Three Bacteriocin Peptides from a Lactic Acid Bacterium Weissella confusa MBF8-1 with Spermicidal Activity.

BACKGROUND: The development of antibiotic resistance amongst bacterial pathogens and a population explosion, e.g. in countries such as Indonesia, are two issues the world is facing today. These issues have stimulated interest in the development of new antimicrobial therapeutic agents and contraceptive strategies, such as novel spermicides. Bacteriocins, which are bacterially-derived antimicrobial peptides, may fulfill some of the criteria for these new agents. METHODS: Weissella confusa MBF8-1, originally isolated from a homemade soy product, exhibits antibacterial activity that was subsequently found to be plasmid-encoded, presumably by three peptides Bac1, Bac2 and Bac3. In the present study, we tested cell-free MBF8-1 bacteriocin preparations and chemically-synthesized versions of Bac1, Bac2 and Bac3 peptides for (i) its antibacterial activity against the indicator bacterium Leuconostoc mesenteroides and (ii) its ability to affect the motility of spermatozoa. Nisin, a known lantibiotic bacteriocin, was used as the control. RESULTS: Here, we demonstrate that synthetic Bac1, in combination with synthetic Bac2, was sufficient to inhibit the growth of L. mesenteroides and affect sperm motility. However, the presence of all three synthetic peptides, s-Bac1, s-Bac2 and s-Bac3, was required for full potency. CONCLUSION: In summary, the bacteriocin-like peptides of W. confusa MBF8-1 have the potential to be developed as a narrow-spectrum antimicrobial agent and a novel spermicidal agent.

Selective recovery of acidic and lactonic sophorolipids from culture broths towards the improvement of their therapeutic potential.

Sophorolipids (SLs) were produced by Starmerella bombicola. The separation and purification of SLs are a complex process, since they are produced as a mixture of compounds with few structural differences. Solvent extraction is commonly used in downstream processing. In this work, an environmental friendly approach was developed for SLs recovery and purification, based on neutral polymeric sorbents, Amberlite XAD16NTM, XAD18TM, and XAD1600NTM. In batch microassays, key parameters of sorption/desorption process (e.g., contact time, temperature, sorbents, and SLs concentrations) were optimized for separation of acidic and lactonic SLs. Sorption equilibrium was reached after 2-3 h, for all the sorbents tested. Among them XAD1600NTM showed a higher sorption capacity (q max 230 mg g-1), a higher removal (≈100 %) of acidic and lactonic SLs [1 and 2.5 % (w/v)], and the best selectivity. Methanol, ethanol, and acetone were suitable for SLs elution. A selective desorption of SLs was attained with acetonitrile aqueous solutions (v/v): (1) 25 % led to 88.3 % of acidic SLs and (2) 55 % followed by methanol solution (100 %) led to 93.2 % of purified lactonic SLs. This achievement was particularly important regarding SLs potential therapeutic applications, since acidic and lactonic SLs show different biologic activities. In fact, acid SLs show higher virucidal and pro-inflammatory cytokine activity, while lactonic SLs show stronger spermicidal and anti-cancer activity.

Effects of Heliopsis longipes ethanolic extract on mouse spermatozoa in vitro.

CONTEXT: Heliopsis longipes (A. Gray) Blake (Asteraceae), a plant native to Mexico, is used in traditional medicine as analgesic and microbicide. The main component in the H. longipes ethanolic extract (HLEE) is affinin, as determined by HPLC/UV-visible and NMR measurement. To date, there is no documented evidence on the spermicidal activity of this extract. OBJECTIVE: The objective of this study was to assess in vitro the effectiveness of HLEE as spermicide. MATERIALS AND METHODS: The spermicidal activity of HLEE was evaluated by the Sander-Cramer assay. Spermatozoa were incubated for 20 s with HLEE in concentrations ranging from 75 to 2000 µg/mL to determine the minimum effective concentration (MEC) value. The 50% effective concentration (EC50) of HLEE was estimated by assaying serial dilutions from the MEC. Additionally, sperms were incubated with 125, 250, or 500 µg/mL of HLEE to evaluate the viability and the integrity of sperm membrane. Lipid peroxidation was assessed by the thiobarbituric acid reactive substances assay. RESULTS: HLEE caused an inhibition of 100% in spermatozoa motility at a MEC value of 2000 µg/mL; the EC50 value was 125 µg/mL. Additionally, exposure to HLEE at 125, 250, or 500 µg/mL for 30 min decreased sperm viability to 27%, 8%, and 2% of the control value, respectively, and significantly increased the percentage of sperms with structurally disorganized membrane. HLEE also increased significantly the level of lipid peroxidation in sperms with respect to controls. DISCUSSION AND CONCLUSION: The results demonstrate the spermicidal activity of HLEE in vitro and suggest that this action is caused by oxidative damage and alterations in the spermatozoal membrane.

Search for a potent microbicidal spermicide from the isolates of Shorea robusta resin.

BACKGROUND: An alarming increase in global population is the root cause of poverty, malnutrition, sexually transmitted infections (STIs) and many other social problems. Microbicidal spermicides possessing dual function of contraception and STI protection can effectively combat this problem, and their development is of utmost importance at present. STUDY DESIGN: A major metabolite isolated from Shorea robusta resin was spectroscopically characterized as asiatic acid. Spermicidal efficacy of the isolate was evaluated in vitro by a modified Sander-Cramer test. The mode of spermicidal action was assessed by (a) double fluoroprobe staining, (b) hypoosmotic swelling test and (c) scanning electron microscopy. Antimicrobial efficacy was assessed by disc diffusion and broth dilution methods using human isolates of bacteria (Escherichia coli ATCC 25938 and Pseudomonas aeruginosa 71) and fungus (Candida tropicalis). RESULTS: The minimum effective concentration of asiatic acid that induced instantaneous immobilization of rat spermatozoa in vitro was 125 mcg/mL. The mechanism of action involved disruption of sperm plasma membrane. The microbicidal efficacy was found to be moderate for vaginal pathogens, with no effect on normal vaginal flora. CONCLUSION: Asiatic acid possesses appreciable spermicidal and microbicidal potential and may be explored as an effective microbicidal spermicide.

Detection of the spermicide nonoxynol-9 via GC-MS.

The spermicide nonoxynol-9 is actually a complex mixture of dozens of closely related amphiphilic compounds, and the chemical properties of this assortment significantly hamper its characterization by GC-MS. The inability to perform routine GC-MS testing on nonoxynol-9 has limited its evidentiary value in forensic casework, which relies heavily on this technique for analysis. A disturbing trend in sexual assault is the use of condoms by assailants, to avoid leaving behind DNA evidence that can connect a perpetrator to a victim. This observation necessitates the development of alternative methods for the analysis of trace evidence that can show causal links between a victim and a suspect. Detection of lubricants associated with sexual assault is one such way to establish this connection. The development of GC-MS methods that permit facile detection of both nonoxynol-9 alone and nonoxynol-9 extracted from other complex matrices that have potential as trace evidence in sexual assault is reported. A detection limit of 2.14 µg of nonoxynol-9 is demonstrated, and a detailed mass spectral profile that elaborates on what is known of its structure is provided.

In vitro determination of the contraceptive spermicidal activity of essential oil of Trachyspermum ammi (L.) Sprague ex Turrill fruits.

This study was conducted to determine the spermicidal and contraceptive efficacy of essential oil of Trachyspermum ammi on human sperm in vitro. Chemical compositions of the oil were analyzed by GC-MS. Nearly 30 compounds representing 91.39% of the total oil were identified. The minimum effective dose (MED) of essential oil of T. ammi that induced instant immobilization of human spermatozoa in vitro was 125 µg/mL. The motility was also irreversible. All of the human sperms were found to be non viable within 10 min at this concentration. The activity of acrosomal enzyme was reduced and a significant releases of 5'-nucleotidase into the surrounding medium was noted after treatment with MED concentration of essential oil, indicating the plasma membrane degradation of the sperm. The maximum number of human sperm failed to decondense when treated with MED concentration of essential oil. The morphological deformities of sperm plasma membrane were evidenced by SEM, which showed vaculation, detachment of heads and tail coiling. The present research indicates that essential oil of T. ammi possesses appreciable spermicidal potential, which may be explored as an effective constituent of vaginal contraceptive.

Evaluation of spermicidal activity of MI-saponin A.

The seed extracts of Madhuca latifolia were reported to have spermicidal activity. The current investigation identified the spermicidal component of the extracts and evaluated its spermicidal potential in vitro. As characterized by infrared, mass, and nuclear magnetic resonance (NMR) spectral analyses, Mi-saponin A (MSA) was found to be the most potent component among a mixture of saponins. The mean effective concentrations of MSA that induced irreversible immobilization were 320 microg/mL for rat and 500 microg/mL for human sperm, as against the respective concentrations of 350 and 550 microg/mL of nonoxynol 9 (N-9). The mode of spermicidal action was evaluated by a battery of tests including (a) double fluoroprobe staining for sperm viability, (b) hypoosmotic swelling test and, assays for 5' nucleotidase and acrosin for physiological integrity of sperm plasma membrane, (c) scanning and transmission electron microscopy for sperm membrane ultrastructure, and (d) plasma membrane lipid peroxidation (LPO). The observations, taken together, were interpreted to mean that the spermicidal effect of MSA involved increased membrane LPO leading to structural and functional disintegration of sperm plasma membrane and acrosomal vesicle. A comparative in vitro cytotoxicity study in human vaginal keratocyte (Vk2/E6E7) and endocervical (End/E6E7) cell lines demonstrated that the 50% cell cytotoxicity (CC(50)) values, and consequently the safety indices, for MSA were >or= 8-fold higher as compared to those of N-9. In conclusion, MSA is a potent spermicidal molecule that may be explored further for its suitability as an effective component of vaginal contraceptive.

Acaciaside-B-enriched fraction of Acacia auriculiformis is a prospective spermicide with no mutagenic property.

As a part of our continued venture to develop a safe and effective spermicide, we have identified a triterpene glycoside (Acaciaside-B (Ac-B))-enriched fraction (Ac-B-en) isolated from the seeds of Acacia auriculiformis and evaluated its spermicidal potential in vitro. Sperm motility was completely inhibited within 20 s at a minimum effective concentration (MEC) of 120 microg/ml. Tests for sperm viability by dual fluoroprobe staining showed the effect to be spermicidal with an EC(50) of 35.20 microg/ml. A series of investigations including tests for hypo-osmotic swelling, membrane lipid peroxidation, and electron microscopy document that the spermicidal effect of the fraction involves loss of sperm plasma membrane integrity and dissolution of the acrosomal vesicle--the two most important structural components that play diverse roles in physiological functions of sperm including fertilization. The fraction at 10 x MEC exerted no detrimental effects on in vitro growth of Lactobacillus acidophilus, which is considered the major constituent of vaginal microflora that maintains vaginal health. Ames tests performed with different strains of Salmonella typhimurium including TA 97a, 98, 100, and 102, which detect mutagens causing bp substitution or frameshifting at G-C or A-T bp, demonstrate no mutagenic potential of the fraction. Significant spermicidal potential with no possible mutagenic effect and adverse impacts on lactobacilli growth attests to the credential of Ac-B-en as a prospective future spermicide for the development of a safe and effective vaginal contraceptive formulation.

Spermicidal activity of bivittoside D from Bohadschia vitiensis.

BACKGROUND: A recent revelation about increased susceptibility to HIV by use of nonoxynol-9 (N-9) has called for identification of novel molecules with potent sperm-attenuating activity and lower side-effect profile, as suitable alternatives. The present study was designed to investigate spermicidal activity in Bohadschia vitiensis whole-body extracts followed by isolation and characterization of bioactive molecule. METHODS: Bohadschia vitiensis (Semper) was collected from the Southern Andaman coast of India. Freshly collected marine animals were extracted with methanol. A portion of the crude extract was fractionated into four fractions by macerating with hexane, chloroform, and n-butanol successively. All fractions were evaluated for spermicidal activity. Because maximum activity was localized in the n-butanol soluble fraction, it was chromatographed over a silica gel column, and elution with chloroform-methanol-water (35:10:2, v/v) yielded the major compound bivittoside D (400 mg). Bivittoside D [molecular weight (MW) 1426] is a lanostane triterpenoid with six monosaccharide units. The structure of the compound was established on the basis of physicochemical data, acid hydrolysis of saponin, identification of sugar units and aglycon, melting point, and by comparison with data reported in the literature. RESULTS: The aqueous methanol extract of the Bohadschia vitiensis caused 100% mortality of human sperm at 0.01% concentration in vitro, whereas N-9 (reference control) exhibited an equivalent activity at 0.05%. On further fractionation, activity was localized in n-butanol soluble fraction from which the major compound purified was a lanostane triterpenoid called bivittoside D. Bivittoside D was found to be a more potent spermicide (approximately 2.3 times) than N-9 and killed 100% human sperm at the concentration of 350 muM in approximately 20 sec in vitro. Supravital staining and hypoosmotic swelling test revealed sperm membrane permeabilization by bivittoside D as the major mode of spermicidal action. However, bivittoside D was much safer than N-9 towards normal vaginal flora (Lactobacillus) in vitro, although it affected the viability of HeLa cells like other surfactants. CONCLUSION: Bivittoside D from B. vitiensis can adequately replace N-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly.

Potato aspartic proteases (StAPs) exert cytotoxic activity on bovine and human spermatozoa.

OBJECTIVE: To evaluate the in vitro spermicidal activity of Solanum tuberosum aspartic proteinases (StAPs) on bovine and human sperm. DESIGN: Controlled laboratory study. SETTING: Three research laboratories at a university of biologic science. ANIMAL(S) AND DONOR(S): Frozen semen from five Aberdeen Angus bulls and six proven fertile men volunteers. INTERVENTION(S): The effect of StAPs on sperm motility was studied in vitro by incubation of different concentrations of StAPs with sperm suspensions, and motility was assessed by direct microscopic observation. Membrane integrity was analyzed by SYTOX Green uptake after incubation with different StAP concentrations. The effect of StAPs was evaluated by human erythrocyte lysis, as a control in somatic cells. The StAPs binding was monitored by fluorescence. MAIN OUTCOME MEASURE(S): Total and progressive sperm motility; hypoosmotic swelling test and SYTOX Green uptake as a measure of membrane damage; fluorescein isothiocyanate-labeled StAP binding by an optical microscopy. RESULT(S): The StAPs reduced sperm motility in a dose-dependent manner, and 25 microM of StAP1 and 35 microM of StAP3 completely abolished the progressive motility. The StAPs were able to bind in the postacrosomal and midpiece region only in bovine sperm. Also, StAPs caused spermatozoa agglutination. In vitro cell toxicity was observed by a dose-dependent increase in hypoosmotic swelling negative sperm and SYTOX Green uptake in both human and bovine spermatozoa; however, no toxic effect was observed on erythrocytes. CONCLUSION(S): The spermicidal effect of StAPs involves plasma membrane permeabilization.

Molluscicidal saponins from Phytolacca icosandra.

Five monodesmosidic serjanic acid saponins and a monodesmosidic spergulagenic acid saponin were isolated from an aqueous extract of the berries of Phytolacca icosandra. A methanol extract of the berries furnished three bidesmosidic serjanic acid glycosides. Their structures were established by spectroscopic (ES-MS, 1H NMR, COSY, HSQC, HMBC, 13C NMR) and chemical methods. The molluscicidal, spermicidal and haemolytic properties of the saponins were investigated.

Spermicidal activity of an antifungal saponin obtained from the tropical herb Mollugo pentaphylla.

The ethyl acetate fraction of Mollugo pentaphylla, a tropical herb, contains an antifungal saponin (mollugogenol-A). We report here the spermicidal effects of this saponin. Washed sperm (> 100 x 10(6) with > 50% motility) from normal volunteers were incubated with varying concentrations (0-300 micrograms/ml) of mollugogenol-A at 30 degrees C. Sperm motility, velocity and viability were assessed at 0, 30, 60 minutes both manually and by using computer assisted semen analysis (CASA). Samples collected at 0 and 60 minutes were evaluated for membrane lipid peroxidation, superoxide dismutase (SOD) activity and transmission electron microscopy. A dose- and time-dependent effect of this saponin on sperm motion and viability was observed. The maximal spermicidal effect (4-5 fold decrease in motility and viability) was observed with 300 micrograms/ml dose of saponin. A three-fold increase in sperm membrane lipid peroxidation with corresponding inhibition of SOD activity were observed after 60 minutes incubation with this spermicidal agent. Transmission electron microscopy of saponin-treated samples revealed significant damage to the sperm membrane in both head and tail regions, and the acrosomal membranes were notably swollen and disrupted. These results indicate that this natural saponin has a potential spermicidal effect besides its known antifungal activity. The likely mechanism of its action involves sperm membrane damage by increased lipid peroxidation.

Male antifertility compounds from Tripterygium wilfordii Hook f.

Extracts of the Chinese medicinal plant, Tripterygium wilfordii, cause reversible infertility in male animals. Sub-fractionation studies have now revealed that the plant extracts contain a number of compounds which are potent antifertility agents in male mammals, including the diterpenes triptolide and tripdiolide and an isomer of the latter. A triptolide, 12,13-chlorohydrin, which is a transformation product formed reversibly by interaction of triptolide with HCl, was also found to be active.

Extraccion, estudio quimico y determinación de la actividad esparmicida de las saponinas de sapindus saponaria L. (boliche) / Extraction, chemical study and determination of spermicid activity of saponines of sapindus saponaria L. (boliche)

En el presente trabajo se ha realizado la extracción, estudio que por determinación de la actividad espermicida de las saponinas de sapindus saponena L. Boliche. La extracción se llevó a cabo con una macerración previa utilizando una solución hidroalcoholica al 50 porciento luego del desangrado y del despegmentado se extraen las saponinas con n-butonol, obteniendose un rendimiento del 34.08 porciento sobre la muestra deseada. Las saponínas crudas aisladas tienen naturalezas twterpénicos según los procedimientos, estanderes de identificación; el aspecto inflarojo indica que las germinas permanecen al grupo coliano. Los restos azucarados fueron identificados como L (-) ramnosa, L(+) arabinosa y D (+) glucosa utilizando la tecnica cromatográfica en papel. La actividad espermicidad se determino utilizando una modificación de la tecnica de la Baker y del test de Harris, se hallo que las saponinas tiene un primer efecto espermostatico y un posterior efecto espermicida. Tambien se encontró relación en la actividad espermicida y el número de espermatozoides. Los valores de la edad varian según el tiempo de contacto de los espermatozoides con las saponinas.

High-performance liquid chromatographic (HPLC) analysis of oligomeric components of the spermicide nonoxynol-9.

The commercially available Nonoxynol-9 spermicide is a multicomponent mixture of oligomers. When Nonoxynol-9 was separated by normal phase gradient HPLC, 17 components were shown to exist in the commercial mixture. These oligomeric components follow a Poisson distribution around the most abundant oligomer, EO 8 (11.7%). Select oligomers were isolated by preparative HPLC (Rt = 19.6, 34.0, 45.6, 51.2, 61.6, and 79.2 min) and purified by HPLC. These were identified by FAB-MS and NMR to be the oligomers EO 3, EO 6, EO 8, EO 9, EO 11, and EO 16, respectively.

A new spermatostatic glycoside from the soft coral Sinularia crispa.

The steroid glycoside 1 has been isolated from the Sri Lankan soft coral Sinularia crispa. The structure has been determined by spectroscopic (1H and 13C nmr) techniques. Glycoside 1 showed spermatostatic activity on rat cauda epididymal spermatozoa.