 54.71%, respectively.5. The sample solution were analyzed by a Phenomenex C18 column (250mm×4.6mm,5μm) with a gradient mobile phase composed of methanol and 0.1% formic acid solution. Both DAD and mass spectrometry detector were used simultaneously, full-scan detection mode was evaluated for the identification of LC peaks. As a result, gallic acid, caffeic acid, isofraxidin and rosmarinic acid were identified; in addition, the contents of gallic acid, caffeic acid, and rosmarinic acid were higher in the methanol extract of C. multistachys (0.372mg/g, 0.111mg/g, and 2.073mg/g) than those of C. japonicus (1.173mg/g,0.173mg/g, and 7.228mg/g). The amount of isofraxidin in C.japonicus and C. multistachys were 0.507 mg/g and 0.292mg/g, respectively. Significant difference was found in contents of phenols and counarins in the different parts of C. japonicus and C. multistachys. The contents of gallic acid in C. japonicus and C. multistachys were both stems>roots>leaves, and the content in the stems of C. multistachys were much more than those in C. japonicus. The contents of caffeic acid and rosmarinic acid in C. japonicus and C. multistachys were both roots>leaves>stems, and the rosmarinic acid content were 34.623 mg/g and 1.432 mg/g in the roots and lesves of C. japonicus, respectively, but 4.582 mg/g and 23.572 mg/g in C. multistachys. The contents of isofraxidin in C. japonicus and C. multistachys were both roots>stems >leaves,0.601mg/g and 0.094 mg/g in the roots of the two plants, respectively.6. Ultrasound-assisted extraction response surface method (RSM) was applied to optimize the extraction condition of the total flavonoids in both C. japonicus and C. multistachys. The influence factors of methanol concentration, extraction temperature, extraction time, and ratio of material to solvent were evaluated using a fractional factorial design. The experimental data were disposed by Design-Expert 7.1.3 program. The optimum extraction conditions were confirmed as follows:In C. japonicus, methanol concentration 57%, ratio of material to solvent 1:17, extraction time 6 min, extraction temperature 60℃; In C. multistachys, methanol concentration 70%, ratio of material to solvent 1:8, extraction time 28 min, extraction temperature 27℃. The extraction rate of flavonoids under this condition agreed with the experimental values.7. The microwave digestion and flame atomic absorption spectrophotometry (FAAS) were employed to determine the macro and trace elements (iron, zinc, copper, manganese, calcium, potassium, sodium and magnesium) in C. japonicus and C. multistachys. The results revealed that the order of concentrations of macro and trace elements in C. japonicus and C. multistachys were K>Ca>Na>Mg>Fe>Zn>Mn>Cu and K>Ca>Mg>Na>Fe>Mn>Zn>Cu, respectively. On the whole, the contents of these elements in C. japonicus were higer than those in C.multistachys.8. Antimicrobial properties of the essential oils of C. japonicus and C. multistachys were evaluated in vitro via disc-diffusion and microbroth dilution assays. Activities were strong against 15 tested microorganisms (8 gram-positive bacteria,3 gram-negative bacteria,3 yeasts). Oil from C. japonicus was generally more efficient for inhibiting bacterial growth. MIC and MBC determinations indicated that gram-positive bacteria and yeasts were more sensitive to both oils than were gram-negative bacteria. The oil of C. japonicus showed the strongest bactericidal activity against B. cereus, as evidenced by the lowest values for MIC (0.39 mg/mL) and MBC (0.78 mg/mL). In contrast, oil from C. multistachys exerted the strongest bactericidal activity against B. cereus and Candida lipolytica, with the lowest MIC (0.78 mg/mL) and MBC (1.56 mg/mL), respectively. C. japonicus oil was stronger against B. cereus, B. coagulans, and Sarcina lutea than was C. multistachys, but the former oil was weaker against Rhodotorula glutinis.9. The antioxidant activities of different extracts of C. japonicus and C. multistachys were evaluated with several antioxidant testing systems such as DPPH radical scavenging assay, superoxide radical scavenging assay,β-carotene-linoleic acid assay (β-CLA), and a plasmid DNA damage protection potential assay. The results revealed that the methanol extracts of the two plants showed higher antioxidant activity than other extracts.10. Karber's method, histological and immunohistochemical method were used to explore the acute toxicity of the methanol extracts of C. japonicus and C. multistachys in mice. The results showed that the 50% lethal dose (LD50) were 50.91g/kg (42.25-61.34g/kg) and 45.74 g/kg (42.60-49.04 g/kg), respectively. According to the toxicity grading criteria, C. japonicus and C. multistachys were slightly and moderately poisonous medicinal plants. The main acute toxic effects were enhanced in a dose-dependent manner. The methanol extracts of C. japonicus and C. multistachys acted on target organs and tissues including kidney, liver, heart, spleen and systemic blood-vessel.11. To investigate the analgesic and anti-inflammatory activity of the methanol extracts of C. japonicus and C. multistachys, the researcher used the acetic acid induced writhing test, hot p late test and formalin test to determine this effect and its analgesic type. The anti-inflammatory effect was determined by using the model of xylene-induced mice ear edema and increased vascular permeability induced by histamine phosphate in mice. The results showed that oral administration of extraction from C. japonicus and C. multistachys could improve the mice's pain threshold, significantly inhibit the inflammatory edema induced by various inflammatory agents and reduce the times of body writhing induced by acetic acid in mice. In addition, the extraction was remarkably active in the second phase of formalin-induced pain. C. japonicus and C. multistachys might have peripheral and central analgesic properties and have marked anti-inflammatory activity both on the acute phase.