The uterotonic activity of compounds isolated from the supercritical fluid extract of Ekebergia capensis

The wood of Ekebergia capensis Sparrm. is used by the local Zulu community in KwaZulu-Natal Province, South Africa to facilitate childbirth. In this investigation, the uterotonic properties of extracts from this tree were evaluated using both pregnant and non-pregnant guinea pig uterine smooth muscle in vitro. The extracts were prepared using water modified supercritical carbon dioxide at 400 atm and 80 degrees C. As samples of these extracts displayed positive results when screened for uterotonic activity, gravity column chromatography followed by NMR spectroscopy was performed in an attempt to isolate and elucidate the structures of the compounds that were present in the extract. The extract yielded five known compounds of which only two, viz. oleanonic acid and 3-epioleanolic acid, displayed uterotonic activity. Receptor binding assays were subsequently performed with 3-epioleanolic acid to ascertain its mode of action. Bradykinin (30 ng/100 microl) and acetylcholine (1 microg/100 microl) were used as the B2 and cholinergic receptor agonists respectively with icatibant (HOE 140) (30 ng/100 microl) and atropine (60 micro/100 microl) as their corresponding antagonists. 3-epioleanolic acid was observed to mediate its effect through the cholinergic receptor. The results of this study show that two compounds from the extract of this tree possess varying degrees of agonist activity on uterine smooth muscle with minor changes in the molecular structure affecting its intrinsic activity on uterine muscle.     

Efficiency of supercritical fluid extraction for the production of non-volatile terpenoids from Taraxaci radix

Supercritical fluid extraction (SFE) is an extraction technique which exploits the solvent properties of fluids above their critical point. As a result supercritical fluid extraction was used to gain various active substances from plants. The use of SFE to obtain bioactive substances from medicinal plants over the past twenty years has been proved to be a viable alternative for the extraction of natural compounds. Dandelion (Taraxacum officinale Wiggers et Webers, Asteraceae) is one of the best known European medicinal plants, not only as a traditional medicine but perspective raw material for modern phytopharmaceuticals. From the characteristic principles our attention has been directed to triterpenes and phytosterols with anti-inflammatory activity, which were extracted with supercritical carbon dioxide. Designed experiments were carried out to determine the optimal extraction parameters. The products obtained by supercritical fluids extraction were compared to extracts prepared by traditional extraction method (Soxhlet) with n-hexane and ethyl alcohol solvents. The content of triterpenes and sterols was monitored after saponification by thin layer chromatography-densitometry. The products gained by supercritical fluid extraction were different from the traditional ones both in their appearance and composition. Triterpenes and their esters were extracted quantitatively by supercritical fluid extraction using CO2 as solvent and the extraction dynamic for triterpenes and phytosterols was different. Triterpenes had a higher concentration in the SFE product then in the extracts prepared by traditional methods.     

川芎挥发油提取工艺的研究进展

川芎是一种重要的药用植物,其主要成分是挥发油。本文综述了川芎挥发油的各种提取方法,包括水蒸气蒸馏法、溶液萃取法、提取-共沸精馏耦合新工艺、超临界流体萃取法和超声强化超临界CO2萃取法,从得率、提取得到的成分及含量等方面对各种方法的优缺点进行了比较和分析,并对其发展方向提出展望。     

Discrimination between the functional and biochemical effects of two herbal oxytocics on the rat myometrium

This study on the rat myometrium is the first report where the effects of herbal extracts used as oxytocics in traditional medicine have been systematically analysed in the same preparation at the level of functional (contractile) and biochemical (second messenger generation) responses. Extracts of Agapanthus africanus and Clivia miniata (used in South African traditional medicine) were compared with other uterotonic agents with regard to their ability to stimulate phosphoinositide metabolism in the rat myometrium and cause accumulation of [3H]inositol phosphates. The maximal contractile response of the isolated rat myometrium in response to stimulation by the herbal extracts and agonists was compared with the maximal contractile response to cumulative addition of acetylcholine. The rank order of intensity of stimulation of [3H]inositol phosphate generation was: oxytocin > Agapanthus > prostaglandin F2alpha(PGF2alpha) > serotonin > acetylcholine > Clivia > ergometrine. This differed from the rank order of maximum contractile response: oxytocin > acetylcholine > PGF2alpha > serotonin approximately Clivia > Agapanthus > ergometrine. Activity was also identified in chemical fractions of the plants and components common to both plants have been identified in the isolated active fractions. These results have identified that the uterotonic activity of Agapanthus is linked to increased turnover of phosphoinositides as a signal transduction mechanism, whereas this appears to play a less significant role in the uterotonic activity of Clivia. This study illustrates the benefits of using the measurement of stimulation of phosphoinositide metabolism as a bioassay in phytomedical research.     

Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria

Various extracts prepared from the traditional dye and medicinal plant Isatis tinctoria L. were submitted to a broad in vitro screening against 16 anti-inflammatory targets. Dichloromethane (DCM) extracts from dried leaves showed a marked cyclooxygenase (COX) inhibitory activity with a preferential effect on COX-2 catalysed prostaglandin synthesis. A supercritical fluid extraction (SFE) procedure employing CO2-modifier mixtures was developed by which the bioactivity profile and chromatographic fingerprint of the DCM extract could be reproduced. High-resolution activity directed on-line identification of the COX-2 inhibitory principle, using a combination of LC-DAD-MS with a microtitre-based bioassay, led to the identification of tryptanthrin (1) as the constituent responsible for essentially all COX-2 inhibitory activity in the crude extract. Following on-line identification, 1 was isolated at preparative scale and its structure confirmed by comparison with synthetic tryptanthrin. In an assay with lipopolysaccharide stimulated Mono Mac 6 cells, tryptanthrin (1) was of comparable potency (IC50 = 64 nM) than the preferential COX-2 inhibitors nimesulide (IC50 = 39 nM) and NS 398 (IC50 = 2 nM). The SFE extract and 1 showed no cytotoxicity in Mono Mac 6 and RAW 264.7 cells when tested at 100 microg/ml and 10 microM, respectively.     

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K⁺ channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K⁺ conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.     

Mammalian tachykinins and uterine smooth muscle: the challenge escalates

We review the actions of mammalian tachykinins on uterine smooth muscle. Derived from sensory neurones and non-neuronal cells within the female reproductive tract, tachykinins are potent uterotonic agents. Three tachykinin receptor genes, and the gene encoding neprilysin, the enzyme that inactivates tachykinins, are present in rat, mouse and human myometrium. In rat and human, the tachykinin NK₂ receptor is important in mediating the uterotonic effects of tachykinins; actions at this receptor remain relatively stable or vary only slightly in the face of changing hormonal and gestational status. In contrast, ovarian steroids and pregnancy regulate expression of the tachykinin NK₃, and to a lesser extent, the tachykinin NK₁ receptor, as well as the activity of neprilysin. In the oestrogen primed mouse uterus, the tachykinin NK₁ receptor primarily mediates tachykinin uterotonic effects, but there is a switch to the tachykinin NK₂ receptor by late pregnancy. The possible physiological and pathological roles of tachykinins, including hemokinins and endokinins, in normal and premature labour, stress-induced abortion and menstrual disorders are briefly discussed.     

超临界CO2流体技术在聚合物颗粒制备中的应用

超临界CO2流体是指当温度和压力处于临界值以上时CO2所表现出的一种状态。处于超临界状态下的CO2与传统有机溶剂相比，在物理和化学性质方面都表现出了很大的不同，这种差异被开发用于各行各业。本文着重介绍了超临界CO2流体在制备聚合物颗粒方面的应用，特别介绍了超临界CO2流体技术应用于药物制剂方面的研究和开发。     

款冬花不同提取物对豚鼠离体回肠收缩作用的研究

目的研究比较款冬花各个不同提取物对豚鼠离体回肠收缩的作用。方法通过石油醚、乙酸乙酯、CO2超临界萃取等方法制备款冬花多个提取物,将离体豚鼠回肠悬吊于克-亨试液中,观察给药后豚鼠回肠的收缩作用,计算豚鼠回肠收缩幅度抑制率。结果款冬花各个不同提取物均能降低组胺引起的豚鼠离体回肠收缩幅度。结论款冬花各个提取物能明显抑制豚鼠回肠收缩运动。     

Analytical and preparative supercritical fluid extraction of chamomile flowers and its comparison with conventional methods

Supercritical fluid extraction (SFE) was compared with Soxhlet extraction, steam distillation and maceration for the isolation of the active components present in chamomile flowerheads. The obtained fractions were analysed by GC-MS and reversed-phase HPLC. The yield of essential oil achieved by a 30-min extraction with pure CO2 at 90 atm and 40 degrees C was 4.4 times higher than that produced by steam distillation performed for 4 h. The recovery of the flavonoid apigenin obtained by supercritical CO2 after a 30-min extraction at 200 atm and 40 degrees C was 71.4% compared to Soxhlet extraction performed for 6 h and 124.6% compared to maceration performed for 3 days. However, the highly polar flavonoid apigenin-7-glucoside was not extracted by 100% CO2 (recovery values < 1.1%). Its extraction efficiency was markedly improved by the addition of the polar modifier methanol (5%, v/v) to the CO2 fluid, yet the obtained recoveries were unsatisfactory (14.6-19.5%). The SFE method was scaled-up for preparative applications using a pilot plant with three separation stages operating in series. Large-scale SFE was technically feasible with pure CO2 as the extracting fluid. However, the use of CO2 modified with organic solvents was not effective at the pilot-plant scale.     

S-腺苷蛋氨酸对子宫平滑肌收缩的影响

目的 探究S-腺苷蛋氨酸对子宫平滑肌收缩的影响,为寻找安全有效的新型子宫收缩调节药物提供理论依据。方法 取未妊娠、妊娠早期(6.5d)、妊娠中期(12.5~13.5d)和妊娠晚期(16.5~17.5d)ICR小鼠,分离子宫肌条,两端分别固定于张力换能器,采用氨甲酰胆碱诱导收缩,累积给药法给予沙丁胺醇、S-腺苷蛋氨酸、蛋氨酸、牛磺酸等药物,记录、测量并分析离体子宫收缩曲线。结果 沙丁胺醇和S-腺苷蛋氨酸对未孕、妊娠早期(6.5d)、妊娠中期(12.5~13.5d)和妊娠晚期(16.5~17.5d)四个时间点的小鼠离体子宫平滑肌的收缩均有较强的抑制作用,分别在10-7mol/L和1.0mmol/L的溶液浓度下显著抑制收缩。蛋氨酸、牛磺酸在实验浓度下对ICR小鼠离体子宫平滑肌的收缩影响不明显。结论 S-腺苷蛋氨酸可有效抑制不同阶段离体子宫的收缩,提示S-腺苷蛋氨酸可能是一个有效且安全的子宫收缩调节药物。     

Influence of extraction methods on the composition of essential oils

The aim of this work is to demonstrate our results on comparison of composition of essential oil fractions obtained by traditional steam distillation and supercritical fluid extraction. The plant materials for the various extraction methods were selected from the Lamiaceae, Apiaceae and Asteraceae families. For the supercritical fluid extraction (SFE) carbon dioxide was used as supercritical solvent. The extracts were collected by stage wise precipitation in two separators. The waxy product and extract rich in essential oil were collected in the 1st and in the 2nd separator respectively. The traditional water steam distillation (SD) was carried out in the special apparatus of the Hungarian Pharmacopoea (7th ed.). GC analysis was carried out on capillary silica fused columns coated with DB-1701 and the specific chiral columns coated with Rt-beta DEX m or Rt-beta DEX sm. Comparing the composition of steam distilled oils with that of volatile SFE fractions the following general characteristics were established. The SFE fractions were richer in monoterpene-esters and poorer in alcohols than the traditional essential oils (clary sage, lavander, moldavian dragonhead). Regarding the distribution of the monoterpene and sesquiterpene compounds, the SFE fractions contained sesquiterpenes in higher percentage than the distilled oils (Salvia fruticosa). Furthermore, the proportion of sesquiterpenes increased in SFE fractions collected successively with time (Salvia officinalis) similar to the ratio of oxygenated monoterpenes to monoterpene hydrocarbons (Rosmarinus officinalis). The phtalides of lovage (Satureja hortensis) did not show regular change during the supercritical extraction. In other cases it was verified that part of the mono- and sesquiterpenes were present originally in bound form (glycosides) in plants. Thus they appeared only in essential oil fractions after previous acidic treatment (Thymus, Origanum, Satureja species). During the super-critical extraction the azulenogene sesquiterpene lactones did not transform to azulenes (chamomile, yarrow), but SFE fractions of some Asteraceae plants contained sesquiterpene-gamma-lactones of unchanged structure.     

Effect of ethanol extracts of three Chinese medicinal plants with laxative properties on ion transport of the rat intestinal epithelia

The effects of ethanol extracts of three Chinese medicinal plants Dahuang (Rheum palmatum L.), Badou (Croton tiglium L.), and Huomaren (Cannabis sativa L.), on ion transport of the rat intestinal epithelia were studied. Rat intestinal epithelia mounted in an Ussing chamber attached with voltage/current clamp were used for measuring changes of the short-circuit current across the epithelia. The intestinal epithelia were activated with current raised by serosal administration of forskolin 5 microM. Ethanol extracts of the three plants all augmented the current additively when each was added after forskolin. In subsequent experiments, ouabain and bumetanide were added prior to ethanol extracts of these medicinal plants to determine their effect on Na(+) and Cl(-) movement. The results suggest that ethanol extracts of the three medicinal plants may affect the Cl(-) movement more directly than Na(+) movement in the intestinal epithelial cells. The results provide evidence for the pharmacologic mechanism of the three Chinese medicinal plants on the intestinal tract.     

Pregnancy-associated changes in responsiveness of the porcine myometrium to bioactive substances

To determine the pregnancy-associated changes in the porcine uterine contractility, the spontaneous contraction and the mechanical responses to bioactive substances of uteri in nonpregnant proestrus and pregnant pigs (25-60 days of gestation) were compared in vitro. Longitudinal muscle (LM) and circular muscle (CM) of the uterus exhibited spontaneous contraction, but the frequency in pregnant pigs was lower than that in the nonpregnant pigs. The duration and force of spontaneous contraction in the pregnant pigs were long and large compared with both in the nonpregnant pigs. L-Nitroarginine methylester (L-NAME) and 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,5-tetrahydro-benzo[cd]indol-2(1H)-one (DR4004) did not change the spontaneous contraction in the uteri of nonpregnant pigs but increased its amplitude in the uteri of pregnant pigs. Isoprenaline inhibited the uterine spontaneous contraction of the nonpregnant and pregnant pigs, and the inhibition was stronger in the pregnant than in the nonpregnant pigs. 5-Hydroxytryptamine also caused inhibition of spontaneous contraction in the uteri of nonpregnant pigs (CM>LM). In the pregnant pigs, sensitivity to 5-hydroxytryptamine increased in a muscle layer-dependent manner (LM>CM) and difference in the responsiveness between LM and CM decreased. Acetylcholine contracted the uterine LM and CM strips of the pregnant and nonpregnant pigs. The responsiveness of CM increased slightly during pregnancy, but that of the LM did not change. 5-Bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine (UK14304) caused contraction of only LM in the uteri of nonpregnant pigs, but contracted both LM and CM strips in the pregnant pigs. Oxytocin and prostaglandin F(2 alpha) also contracted the uteri of nonpregnant pigs (LM>CM). Pregnancy increased the contraction of both agents in the LM and CM, but the increment was marked in the CM. The contractile forces induced by all stimulants were increased (by 1.7- to 2.5-fold) in the LM and CM of pregnant pigs. In conclusion, (1) low frequency, slow kinetics and large force of spontaneous contraction are characteristics of the pregnant porcine uteri, and nitric oxide and 5-hydroxytryptamine are supposed to be partially involved in the regulation of spontaneous contraction, and (2) responses to both contractile and inhibitory agents are increased in the pregnant pigs. Increment of the responsiveness is conspicuous in the muscle layer that is less sensitive to each agonist in the uteri of nonpregnant pigs. According to the pregnancy-associated changes, muscle layer-related differences of responsiveness to bioactive substances in the nonpregnant pigs tend to decrease in the pregnant pigs.     

超临界CO2流体萃取丹参素的工艺研究

目的研究萃取丹参素的最佳工艺条件。方法通过正交设计,用超临界CO2流体萃取,优化出合理工艺条件,并与传统溶剂提取工艺相对照。结果超临界CO2流体萃取率为传统工艺萃取率的1．1倍。结论超临界CO2流体萃取丹参素具有开发价值。     

超临界二氧化碳萃取法与水蒸气蒸馏法提取缬草油的化学成分比较

摘要目的对不同提取方法提取的缬草油化学成分进行比较研究。方法采用超临界二氧化碳（CO2）萃取法和水蒸气蒸馏法从缬草中提取缬草油,用气相色谱 质谱联用（GC MS）法进行化学成分定性与相对含量的比较。结果共鉴定了118种成分,超临界CO2萃取法提取物共鉴定98种,水蒸气蒸馏法提取鉴定了67种,共有成分47种;超临界CO2萃取所得缬草油的收率约为水蒸气蒸馏收率的1.8倍。结论超临界CO2萃取法是缬草油较好的提取方法。     

Reproduction physiology and prostanoids

Prostanoids, which consist of prostaglandins (PGs) and thromboxane, are produced from arachidonic acid by cyclooxygenases (COXs) as a rate-limiting step, and they exert various biological actions. Classically, prostanoids are suspected to be closely related to female reproductive processes such as ovulation, luteolysis and uterine contraction, as well as pathological processes such as fever generation and pain modulation. Recently the cDNA cloning of a series of prostaglandin-synthesizing enzymes and receptors enabled us to clarify which isoform or subtype is involved in each reproductive process by generating individual gene-deficient mice. In late pregnancy, PGF2 alpha synthesized by COX-1 is essential for induction of parturition via luteolysis. Furthermore, impaired induction of COX-2 in the myometrium of PGF2 alpha receptor-deficient mice is accompanied with loss of parturition, suggesting that COX-2 is presumably responsible for producing uterotonic PGs. In early pregnancy, PGE2 synthesized by COX-2 induces the expansion of cumulus cells through EP2 receptor and contributes to ovulation and fertilization. These results may be useful in not only developing novel drugs in the reproductive area but also understanding and overcoming harmful reproductive side effects of classical and novel drugs in non-reproductive areas.     

益母草不同提取物调经作用研究

目的:探讨益母草不同提取物对正常大鼠离体子宫平滑肌收缩功能的影响,寻找调经活性部位。方法:从益母草中提取分离3个不同提取物,分别为益母草水部位(W1)、乙酸乙酯部位(Et)和正丁醇部位(Bu);采用未孕大鼠离体子宫模型观察3种提取物对子宫收缩的活动力、张力和频率的影响,用BL-420E生物机能系统记录子宫收缩曲线。结果:W1、Bu均使子宫活动力、收缩张力均值和最小值明显增加(P0.05或P0.01),且呈一定量效关系;3种提取物均可抑制缩宫素对子宫的收缩作用,使子宫活动力、收缩张力的均值和最大值显著降低(P0.05或P0.01)。结论:提取物对未孕大鼠正常子宫具有兴奋作用,而对缩宫素所致痉挛子宫具有舒张作用;提示益母草对子宫活动具有双向调节作用,活性物质主要是益母草水部位和正丁醇部位。     

17味超临界CO2萃取中药配方颗粒急性毒性研究

目的对比观察17味同批次中药饮片所制备的标准煎剂、膏粉(水提物)、超临界CO2萃取技术制备的中药配方颗粒和超临界CO2萃取物等四种制剂的急性毒性,明确超临界CO2萃取技术制备配方颗粒的安全剂量范围。方法将药物制成最大可溶浓度的水溶液,小鼠按照0.4 ml/10 g体重灌胃给药,空白对照组以同样容积的去离子水灌胃,24 h内给药3次,连续观察14 d动物出现的反应。结果在观察的14 d内,动物均未出现任何异常行为变化;体重未有减轻现象,与对照组相比,无显著差异;也无任何动物死亡;17味中药饮片不同工艺的最大给药量的临床用药剂量倍数在75.39～2 629.05倍之间。结论 17味同批次中药饮片所制备的标准煎剂、膏粉剂、超萃物和配方颗粒,均为无毒性药物,超临界萃取中药配方颗粒临床可安全使用。