Inhibition of CCl4-induced liver fibrosis by Piper longum Linn.?

This study was carried out to evaluate the antifibrotic effect of ethanol extract of the fruits of Indian herb Piper longum Linn. Liver fibrosis was induced in rats by CCl(4) administration. The extent of liver fibrosis was assessed by measuring the level of liver hydroxy proline (HP) and serum enzyme levels. Following CCl(4) administration HP was significantly increased and serum enzyme levels were elevated. Treatment with the ethanol extract of Piper longum Linn. reduced the HP and also the serum enzymes. The liver weight that increased following CCl(4) administration due to the deposition of collagen was reduced by the ethanol extract. Hence, it is concluded that this extract inhibits liver fibrosis induced by CCl(4).     

Enzymatic and non-enzymatic antioxidants in selected Piper species

Piper species, commonly used in diet and traditional medicine were assessed for their antioxidant potential. Catalase activity was predominated in Piper longum, followed by Piper cubeba, green pepper, Piper brachystachyum and Piper nigrum. P. nigrum was richest in glutathione peroxidase and glucose-6-phosphate dehydrogenase, green pepper was richest in peroxidase and vitamin C while vitamin E was more in P. longum and P. nigrum. P. brachystachyum and P. longum were rich sources of vitamin A. All the Piper species had GSH content of around 1 to 2 nM/g tissue. The antioxidant components of Piper species constitute a very efficient system in scavenging a wide variety of reactive oxygen species. Antioxidant potential of Piper species was further confirmed by their ability to curtail in vitro lipid peroxidation by around 30-50% with concomitant increase in GSH content.     

Analgesic activity of Piper longum Linn. root

Piper longum root, commonly called Kandantippili, is traditionally used to treat rheumatism, insomnia, palsy and epilepsy. But a scientific study on its central actions is not available. This study screens P. longum root for opioid type analgesia using rat tail-flick method and for NSAID type analgesia using acetic-acid writhing method. Pentazocine (ip) and ibuprofen (oral) are used as respective drug controls. An aqueous suspension of P. longum root powder is given orally to mice and rat in doses of 200, 400 and 800 mg/kg. The delay in reaction time for thermal stimulus in rats and the number of writhings to chemical stimulus in mice are determined in each group. The results are analysed statistically. The 400 and 800 mg/kg doses of P. longum show significant NSAID type of analgesia (P < 0.001). Both Ibuprofen (40 mg/kg) and P. longum (800 mg/kg) show 50% protection against writhing. The delay in reaction time to thermal stimulus was less than 6% for different doses of P. longum as against 100% for pentazocine. This indicates that P. longum root has weak opioid but potent NSAID type of analgesic activity.     

Molecular docking analysis of vascular endothelial growth factor receptor with bioactive molecules from Piper longum as potential anti-cancer agents

It is known that vascular endothelial growth factor receptor (VGFR) is linked with cancer. Therefore, it is of interest to document the molecular binding features of bioactive molecules from Piper longum as potential anti-cancer agents with VGFR2 for further consideration. Thus, we document the binding features of four compounds (sesamin, fargesin, longamide and piperlonguminine) with VGFR2 for further consideration in drug discovery.     

论紫堇属的系统演化与区系发生和区系分区的关系

紫堇属 Corydalis DC.是紫堇科 Fumariaceae 的最大属,也是北温带分布类型的大属之一。本属自建立以来,迄今尚无完整的分类系统,甚至连属的命名人、后选属模式以及属下分类单位的划分均存在许多争议,本文根据作者们二十多年来在国内外有关标本室收集的模式标本和有关东亚、南亚标本,经过鉴定整理,首次将本属划分为两大类群,即Corydalis群和Pistolochia群和40个组,并初步建立了一个较全而新的进化系统。     

Effect of ayurvedic medicines on beta-glucuronidase activity of Brunner's glands during recovery from cysteamine induced duodenal ulcers in rats

Biochemical and histochemical studies revealed decreased beta-glucuronidase activity in the Brunner's glands of duodenal ulcerated rats. The enzyme activity showed gradual increase during recovery. Rats treated with a mixture of Ayurvedic medicines (Glycyrrhiza glabra, Terminalia chebula, Piper longum and Shanka Bhasma) recovered faster with concomitant increase in beta-glucuronidase activity in the Brunner's glands. It can be concluded that Ayurvedic medicines used do not act as antacid but improve the secretory status of Brunner's glands involved in the protection against duodenal ulcer.     

我国胡椒属植物区系地理研究

胡椒属(Piper)为胡椒科(Piperaceae)重要的泛热带组成成分,约有1 000 ～2 000个植物种类,中国是其天然分布的北缘.本文对我国产胡椒属植物区系地理进行了分析,揭示其区系特征、物种起源和散布途径等.结果表明:云南是我国胡椒属植物的重要分布地,并以此为中心向我国东部和北部扩散,物种数量逐渐减少.各省胡椒属物种组成相似性较低,大致可被聚为热带地区、亚热带地区和台湾地区3大分支.在区系成分上,国产胡椒属植物中的中国特有比例最高,其次是热带亚洲分布,只有个别种为泛热带分布和东亚分布.我国胡椒属区系与中南半岛、印度半岛及爪哇群岛存在一定联系,但并不紧密.分析认为,云南是胡椒属植物的起源中心或分化中心之一,而中国台湾岛种类主要由菲律宾群岛等传入并分化.本结果对于研究该属系统分类和起源演化将具有一定意义.     

Floristic Geography of Piper (Piperaceae) in China

Piper is the largest genus and important Pantropical components of the Piperaceae family with approximately 1000-2000 species in the world. China is in the northern verge of its natural distribution. In this study, floristic geography of the Piper genus in China was analyzed to reveal its botanical characteristic, origins of speciation and its dispersal channels. The study results showed Yunnan province is one of the very important distribution regions for Piper species, served as the center of the Piper species dispersal toward to the eastern and northern regions of China with gradually reduced species numbers. The similarity level appeared to be low for its species among different provinces, and the Piper distribution regions could be approximately clustered into three clades: tropical clade, subtropical clade and Taiwan clade. Cladistically, Piper species found in China have the highest proportions which were endemic to China, and then followed by Piper species distributed in Tropical Asia, only few species belonged to Pantropical and East Asia areal types. The floristic geography of Piper genus in China is related to that of Indo Chinese Peninsula, Java Islands and Indian Peninsula in some degrees, but not closely related. This study revealed Yunnan province is one of the central of origins for the Piper species, and or one of the central of regions for its species diversification, and the Piper species of Taiwan mainly came from Philippine islands and further diversified into Taiwan clade. Our study results presented a useful method for the systematic studying of Piper genus taxonomy and the evolution of Piper speciation.     

Alkamides from the fruits of Piper longum and Piper nigrum displaying potent cell adhesion inhibition

Eight alkamides 1-8 were isolated by bioassay-guided isolation of EtOH extracts of the fruits of Piper longum and Piper nigum (Piperaceae). Their structures were elucidated by spectroscopic analysis ((1)H, (13)C NMR, and ESI-MS) as follows: guineensine (1), retrofracamide C (2), (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (3), pipernonaline (4), piperrolein B (5), piperchabamide D (6), pellitorin (7), and dehydropipernonaline (8). Their compounds 3-5, 7, and 8 inhibited potently the direct binding between sICAM-1 and LFA-1 of THP-1 cells in a dose-dependent manner, with IC(50) values of 10.7, 8.8, 13.4, 13.5, and 6.0 microg/mL, respectively.     

Liquid chromatographic method for determination of piperine in rat plasma: application to pharmacokinetics

Piperine, a major alkaloid of Piper longum and Piper nigrum has been reported to have several pharmacological/toxicological effects. Though a number of methods for analysis of this omnipresent food component in pepper fruits are available, its analysis in body fluids has been largely neglected. A high-performance liquid chromatography method for the analysis of piperine in rat plasma is presented in this communication. Analysis was performed using a Symmetry C(18) column (250x4.6 mm) by isocratic elution with 25 mM KH(2)PO(4) (pH 4.5)-acetonitrile (35:65) and UV detection at 340 nm. The calibration plot was linear over the range studied (2-2000 ng) with correlation coefficient of 0.9984. Limit of detection and limit of quantitation were 1 ng/ml and 3 ng/ml, respectively. Good overall recovery (85.5+/-6%) was obtained with 4 ml ethyl acetate and extraction time of 3 min. Intra- and inter-assay coefficient of variation was found to be less than 7.5%. Plasma concentration-time profile of piperine in a conscious rat implanted with jugular vein cannula was obtained using this method. The method is simple, sensitive and reproducible.     

Novel aromatic ester from Piper longum and its analogues inhibit expression of cell adhesion molecules on endothelial cells

We report here the isolation and characterization of two active principles, ethyl 3',4',5'-trimethoxycinnamate (1) and piperine (2), from the combined hexane and chloroform extracts of Piper longum. Using primary human umbilical vein endothelial cells, we evaluated the activities of compound 1 on TNF-alpha-induced expression of cell adhesion molecules, viz., ICAM-1, VCAM-1, and E-selectin, which play key roles in controlling various inflammatory diseases. Both compounds 1 and 2 inhibited the TNF-alpha-induced expression of ICAM-1 in a dose- and time-dependent manner; however, the activity of ethyl 3',4',5'-trimethoxycinnamate (1) was approximately 1.3 times higher than that of piperine (2). As ethyl 3',4',5'-trimethoxycinnamate (1) has been isolated for the first time from a natural source, Piper longum, and it exhibited higher activity, we carried out further studies on it. To correlate its cell adhesion molecule inhibitory activity with its functional consequences, we showed that it significantly blocked the adhesion of neutrophils to endothelium in a time- and concentration-dependent manner. Importantly, the inhibitory effect of cinnamate 1 was found to be reversible. To elucidate its structure-function-activity relationship, we synthesized nine different analogues of ethyl 3',4',5'-trimethoxycinnamate, i.e., compounds 3-11, and compared the ICAM-1 inhibitory activity of compound 1 with those of its synthetic analogues as well as the corresponding acids 12-15. The structure-activity studies indicate that the chain length of the alcohol moiety, substituents in the aromatic ring, and alpha, beta-double bond of the cinnamic acid ester have significant effects on the inhibition of TNF-alpha-induced expression of ICAM-1 on endothelial cells. These findings have implications in developing compounds with a better therapeutic index against various inflammatory diseases.     

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae).

Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P. sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage, while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides used for mosquito control.     

大肠埃希菌—双歧杆菌穿梭表达载体的构建及白介素-10基因的表达

目的构建能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因的载体,并用此载体在大肠埃希菌和双歧杆菌中表达人白介素-10基因（hIL-10）的蛋白产物;为hIL-10基因重组双歧杆菌治疗炎症性肠病做前期准备。方法以质粒pDG7为模板扩增pMB1片段,构建表达质粒pET28B1。用PCR法扩增hIL-10基因,将此目的基因以及pET28B1经酶切后用连接酶连接,形成重组质粒pET28B1-hIL10。pET28B1-IL10转染大肠埃希菌BL21和长双歧杆菌。最后用Western blot检测hIL-10基因在大肠埃希菌和长双歧杆菌中的表达情况。结果pET28B1-hIL10阳性克隆扩增后提取质粒并进行基因测序,结果显示插入片段为hIL-10,序列正确且无突变。hIL-10基因在大肠埃希菌、长双歧杆菌中的诱导表达产物通过Western blot检测验证为IL-10蛋白,显示该hIL-10表达载体在大肠埃希菌阳性克隆中经诱导可高量表达,在长双歧杆菌体中有少量表达。结论成功构建质粒pET28B1,该质粒能够在大肠埃希菌和双歧杆菌中穿梭表达目的基因hIL-10。     

Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.     

双歧杆菌原生质体的制备与回复研究

进行了双歧杆菌原生质体的制备与回复相关技术研究 ,为其基因操作及相关研究提供技术基础。采用浓度分别为 1 ,5 ,1 0mg/LMutanolysin(变溶菌素 )对长双歧杆菌进行脱壁处理 ,以探讨其原生质体形成与时间和酶浓度的关系 ,然后选用较适宜的酶浓度 ( 5mg/LMutanolysin)制备其原生质体 ,并将其倾入自制的双层再生培养基上 ,观察其在不同环境条件下培养时的回复生长情况。结果表明 ,长双歧杆菌的细胞壁对Mu tanolysin较为敏感 ,用浓度为 5mg/L的Mutanolysin处理长双歧杆菌 40min ,在普通光学显微镜下即可见90 %的原生质体形成 ,当Mutanolysin浓度为 1 0mg/L时 ,只需 2 5min其原生质体形成率就达此值。制备的长双歧杆菌原生质体倾入自制的双层再生培养基中 ,在厌氧条件下能很好地回复生长。     

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: Influence on brush border membrane fluidity, ultrastructure and enzyme kinetics

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.     

Cloned cytosine deaminase gene expression of Bifidobacterium longum and application to enzyme/pro-drug therapy of hypoxic solid tumors

Bifidobacterium longum is a nonpathogenic anaerobic bacterium among normal bacterial flora. Recently, it was reported that B. longum accumulated in hypoxic solid tumors. The gene of interest was expressed in transfected B. longum by the shuttle vector pBLES100 in solid tumors. In this report, we constructed pBLES100-S-eCD, which included the cytosine deaminase gene. We confirmed by western blotting that transfected B. longum produced cytosine deaminase. In addition, transfected B. longum produced cytosine deaminase that converted 5-fluorocytosine into 5-fluorouracil. B. longum could be useful for enzyme/pro-drug therapy of hypoxic solid tumors.     

Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy

In our previous studies, a strain of the nonpathogenic, anaerobic, intestinal bacterium, Bifidobacterium longum (B. longum), was found to be localized selectively and to proliferate within solid tumors after systemic administration. In addition, B. longum transformed with the shuttle-plasmid encoding the cytosine deaminase (CD) gene expressed active CD, which deaminated the prodrug 5-fluorocytosine (5-FC) to the anticancer agent 5-fluorouracil (5-FU). We also reported antitumor efficacy with the same plasmid in several animal experiments. In this study, we constructed a novel shuttle-plasmid, pAV001-HU-eCD-M968, which included the mutant CD gene with a mutation at the active site to increase the enzymatic activity.In addition, the plasmid-transformed B. longum produces mutant CD and strongly increased (by 10-fold) its 5-FC to 5-FU enzymatic activity. The use of B. longum harboring the new shuttle-plasmid increases the effectiveness of our enzyme/prodrug strategy.     

大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。