| Paridis rhizoma recorded in Chinese pharmacopoeia (2010) are dry rhizomes derived from Paris polyphylla Smith var. yunnanensis (Franch.) Hand.-Mazz. or Paris polyphylla Smith var. chinensis (Franch.) Hara. belonging to genus Paris in the Liliaceae family. Rich chemical components existed in the plants of genus Paris, including sterol, steroline, flavonoid glycoside, steroidal saponins, amino acid, fatty acid, microelements, polysaccharide, and etc. Among of them, steroidal saponins are the main chemical components, which have important physiological activities related to antitumor, inflammation, bacteriostasis, sedation, hemostasis, and used to treat many type of cancers, such as uterine hemorrhage, chronic bronchitis, epidemic parotitis, and etc. In order to clarify the differences of chemical constituents and biological activities between the two varieties of P. polyphylla, the characteristic of morphological structure, chemical compositions and biological activities, and properties of DNA fingerprinting and chemical fingerprinting for both of P. polyphylla Smith var. yunnanensis and P. polyphylla Smith var. chinensis were studied, using phytohistochemistry, pharmacology, DNA fingerprinting and chemical fingerprinting technology. The results could make clarify chemical compositions and biological activities, and be beneficial to the further exploitation and utilization of the Paridis rhizome resources. The main results are as follows:1. The compositions and contents of fat-soluble constituents of P. polyphylla Smith var. yunnanensis and P. polyphylla Smith var. chinensis were studied. The results showed that the composition of two varieties of P. polyphylla were consistent, containing 11 kinds of common components, such as palmitic acid, stearic acid, and etc., but different on relative contents. Fatty acid was the main component, and the contents of UFA (75.55% and 66.64%) were higher than SFA (22.81% and 30.13%) in both kinds of P. polyphylla.2. The contents of 7 inorganic elements in two varieties of P. polyphylla were determined by FAAS (Flame Atomic Absorption Spectrometry) with microwave digestion. The results showed that the contents of the macro element in P. polyphylla Smith var. yunnanensis (Ca>K>Mg) were higher than those of P. polyphylla Smith var. chinensis (K>Ca>Mg). In addition to Cu, the trace elements from the former were higher than the latter, and the order of the contents were Fe>Zn>Mn>Cu.3. Total polyphenols were determined by the Folin-Ceocalteu method for P. polyphylla Smith var. yunnanensis and P. polyphylla Smith var. chinensis. The results showed that the total polyphenols contents of two varieties of P. polyphylla were as follows:EAF>CME> WF>BF. And the total polyphenols contents of the latter were higher than those of the former, and the content of total polyphenol in EF of P. polyphylla Smith var. chinensis is 22.15±0.08 mg/g which is the highest one in all of the extracts.4. The polyphyllin were extracted by the heating reflux method, the ultrasonic extraction method and the Soxhlet extraction method, from both P. polyphylla Smith var. yunnanensis and P. polyphylla Smith var. chinensis. The results showed that three kinds of extraction methods could obtain similar extraction ratio, but the heating reflux method was the best method. The polyphyllins extracted by the heating reflux method P. polyphylla Smith var. yunnanensis were 1.81 times than that of P. polyphylla Smith var. chinensis.5. In order to clarify the distribution of polyphyllin in P. polyphylla, we used P. polyphylla Smith var. chinensis as an example to investigate the morphological structure and histochemical location of polyphyllin. The results showed that the morphological structures of P. polyphylla Smith var. chinensis was similar to those of P. polyphylla Smith var. yunnanensis, and polyphyllin existed in all of vegetative organs, and the concentration of polyphyllins in vegetative organs were as follows:rhizome> stem> adventitious root>leaf.6. The antioxidant activities in vitro of the extracts obtained from two varieties of P. polyphylla were evaluated, by use of DPPH-radical scavenging activities,β-carotene-linoleic acid assay, ferric reducing/antioxidant power assay and superoxide radical scavenging activities. The results showed that different extracts from two varieties of P. polyphylla exhibited antioxidant activities, and the antioxidant activities of P. polyphylla Smith var. chinensis were higher than those of P. polyphylla Smith var. yunnanensis.7. The antimicrobial activities in vitro of the extracts obtained from two varieties of P. polyphylla were evaluated via disc-diffusion and microbroth dilution. The antimicrobial activities of EAF from P. polyphylla var. chinensis were stronger than those of P. polyphylla Smith var. yunnanensis. The antimicrobial activities in P. polyphylla Smith var. yunnanensis were as follows:Klebsiella pneumoniae (MIC=50.00mg/mL)> Bacillus subtillis>Escherichia coli. Otherwise, the antimicrobial activities of EAF from P. polyphylla var. chinensis were as follows:B. subtillis (MIC=0.78mg/mL, MBC=50.00mg/mL)>Bacillus coagulans (MIC=0.78mg/mL, MBC=50.00mg/mL)>Staphylococcus aureus>E. coli. (MIC=1.56mg/mL, MBC=50.00mg/mL)>K. pneumoniae (MIC=1.56mg/mL, MBC=50.00mg/mL).8. The effects of total saponin from two varieties of P. polyphylla on proliferation and apoptosis of Caco-2 cell line were discussed. The results showed the effect of total saponin from P. polyphylla var. chinensis on proliferation of Caco-2 cell line was weaker than that of P. polyphylla Smith var. yunnanensis, and the inhibitory action of the latter enhanced with the extension of time and increasing of concentration of total saponin. From a series of experiment results, including morphological structure observation, cell cycle analysis, Caspase-3 activities assay, mitochondrial membrane potential determination, it was hypothesised that apoptosis of Caco-2 cells could be induced via mitochondria way by the total saponin of P. polyphylla Smith var. yunnanensis.9. The AFLP fingerprint of P. polyphylla germplasms was established. The results showed that 1059 bands containing 1019 polymorphic bands were generated by AFLP from the 27 P. polyphylla germplasms using the 8 pairs of primer, and all of the 27 P. polyphylla germplasms shared 40 common bands. The same primer combinations generated 63,60 and 31 specific bands in the germplasms of P. polyphylla Smith var. yunnanensis, P. polyphylla var. chinensis and P. polyphylla Smith var. polyphylla, respectively. The AFLP fingerprints could effectively distinguish 3 kinds of class groups. And the highest percentage of polymorphic marks was 90.80%for P. polyphylla Smith var. yunnanensis, P. polyphylla var. chinensis (89.01%) was the second, and the last one was P. polyphylla Smith var. polyphylla (81.90%). The results of cluster analysis showed that the genetic similarity was higher (0.51-0.79) for 3 kinds of class groups, which supported the results of morphological classification.10. The ISSR fingerprint of P. polyphylla germplasms was established. The results showed that 71 bands containing 70 polymorphic bands were generated by ISSR from 27 P. polyphylla germplasms using 8 primers, which displayed abundant genetic diversity. The ISSR fingerprints could distinguish effectively 3 kinds of class groups. The highest percentage of polymorphic marks was 96.77% for P. polyphylla var. chinensis, P. polyphylla Smith var. polyphylla (96.23%) was the second, and the third one was P. polyphylla Smith var. yunnanensis (93.55%). The results of cluster analysis showed that the levels of hereditary variation in species of P. polyphylla var. polyphylla (0.57) were higher than those of P. polyphylla Smith var. yunnanensis (0.67) and P. polyphylla var. chinensis (0.61), and they were similar for hereditary variation.11. The HPLC fingerprint of 44 germplasms of P. polyphylla from Shaanxi, Hunan, Sichuan and Yunnan provinces were established. The results illustrated that the similarity coefficient of 44 batches of paridis rhizoma were between 0.854 and 0.998, and the polyphyllin contents of all paridis rhizomas exceeded the standard of Chinse pharmacopoeia (2010). The polyphyllin contents of paridis rhizomas obtained in Shaanxi (4.66%) were lower than those of Hunan (8.58%), Sichuan (11.90%) and Yunnan (9.24%). The polyphyllin content of the paridis rhizomas in Sichuan was the highest one. The saponin contents of P. polyphylla Smith var. yunnanensis (9.97%) were higher than those of P. polyphylla Smith var. chinensis (5.40%). The HPLC fingerprint established in this experiment was more information, reliable and rapid, could reflect roundly the characters of chemical fingerprint, and could be used to control and evaluate the quality of paridis rhizomas. |
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