Vinegar processing attenuates toxicity on IEC-6 cells caused by chloroform extraction of Daphne genkwa / 中国中药杂志

To screen the toxic polar fractions of Daphne genkwa, compare the toxicity of D. genkwa on crypts epithelial cells IEC-6 before and after vinegar processing, and preliminarily investigate the mechanism of D. genkwa vinegar processing on toxicity reducing. The proliferation of IEC-6 cells was observed by MTT. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH), as well as the enzyme activity of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were determined in IEC-6 cells to evaluate the oxidative damages degree of IEC-6 cells. The apoptosis and cell cycle were analyzed by Flow Cytometry. The results showed that the dichloromethane extraction was the toxic polar fraction of D. genkwa, and after vinegar processing, the toxicity of dichloromethane fraction was significantly reduced (<0.01). As compared with the blank control group, the dichloromethane fraction of D. genkwa can obviously decrease the levels of SOD, Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase (<0.01) and content of GSH, but increase the level of LDH and MDA in cell supernatant (<0.01). Besides, it obviously increased the early and late apoptotic rate of IEC-6 cells, obviously decreased the proportion of G₁stage cells, increased the ratio of S stage cells and M stage cells (<0.01). After vinegar processing, as compared with D. genkwa groups of various doses, it can significantly increase the levels of SOD, Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase (<0.01) and content of GSH, decrease the level of LDH, MDA(<0.01), significantly decrease the early and late apoptosis rate of IEC-6 cells (<0.01), increase the proportion of G₁stage cells, and decrease the ratio of S stage cells and M stage cells (<0.01). Vinegar processing can reduce the toxicity of dichloromethane fraction of D. genkwa, and its mechanism may be associated with improving the activity of antioxidant enzymes and permeability in IEC-6 cells, and decreasing the oxidative damage.

Morphological and microscopical identification of Genkwa Ramulus and its adulterants / 中国中药杂志

The purpose of this article is to identify Daphne genkwa and its adulterants, Wikstroemia chamaedaphne, according to the morphological and microstructure characteristics of their stem and foliage. The root of D.genkwa was studied simultaneously. The results indicated that the crude drug and processed pieces of Genkwa Ramulus were mainly composed of stems and branches where obvious opposite petiole scars and branch marks were able to be seen on their nodes. Otherwise, foliage or peduncles generally couldn't be found. Moreover, the fine silver flocculent fibers could be observed in the bark of fracture surface. The adulterants were the plant segments which were composed of stems, foliage and peduncles with spikelet-pedicel scars. There existed microstructures differences between Genkwa Ramulus and its adulterants. In the former, single thick lignified phloem fibers were interspersed in the stem phloem of the transverse section with very thick wall and unicellular non-glandular hairs could be observed on the lower epidermis of foliage. Nevertheless, in the latter, there was no thick lignified phloem fibers in cross section of stem phloem, the outer wall of epidermal cells of foliage hadthick cuticles and no non-glandular hairs in lower epidermis of foliage. The results can be used for the identification and the quality standard of the crude drug and processed pieces of D.genkwa.The characteristics of the microstructures and the transverse section can be used to identify the radix D.genkwa.

Study of Liver Damage Mechanism in Mice Caused by the Incompatibility of Daphne genkwa Combined with Glycyrrhiza uralensis / 中国药房

OBJECTIVE:To study the liver damage mechanism in mice caused by the incompatibility of Daphne genkwa com-bined with Glycyrrhiza uralensis in aspect of liver transporter. METHODS:40 mice were equally randomized into a normal control (isometric normal saline)group,a group of G. uralensis [15 g(medicinal matierias)/kg],a group of D. genkwa [15 g(medicinal tatierias)/kg],a group of D. genkwa with G. uralensis in the ratio of 1∶1 [15 g(crude drug)/kg],a group of D. genkwa with G. uralensis in the ratio of 1∶3 [15 g(medicinal matierias)/kg](n=8). The mice were given the corresponding drug,ig,once a day for 7 consecutive days. HE staining was performed and then the pathomorphology of liver tissues were observed under the light mi-croscope,and calculation was made for pathological grading. Western blot method was employed to determine the protein expre-ssion of the transporter Ntcp protein in the livers of mice. The contents of total bile acids(TBA)in livers of mice were determined. RESULTS:Compared to the mice in the normal control group,those in the group of 1∶1 and 1∶3 demonstrated higher protein ex-pression of Ntcp. In the group of 1∶1,the mice with grade“+++”hepatocyte degeneration were more (8). The mice with grade“+++”and“++”hepatocyte degeneration in the groups of 1∶3 were more (2 and 8 respectively). CONCLUSIONS:D. genkwa combined with G. uralensis can induce liver damage in mice by a mechanism which may be related to the accumulation of a large amount of TBA in the liver as a result of the increase in the expression of Ntcp in mice.

Anti-herpes Activity of Vinegar-processed Daphne genkwa Flos Via Enhancement of Natural Killer Cell Activity

Herpes simplex virus (HSV) is a common causative agent of genital ulceration and can lead to subsequent neurological disease in some cases. Here, using a genital infection model, we tested the efficacy of vinegar-processed flos of Daphne genkwa (vp-genkwa) to modulate vaginal inflammation caused by HSV-1 infection. Our data revealed that treatment with optimal doses of vp-genkwa after, but not before, HSV-1 infection provided enhanced resistance against HSV-1 infection, as corroborated by reduced mortality and clinical signs. Consistent with these results, treatment with vp-genkwa after HSV-1 infection reduced viral replication in the vaginal tract. Furthermore, somewhat intriguingly, treatment of vp-genkwa after HSV-1 infection increased the frequency and absolute number of CD3-NK1.1+NKp46+ natural killer (NK) cells producing interferon (IFN)-gamma and granyzme B, which indicates that vp-genkwa treatment induces the activation of NK cells. Supportively, secreted IFN-gamma was detected at an increased level in vaginal lavages of mice treated with vp-genkwa after HSV-1 infection. These results indicate that enhanced resistance to HSV-1 infection by treatment with vp-genkwa is associated with NK cell activation. Therefore, our data provide a valuable insight into the use of vp-genkwa to control clinical severity in HSV infection through NK cell activation.

Anti-inflammatory constituents from the stems of Daphne genkwa / 第二军医大学学报

Objective: To study the anti-inflammation contents in the stems of Daphne genkwa. Methods: The anti-inflammation contents were obtained from Daphne genkwa by bio-assay guide isolating method. The pharmacological model of dimethylbenzene-induced ear swelling was used for pharmacological study. The fractions of Ligroine and chloroform part of the the EtOH extraction were isolated and purified by column chromatography on silica gel and Sephadex LH-20 and recrystallization. Their structures were studied by using UV, IR,1 H-NMR,13 C-NMR, and MS, techniques. Results: Nineteen compounds were isolated from the stems of Daphne genkwa and were identified as 2, 3-dihydroxypropyl hexadecoate(1), β-sitosterol(2), dueicosanyl caffeate(3), docosyl caffeate(4), octadecyl caffeate(5), daucosterol(6), genkwanin(7), luteolin(8), (+) lariciresinol (9), apigenin (10), kaempferol (11), daphnodorin B (12), genistein (13), dihydrokaempferol (14), p-hydroxybenzonic acid (15), quercetin (16), syringin (17), syringaldehyde (18), ethyl 4-hydroxybenzoate (19). Conclusion: Compounds 1, 3-5, 7, 15, 17-19 have been isolated from the stems of Daphne genkwa for the first time. The high dosage of petroleum ether extract, chloroform extract and compound 4 have significant anti-inflammation activity in mice with ear swelling.

Three biflavonoids from ethanol extract of the root of Daphne genkwa / 药学学报

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.

Effect of serum containing total flavonoids from roots of Daphne genkwa on cell immunity in mice / 中草药

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.

Analgesic activity of ethanol extracts from root of Daphne genkwa / 中草药

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.