| Hibiscus Sabdariffa L.(Roselle) is a member of natural plant which has been used as both common food and herbal medicine. Besides, it is also a kind of traditional economic crops, whose calyces, seeds, stems and leaves are all available for use, among which the dried or sold calyx is the most valuable. The main constituents of Hibiscus Sabdariffa L. are organic acids, anthocyanins, phenols, flavonoids, polysaccharides and volatile compounds. Hibiscus Sabdariffa L., who is cold-natured, has been widely consumed in the indigenous areas as folk drink for their distinctive tastes of sour and refreshing, with those effects on qingre, removing the heat, spot elimination, lowering lipid, losing weight, detoxification hangover and sober-up. In addition, Hibiscus Sabdariffa L. is clinically a potential medicine used to treat hypertension, cancer and scurvy, to relieve bronchitis and coughing, and to prevent neurological and cardiovascular diseases. However, there are relatively lack of researches on essential oils and polysaccharides of Hibiscus Sabdariffa L. at present. This project systematically explores both the extraction and chemical composition analysis for the essential oils and the extraction, separation, purification and structure identification for the polysaccharides isolated from Hibiscus Sabdariffa L.. Moreover, the evaluation of the anti-inflammatory and immune activity of essential oils and polysaccharides, respectively, as well as some other biological activities was explored. The main results are as follows:The essential oils of Hibiscus Sabdariffa L. calyces(HSO) were extracted by two extraction methods of steam distillation(SD) and solid phase micro-extraction(HS-SPME), and the sample extracted by the former method was re-treated by ultrasound or microwave. As a whole, there received four kinds of HSO samples. The chemical composition analysis in these four HSO was conducted by gas chromatography-mass spectrometry(GC-MS). As a result, there are significant differences in compounds content and species between the two kinds of extraction methods. A total of 20 kinds of component was detected in HSO-Ⅰextracted by SD, in which the content of n-Hexadecanoic acid(39.19%) is the highest, while HSO- Ⅳ was represented by Androst-4-ene-3,17-dione(8.68%) and Methyl hexadecanoate(5.65%) with relative less categories. On the contrary, there was almost no change in the species of the main chemical composition, but just slight content differences after retreatment using ultrasound or microwave.Antioxidant, antitumor, antibacterial and anti-inflammatory activities of HSO-Ⅰ extracted by SD was investigated. Results showed that the FRAP value of the whole tested concentration of HSO-Ⅰ were very low, and so were the scavenging rate of both DPPH and ABTS free radicals, suggesting its relative poor antioxidant capacity. The inhibition rate on both MCF-7 and PC3 cells proliferation intervened by HSO-Ⅰ was increased in a dose dependent manner at first, and then tended to smooth after reaching a certain concentration. At the concentration of 500 μg/m L, the inhibition rate could be up to 92.41% and 86.72%, respectively, that was much better than the positive control of 5-fluorouracil(5-FU), while the effect on HepG2 was relatively poor. In the antibacterial experiment, the bacteriostasis assay results showed there were different antibacterial effect on those tested strains, among which, HSO was moderately sensitive to Staphylococcus aureus strain, low sensitive to Escherichia coli and Salmonella typhi strains, and insensitive to Pseudomonas coli and Salmonella typhi strains. Cell viability assay and the inhibition on NO release showed HSO-Ⅲ was both non-toxic and had a remarkable inhibition effect on NO release. the further research found that it could also effectively restrain the TNF-α、iNOS、IL-1、IL-6 and COX-2 mRNA expression and cytokines secretion of IL-6 and IL-1, additionally, HSO-Ⅲ could significantly suppress the protein expression of P-ERK1/2, P-JNK and P-P65 activated in LPS-induced RAW264.7 macrophages, suggesting that HSO-Ⅲ might play the potential and significant role in anti-inflammatory effect through effectively suppressing and blocking the NF-κB P65 and MAPK signaling pathways.The pale brown HSP was isolated from dry calyx of Hibiscus Sabdariffa L. in water-extraction and alcohol-precipitation method, and yielded at 9.782% after removing pigment, protein, and impurities. Four polysaccharides components were gained using DEAE-52 anion exchange column chromatography at the eluent of H2 O, 0.1, 0.2 and 0.3mol/L NaCl, respectively. Physical and chemical indexes assessed that HSP-Ⅰ, HSP-Ⅱ, HSP-Ⅲ and HSP-Ⅳ were all no starch, the total sugar content was: 86.21%, 83.16%, 89.36%, 78.99%, respectively, the protein content was: 10.76%、7.78%、4.96%、0.33%, respectively; the uronic acid content was: 21.08%, 39.73%, 61.64%, 72.75%, respectively. Seen from the GPC spectrogram, the four HSP were all the single peak symmetry. PW was: 8733Da、14190Da、444080Da、137448Da, respectively. IR spectroscopy showed that the four kinds of polysaccharides all existed O-H stretching vibration within the wave-number range of 3393~3398 cm-1, the C-H stretching vibration within the wave-number range of 2927~2932 cm-1, and the C-O stretching vibration in the wave number 1417 cm-1. All the above results proved the four polysaccharides to be homogeneous. Structure analysis showed that HSP-Ⅲ contained uronic acid, mannose, rhamnose, glucose and galactose, and the mole ratio was 1∶10.98∶3.28∶1.23∶8.68. Periodate oxidation and Smith degradation results revealed that HSP-Ⅲ contained 65.71% of(1'6),(1'),(1'2) glucosidic bond which can be oxidized, and 34.29% of(1'3)) that can’t be oxided. 13C-NMR and 1H-NMR results showed that HSP-Ⅲ contained α, β-pyranoside configurations, and was composed of four kinds of monosaccharide units, and(1') and(1'6) should be the main chain.Anti-tumor, influence on mice spleen cell proliferation and RAW264.7 immune activities of HSP were investigated. Results showed that HSP-Ⅱ, HSP-Ⅲ and HSP-Ⅳ could significantly and dose-dependently inhibited MCF-7 and HepG2 cells proliferation, but only HSP-Ⅳ had preferable inhibition effect on PC3. On the contrary, HSP-Ⅰcould greatly promote all the three cells proliferation. HSP-Ⅱ, HSP-Ⅲ and HSP-Ⅳ had closely promoter effects on the spleen cell proliferation. When coordinated with ConA, they all could significantly promote the spleen T lymphocyte proliferation. Additionally, The synergistic effect together LPS with HSP-Ⅰor HSP-Ⅳ on the spleen B lymphocyte was also remarkable significant(P<0.001), suggesting that the synergy of HSP together with ConA or LPS could enhance lymphocyte immune regulating function through promoting the spleen T and B lymphocytes proliferation, thus improving the body immunity levels. In addition, HSP-Ⅱ could significantly promote the release of NO, TNF-α and IL-6, accelerated iNOS、IL-1β and IL-6 mRNA expression in different degrees of RAW264.7 macrophage, suggesting HSP-Ⅱ might activate the NF-κB signaling pathways to play the valid immune activity. |
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