AIM: To establish ()~1H NMR fingerprint of Radix Daphne genkwa to disclose its chemical composition of the secondary metabolites and provide a quick and credible assay for the detoxicated extraction of the active constituents. METHODS: The ()~1H NMR spectrum of petrol-acetone-methanol extract from Radix Dephne genkwa was detected using CDCl_3/DMSO-d_6(1∶1) as the deuterium reagent. The resonance intensity of the proton in the spectrum was indicated in relative integral area of peaks referenced by the resonance intensity of methyl signal in DMSO. The ()~1H NMR of cascade extraction of Radix Dephne genkwa by the reagent in sequence of petrol→acetone→methanol or chloroform→aether→acetyl acetate→methanol was utilized for observing the variation on their chemical compositions. RESULTS: The ()~1H NMR of Radix Dephne genkwa clearly expressed the information of the protons from long chain aliphatics or genkwadaphnin derivatives, aromatic coumarins or flavonoids as well as glycosides with moiety(ies) of pyran saccharide, symbolizing the existence of aliphatics, genkwadaphnin derivatives, coumarins and flavonoids. Remarkable difference was observed in ()~1H NMR spectrum of the extract by different cascade reagent. As the increase in the polarity of reagent, the intensity of the proton signals in upper field was quickly reduced concomitantly with the rapid enhancement of active proton signals from hydroxyls and saccharides in glycosides in down field. CONCLUSION: The ()~1H NMR fingerprint of Radix Dephne genkwa possesses its own characteristics and can be used as a reliable assay for studying the extraction of active constituents with minimum content of toxic diterpenoids. (Key

Aim To investigate the chemical constituents of the secondary metabolites of the roots of Daphne genkwa. Methods The roots of D. genkwa were extracted with 95% ethanol at 60 - 70 ℃ for 7 days to obtain the crude extract. The crude extract was purified by silica gel and Sephadex LH-20 column chromatography as well as the HPLC techniques. The structures of the isolates were elucidated by combined spectroscopic methods including 1D and 2D NMR, MS, UV, IR and CD. Results Three new biflavonoids were isolated from the ethanol extract of the roots of D. genkwa and their structures were identified as daphnodorin H-3-methyl ether (1), daphnodorin H-3"-methyl ether (2) and daphnodorin G-3"-methyl ether (3). Conclusion Compounds 1, 2 and 3 are three new biflavonoids.

Objective To study the factors affecting the accumulation of hydrolysable tannins in cultured mycelia of Inonotus obliquus. Methods Taking dry weight of mycelia and hydrolysable tannin content as index, different carbon and nitrogen sources, pH levels, and metal ions were evaluated for the accumulation of hydrolysable tannins in the submerged culture of I. obliquus. Results The optimal carbon and nitrogen sources were glucose and peptone. Optimal initial pH value was 5.5. The accumulation of hydrolysable tannins was greatly enhanced in the medium with the presence of Cu2+ at 0.8 mmol/L, Co2+ and Zn2+ at 1.6 mmol/L, Mn2+ at 1 mmol/L, and NH4+ at 4 mmol/L when compared to the control. Conclusion The accumulation of hydrolysable tannins is maximized in the medium containing glucose and peptone with pH value at 5.5. Supplementation of Cu2+, Co2+, Zn2+, and Mn2+ into the medium is an effective method for further increasing the accumulation of hydrolysable tannins in cultured mycelia of I. obliquus.

Objective To elucidate the antitumor activity of exopolysaccharide from Aphanothece halophytica(EPAH).Methods The in vivo inhibition of EPAH on growth of tumor was performed by inoculation of S_(180) sarcoma cells into ICR mice.While in vitro activity against tumor cells was assayed by the growth inhibition of cell lines of S_(180) sarcoma,Smcc7721,and HeLa.The effects of EPAH on immune function were evaluated by the influence on the thymus,spleen,and the number of lymphocytes in blood stream,the influence on proliferation of lymphocytes,the killing activity of NK cells,and the production of NO,IL-1?,and TNF-?. Results EPAH inhibited in vivo S_(180) sarcoma growth with the highest inhibi-(tory) rate of 66.79% and 47.93% in the test mice of pretreatment and simultaneous treatment,respectively.EPAH also displayed in vitro activity against the test cell lines with the highest inhibitory rate being more than 60% at a concentration of 100 ?g/mL.EPAH was found to affect the immune function in mice including increasing the weight of thymus,spleen,and the number of lymphocytes in the blood stream,accelerating the proliferation of lymphocytes,enhancing the killing activity of NK cells,and stimulating the production of NO,IL-1?,and TNF? by macrophages.Conclusion EPAH is an effective antitumor(agent.) It inhibites the tumor cells directly and hence the growth of tumor.Its antitumor activity is probably realized by increasing the weight of immune organs and the number of immunocytes as well as lymphocyte proliferation,enhancing the killing activity of NK cells,facilitating the production of NO and related(cytokines) in tumor-bearing mice.

Objective To elucidate the analgesic activity of the ethanol extracts from the root of Daphne genkwa (EERD). Methods The analgesic activity of EERD was evaluated by the effects on adjuvant-induced nociceptive response and paw swelling, the formation of PGE_2 and IL-1? in adjuvant arthritis (AA) rats, the activities of SOD and CAT, the levels of NO/iNOS in serum and brain tissue as well as by the effects on c-Fos protein expression in spinal cord of AA rats. Results EERD at used doses significantly delayed the adjuvant-induced nociceptive response and eased the paw swelling in AA rats. EERD also evidently inhibited the production of PGE_2 and IL-1?, and enhanced the activities of SOD and CAT in the tissue of paws being injected by adjuvant. Furthermore, it remarkably reduced the content of NO and inactivated the activity of iNOS in brain tissue of AA rats. In addition, EERD at used doses exhibited prominent inhibition on adjuvant-induced expression of c-Fos protein in the spinal cord of AA rats. Conclusion EERD is an effective agent for analgesia. The possible mechanisms for its analgesia might be the actions of inhibiting the production of PGE_2 and the release of IL-1?, reducing the activity of iNOS and hence the generation of NO in brain tissue, and blocking superoxidation through enhancing the activity of SOD and CAT.

Objective To establish HPLC method for assaying total flavonoids from the roots of Daphne genkwa(TFRD) in serum of mice and to elucidate the effect of mice serum containing TFRD on cell immunity in mice.Methods TFRD Concentration in serum was determined from the mice received single ig TFRD at certain time intervals using HPLC method.The effects of TFRD serum on lymphocyte proliferation,killing activities of NK and LAK cell,and phagocytic activity of macrophage were detected by MTT method.Results TFRD in serum reached its highest concentration in 20—30 min after ig admi-(nistration.) TFRD-containing serum significantly improved the proliferation of lymphocyte,enhanced the killing activities of NK and LAK cells,and enforced the phagocytic activity of macrophage.Conclusion(TFRD-)containing serum is an effective agents for enhancing cell immunity in mice.

Objective To investigate the protective effects of ethyl docosahexaenoate on brain oxidative injury and edema induced by cerebral ischemic/reperfusion in gerbils. Methods The gerbils were subjected to both common carotid arteries occlusion. The contents of MDA and GSH, the activities of GSH-PX, CAT, SOD and ATPase, the water content and the concentrations of Na + and Ca 2+ were measured. Histopathological examination was also done. Results The pretreatment with E-DHA significantly prevented the raise of MDA level, the decline of GSH content, the activities of GSH-Px, CAT, ATPase and the increases of Ca 2+, Na + and water content. Conclusion E-DHA has protective effect on brain ischemic injury and edema, which may be due to an inhibitory action on hydroxyl radical formation and brain edema.

Sterols are one of the active classes of compounds in Inonotus obliquus for their effective therapy of many diseases. In field environment, this fungus accumulates large amount of sterols. In cultured mycelia, however, this class of compounds is less accumulated. For analyzing the factors responsible for differing sterol composition, the field-grown and cultured mycelia were extracted with 80% ethanol at room temperature and total sterols were prepared using silicon gel column chromatography followed by identification using either GC-MS or spectroscopic methods. For culturing Inonotus obliquus, the seed culture was grown either in basic medium consisting of glucose (2%), yeast extract (0.5%), KH2PO4 (0.01%), MgSO4·7H2O (0.05%) and distilled water at pH 6.5, or the basic medium supplemented with serial concentrations of AgNO3. The results indicated that field-grown mycelia contained lanosterol and inotodiol (comprised 45.47% and 25.36% of the total sterols, respectively) and other 10 sterols (comprising the remaining 30.17%) including ergosterol biosynthetic intermediates such as 24-methylene dihydrolanosterol, 4,4-dimethylfecosterol, 4-methyl fecosterol, fecosterol and episterol. Column chromatography also led to the isolation of lanosterol, Inotodiol, trametenolic acid, foscoparianol B and a new triterpenoid foscoparianol D in field-grown mycelia. In comparison, the cultured mycelia only contained three sterols with ergosterol as the predominant one (82.20%). Lanosterol only accounted for 3.68%. Supplementing Ag+ into the culture at 0.28 μmol·L-1 greatly enhanced content of lanosterol (accounting for 56.81%) and decreased the content of ergosterol (18.5%) together with the presence of intermediates for ergosterol biosynthesis. These results suggested that the sterol composition in mycelia of the fungus can be diversified by supplementing substances inhibiting enzymatic process towards the synthesis of ergosterol. Harsh growth conditions in field environment (I.e. temperature variation, UV irradiation etc.) can delay the synthesis of ergosterol and hereby diversify the sterol composition in the mycelia of Inonotus obliquus.

Objective: To elucidate antiviral fractions from ethanol extract of Euphorbia kansui .Methods: Initial separation of ethanol extract from Euphorbia kansui was performed by column chromatography. The test mice were infected with flu virus mouse pneumonia adaptive strain (FM 1) and then treated with separated fractions intragastrally for determining lung index inhibition rate. The T lymphocyte proliferation enhancement was observed by the dosimetry of 3 H TdR infiltrated into DNA of the cell.Result: Initial separation of the extract afforded to 15 fractions. Fractions 8～15 with the elution volume from 29500 to 95000mL were highly effective to the therapy of the mice pneumonia. The enhancement to the lymphocyte proliferation of the 8 fractions under low concentration were 2～3 times higher than that of the control.Conclusion: Fractions 5 and 8～15 exhibited strong in vivo antiviral activity to the test virus, which might be realized by stimulating lymphocyte proliferation, enhancing the capacity of killing the cells infected by the virus.