Patent Description:
At other stages of the cycle, the CF is thick and more acidic due to the effects of progesterone. This "infertile" CF acts as a barrier to spermatozoa thus preventing them from entering the uterus. Thick CF also prevents pathogens from interfering with a nascent pregnancy. A CF plug, called the operculum, forms inside the cervical canal during pregnancy. This provides a protective seal for the uterus against the entry of pathogens and against leakage of uterine fluids. The operculum is also known to have antibacterial properties. This plug is released as the cervix dilates, either during the first stage of childbirth or shortly before. It is visible as a blood-tinged mucous discharge.

CF has many potential practical uses. CF is used in sperm motility testing. The ability of CF to maintain the viability of spermatozoa indicates that it could be a useful storage or insemination medium. The selective permeability of CF indicates that it could be useful to separate spermatozoa from semen. As natural CF is difficult to collect in large volumes, and as CF with useful properties is only present at certain times during the menstrual cycle, there is a need in the art for an artificial CF with properties like those of CF that is present during ovulation. <CIT> is a Chinese patent application in the technical field of Chinese herbal medicine and discloses okra-containing antivirus granular electuary. The okra-containing antivirus granular electuary is characterized by comprising the following components in percentage by weight: <NUM>-<NUM>% of okra extract and <NUM>-<NUM>% of auxiliary material. The okra-containing antivirus granular electuary is prepared by repulping and extracting pure natural tender okra pod or tender stem. The authors state that the okra-containing antivirus granular electuary has the effects of protecting people and livestock, poultry and animals against influenza, viruses, bacteria and fungus, and is simultaneously capable of regulating immune system functions, controlling malignant proliferation of cells and preventing viral influenza and has the advantages of simplicity and convenience in processing method, less equipment investment, guaranteed material sources, energy conservation, safety in storage and application, low preparation cost, naturalness, environmental friendliness and no toxic or side effects. It discloses that stereochemically well-defined flavonoids present in okra seed extracts were used to identify their potential to inhibit HCV replication by targeting the viral polymerase. In preliminary experiments the authors identified the potency of okra seed extract to inhibit HCV replication by targeting the viral polymerase NS5B. It discusses previous work and discloses one-and two-dimensional NMR studies of flavonoids present in okra seed extracts which exhibited antiviral activity against hepatitis C virus at a low micromolar concentrations. The okra seed extract exhibited an IC50 value of <NUM> OM against HCV NS5B RNA-dependent RNA polymerase in vitro.

It has been unexpectedly discovered that a mucilaginous extract from the fruit of the okra plant (A. esculentus) can be produced with similar physical and biological properties to human CF, including the ability to be selectively penetrated by motile spermatozoa. This extract finds use for many of the same purposes as natural human CF, as further described below.

It has been unexpectedly discovered that an okra-derived composition can be produced with antiviral properties. This okra-derived composition finds use for many antiviral applications.

Disclosed herein is a mucilaginous extract of the fruit of A. esculentus, that is the product of the process comprising: (a) extracting a fruit of A. esculentus in an aqueous medium, to produce a first extract; and (b) separating a substantially clear mucilaginous fluid from the first extract; wherein the substantially clear mucilaginous fluid has properties similar to those of CF. Examples of such properties include: a Spinnbarkeit of at least about <NUM> when measured according to the test disclosed herein; displays ferning when subjected to the fern test; lacks visible green coloration; and when a semen sample is subjected to a sperm-mucus penetration test using the clear mucilaginous fluid in place of cervical mucus, spermatozoa that penetrate into the clear mucilaginous fluid have a significantly better indication of fertility than do spermatozoa in the semen sample.

The invention is as set forth in the appended claims.

In a first aspect, an in vitro method of decreasing the endocytosis of a virus into a eukaryotic cell is provided, the method comprising: (a) extracting a fruit of A. esculentus in an aqueous medium, to produce a first extract; and separating a substantially clear fluid from the extract to produce an okra-derived composition and (b) contacting the cell with the okra-derived composition in the presence of the virus.

In a second aspect, an in vitro method of decreasing replication of viral DNA in a eukaryotic cell infected by a virus is provided, the method comprising: contacting said cell infected with said virus with an okra-derived composition, wherein the okra-derived composition is a product of a process comprising extracting a fruit of A. esculentus in an aqueous medium and separating a substantially clear fluid from the extract.

In a third aspect, a method of reducing the likelihood of viral transmission in a sample of spermatozoa from a donor is provided, the method comprising (a) extracting a fruit of A. esculentus in an aqueous medium and separating a substantially clear fluid from the extract to produce an okra-derived composition; (b) providing the sample from the donor, wherein said donor is infected with or at risk for infection of a sexually transmitted virus; and (c) introducing the sample to a storage medium comprising the okra-derived composition.

In a fourth aspect, a method of isolating spermatozoa from semen obtained from a subject having or at risk for infection of a sexually transmitted virus while reducing the likelihood of transmission of said virus is provided, the method comprising: (a) extracting a fruit of A. esculentus in an aqueous medium and separating a substantially clear fluid from the extract to produce an okra-derived composition; (b) contacting a semen sample with a volume of the okra-derived composition; (c) allowing sufficient time for a multiplicity of spermatozoa in the semen sample to penetrate into the okra-derived composition to create a sperm suspension in the okra-derived composition; and (d) separating the sperm suspension from the semen sample.

The above presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art of this disclosure. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity or clarity.

As used herein, the singular forms "a", "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The terms "first," "second," and the like are used herein to describe various features or elements, but these features or elements should not be limited by these terms. These terms are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element discussed below could be termed a second feature or element, and similarly, a second feature or element discussed below could be termed a first feature or element without departing from the teachings of the present disclosure.

The term "consisting essentially of" means that, in addition to the recited elements, what is claimed may also contain other elements (steps, structures, ingredients, components, etc.) that do not adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure. Importantly, this term excludes such other elements that adversely affect the operability of what is claimed for its intended purpose as stated in this disclosure, even if such other elements might enhance the operability of what is claimed for some other purpose.

The terms "about" and "approximately" shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within <NUM>%, preferably within <NUM>%, and more preferably within <NUM>% of a given value or range of values. For biological systems, the term "about" refers to an acceptable standard deviation of error, preferably not more than <NUM>-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated.

The terms "prevention," "prevent," "preventing," "suppression," "suppress," and "suppressing" as used herein refer to a course of action initiated prior to the onset of a clinical manifestation of a disease state or condition so as to reduce the likelihood or severity of such clinical manifestation of the disease state or condition. Such reduction of the likelihood or severity need not be absolute to be useful. The terms also refer to inhibiting the full development of a disease state or condition in a subject who is at risk of developing the disease state or condition.

The terms "treatment", "treat," and "treating" as used herein refer to a course of action initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.

The terms "in need of treatment" and "in need of prevention" as used herein refer to a judgment made by a caregiver that a patient requires or will benefit from treatment or prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method or composition of the present disclosure.

The term "individual," "subject," or "patient" as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

Terms such as "comprise" and "include" as used herein are inclusive, and non-exclusive, and should therefore be construed to mean "comprise/include but are not limited to. " Permissive and optional terms such as "may" or "some embodiments" as used herein are also inclusive and non-exclusive.

The terms "inhibit," "decrease," and/or "reduce the likelihood of" (and like terms) generally refers to the act of reducing, either directly or indirectly, a function, activity, or behavior relative to the natural, expected, or average or relative to current conditions. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. Such terms can include complete inhibition, complete reduction, or elimination of the likelihood of a function, activity, or behavior relative to the natural, expected, or average or relative to current conditions.

The term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances that does not cause significant irritation to a human or other vertebrate animal and does not abrogate the biological activity and properties of the administered compound. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier). Typically, the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use. The pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.

The term "therapeutically effective amount" as used herein refers to an amount of an agent, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state or condition. Such effect need not be absolute to be beneficial.

The terms "site susceptible to viral infection" and/or "site of anticipated viral infection" refer to any bodily site known to be a route for transmission of a virus and having cells competent to host the virus.

Okra-derived compositions are provided, that can be produced through aqueous extraction of the fruit of A. esculentus (referred to herein by its common name, okra), followed by the removal of at least a fraction of the particulate material in the resulting extract. In an embodiment of the okra-derived composition, an artificial CF is provided, made of a mucilaginous extract of the fruit of A. esculentus (referred to herein by its common name, okra). Like CF itself, the extract finds many uses related to fertility and sex, including such uses as a sperm storage medium. The okra-derived composition finds uses related to preventing transmission of viruses, such as sexually transmitted viruses, and inhibiting replication of a virus.

In a general embodiment of the extract, it is the product of the process comprising: (a) extracting an okra fruit in an aqueous medium, to produce a first extract; and (b) separating a substantially clear mucilaginous fluid from the first extract. In this context "substantially clear" means that macroscopic (visible) particles have been removed from the extract. Some embodiments of the mucilaginous fluid are completely clear, meaning that the particulate fraction has been removed to the extent that the mucilaginous fluid does not appear cloudy to the unaided eye. Such removal may be achieved by any means known in the art. For example, a substantially clear mucilaginous fluid may be produced by straining the first extract through one or more pores of about <NUM> diameter or less. In a specific embodiment, the first extract is drained through a single pore of about <NUM> diameter under the force of gravity, which results in a substantially clear mucilaginous fluid. Other means include filtration, centrifugation, settling (with or without flocculants), and vortex separation.

The rate and efficiency of extraction may be improved by chopping the fruit into two or more pieces. In an exemplary embodiment the fruit is chopped into slices about <NUM>-<NUM> thick. Prior to any chopping steps the seeds may be removed, or the ribs may be removed, or both (doing so has been observed to eliminate a greenish color in the extract that is otherwise present -although this color is not known to affect the properties of the extract).

The outer surface of the fruit may be sanitized prior to extraction to reduce the likelihood of microbial contamination of the extract. Such sanitation may be performed using chemical sanitizers, such as an ethanol solution (for example, <NUM>% v/v ethanol in water). Ethanol has the advantage of low toxicity and high partial vapor pressure that results in rapid evaporation. Other chemical sanitizers could also be used. It is contemplated that other microbicidal agents could be used, such as gamma radiation or combined heat and pressure.

The extraction step may be carried out at elevated temperature. For example, the extraction may be performed by boiling the fruit in the aqueous medium. Water (as from a municipal water supply) has been found to be particularly suitable for use when the fruit is boiled. Boiling has the advantage of completing the extraction at a rapid rate. Alternatively, extraction may be performed at a lower temperature. At such lower temperatures extraction may take longer than at a boiling temperature; however, lower temperature extraction has the advantage of producing an extract without the green color. For example, in a specific embodiment extraction is performed at <NUM>° C for <NUM> hours. Agitation may be used to increase the rate of extraction as well. Extraction at lower temperature may also be performed with water, and it has also been found that lower temperature extraction may be performed with human tubal fluid (HTF) or HTF medium. HTF medium is a synthetic defined medium that is commercially available (for example, from Irvine Scientific, Santa Ana, California USA). Typically HTF medium is buffered against pH drift using a carbonate-CO<NUM> buffer system, in which case it is referred to in this disclosure as "bicarbonate buffered HTF medium. " Other kinds of HTF media are available, for example, HEPES buffered HTF medium is available from Irvine Scientific, Santa Ana, California USA. In further embodiments of the mucilaginous fluid the HTF may contain albumin, such as human serum albumin, to facilitate extraction. A specific embodiment of the aqueous extractant contains <NUM>% w/v human serum albumin or bovine serum albumin. In still another embodiment, the extraction step may be performed with an antibiotic. For example, the fruit may be contacted with an aqueous medium including an antibiotic. Suitable antibiotics include, but are not limited to, penicillin, streptomycin, and combinations thereof. In one embodiment, the extraction step may be performed with an antibiotic in an amount of about <NUM>/L to about <NUM>/L. In another embodiment, the antibiotic may be used in an amount of about <NUM>/L to about <NUM>/L. For instance, the antibiotic may be used in an amount of about <NUM>/L to about <NUM>/L.

As described above, the okra composition is the product of an aqueous extraction step followed by coarse particle separation. The extraction and separation steps may employ any technique known in the art. For instance, the separation step may employ any technique for particle separation known in the art. Suitable separation techniques include, but are not limited to, extraction, evaporation, distillation, filtration, centrifugation, chromatography, drying, and/or freezing.

As stated above, the substantially clear mucilaginous fluid has properties similar to those of CF. Examples of such properties include: a Spinnbarkeit of at least about <NUM> when measured according to the test disclosed herein; displays ferning when subjected to the fern test; lacks visible green coloration; and when a semen sample is subjected to a sperm-mucus penetration test using the clear mucilaginous fluid in place of cervical mucus, sperm that penetrate into the clear mucilaginous fluid have a significantly better indication of fertility than do sperm in the semen sample. Each of these properties, on its own, confers significant utility to the mucilaginous fluid.

Spinnbarkeit is the elastic quality of a fluid, and it is specifically measured as a characteristic of mucus of the uterine cervix, especially shortly before ovulation. As is known in the art, the Spinnbarkeit of CF decreases shortly before ovulation. Functionally, this elastic quality contributes to the lubricating qualities of the mucilaginous fluid. It also presents a mechanical barrier to non-motile semen components, such as prostaglandins, which acts as a selective barrier that admits spermatozoa but excludes other components. This property is advantageous in a medium for assisted reproduction procedures and as a medium for use in a sperm penetration test. Spinnbarkeit is measured by contacting the surface of the fluid with an object, such as a glass pipette or polypropylene centrifuge tube, and raising the object above the level of the fluid. A thread of adhered fluid will form, and the object is progressively raised until the thread breaks. Spinnbarkeit is thus measured as a distance. Some embodiments of the clear mucilaginous fluid have a Spinnbarkeit of at least about <NUM>. In further embodiments the clear mucilaginous fluid has a Spinnbarkeit of at least about <NUM>.

The fern test detects solutes in CF, which crystalize as the fluid evaporates, forming microscopic structures reminiscent of the leaves of a fern on a stem that can be viewed under a low-power microscope. The fern test is often used to provide evidence of the presence of amniotic fluid in CF, and is used in obstetrics to detect rupture of membranes and onset of labor. Ferning is due to the presence of sodium chloride in the CF, which is frequently due to estrogen's effects in vivo. The artificial CF described herein under some conditions displays ferning when observed by the fern test. The ferning that has been observed indicates similar composition to the follicular phase of the menstrual cycle.

Spermatozoa that penetrate into certain embodiments of the clear mucilaginous fluid have a significantly better indication of fertility than do spermatozoa in the semen sample. These qualities can be measured using the cervical mucus penetration test (CMPT - also called the sperm penetration test). The CMPT is a widely used method known by those of ordinary skill in the art. As referred to in this disclosure, the CMPT is the standard protocol of <NPL>). All media used in this test must be pre-equilibrated to <NUM>° C. Circular cross-section capillary tubes are recommended for use. Each tube is filled by aspiration using a <NUM> syringe and a plastic tube attached to the upper end of the capillary while the lower end is dipped into a pool of cervical mucus, mucilaginous extract, or other media. A column of medium is aspirated into the tube so that the upper meniscus is close but not at the top of the tube. Great care should be taken to avoid trapping air bubbles within the column. The top of the tube is then sealed with PLASTICINE and any trailing medium should be cut off the lower end to produce a flat interface. Approximately <NUM>µL of liquefied semen should be placed in the bottom of a small conical plastic tube and a capillary tube containing the cervical mucus or other media placed with its open end in the semen. Two semen reservoirs and capillary tubes will then be mounted on a microscope slide as shown in the WHO manual (<NPL>), following which, the slides are placed in a Petri dish containing damp sponges to maintain humidity and prevent drying of the semen and media. It is recommended that capillary tubes be incubated in the horizontal position for <NUM> minutes. Subsequently, the slides are removed from the Petri dishes and the capillary tube viewed under bright field illumination with a 20X phase contrast objective lens and 10X oculars. The microscope stage should be adjusted to select a focal plane incorporating the central axis of the capillary and, at this magnification, the microscope field width approximates the inner diameter of the capillary tube. The length of the tube is then scanned to establish the distance furthest from the semen reservoir attained by spermatozoa.

In addition, the mucilaginous fluid may have one or more properties that are found not necessarily present in human CF. For example, okra extracts have been observed to have anti-adhesive properties against certain microbes, specifically Helicobacter pylori. Consequently it is contemplated that the mucilaginous fluid may have anti-adhesive properties against viruses and microorganisms.

The mucilaginous fluid provided herein serves as an effective substitute for CF in the CMPT, as it is selectively penetrated by spermatozoa with high viability. When a semen sample is subjected to a sperm-mucus penetration test using some embodiments of the clear mucilaginous fluid in place of cervical mucus, sperm that penetrate into the clear mucilaginous fluid have a significantly higher rate of normal morphology as compared to spermatozoa in the semen sample. When a semen sample is subjected to a sperm-mucus penetration test using further embodiments of the clear mucilaginous fluid in place of cervical mucus, sperm that penetrate into the clear mucilaginous fluid have a significantly higher rate of progressive motility as compared to spermatozoa in the semen sample. When a semen sample is subjected to a sperm-mucus penetration test using still further embodiments of the clear mucilaginous fluid in place of cervical mucus, sperm that penetrate into the clear mucilaginous fluid have a significantly higher grade of motility as compared to spermatozoa in the semen sample. In this context, the property is "significantly higher" if a person skilled in the art, such as a board-certified andrologist, would note that the property is markedly higher when using standard clinical tests.

Without wishing to be bound by any hypothetical model, it is believed that the mucilaginous extract contains four main polysaccharides and protein. The polysaccharides are dominated by rhamnose, galactose, and uronic acid monomers. Some embodiments of the extract comprise <NUM>-<NUM>% mol/mol rhamnose, <NUM>-<NUM>% mol/mol galactose, <NUM>-<NUM>% mol/mol uronic acids, and <NUM>-<NUM>% mol/mol protein (in which mole percent refers to the molarity of the specific fraction divided by the combined molarity of all sugar monomers, uronic acids, and proteins after hydrolysis of the polysaccharides). Further embodiments of the extract comprise one or more of <NUM>-<NUM>% mol/mol rhamnose, <NUM>-<NUM>% mol/mol galactose, <NUM>-<NUM>% mol/mol uronic acids, and <NUM>-<NUM>% mol/mol protein. Still further embodiments comprise one or more of <NUM>% mol/mol rhamnose, <NUM>% mol/mol galactose, <NUM>% mol/mol uronic acids, and <NUM>% mol/mol protein. Further embodiments of the extract further comprise one or more of <NUM>-<NUM>% mol/mol arabinose, <NUM>-<NUM>% mol/mol xylose, <NUM>-<NUM>% mol/mol mannose, and <NUM>-<NUM>% glucose. Still further embodiments of the extract may comprise one or more polysaccharides having any one of the structures shown in <FIG>.

The artificial CF described above finds use as cryopreservation medium for sperm. The cryopreservation medium comprises any embodiment of the artificial CF described above, and may further comprise a plurality of spermatozoa. The medium may also include any of several useful cryopreservative components, including one or more components such as glycerol, citrate, egg yolk, and antibacterial agents. Glycerol may be present at any concentration known in the art to preserve cellular viability upon freezing; examples of such concentrations include about <NUM>-<NUM>% v/v, more specifically about <NUM>-<NUM>% v/v, <NUM>% v/v, and <NUM>% v/v. Citrate and egg yolk can be used to reduce osmotic stress on the cells during penetration by glycerol. Antibiotics can be used to reduce the likelihood of microbial contamination of the sample. Such components may be used at concentrations that would be used in standard glycerol and glycerol-egg yolk cryopreservatives, in which the balance of the composition is the artificial CF.

The cryopreservative can be used in a method of sperm preservation. The method involves providing a composition comprising the cryopreservative in combination with a plurality of spermatozoa, and freezing the composition. In some embodiments of the method the composition is frozen at a temperature of about -<NUM>° C or below; in further embodiments the composition is frozen at -<NUM>° C or below; in still further embodiments the temperature is about -<NUM>°, -<NUM>°, -<NUM>°, -<NUM>°, -<NUM>°, or -<NUM>° C or below. Any embodiment of the cryopreservative disclosed above may be used. The method may further comprise thawing the composition as needed.

Various methods of using the extract and the cryopreservation medium are provided.

Another general embodiment of a method of assisted fertility comprises recovering viable spermatozoa from semen into the mucilaginous extract of the fruit of A. esculentus with the apparatus described below, contacting the spermatozoa to an unfertilized egg in vitro to create a fertilization mixture, incubating the fertilization mixture for a period of time sufficient for fertilization to occur and create an embryo, and transferring the embryo to the uterus of a subject. The method may also comprise separating the extract from the spermatozoa, for example by centrifugal washing.

In another embodiment, the okra composition described herein can be used, for example, to prevent transmission of viruses, such as sexually transmitted viruses.

The okra composition disclosed herein has been found to inhibit viral transmission and replication, such as HIV replication. The human vaginal epithelium is the first line of defense against HIV-<NUM> sexual transmission. It is believed that HIV-<NUM> may enter the epithelial cells through endocytosis and accumulate inside of the cells (as shown in <FIG>). The intracellular HIV particles then get released to the exterior of the cells through the endosomal recycling pathway. Without being bound by any particular theory, it is believed that the okra composition blocks HIV-<NUM> transcytosis through vaginal epithelial cells and inhibits viral transmission and replication.

In one embodiment, the present disclosure provides for a method of decreasing endocytosis of a virus into a cell, the method including contacting the cell with the okra composition described herein in the presence of the virus. The cell may be a eukaryotic cell such as an epithelial cell. Examples of eukaryotic cells include, but are not limited to, a blood cell, dendritic cell, T cell, such as CD4+ T cell, vaginal epithelial cell, cervical epithelial cell, uterine epithelial cell, and rectal epithelial cell. In one embodiment, the method involves contacting a vaginal epithelial cell with the okra composition in the presence of the virus. Contact of a cell with the okra composition may result in a decrease of endocytosis of a virus into the cell by <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or at least any of the foregoing.

Viruses that can be prevented by the disclosed compositions include sexually transmitted viruses. Sexually transmitted viruses that may be prevented by the disclosed compositions include, but are not limited to, human immunodeficiency virus (HIV), such as human immunodeficiency virus type I (HIV-I) and human immunodeficiency virus type II (HIV-II), herpes simplex viruses (HSV), human papillomavirus (HPV), hepatitis B virus (HBV), and cytomegalovirus.

In one embodiment, the disclosed okra composition is used to prevent transmission of HIV.

In another embodiment, the okra composition is used to prevent transmission of HBV.

In another embodiment, the present disclosure relates to a method for preventing or inhibiting replication of any one or more of the viruses disclosed above. For example, the present disclosure provides for a method of decreasing replication of viral DNA in a eukaryotic cell infected by a virus, the method including contacting the cell infected with the virus with the okra composition described herein. The okra composition can act as an agent for inhibiting replication of the virus.

Due to the ability of the mucilaginous okra extract to reduce the likelihood of transmission of viruses, such as sexually transmitted viruses, the okra composition may also be used as a blocking agent to prevent a virus from penetrating cervical and uterine epithelium during intra-uterine insemination. For instance, the present disclosure provides for a method of reducing the likelihood of viral transmission in a sample of spermatozoa from a donor, the method including providing the sample from the donor, wherein said donor is infected with or at risk for infection of any of the sexually transmitted viruses discussed above and introducing the sample to any of the storage media described above, for example, the cryopreservation medium, including the okra composition. In still another embodiment, the present disclosure provides for a method of isolating spermatozoa from semen obtained from a subject having or at risk for infection of any of the sexually transmitted viruses described above while reducing the likelihood of transmission of said virus, the method including contacting a semen sample with a volume of the okra composition; allowing sufficient time for a multiplicity of spermatozoa in the semen sample to penetrate into the okra composition to create a sperm suspension in the okra composition; and separating the sperm suspension from the semen sample.

An <NUM>" x <NUM>" aluminum foil sheet was sterilized with <NUM>% ethanol and wiped dry with a task wipe, such as KIMWIPES. Six okra fruits were thoroughly washed with a mild soap solution prior to being thoroughly rinsed with tap water (<NUM> x <NUM>) followed by DI water (<NUM> x <NUM>), and sterile wiped with a task wipe pre-soaked in <NUM>% ethanol. After allowing ethanol film on okra fruits to dry (<NUM>-<NUM>), the fruits were placed on the sterile aluminum foil and okra slices (<NUM>-<NUM>) were prepared with a sterile scalpel blade. Ten grams of sliced okra fruit portions were placed in each of three <NUM> centrifuge tubes containing <NUM>, <NUM>, or <NUM> of HTF-bicarbonate solution (<NUM> of Na<NUM>CO<NUM> in buffered human tubal fluid [HTF] containing <NUM>% human serum albumin). Extraction tubes were rocked at <NUM>° C on a tissue rocker/shaker for <NUM> hours. Each extraction centrifuge tube was inverted and the bottom sterilized with a <NUM>% ethanol wipe. Using a pre-heated <NUM> needle, a pinhole was created at the center of the bottom of the tube with a pore size of approximately <NUM> (large enough to permit fluid escape but small enough to prevent okra seeds escape -- <FIG>). Each extraction tube was inverted (cap side up) over another uncapped <NUM> centrifuge tube (<FIG>). The cap to the extraction tube was loosened to permit the clear extracted okra mucus to drip under gravitational force into the collection tube (<FIG>). The extracted mucus was clear and devoid of chlorophyll pigment.

A ruler was placed by the side of the collection tube and the length of each mucus string was measured before breaking, as shown in <FIG>. This measurement corresponded to the stretchability or Spinnbarkeit of the mucilaginous extract. The extraction efficiency of the mucus was based on the amount of mucus recovered relative to the amount of HTF-bicarb used to extract the okra mucilage and did not differ significantly among preparations (<NUM>-<NUM>%). The Spinnbarkeit (stretchability) was similar among preparations (greater than <NUM>). The ferning pattern of mucilaginous extract was similar among preparations and mirrors the pattern observed in the follicular phase of the menstrual cycle (see <FIG>). The mucilaginous extract was stored at -<NUM>° C for <NUM> weeks and longer.

The sperm penetration test protocol used in this application is a modification of the method of Tang et al. (<NUM>), supra. Unless otherwise specified, all media were pre-equilibrated to <NUM>° C. Non-treated Kimble circular cross-section capillary tubes, <NUM> in length and inner diameter <NUM> (Fisher Scientific, PA) were used. Each tube was filled by aspiration using a <NUM> tuberculin syringe and a plastic tube attached to the upper end of the capillary while the lower end was dipped into the A. esculentus mucus (<NUM>µL) or equivalent volume of control medium (bicarbonate-buffered HTF containing <NUM>% human serum albumin). An approximately <NUM> column of A. esculentus mucus or control medium was aspirated into the tube so that the upper meniscus was <NUM>-<NUM> from the top of the tube, while being careful not to trap air bubbles within the column. The top of each tube was sealed with modeling clay, such as PLASTICINE, and any trailing medium was cut off the lower end to produce a flat interface. Approximately <NUM>µL of liquefied semen was placed in the bottom of a small conical plastic tube and a capillary tube containing A. esculentus mucus or control medium placed with its open end in the semen. This preparation was replicated five times with semen samples from different donors. The two semen reservoirs and capillary tubes were mounted on a microscope slide as demonstrated in the WHO manual (WHO, <NUM>). The slides were then placed in a Petri dish and incubated in the horizontal position for <NUM> minutes at <NUM>° C in an atmosphere of <NUM>% CO<NUM> in humidified air.

After <NUM>, the motility of the spermatozoa was determined for the control and the mucilaginous extract sample by viewing the capillary tubes with a phase contrast microscope at 200X magnification. A significant mean number of spermatozoa (><NUM> ± <NUM> spermatozoa under 200X magnification) penetrated the mucilaginous extract and traveled approximately <NUM> (migration distance) within <NUM> of initiation of interaction with semen versus <NUM> ± <NUM> spermatozoa in the control preparation.

Approximately <NUM>µL of separated motile spermatozoa in mucilaginous extract was withdrawn from each capillary tube diluted in <NUM>µL of human tubal fluid (HTF) medium containing <NUM>% human serum albumin (HSA) and <NUM> HEPES (HTF-HEPES; pH, <NUM>), and a drop of the resulting solution was placed on a microscope slide. The percentage sperm motility was quantified manually with a phase contrast microscope. Spermatozoa were considered to be progressively motile if they moved in a linear manner from one point to another. The percentage of progressively motile spermatozoa was determined twice for each sample by a certified clinical andrologist on <NUM> cells in different fields, with a laboratory counter and averaged. Counts were accepted as accurate if each of the two counts did not differ by <NUM>%. The grade of motion on a scale of <NUM> to <NUM> was determined subjectively. The grade and percentage progressive motility of mucilaginous extract-penetrated spermatozoa were <NUM>-<NUM> and <NUM>%, respectively, compared with those of the control (<NUM>-<NUM>, <NUM>%, respectively). The above procedure was carried out using the sperm from patients (N=<NUM>) undergoing fertility evaluation to determine whether their spermatozoa would penetrate the mucilaginous extract. <FIG> shows that the spermatozoa from an infertile patient that did not penetrate the mucilaginous extract. These data suggest that mucilaginous extract can substitute for CF for testing sperm function.

Sperm morphology assessment of spermatozoa in mucilaginous extract was conducted by a certified clinical andrologist. Feathered smears of approximately <NUM>µL each of undiluted mucilaginous extract containing human spermatozoa and matching semen (control; N=<NUM>/group) were prepared on ethanol-cleaned glass slides and left to dry in a dust free chamber. Subsequently, smears were fixed and stained with reagents in a modified Papanicolaou staining kit (Spermac, Stain Enterprises, South Africa) according to the manufacturer's instructions and sperm morphologies assessed using the Kruger strict criteria with a phase contrast microscope equipped with a scaler. A spermatozoon was considered normal if the head had a smooth, oval shape with a well-defined acrosome comprising approximately <NUM>-<NUM>% of the head area. Morphologically, the sperm head must be <NUM>-<NUM> long and <NUM>-<NUM> wide to be considered normal. In addition, a spermatozoon was considered normal if it was devoid of neck, midpiece, or tail defects and no cytoplasmic droplet of more than half the size of the head. All borderline morphologic forms of spermatozoa were considered abnormal. The slides are shown in <FIG>. The mean percentage of normal morphological form of spermatozoa in the mucilaginous extract samples is much greater than in the semen sample (control; see <FIG>).

Okra mucus extraction: Fresh okra was purchased from local grocery stores and mucus was extracted as described above.

Semen and processing: Discarded de-identified semen samples that met criteria of WHO for normal semen samples (<NUM>-<NUM> volume, <NUM>-<NUM> pH, <NUM>% or greater motility, concentration = <NUM> x <NUM> million/mL) were used in the freezing protocol with okra mucus. Each semen sample was divided into halves to be frozen in conventional freezing medium consisting of TEST Yolk Buffer containing <NUM>% glycerol (volume/volume) and <NUM>µg gentamicin/ml (Irvine Scientific, Santa Ana, CA) or in the presence of okra mucus. Each half of semen samples designated for freezing in the presence of okra mucus was diluted with equivalent volume of human tubal fluid (HTF) buffer containing <NUM> sodium bicarbonate (HTF-buffer) and <NUM>% okra mucus (volume/volume) from a <NUM>% okra extract. The remaining half was similarly diluted with HTF medium only. Subsequently, an equivalent volume of TEST-Yolk buffer to each diluted semen sample was added dropwise with gentle mixing to a homogenous suspension with a final glycerol concentration of <NUM>% (volume/volume), final okra dilution in ready to freeze preparation = <NUM>% (volume/volume of the <NUM>% extract) and placed on ice for approximately <NUM> hours. Prior to subjecting each sample to freezing conditions, <NUM>µL of cooled TEST-Yolk-extended semen sample was withdrawn and placed on a clean glass slide, covered with cover glass and allowed to warm to room temperature (<NUM>) before post cooling evaluation of percentage sperm motility was conducted with a phase contrast microscope and a laboratory counter. Cooled TEST-Yolk-extended semen samples were then subjected to liquid nitrogen (LN) vapor phase freezing for <NUM> minutes and subsequently stored immersed in LN. Samples were allowed to remain in LN storage for at least <NUM> hour before thawing. Samples were thawed to room temperature and subsequently diluted with two volumes of warm (<NUM>) HTF-buffer in order to elute the cell penetrating cryoprotectant (glycerol) from the cytoplasm of the cryopreserved spermatozoa. Frozen-thawed human spermatozoa were subjected to phase contrast microscopy to determine the motility status of each sample as described above.

Acrosomal Status of Post-thaw Motile Spermatozoa and Longevity: After the assessment of post-thaw sperm motility, control and okra mucus-exposed samples subjected to gradient (ISOLATE, Irvine Scientific; <NUM>%) centrifugation to separate motile from non-motile spermatozoa. Subsequently, motile spermatozoa were permeabilized in cold (-<NUM>) <NUM>% ethanol and stained with FITC-conjugated lectin (Vector Labs, Burlingame, CA). Following FITC-lectin staining, spermatozoa were subjected to epi-fluorescence microscopy. Spermatozoa with green even fluorescence in the acrosomal region were considered acrosome intact and therefore to lack damage to the membrane. Spermatozoa with fluorescence in the equatorial region of the cell were considered acrosome reacted and therefore to have sustained membrane damage.

Longevity of Non-frozen Human Spermatozoa in Okra Mucus: Liquefied freshly ejaculated human semen samples were washed with HTF containing <NUM> HEPES and <NUM>% BSA (HTF-HEPES; pH <NUM>) following which each sperm suspension was subjected to a continuous gradient wash as described above for the purpose of harvesting highly motile cells. Each sample of highly motile cells was divided into two to be cultured in HTF-buffer or HTF-buffer + <NUM>% okra mucus (volume [<NUM>% okra mucus in HTF-buffer]/volume [HTF-buffer]) for <NUM> hours in an atmosphere of <NUM>% CO<NUM> in air in a humidified incubator.

During the pre-freezing period (PF), cooled spermatozoa in the base freezing buffer (control; PF control; FB) sustained a significant reduction in mean percentage motility (p<<NUM>) compared with those in cooled base freezing buffer supplemented with okra mucus (PF OK) or those in raw semen (RS). Further, supplementation of base freezing buffer with okra mucilage maintained a similar percentage motility rate observed in RS. (Graphs with different superscripts are statistically different; <FIG>; graphs with different superscripts are statistically different). Freezing human semen in extracted Abelmoschus esculentus mucilage (EAEM) increased the percentage of post-thaw motile sperm population compared with FB (P<<NUM>; <FIG>; graphs with different superscripts are statistically different). <FIG> shows a micrographic image of the frozen-thawed spermatozoa.

This observation suggests that more post-thaw motile spermatozoa can be harvested after freezing in EAEM for increased efficacy of affordable assisted reproductive technologies (ART) such as intra uterine insemination (IUI). Interestingly, spermatozoa in semen samples frozen in EAEM had a higher (P<<NUM>) percentage motility (<NUM> + <NUM> %) compared with those frozen in FB (<NUM> + <NUM>%), <NUM> hr post-thaw.

Regardless of the freezing medium, the highly motile spermatozoa harvested via gradient centrifugation maintained their membrane integrity. This suggests that if more motile spermatozoa are harvested based on higher percentage motility observed among cells frozen in EAEM, freezing in EAEM can facilitate the collection of a significant number of motile spermatozoa.

Highly motile spermatozoa (<NUM> ± <NUM>%) harvested via continuous gradient centrifugation that were cultured for <NUM> hours in HTF-buffer supplemented with okra mucus had a higher percentage of motile spermatozoa (P<<NUM>; <FIG>) than their counterparts cultured in HTF-buffer-only (graphs with different superscripts are statistically different). The result of this experiment indicates that culturing spermatozoa in the presence of okra mucus protects more spermatozoa to remain motile during a protracted period. The ability of okra mucus to maintain longevity in more cells than their counterparts cultured in HTF-only indicates that it has the potential to maintain more cell in motile state in women with a compromised cervix.

A test was conducted to determine whether okra mucilage functions as an effective medium to measure sperm penetration. Microscope slides were cleaned with <NUM>% ethanol and thoroughly wiped dry with a task wipe. Subsequently, double-sided tape was applied to the clean glass slide, leaving a longitudinal strip uncovered approximately <NUM> x <NUM> (the "sperm lawn"). This was followed by the application of cover glass and pressed gently to eliminate air bubbles and firmly secure the cover glass. Thereafter, okra mucus extracted as described above was applied steadily to the <NUM>° angled uncovered side of the glass slide to permit the mucus to penetrate the sperm lawn without air bubbles. The mucus loaded slide was placed in a <NUM> x <NUM> FALCON style polystyrene nonpyrogenic Petri dish (Corning, Inc. , Corning, NY) and placed in a humidified incubator with an atmosphere of <NUM>° C and <NUM>% CO<NUM> for about <NUM> minutes for the mucus to attain zero movement (acquire static status). Subsequently, <NUM>µL of semen was carefully placed in the okra mucus, just before the edge of the cover glass and incubated as described above for <NUM> minutes. Post incubation, the preparation was subjected to phase contrast microscopy at a magnification of 200X for sperm penetration of the mucus, phalange (finger-like projections of the seminal fluid into the okra mucus creating an interface between the two substances) formation and distance travelled down the sperm lawn from the loading zone.

All preparations developed phalanges. However, spermatozoa in semen samples (N=<NUM>) that did not meet the WHO criteria for normalcy had zero to poor transition and travelling distance (mean = <NUM>± <NUM>) with the number of motile spermatozoa in first microscopic field (F1) from the tip of a phalange showing <NUM> cells, F2 showing <NUM> cells, and F3 showing <NUM> cells. Spermatozoa in semen that met the WHO criteria for normalcy were superior (P < <NUM>) in their transition and travel distance in the sperm lawn. The mean distance travelled by spermatozoa in normal samples (N=<NUM>) was <NUM> ± <NUM> in <NUM> minutes. The F1, F2, and F3 for spermatozoa in normal semen samples were <NUM> cells, <NUM> cells, and <NUM> cells, respectively.

Implications: The observations made in this study indicate that the preparation described above has the potential to be utilized as a replacement cervical mucus kit. Okra mucus-loaded slides as described above can be prepared and snap frozen and used when needed by thawing in a humidified chamber as described above followed by semen application, incubation as described above and evaluated for phalange formations, transition of spermatozoa into okra mucus, evaluation of the number of motile spermatozoa in microscopic fields from the phalanges and distance travelled.

An <NUM>" x <NUM>" aluminum sheet was sterilized with <NUM>% ethanol and wiped dry with a task wipe, such as KIMWIPES. Six okra fruits were thoroughly washed with a mild soap solution prior to being thoroughly rinsed five times with <NUM> tap water (each rinse). Subsequently, the okra fruits were rinsed as described above with deionized water prior to being place in a <NUM> liter Pyrex dish to which Sterile Dulbecco Phosphate Buffered Saline (DPBS) containing <NUM> penicillin/I and <NUM> streptocysin/I was added to cover the okra fruits and rocked on a rocker platform for <NUM> hours. The okra fruits were washed with deionized water and dried as described above. The okra fruits were then placed on sterile aluminum foil and sliced into <NUM>-<NUM> okra sections with a sterile scalpel blade. Aliquots of <NUM> sliced okra fruit portions were each placed in a <NUM> centrifuge tube and subjected to <NUM>% extraction with <NUM> DPBS containing <NUM> penicillin and <NUM> streptomycin/ml. Extraction tubes were rocked at <NUM> on a tissue rocker/shaker for <NUM> hours. Each extraction centrifuge tube was inverted and the bottom sterilized with <NUM>% ethanol wipe. Using a pre-heated <NUM> needle, a pinhole was created at the center of the bottom of the tube with a pore size of approximately <NUM> (large enough to permit fluid escape but small enough to prevent okra seed escape - <FIG>). Each extraction tube was inverted over another uncapped <NUM> centrifuge tube (<FIG>). The cap to the extraction tube was loosened to permit the clear extracted okra mucus to drip under gravitational force into the collection tube (<FIG>).

A ruler was placed by the side of the collection tube and the length of each mucus string was measured before breaking, as shown in <FIG>. This measurement corresponded to the stretchability or Spinnbarkeit of the okra composition. Ferning was conducted to establish similarity of the DPBS extract with cervical mucus by preparing feathered smears of okra extract from each preparation on <NUM>% ethanol cleansed microscope slides and dried overnight in a dust free chamber. The extracted mucus was clear and devoid of chlorophyll pigment. The Spinnbarkeit (stretchability) was similar among preparations and was ><NUM>-<NUM>. The ferning pattern of extracted okra mucilage in DPBS mirrored the pattern observed in the follicular phase of the menstrual cycle.

To test whether the okra composition has the potential to block HIV transmission, VK2/E6E7 cells, a human vaginal cell line, were pre-treated with concentrations of ½ diluted okra composition, ¼ diluted okra composition, and ⅛ diluted okra composition for <NUM> hour. Subsequently, HIV-<NUM> IIIB virus (60ng p24 content) was added to the VK2/E6E7 cells for <NUM> hours, following which, <NUM>% Trypsin was used to remove the HIV particles attached to the surface of the cells (outside the cells). After washing three times with PBS, the cells were subjected to Western-blot analysis. The antigen targeted and detected in the Western-blot was HIV-<NUM> capsid protein p24.

As shown in <FIG>, the okra composition dramatically reduced the amount of HIV in the cells. As can be seen, all concentrations of the okra composition nearly completely inhibited the accumulation of HIV inside the VK2/E6E7 cells. These data demonstrate that the okra composition is able to block the transcytosis of HIV through vaginal epithelial cells and suggest that the okra composition has the potential for development as an anti-HIV/AIDS agent.

CD4+T cells are the primary cell type supporting HIV replication. In order to evaluate the inhibitory effect of the okra composition on HIV replication, SupT1 cells, a human T cell lymphoblastic Lymphoma cell line, were pre-treated with concentrations of ½ diluted okra composition, ¼ diluted okra composition, and ⅛ diluted okra composition for <NUM> hour. Subsequently, HIV IIIB (<NUM> ng p24 content) was added to the cell culture to infect the SupT1 cells for <NUM> hours, following which, the cells were washed three times with PBS. The SupT1 cells were then subjected to in vitro cell culture for eight days. Aliqouts of conditioned media were harvested on day <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and subjected to qRT-PCR to measure viral release. The target sequence for the qRT-PCR was in the HIV-<NUM> LTR region.

As shown in <FIG>, the okra composition dramatically inhibited HIV replication (<FIG>) and viral release (<FIG>) in SupT1 cells. These results indicate that the okra composition not only inhibits HIV transmission in vaginal epithelial cells, but also inhibits viral replication in CD4+ T cells.

To test the cytotoxicity of the okra composition on SupT1 cells, ¼ diluted okra composition was used to treat SupT1 cells for <NUM> hours. After treatment, the SupT1 cells were subjected to a cell viability assay using LIVE/DEAD® Cell Vitality Assay Kit (Invitrogen).

As shown in the graphs of <FIG>, the okra composition did not alter the viability of SupT1 cells.

Claim 1:
An in vitro method of decreasing the endocytosis of a virus into a eukaryotic cell, the method comprising: (a) extracting a fruit of Abelmoschus esculentus in an aqueous medium and separating a substantially clear fluid from the extract to produce an okra-derived composition and (b) contacting the cell with the okra-derived composition in the presence of the virus, wherein the eukaryotic cell is preferably selected from the group consisting of: dendritic cell, T cell, vaginal epithelial cell, cervical epithelial cell, uterine epithelial cell, and rectal epithelial cell.