Protective effects of escin against indomethacin-induced gastric ulcer in mice

Escin, a natural mixture of triterpenoid saponin isolated from the seed of the horse chestnut, is reported to have a potent antiulcer activity against ethanol-induced gastric mucosal lesions. This study investigated the possible mechanisms underlying the gastroprotective effect of escin against indomethacin-induced gastric ulcer in mice. Gastric ulceration was induced by a single intragastric administration of indomethacin (18?mg/kg). The mice underwent intragastric treatment with escin at doses of 0.45, 0.9 or 1.8?mg/kg. Gastric lesion was estimated morphometrically and histopathologically 6?h after the indomethacin administration. The antioxidative parameters in gastric mucosa were measured. Moreover, the activity of myeloperoxidase and the contents of TNF-α, P-selectin and VCAM-1 in gastric tissues were determined. The results showed that escin protected gastric tissues against indomethacin-induced gastropathy as demonstrated from a reduction in the ulcer index and an attenuation of histopathologic changes. Escin caused significant reductions of the contents of malondialdehyde, TNF-α, P-selectin, VCAM-1 and myeloperoxidase activity. The altered activities of superoxide dismutase, catalase and glutathione peroxidase in the stomach tissues were also ameliorated by escin treatment. The present study demonstrated that escin had a protective effect against indomethacin-induced gastric ulcer in mice, not only by virtue of its antioxidant potential, but also due to its anti-inflammatory effect.     

Comparative Study Of Anthraquinones From Embryogenic Callus Tissue And Zygotic Embryos Of Frangula Alnus And Rhamnus Catharticus

Organogenesis and somatic embryogenesis were achieved in cotyledon cultures of Frangula alnus Mill. (alder buckthorn) and Rhamnus catharticus L. (common buckthorn), and in zygotic embryo cultures of F. alnus. Histological examination of cotyledonary segment cultures revealed that adventitious buds and somatic embryos arose indirectly through an intervening callus phase. In comparison to zygotic embryos, embriogenic callus of Frangula alnus contained significantly more anthraquinones in the form of free aglycones and different participation of chrysophanol and physcion within synthesized complex of metabolites. Also, in comparison to shoots, root cultures and in particular undifferentiated callus of Frangula alnus, embryogenic callus contained more chysophanol and aloe emodin and less physcion derivatives. Production of physcion derivatives was significantly increased in embryogenic calluses of R. catharticus compared to zygotic embryos. On the other hand, the emodin content was lower than in zygotic embryos. In comparison to shoot, root cultures and in particular undifferentiated callus of common buckthorn, embryogenic callus contained significantly more derivatives of physcion and emodin. The content of chrysophanol and its derivatives was decreased proportionally.     

Effects of escin on acute inflammation and the immune system in mice

Escin has been used extensively to treat chronic venous insufficiency, hemorrhoids, and edema resulting from cerebral ischemic damage, trauma or operation. However, no studies have looked at the anti-inflammatory properties of escin administered by intravenous injection, and it is still not clear whether escin has an effect on the immune system. This study seeks to investigate the time dependent anti-inflammatory properties of escin and its effect on the immune system. The anti-inflammatory effect of escin was observed in carrageenan-induced paw edema and acetic acid-induced capillary permeability in mice. The immunopharmacological effects of escin were evaluated by spleen index (SI), thymus index (TI), proliferative capacity of splenocytes (PS), lymphocyte count (LC), serum TNF-α levels, and phagocytic rate (PR) in mice. Escin treatment showed a significant anti-inflammatory effect, similar to that seen with dexamethasone treatment. However, the duration of the anti-inflammatory response was longer with escin treatment than with dexamethasone treatment. The results also demonstrated that escin had no significant effects on SI, TI, LC, PS, TNF-α levels, and PR. The findings suggest that escin is a potent anti-inflammatory drug with long-lasting anti-inflammatory effects and without any immunosuppressive effects.     

Escin attenuates cerebral edema induced by acute omethoate poisoning

Organophosphorus exposure affects different organs such as skeletal muscles, the gastrointestinal tract, liver, lung, and brain. The present experiment aimed to evaluate the effect of escin on cerebral edema induced by acute omethoate poisoning. Sprague-Dawley rats were administered subcutaneously with omethoate at a single dose of 60 mg/kg followed by escin treatment. The results showed that escin reduced the brain water content and the amount of Evans blue in omethoate-poisoned animals. Treatment with escin decreased the levels of tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), and prostaglandin E? (PGE?) in the brain. Escin also alleviated the histopathological change induced by acute omethoate poisoning. The findings demonstrated that escin can attenuate cerebral edema induced by acute omethoate poisoning, and the underlying mechanism was associated with ameliorating the permeability of the blood-brain barrier.     

Escin attenuates cerebral edema induced by acute omethoate poisoning

Organophosphorus exposure affects different organs such as skeletal muscles, the gastrointestinal tract, liver, lung, and brain. The present experiment aimed to evaluate the effect of escin on cerebral edema induced by acute omethoate poisoning. Sprague-Dawley rats were administered subcutaneously with omethoate at a single dose of 60?mg/kg followed by escin treatment. The results showed that escin reduced the brain water content and the amount of Evans blue in omethoate-poisoned animals. Treatment with escin decreased the levels of tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase-9 (MMP-9), cyclooxygenase-2 (COX-2), and prostaglandin E₂ (PGE₂) in the brain. Escin also alleviated the histopathological change induced by acute omethoate poisoning. The findings demonstrated that escin can attenuate cerebral edema induced by acute omethoate poisoning, and the underlying mechanism was associated with ameliorating the permeability of the blood–brain barrier.     

生长抑制因子2在植入前小鼠胚胎发育中的作用

目的探讨生长抑制因子2(Ing2)在小鼠植入前胚胎中的表达及生物学功能。方法使用ICR小鼠进行促进排卵以获得植入前胚胎。采用免疫荧光方法对Ing2蛋白在小鼠植入前胚胎中的定位进行研究,采用显微注射Ing2-esiRNA实验对其在早期胚胎中的功能进行研究。抽提小鼠囊胚期合子的RNA反转录为cDNA,进行实时PCR分析。结果免疫荧光结果显示Ing2蛋白在小鼠植入前胚胎中定位于胞浆及胞核。从4-cell期胚胎开始,胞核中的表达量明显增高。Ing2-esiRNA显微注射实验显示在小鼠合子中干扰Ing2后,各期胚胎形成率均显著低于对照组(4-cell期胚胎比率:82.7%vs.94.7%;桑葚胚比率:67.3%vs.89.4%;囊胚形成率:48.7%vs.75.8%)(P<0.01)。结论 Ing2的下调,引起植入前小鼠胚胎发育阻滞。     

Ammi visnaga: Somatic Embryo Induction and Furanochromone Production in Embryos, Seedlings, and Plants

AMMI VISNAGA (L.) Lam. globular, heart, and torpedo shaped embryos were induced from the calli of sterile seedling hypocotyls in Murashige and Skoog's medium with or without either 0.5 ppm benzyladenine or 1.0 ppm 2,4-D. After transfer of the calli into liquid media supplemented with 10 mM L-glutamine, all three embryo stages were observed after eight weeks. Liquid media lacking growth regulators appeared to enhance the development into torpedo stage embryos, especially at four weeks. Eight weeks after the transfer of the callus into liquid media, flasks containing predominately one microscopic stage were pooled, transferred into fresh liquid media, and the production of the furanochromones, khellin and visnagin, in the embryos were determined by a normal phase HPLC method. All three embryo stages produced similar amounts of khellin and visagin. Maximum production was reached at six weeks. The six-week-old torpedo shaped embryos produced 0.57% khellin and 0.72% visnagin on a dry weight basis. The embryo cultures produced significantly higher concentrations of the furanochromones than A. VISNAGA cell suspensions (reported previously) that produced approximately 0.1-0.3% visnagin and no detectable khellin. In addition, the production of khellin and visnagin in the fruits, seedlings, young plants, and flowering or fruiting AMMI VISNAGA (L.) Lam plants was determined. Upon germination of the fruits, a rapid decline in the content of the furanochromones was observed. Subsequent experiments showed that the majority of the furanochromone content in fruits was released into the media within one to two days. Total visnagin content (media + seedlings) was unchanged, however, a loss of 40% of the khellin content was found after 2-4 weeks. When media containing seedlings was spiked with exogenous khellin and visnagin, the concentrations of both furanochromones declined, apparently due to uptake by the roots and subsequent catabolism in the plant. No evidence for degradative enzymes in media exposed to seedlings was observed. During vegetative growth of the plants, relatively low levels of furanochromones were found. High concentrations were observed at early flowering, followed by a decline, and a gradual rise at fruiting as was previously reported by other investigators. In the vegetative stages, visnagin concentrations were higher than those for khellin, whereas in fruiting plants and fruits, the ratio was reversed.     

Growth and ginsenoside production in hairy root cultures of Panax ginseng using a novel bioreactor

We tested the effect of three variables: the bioreactor system (Wave or Spray reactor), medium exchange and culture period, on the capacity of a selected hairy root line of Panax ginseng to produce ginsenosides. Among the reactors, the Wave bioreactor appeared to be the most efficient in promoting hairy root line growth. Periodic exchanges of the medium and a longer culture period increased the growth rate of cultured hairy root line and, consequently, its capacity to produce ginsenosides. Under established optimum conditions (medium exchange every 14 days over a culture period of 56 days using the Wave bioreactor), the initial root fresh weight was enhanced more than 28-fold, giving a root biomass of 284.9 g L(-1) and a ginsenoside content of 145.6 mg L(-1). It is noteworthy that this ginsenoside production exceeded by almost 3-fold that obtained during the shake flask culture of our hairy root line, although it often happens that the scale-up from shake flask to a bioreactor culture results in reduced productivities. To our knowledge this is the first time that a Wave bioreactor has been used for hairy root culture.     

Oxidative stress status and development of late organogenesis stage rat whole embryos cultured from gestational days 13.5 to 14.5.

A new method for culturing rodent whole embryos at the late organogenesis stage was developed using a roller-bottle system with intermittent gassing. Rat embryos were cultured for 24 h from gestational day (GD) 13.5 to 14.5. Growth and metabolic comparisons were made between in vivo embryos and embryos of the same GD cultured under various media and conditions. Crown-rump length, head length and protein content were used as growth indicators. Biologic markers such as embryonic tissue concentration of glutathione (GSH), glutathione disulfide (GSSG) and lipid peroxidation were used as assessments of metabolic activity in terms of oxidative stress. Embryos cultured with media consisting of either 15% or 20% male rat serum and balanced with Dulbecco's Modified Eagle Medium (DMEM) were found to most closely match in vivo embryos. 6 h gassing intervals and 5 mL medium volume/embryo provided optimal conditions for cultured embryos. By shortening the 24 h embryo culture period to 12 h, embryonic haemorrhaging was avoided. Moreover, the 12-h cultured embryos showed similar redox GSH/GSSG ratios and similar GSH content to the in vivo embryos, which was not observed in the embryos cultured under 24 h culture conditions. The present work demonstrates the utility of late organogenesis stage embryo culture as a model for the assessment of in vivo embryonic growth and oxidative stress indices.     

Somaclonal variation in transformed roots and protoplast-derived hairy root clones of Hyoscyamus muticus

Substantial somaclonal variation in growth rate, morphology, and alkaloid production of Hyoscyamus muticus L. hairy root clones obtained by transformation with four Agrobacterium strains was shown. The hyoscyamine content of the root clones (n = 100) obtained from the same origin varied from 0.03 to 0.59% of dry weight. The clones produced 25-320 times less scopolamine than hyoscyamine. The best producing root clone was used as a starting material for protoplast isolation. The hyoscyamine content of protoplast-derived hairy root clones (n = 171) ranged from 0.04 to 1.45 % of dry weight. Most clones showed improved alkaloid production in relation to the parent clone, but the mean hyoscyamine content of the clones was the same as that of the parent clone. All the studied hairy root clones showed relatively stable alkaloid production during long-term cultivation. No correlation was found between alkaloid production and growth rate or morphology of the clones.     

甘草毛状根中甘草总黄酮和甘草酸的检测和分析

目的检测甘草毛状根中总黄酮和甘草酸的含量。方法采用紫外分光光度法检测甘草总黄酮含量,采用HPLC检测甘草酸含量。结果甘草毛状根中的总黄酮含量在不同根系中有很大差异,最高可达0.803%,接近生药根;大多数根系高于正常组培根;总黄酮含量随着培养时间的延长呈增加趋势,至9周时增加了68.5%。不同甘草毛状根系中的甘草酸含量差异显著,部分根系未检出甘草酸,能检出的根系最高干重含量为0.69mg·g^-1,远低于生药根（干重含量32.13mg·g^-1）,且含量不受培养时间影响。结论甘草毛状根在目前情况下不适于作为甘草酸的获取资源,但作为甘草总黄酮获取的新资源,是值得进一步研究的。     

黄花蒿发根的液体培养及青蒿素合成的动态特征研究

目的:探讨黄花蒿发根生长及青蒿素生物合成的动态特征,建立高效、稳定的黄花蒿发根液体培养体系。方法:测定不同培养基以及MS培养基中不同营养元素对黄花蒿发根生物量和青蒿素含量的影响。结果:筛选出优化的黄花蒿发根液体培养基,获得拟合的黄花蒿发根生长和青蒿素合成的Logistic方程。结论:在优化的MS培养基中黄花蒿发根生长迅速且能稳定合成青蒿素,为工业化大规模生产青蒿素提供了可能。     

Variations in the Leaf Alkaloid Content of Androgenic Diploid Plants of Datura innoxia

Anther culture of DATURA INNOXIA Mill, has permitted the obtention of spontaneous diploid androgenic plants which produced the tropane alkaloids. The source plants (zygotic diploid) showed no significant variations in the leaf alkaloid content. On the contrary, androgenic diploid plants obtained after the first cycle of androgenesis showed important quantitative and qualitative variations in the leaf alkaloid content. Thus, androgenesis was found to induce a large variation in the accumulation of these secondary metabolites in the leaves. It has also permitted the obtention of tropane alkaloid-overproductive plants, particularly rich in scopolamine. The analyses of zygotic plants obtained from seed germination of the first cycle androgenic plants have shown that this variability is transmissible by simple cross-pollination. The analyses of androgenic diploid plants obtained after the second cycle of androgenesis also showed variations in the leaf alkaloid content. IN VITRO androgenesis, therefore, clearly induced variability in the leaf alkaloid content of the androgenic plants. The role of IN VITRO androgenesis in inducing variability has been discussed.     

秦巴山区不同产地娑罗子中七叶皂苷的含量比较

目的:对秦巴山区10个不同产地娑罗子药材中的七叶皂苷成分进行分析比较。方法:采用高效液相色谱法。色谱柱为Hypersil ODSC18(100mm×4.6mm,5μm),检测波长为220nm,流动相为乙腈-0.02%磷酸溶液(35:65,V/V),流速为1mL·min-1,柱温为20℃。结果:10个不同产地样品中七叶皂苷A的含量均高于七叶皂苷B,且均符合2010年版《中国药典》标准(七叶皂苷A>0.7%)。产自略阳的样品中七叶皂苷A和七叶皂苷B的含量最高,分别为28.46和19.77mg·g-1(生药);其次为康县、留坝的样品,宁强、勉县、宁陕、镇坪的样品含量居中,而平利、西乡、洋县的样品含量则相对较低。10个产地样品中七叶皂苷A含量在1.16%～2.85%之间,七叶皂苷B含量在0.89%～1.98%之间,平均值分别为1.92%和1.43%,其中七叶皂苷A与七叶皂苷B的含量比在1.14～1.45之间。结论:秦巴山区不同产地娑罗子中七叶皂苷含量和组成存在明显差异,种源、生态环境等因素对娑罗子药材有效成分的积累过程影响较大,控制药材产地来源对保障娑罗子药材质量的稳定性有着重要意义。     

Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D. leichhardtii hybrid

Co-culture conditions for Duboisia myoporoides-D. leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834. The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria. One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA). The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark. The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis. Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA). Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem. In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots. In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light. In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA. On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.     

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.     

Successful in vitro growth of rat two-cell embryos to blastocysts using a simple chemically defined medium

The aim of the present study was to formulate a simple chemically defined medium for the in vitro growth of rat two-cell embryos to blastocysts. Embryos from day 2 pregnant rats were retrieved and placed in paraffin oil-covered droplets of "rat two-cell embryo culture medium" (R2ECM) containing combinations of various serum supplements, glucose, L-glutamine, and cultured up to 96 h in a CO(2) incubator. Embryos cultured in the basic medium (R2ECM), as well as those supplemented either with fetal bovine serum (FBS) or male rat serum (MRS) did not develop beyond the two- to four-cell stage. In R2ECM with 0.3% bovine serum albumin (BSA) and 7.5 mM glucose, 44% of embryos reached the blastocyst stage by 96 h in culture, and the blastulation rate increased to about 83% when 1 mM of L-glutamine was added. To evaluate the effects of varying doses of glucose, two-cell embryos were cultured in R2ECM supplemented with 0.3% BSA, 1 mM L-glutamine, and 2.5, 5.0, or 7.5 mM of glucose. The percentage of embryos reaching the blastocyst stage for 2.5, 5.0, and 7.5 mM glucose was 64.6%, 65.3%, and 82.9%, respectively. The present study showed that the modified medium (R2ECM) is a simple chemically defined medium that is capable of supporting in vitro growth of rat two-cell embryos to blastocysts in high proportion (greater than 80%) without the need for change of medium within 96 h of culture.     

唐古特山莨菪毛状根对去氢表雄酮的生物转化

目的通过生物转化对去氢表雄酮进行结构修饰，以期获得更有意义的转化产物。方法利用唐古特山莨菪毛状根液体悬浮培养体系对去氢表雄酮进行生物转化，通过柱色谱及制备薄层色谱进行分离纯化，利用核磁共振、ESI-MS等光谱鉴定结构。结果分离得到了5个转化产物，即：androst-4-ene-3,17-dione ( I)； 6α-hydroxyandrost-4-ene-3, 17-dione (II)；6α,17β-dihydroxyandrost-4-ene-3-one(III)； androst-4-ene-3,6,17-trione (IV)；17β-hydroxyandrost-4-ene-3-one (V)。结论所得到的5个化合物均为首次通过植物组织培养生物转化的方法从唐古特山莨菪毛状根液体培养体系中分离得到。     

重组人Neurturin对体外培养的胚胎大鼠多巴胺能神经元的保护作用

目的建立大鼠胚胎多巴胺能神经元原代培养方法 ,研究重组人Neurturin(rhNTN)体外对黑质多巴胺能神经元的营养和保护作用。方法孕 14dWister大鼠 ,取出胎鼠中脑腹侧区组织 ,经胰酶消化、分散 ,接种至涂有多聚左旋赖氨酸基质的 6孔板中 ,37℃培养 2 4h后将培养液更换为B2 7无血清培养基 ,实验组同时加入纯化的rhNTN。继续培养 ,于培养第 7、14、2 1天直接观察及TH免疫细胞化学染色观察结果。结果培养 14、2 1d ,对照组中多数神经元已经萎缩、死亡 ,少量存活的多巴胺能神经元胞体小 ,突起细短 ;实验组中存活的多巴胺能神经元数目较多 ,神经元胞体粗壮 ,突起长 ,生长状况良好。结论rhNTN能促进中脑黑质多巴胺能神经元的存活及突起生长 ,对多巴胺能神经元具营养和保护作用。