Development of a pair of real-time loop mediated isothermal amplification assays for detection of Yersinia pestis, the causative agent of plague.

Yersinia pestis, the causative agent of plague mainly infects rodents, while humans are the accidental host. The conventional diagnostic methods available for Y. pestis exhibit cross-reactivity with other enteropathogenic bacteria which makes its detection difficult. Rapid and reliable point-of-care detection of Y. pestis is essential for timely initiation of medical treatment. In the present study, a pair of loop mediated isothermal amplification (LAMP) assays has been developed for rapid detection of Y. pestis. Two sets of LAMP primers, each containing 6 primers were specifically designed targeting caf1 and 3a genes located on pFra plasmid and chromosome of Y. pestis, respectively. Isothermal amplification was accomplished at 65 °C for 40 min for caf1 target, and at 63 °C for 50 min for 3a choromosomal target. The analytical sensitivity of the assay for the caf1 and 3a targets was found to be 500 fg and 100 fg genomic DNA of Y. pestis, respectively. The caf1 and 3a LAMP assays detected as few as 100 copies of caf1 and 10 copies of 3a gene targets harboured in the respective recombinant plasmids. The amplified products were detected visually under visible and UV light using SYBR Green 1 dye. The assay pair was found to be highly specific as it did not cross-react with closely related and other bacterial species.

BA3338, a surface layer homology domain possessing protein augments immune response and protection efficacy of protective antigen against Bacillus anthracis in mouse model.

AIM: Category A classified Bacillus anthracis is highly fatal pathogen that causes anthrax and creates challenges for global security and public health. In this study, development of a safe and ideal next-generation subunit anthrax vaccine has been evaluated in mouse model. METHOD AND RESULTS: Protective antigen (PA) and BA3338, a surface layer homology (SLH) domain possessing protein were cloned, expressed in heterologous system and purified by IMAC. Recombinant PA and BA3338 with alum were administered in mouse alone or in combination. The humoral and cell-mediated immune response was measured by ELISA and vaccinated animals were challenged with B. anthracis spores via intraperitoneal route. The circulating IgG antibody titre of anti-PA and anti-BA3338 was found significantly high in the first and second booster sera. A significant enhanced level of IL-4, IFN-γ and IL-12 was observed in antigens stimulated supernatant of splenocytes of PA + BA3338 vaccinated animals. A combination of PA and BA3338 provided 80% protection against 20 LD50 lethal dose of B. anthracis spores. CONCLUSION: Both antigens induced admirable humoral and cellular immune response as well as protective efficacy against B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has been evaluated for the first time using BA3338 as a vaccine candidate alone or in combination with well-known anthrax vaccine candidate PA. The findings of this study demonstrated that BA3338 could be a co-vaccine candidate for development of dual subunit vaccine against anthrax.

Development of a novel chimeric PA-LF antigen of Bacillus anthracis, its immunological characterization and evaluation as a future vaccine candidate in mouse model.

Tremendous efforts are being made to develop an anthrax vaccine with long term protection. The main component of traditional anthrax vaccine is protective antigen (PA) with the trace amount of other proteins and bacterial components. In this study, we developed a recombinant PA-LF chimera antigen of Bacillus anthracis by fusing the PA domain 2-4 with lethal factor (LF) domain 1 and evaluated its protective potential against B. anthracis in mouse model. The anti-PA-LF chimera serum reacted with both PA and LF antigen, individually. The chimera elicited a strong antibody titer in mice with predominance of IgG1 isotype followed by IgG2b, IgG2a and IgG3. Cytokines were assessed in splenocytes of immunized mice and a significant up-regulation in the expression of IL-4, IL-10, IFN-γ and TNF-α was observed. The PA-LF chimera immunized mice exhibited 80% survival after challenge with virulent spores of B. anthracis. Pathological studies showed normal architecture in vital organs (spleen, lung, liver and kidney) of recovered immunized mice on 20 DPI after spore challenge. These findings suggested that PA-LF chimera of B. anthracis elicited good humoral as well as cell mediated immune response in mice, and thus, can be a potent vaccine candidate against anthrax.

An ELISA using a recombinant chimera of protective antigen and lethal factor for serodiagnosis of cutaneous anthrax in India.

In this study, an ELISA was developed for simultaneous detection of antibodies against both the important toxins of B. anthracis i.e. protective antigen (PA) and lethal factor (LF). A chimera of PA and LF was made by fusion and cloned and expressed in E. coli. The purified recombinant protein was used in plate ELISA for serodiagnosis of anthrax. The chimera could detect antibodies against both the toxins of Bacillus anthracis. The human serum samples (n = 98) collected from anthrax endemic and non-endemic areas were tested employing ELISA. The ELISA gave sensitivity of 100% (95% Confidence Interval [CI], 92.13 to 100) and specificity of 97.78% (95% Confidence Interval [CI], 88.23 to 99.94) with a J index of 0.97. The efficiency of ELISA was found to be 98.9% with the positive predictive value (PPV) and negative predictive value (NPV) of 97.8% and 100%, respectively. The chimera of PA and LF could be a better diagnostic antigen for serodiagnosis as the assay detects antibodies against both the toxins in early as well delayed infection cases of anthrax. Therefore, it can be a very useful tool for the surveillance as well as for confirmation of cutaneous anthrax cases.

Molecular expression of caprine estrogen receptor gene 1 in reproductive and non-reproductive tissues.

During the last decades, physiological effects of oestrogens have been increasingly explored by scientists and biotechnologists. Estrogens exert a wide range of effects on a large variety of cell types. Oestrogen and its receptors are essential for sexual development and reproduction. Estrogen receptor alpha is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene estrogen receptor gene 1 (ESR1), responsible for better fertility. The ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. Therefore, this is a good startby to study the expression pattern of estrogen receptor 1 gene in high-fertile (G1) and low-fertile (G2) bucks of Jamunapari and Barbari breeds identified on the basis of seminal quality traits and fertility trials. RNA was extracted from the tissues by TRIzol method. The identification and expression pattern of caprine ESR1 gene was analysed by real-time PCR (Roche LC-480). Our work shows that the relative quantification by RT-PCR indicates more fold in head of epididymis as compared to spleen of caprine ESR1 gene. Furthermore, the RT-PCR indicated that fertile bucks of Jamunapari breed have more fold value as compared to Barbari breed in respect of reproductive organ.

Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax.

BACKGROUND & OBJECTIVES: Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. METHODS: Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. RESULTS: The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). INTERPRETATION & CONCLUSIONS: The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers.

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82 % polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03-0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100 % bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.

Molecular Characterization of Vibrio cholerae O1 Reveals Continuous Evolution of Its New Variants in India.

Vibrio cholerae, the causing agent of cholera is still a major health challenge in most of the developing countries. In this study, V. cholerae strains collected from different cholera outbreaks in India over a period of past 7 years were found to have various toxigenic, pathogenic and regulatory genes viz. ctxAB, zot, tcp, hlyA, ace, ompU, ompW, rfbO1, toxT and toxR. The biotype specific genes rstR and rtxC revealed the El Tor biotype in majority of the isolates. However, variants among the isolates were found having genotype of both the biotypes. Sequencing of ctxB gene revealed the presence of altered ctxB of classical biotype with additional variations in isolates of 2007. Mismatch amplification mutation assay PCR also confirmed the isolates belonging to classical biotype. Antibiogram of the isolates revealed resistance for nalidixic acid, co-trimoxazole, streptomycin, and polymyxin B and susceptibility for tetracycline among most of the isolates from India. However, V. cholerae isolates from a recent outbreak in Eastern India were resistant to tetracycline. The study corroborated the continuous emergence and wide-spread of multidrug resistant El Tor variant strains in the Indian subcontinent.

Surface Plasmon Resonance Biosensor for Detection of Bacillus anthracis, the Causative Agent of Anthrax from Soil Samples Targeting Protective Antigen.

Bacillus anthracis, the causative agent of anthrax is one of the most important biological warfare agents. In this study, surface plasmon resonance (SPR) technology was used for indirect detection of B. anthracis by detecting protective antigen (PA), a common toxin produced by all live B. anthracis bacteria. For development of biosensor, a monoclonal antibody raised against B. anthracis PA was immobilized on carboxymethyldextran modified gold chip and its interaction with PA was characterized in situ by SPR and electrochemical impedance spectroscopy. By using kinetic evaluation software, KD (equilibrium constant) and Bmax (maximum binding capacity of analyte) were found to be 20 fM and 18.74, respectively. The change in Gibb's free energy (∆G = -78.04 kJ/mol) confirmed the spontaneous interaction between antigen and antibody. The assay could detect 12 fM purified PA. When anthrax spores spiked soil samples were enriched, PA produced in the sample containing even a single spore of B. anthracis could be detected by SPR. PA being produced only by the vegetative cells of B. anthracis, confirms indirectly the presence of B. anthracis in the samples. The proposed method can be a very useful tool for screening and confirmation of anthrax suspected environmental samples during a bio-warfare like situation.

Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax.

Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 103 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.

Development of a set of three real-time loop-mediated isothermal amplification (LAMP) assays for detection of Bacillus anthracis, the causative agent of anthrax.

Bacillus anthracis, the causative agent of anthrax is a Gram-positive, non-motile, spore forming bacterium. Its spores can persist in soil and water for years and can also be aerosolized. A rapid, sensitive and specific method to detect B. anthracis is important for clinical management and preventing spread of anthrax. Loop-mediated isothermal amplification (LAMP) assay is a rapid technique that amplifies target DNA in isothermal conditions with high sensitivity and specificity. In this study, a LAMP assay set targeting a chromosomal and two plasmid markers was developed. The individual assays of the LAMP set targeting pXO1 plasmid (lef), pXO2 plasmid (capB), and chromosome (BA5345) sequences could detect 10, 250, and 100 fg of genomic DNA and 10, 100, and 50 copies of the DNA targets harboured in recombinant plasmids, respectively. The lef and capB LAMP assays could detect ≥ 1 × 103 CFU per mL of bacteria in spiked human blood samples, while BA5345 LAMP assay could detect ≥ 1 × 104 CFU of bacteria per mL of spiked blood. The amplification was monitored in real-time by turbidimeter, and visual detection was also accomplished under normal and UV light after adding SYBR Green 1 dye on completion of the reaction. The assay set was found to be highly sensitive and did not cross-react with the closely related Bacillus spp. and other bacterial strains used in the study.

Development of a PCR-lateral flow assay for rapid detection of Yersinia pestis, the causative agent of plague.

Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category 'A' agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5' 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.

Molecular characterization of Vibrio cholerae outbreak strains with altered El Tor biotype from southern India.

Forty-four Vibrio cholerae isolates collected over a 7-month period in Chennai, India in 2004 were characterized for gene traits, antimicrobial susceptibility and genomic fingerprints. All 44 isolates were identified as O1 El Tor Ogawa, positive for various toxigenic and pathogenic genes viz. ace, ctxB, hlyA, ompU, ompW, rfbO1, rtx, tcpA, toxR and zot. Nucleotide sequencing revealed the presence of cholera toxin B of classical biotype in all the El Tor isolates, suggesting infection of isolates by classical CTXPhi. Antibiogram analysis showed a broad-spectrum antibiotic resistance that was also confirmed by the presence of resistant genes in the genomes. All isolates contained a class 1 integron and an SXT constin. However, isolates were sensitive to chloramphenicol and tested negative for the chloramphenicol resistant gene suggesting a deletion in SXT constin. Fingerprinting analysis of isolates by ERIC- and Box PCR revealed similar DNA patterns indicating the clonal dissemination of a single predominant V. cholerae O1 strain throughout the 2004 outbreak in Chennai. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11274-009-0171-7) contains supplementary material, which is available to authorized users.

A large cholera outbreak due to a new cholera toxin variant of the Vibrio cholerae O1 El Tor biotype in Orissa, Eastern India.

A total of 32 Vibrio cholerae isolates were collected during a recent large cholera outbreak in Eastern India. Biochemical and serological studies revealed that all of the isolates belonged to serogroup O1, biotype El Tor, serotype Ogawa. Two multiplex PCR assays confirmed the presence of various toxigenic and pathogenic genes - ace, ctxAB, hlyA, ompU, ompW, rfbO1, rtx, tcp, toxR and zot - in all of the isolates. Sequencing of the ctxB gene from the isolates revealed a novel mutation in the gene. Sequencing also confirmed the presence of altered cholera toxin B of the classical biotype in all of the El Tor isolates, suggesting infection of isolates by classical CTXPhi. The molecular diversity of V. cholerae isolates studied by enterobacterial repetitive intergenic consensus sequence PCR, BOX-PCR and randomly amplified polymorphic DNA analysis uniformly showed the clonal relationship among the outbreak V. cholerae O1 isolates. The results of this study suggest that cholera-causing V. cholerae strains are constantly evolving in epidemic areas, highlighting the potential of the emergence of more virulent strains.

Relationship of foetal number and parity in Barbari goats to plasma profile of caprine pregnancy-associated glycoprotein (caPAG) during gestation and the early postpartum period.

This study was conducted to characterise pregnancy-associated glycoprotein (caPAG) in peripheral plasma during gestation and postpartum periods of nulliparous and multiparous does with one or two foetuses using a caPAG specific two-step sandwich ELISA system. Earliest time-points for detection of pregnancy and foetal number with appropriate cut-off values were identified. Plasma samples from 15 pregnant (multiparous: n = 8; nulliparous: n = 7; during pregnancy and postpartum period) and six non-pregnant (during oestrous cycle) goats were collected and analysed. Mean caPAG concentration was greater than the threshold for pregnancy detection (S-N = 0.40) on d22, peaked on d45 and remained unchanged until parturition. From d45 until parturition, caPAG concentration in multiparous does with two foetuses was 1.4 to 1.8 fold greater (P < 0.001) than those with one foetus. For the ELISA, 0.83 (S-N) was the most appropriate cut-off to differentiate does with two from those with a single foetus with an overall sensitivity and accuracy of 88.9% and 84.7%, respectively. Circulating caPAG concentration in multiparous goats was greater (P < 0.05) compared with nulliparous goats during the early pregnancy and postpartum periods. After parturition, caPAG concentrations markedly decreased and were basal within 14 days postpartum. In conclusion, using the caPAG specific ELISA, results indicated there were unique gestational and postpartum profiles for caPAG concentrations that are affected by number of foetuses and parity of the doe. The marked decrease in concentration of caPAG following parturition indicates there would not be compromising of the detection of subsequent pregnancies in goats using this technique.

Development of a real-time loop-mediated isothermal amplification assay for detection of Burkholderia mallei.

Burkholderia mallei is the aetiological agent of glanders, a highly contagious and re-emerging zoonotic disease. Early diagnosis of glanders is critically important to ensure timely treatment with appropriate antibiotics in humans, and to prevent spread of infection in animals. Molecular detection of B. mallei has always been troublesome because of its genetic similarity with Burkholderia pseudomallei, the causative agent of melioidosis. In present investigation, a set of six B. mallei-specific primers were designed and a simple, rapid, specific and sensitive real-time loop-mediated isothermal amplification (LAMP) assay was developed for detection of B. mallei. The LAMP assay could detect as low as 1 pg of B. mallei genomic DNA and 5.5 × 103  CFU/ml of B. mallei in spiked human blood. The assay was highly specific for B. mallei as it did not cross-react with other bacterial strains used in the study. The established LAMP assay is field adaptable and can be a better and viable alternative to PCR-based techniques for detection of B. mallei in glanders endemic areas with resource-limited settings.

Single multiplex polymerase chain reaction for environmental surveillance of toxigenic-pathogenic O1 and non-O1 Vibrio cholerae.

A multiplex PCR assay was developed for the detection of toxigenic and pathogenic V. cholerae from direct water sources using specific primers targeting diverse genes, viz. outer membrane protein (ompW), cholera toxin (ctxB), ORF specific for O1 (rfbG), zonula occludens (zot) and toxin co-regulated pilus (tcpB); among these genes, ompW acts as internal control for V. cholerae, the ctx gene as a marker for toxigenicity and tcp for pathogenicity. The sensitivity of multiplex PCR was 5 x 10(4) V. cholerae cells per reaction. The procedure was simplified as direct bacterial cells were used as template and there was no need for DNA extraction. The assay was specific as no amplification occurred with the other bacteria used. Toxigenic V. cholerae were artificially spiked in different water samples, filtered through a 0.45 microm membrane, and the filters containing bacteria were enriched in APW for 6 h. PCR following filtration and enrichment could detect as little as 8 V. cholerae cells per mL in different spiked water samples. Various environmental potable water samples were screened for the presence of V. cholerae using this assay procedure. The proposed method is rapid, sensitive and specific for environmental surveillance for the presence of toxigenic-pathogenic and nonpathogenic V. cholerae.

Development of a Novel Herbal Formulation To Inhibit Biofilm Formation in Toxigenic Vibrio cholerae.

Vibrio cholerae, a causative agent of the waterborne disease cholera, still threatens a large proportion of world's population. The role of biofilm formation in V. cholerae pathogenesis is well established, as it provides the bacterium enhanced tolerance to antimicrobial agents and increased transmission. In the present study, four medicinal plants used in traditional medicines with antidiarrheal properties were evaluated for its antibiofilm activity. Methanol extracts of these plants (Centella asiatica, Elephantopus scaber, Camellia sinensis, and Holarrhena antidysenterica) showed promising antibiofilm activity against V. cholerae with crystal violet and air-liquid interface coverslip assays. Results revealed that C. asiatica, E. scaber, C. sinensis, and H. antidysenterica extracts significantly inhibited biofilm formation by approximately 75, 76, 78, and 55% at concentrations of 3, 2, 1, and 0.6 mg/mL, respectively. A promising antibiofilm activity of ∼89% inhibition at 1.5 mg/mL concentration was observed when a combination of E. scaber and C. sinensis was used. The herbal extracts were thermostable at a temperature range of 40 to 100°C. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay revealed that the viability of bacteria was not affected by treatment with these plant extracts. Gene expression studies revealed that extracts of H. antidysenterica leaf, H. antidysenterica bark, and the whole plant of E. scaber and C. asiatica down-regulate aphA or aphB, the major regulator genes modulating both virulence and biofilm formation. Hence, we propose that these herbal combinations could serve as a multifaceted approach to combat the pathogen and also, in turn, reduce antimicrobial resistance development.

Bioactivity of marine organisms: part X--screening of some marine fauna from the Indian coasts.

Methanolic and chloroform-methanol extracts of 25 identified species of marine fauna have been screened for a wide range of biological activities. Of these, 2 extracts exhibited diuretic activity, while antibacterial, antiviral, oxytocic and spasmolytic activities, were observed in 1 extract each.

Bioactivity of marine organisms: Part IX--screening of some marine flora from the Indian coasts.

Alcoholic extracts of 48 identified species of marine flora were screened for a wide range of biological activities. Of these, 3 extracts showed diuretic activity while 2 extracts showed hypotensive effect.