CN108096296B

CN108096296B - System and article for prolonged nitric oxide production based on microencapsulated powders and acidic gels of spinach extracts

Info

Publication number: CN108096296B
Application number: CN201710942743.4A
Authority: CN
Inventors: 陈振兴
Original assignee: Hebei Jingding Biomedical Technology Co ltd
Current assignee: Hebei Jingding Biomedical Technology Co ltd
Priority date: 2017-10-11
Filing date: 2017-10-11
Publication date: 2020-10-16
Anticipated expiration: 2037-10-11
Also published as: CN108096296A

Abstract

The present invention relates to a prolonged release system and kit for nitric oxide comprising a microencapsulated spinach extract as nitric oxide donor and an acidic gel having sufficient acidity to convert the spinach extract to nitric oxide. The delayed nitric oxide release system and kit of the present invention can release NO for a long period of time and provide a sustained action on the affected area for a long period of time.

Description

System and article for prolonged nitric oxide production based on microencapsulated powders and acidic gels of spinach extracts

Technical Field

The invention belongs to the field of medicines, and particularly relates to a microencapsulated medicament containing spinach extract as an active ingredient, and a system and an article containing the microencapsulated medicament containing spinach extract.

Background

In mammals, NO is an endogenous, physiologically regulated substance in many of the physiological processes of the nervous system, immune system and cardiovascular system, and its actions include relaxation of vascular smooth muscle, resulting in vasodilation of arteries and increased blood flow. NO is a neurotransmitter, involved in neuronal activity and various functions, ranging from avoidance of learning to genital erection in both men and women (Kim et al, j.nutrition 134(2004) 2873S). NO also has a cytotoxic effect on microorganisms and tumor cells, in part, by regulating macrophages. In addition to mediating normal physiological functions, NO is involved in different pathophysiological states such as septic shock, hypertension, stroke and neurodegenerative diseases.

NO is used pharmacologically in various forms, and topical application of NO can aid in wound healing of wounds and burns, hair growth, impotence, and causing vasodilatation where needed (e.g., to promote circulation of peripheral blood flow and cervical maturation during pregnancy in patients with impaired diabetes or other conditions). However, although NO itself is physiologically active, it is chemically unstable in the air or in the body. Thus, their pharmacological use in the prior art is almost always produced by chemical reactions of various individual stable precursor compounds. Organic and inorganic nitrates are commonly used as NO donors. In the context of topical application, it is desirable that the dosage of NO be low and long-lasting. NO is effective as a powerful bactericide against antibiotic-resistant bacteria. In antibacterial and other topical applications, it is desirable to extend the time that NO is in contact with the skin. In antibacterial applications, the therapeutically effective amount of NO is few, as few as a few parts per million (ppm) (see Ghaffari et al, Nitric Oxide Biology and Chemistry 14(2006)21-29)), but the effectiveness of NO depends on how long contact with the skin is maintained (Ormerod et al, BMCResearch Notes 4(2011) 458-465).

Although there are systems and methods of delayed generation of nitric oxide using microencapsulated nitrite and acidified hydrogels in the prior art (patent applications CN201310355902.2, CN201310356220.3), the NO donor used is nitrite, which is generally toxic, especially at high doses. Furthermore the application of the delay system and the method of operation depends on the volume of water activated or several activated volumes, with certain limitations in the specific operation.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a system for generating nitric oxide based on the spinach extract micro-encapsulation powder and the acidic gel.

The invention also relates to a kit for generating nitric oxide based on a microencapsulated powder and an acidic gel of spinach extract.

The system for generating nitric oxide in a delayed manner can ensure the sustained release of NO under a long-term condition, is simple and convenient to operate, and has the effect of sustained release.

The invention also relates to a kit for prolonged nitric oxide production.

The system and the kit have simple preparation process, and the system has higher biological safety and can exert the physiological activity of NO for a long time.

Drawings

FIG. 1A is a cross-sectional view of a pad containing a mixture of microencapsulated reagents (reacted to form NO) in one embodiment;

FIG. 1B is a cross-sectional view of a pad containing an internal component that holds the particles in place;

FIG. 1C is a cross-sectional view of a pad in which the absorbent layer contains microencapsulated reagents (reacted to produce NO) in one embodiment;

FIG. 2 shows the release of NO from microencapsulated spinach extract and an acidic gel in solution over a period of 5 hours;

FIG. 3 shows the release of NO from microencapsulated spinach extract and acidic gel in a paste over a period of more than 10 hours;

FIG. 4 shows the release process of micro-encapsulated sodium nitrite and acid gel in NO in paste for 8 hours;

FIG. 5 shows the release of NO from a paste of microencapsulated spinach extract and an acidic gel containing a reducing agent for a period of more than 10 hours;

reference numerals:

particle 1, layer 2, layer 3, isolation layer 4, water absorbing material 5, and impermeable layer 6.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

The present invention discloses a prolonged release system for nitric oxide comprising a microencapsulated spinach extract and an acidic gel having sufficient acidity to convert the spinach extract to nitric oxide.

The present invention uses spinach extract as donor for the production of pharmaceutically acceptable NO. The reaction principle of the present invention can be seen from the following reaction formula (1), and nitrous acid is produced by the reaction of nitrite with acid (HA). Nitrous acid is stable in aqueous solution at low temperatures, but readily decomposes to NO and NO at room temperature₂As shown in the reaction formula (2).

In a reducing agent (e.g. ascorbic acid, dihydroxyascorbic acid (Asc (OH)₂) In the presence of NO, NO₂It is easily converted into NO as shown in the following reaction formula (3).

2HA+NaNO₂→2HNO₂+2NaA (1)，

HA is an organic or inorganic acid

2HNO₂→NO+NO₂+H₂O(2)，

Nitrous acid decomposition to nitrogen dioxide

NO+NO₂+H₂O+Asc(OH)₂→2NO+2H₂O+AscO₂(3)，

Ascorbic acid reaction to produce nitric oxide

According to the invention, the spinach extract is used as a donor of NO, so that the potential harm of the toxicity of nitrite to human bodies is reduced.

In one embodiment, the spinach extract retains vitamin C in the spinach, and thus the system and kit of the invention may be able to release NO for a prolonged period of time without the addition of a reducing component. Vitamin C in spinach extract can prevent or slow down the reduction of nitric oxide to nitrogen dioxide and also has the ability to directly reduce NO₂Is NO, such that the gas released from the composition is primarily NO.

In one embodiment, the spinach extract is used for adjusting the pH value of the solution in each extraction step to 3-4 by acid in the extraction process of spinach, so as to ensure the extraction and activity of the spinach-derived vitamin C.

The spinach extract can be extracted by using a conventional method in the field, and the pH is adjusted to 3-4 in the extraction process so as to ensure the extraction and activity of vitamin C in the spinach extract.

In one embodiment, the spinach extract is extracted using spinach, and the acid that adjusts the pH of the solution is oxalic acid.

in one embodiment, the spinach extract has a nitrite content of 5.017 × 10^-3mg/ml, nitrate content of 0.236mg/ml, vitamin C content of 1.21 × 10^-2mg/ml。

In one embodiment, the microencapsulated support is a polymeric matrix. The reagent and matrix are placed together in a sub-millimeter structure (at least one dimension of less than 1 mm). Such structures may be particles, fibers or films.

In one embodiment of the invention, microencapsulated spinach extract is contacted with an acidified hydrogel of sufficient acidity to convert nitrite to nitric oxide. Preferred acidulants employ organic acids, such as citric acid, although inorganic acids, such as boric acid, may also be suitable. Other acidulants may also include lactic acid, glyceric acid, formic acid, ascorbic acid or other organic acids as those known to those skilled in the art. Biologically acceptable mineral acids having appropriate pKa values (e.g., the boronic acids described above) may also be used. Gelling agents include hydroxymethyl cellulose, hydroxyethyl cellulose, gelatin, agar, natural gums, starches, and pectins.

The medium in which the acid is dissolved may be an aqueous medium or a non-aqueous medium. Aqueous media are preferred, and gels are easily prepared. The acid gel composition may additionally contain one or more conjugate bases of the acid used. While the preferred base is the conjugate base of the acid used, other organic or inorganic bases known to those skilled in the art are also possible. Embodiments of the present invention may be applied directly to promote skin circulation, promote wound healing, remain on the scalp for a period of time as a treatment to promote hair growth, and may be applied in other locations where the local release of NO is beneficial.

In one embodiment of the invention, a reducing agent is included in the gel to further assist in maintaining the biological activity of nitric oxide. The acidifying agent may also be a reducing agent, such as ascorbic acid (vitamin C) or an ascorbic acid derivative. The ascorbic acid derivatives include, but are not limited to, 3-O-ethyl ascorbic acid and other 3-alkyl ascorbic acids, 6-O-octanoyl-ascorbic acid, 6-O-dodecanoyl-ascorbic acid, 6-O-tetradecanoyl-ascorbic acid, 6-O-octadecanoyl-ascorbic acid, and 6-O-sebacoyl-ascorbic acid. Preferred reducing agents have the ability to prevent or slow down the oxidation of nitric oxide to nitrogen dioxide together with vitamin C in spinach extracts and also have the ability to directly reduce NO₂is NO, so that the gas released from the composition is mainly NO.

One embodiment of the present invention discloses a packaged acidified gel and microencapsulated spinach extract. Both the acidified gel and the microencapsulated spinach extract are individually packaged in a moisture-proof packaging, which is opened and the contents are mixed together immediately before the mixture is applied. In another alternative embodiment, the microencapsulated spinach extract and the acidulant are packaged together or separately in a moisture barrier. Prior to application, the package is opened and their contents mixed with a measured amount of water or an aqueous gel of neutral pH.

A preparation method of the microcapsule comprises the following steps: a solution of one agent or a polymer solution is spray dried to produce a powder of finely divided individual particles (containing the agent dispersed within the polymer matrix). Other methods of making microcapsules, such as pan coating, air suspension coating, centrifugal extrusion, fiber spinning, fiber extrusion, nozzle vibration, ionic gelation, coacervate phase separation, interfacial crosslinking, in situ polymerization, and matrix polymerization, may also be used.

In one embodiment of the preparation of the encapsulated spinach extract of the invention, a stabilizer may be added to the spinach extract, which stabilizer is capable of maintaining the activity of vitamin C in the spinach extract during the preparation of the microcapsules and for a long period of time during storage, and which stabilizer may be L-cysteine hydrochloride and sodium metabisulfite.

To be suitable for medical indications, the encapsulating polymers disclosed herein are biocompatible polymers. Suitable polymers include ethylcellulose, natural polymers such as zein, a prolamine seed storage protein found in certain grasses such as corn and cereals, chitosan, hyaluronic acid, alginic acid, biodegradable polyesters, polyanhydrides, polyethylene (ortho esters), polyphosphazenes, or polysaccharides (see Park et al, polymers 10(2005) 146-161).

The use of the above-described agent microencapsulated compositions for the delivery of pharmaceutical agents is well known. See Shalabyand Jamiolkowski, US Pat. No.4,130,639; buchholz and Meduski, U.S. Pat. No.6,491,948. However, in all of these compositions, the microencapsulated agent is the therapeutic agent itself, and the therapeutic agent is not produced by the reaction of the microencapsulated agent. Nitric oxide releasing polymers involving nitric oxide adducts/donors have been described in the medical literature, e.g. Arnold, US Pat. No.7,829,553 (carbon-based diazeniumdiolates attached to hydrophobic polymers); knapp, US Pat. No.7,135,189 (precursor of nitrosothiols and nitric oxide donor).

Applications of embodiments of the invention include the direct application of microencapsulated agents to wounds, wound dressings, surgical dressings, bed protectors for decubitus patients (or patients that may develop), socks, other garments suitable for patients with diabetes and other circulatory disorders, orthopedic casts, and for the local delivery of NO for vasodilators in the treatment of sexual dysfunction. The present invention also addresses the need for small and long-lasting NO doses associated with conventional implanted or inserted medical supplies (e.g., vascular stents, catheters, cardiac pacemakers, defibrillators, cardiac assist devices, artificial valves, electrodes, and orthopedic screws and pins).

The invention may be a wound dressing or bandage, a portion of which comprises particles of microencapsulated reagents. This part of the dressing also incorporates a material with water retention properties to retain the appropriate amount of moisture required to keep the particles in a moist environment. Wetting the dressing initiates the reagent reaction and the dressing begins to release NO. The dressing is designed to release NO in the vicinity of the wound.

One embodiment of the present invention discloses a multi-purpose, time release NO technology from a layered pad. Cross section as shown in fig. 1A: the particles 1 are contained between

layers

2 and 3, wherein at least one of the

layers

2 and 3 is a body facing layer for transporting gaseous NO, while at least one is an outward facing layer having moisture impermeable or retentive properties (allowing transport of the applied liquid into the particles and/or maintaining the particles in a moist environment). In applications where it is desired to provide NO on one side of the liner, one of the

layers

2 or 3 is impermeable to NO. The particles at sub-millimeter level comprise microencapsulated spinach extract. In an aqueous environment, the reagents formed from the conversion of the microparticles combine with each other to produce NO. When water is introduced to the pad, the agent begins to be released and NO begins to be produced.

In the embodiment shown in fig. 1B, the

outer layers

2 and 3 are separated by a spacer layer 4, the layer 4 serving to maintain the spacing of the outer layers and to hold the particulate layer in place. The contained reagent particles may be embedded or otherwise immobilized on the barrier layer 4, or on the inner surface of either of the

outer layers

2 or 3.

A liner of the type shown in fig. 1 may be prepared in any given size and shape. The vertical dimensions in fig. 1A-C are not to scale and the bibulous material 5 may be much thicker than the pad containing the reagent.

Such liners have a variety of applications. They can be simply applied by placing over the wound and covering with a layer of suitable adhesive medical tape. They may also be incorporated into pre-made bandages or dressings. Alternatively, the bandage or dressing is provided with a packet containing microencapsulated reagents capable of reacting to form NO. In addition, the agents may be attached to different layers of material and then assembled together to form a complete bandage or dressing.

The pad of other configurations shown in fig. 1 can be used as a long lasting antimicrobial wipe. Such a pad may be re-sized for insertion into a garment such as a sock or panty of a patient with circulatory disorders. The pad itself may also be used as a fabric for socks, gloves and other garments for patients with circulatory disorders by suitable treatment of the edges of the material and the structure of the pad containing the particles. These garments may be activated by natural moisture from the patient's skin or by added moisture.

Another embodiment of the invention is a layered mat as shown in fig. 1C, which consists of the above mentioned particle-containing mat, absorbing or permeable layer 5 and the water-impermeable layer 6 underneath it. Such absorbent pads are suitable for patients who have or are beginning to develop decubitus ulcers. Such patients will produce adequate amounts of moisture through urinary incontinence and perspiration. The moisture will activate the NO producing liner and excess moisture will be absorbed by the absorbent layer underneath the liner. This arrangement bathes the bedsores in NO and nitric oxide stimulates bedsore healing, preventing further enlargement of the ulcer area.

Among the different applications, the topical application of small doses of NO to the penis to rapidly stimulate penile erection in male rats has proven to be very effective (Han et al, Journal of Sexual Medicine 7(2010) 224). The present invention discloses topical application of NO for similar effect in humans. Current systemic drugs of sexual dysfunction have a variety of side effects and take a period of time to become effective. There is a need for such rapid, local therapeutic agents in terms of controllability and absence of systemic side effects. The NO-generating agent may be placed on a dry coating on the dressing of erectile tissue. One embodiment is an internal dressing for use as a male or female condom. The agent is activated by wetting the dressing applied to the erectile tissue, releasing NO over a prolonged period of time.

In another embodiment of the invention, the interior surface of the condom is coated with a coating comprising microencapsulated reagents that react with each other to produce NO when in aqueous solution. The particle size range of this embodiment may be 0.01 to 100 microns, with a preferred range being

And (3) micron. The smaller particle size facilitates the coating to be made adherent to the inner surface of the condom, with the time scale for NO release being minutes rather than hours. In such embodiments, the user applies an aqueous compound such as a K-Y jelly (manufactured by McNEIL-PPC, inc., ft. washington, PA) to the erectile tissue prior to donning the condom. NO is released when the particles come into contact with the aqueous compound. The released NO is trapped within the condom until it is transdermally absorbed by erectile tissue to stimulate and prolong an erection.

Another embodiment of the invention is a sexual arousal gel reagent kit comprising an aqueous gel compound package resembling a K-Y jelly and a moisture resistant package containing microencapsulated reagents which react together in aqueous solution to produce NO. Before use, the package is opened to mix the aqueous gel, which is applied to the external genitalia of a male/female user to stimulate blood flow, thereby promoting penile and clitoral erection. Such a kit may be used to treat sexual dysfunction and to improve sexual satisfaction in both men and women.

Although there is no clinical study in humans, studies in rats have shown that the gel compositions described by Seitz et al (US Pat. No.6,103,275) are capable of stimulating hair growth. It is well known that local vasodilators such as minoxidil are effective in alleviating hair loss and stimulating hair growth in humans, and thus topical application of a sustained low dose of NO (NO is a potent vasodilator) is likely to have a therapeutic effect on hair loss. Thus, another extended release application disclosed herein is in devices and compositions for alleviating hair loss and stimulating hair regrowth. One particular embodiment is a head-shaped cap composed of the material shown in fig. 1 for treating alopecia. The cap is made suitable for application over a bald area of a patient's head and is wetted with water to activate it.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

Example 1 preparation of spinach extract

1.1 extraction of spinach

Washing edible part of spinach with tap water and deionized water, drying, chopping into small pieces, taking 10g, putting into a large beaker, adding 50mL of deionized water, adjusting the pH of an extracting solution to 3-4 with oxalic acid, grinding, putting into a 70 ℃ water bath, incubating for 30min, filtering an extracting solution into a 100mL volumetric flask, diluting to 100mL with deionized water, adjusting the pH of the extracting solution to 3-4 with oxalic acid, concentrating to about 10mL, transferring into a 250mL beaker, adding 5mL of saturated borax solution and l00mL (70-80 ℃) hot water, adjusting the pH of the extracting solution to 3-4 with oxalic acid, putting into a boiling water bath, heating for 15min, shaking continuously, taking out, cooling to room temperature, adding l0mL potassium ferrocyanide solution, l0mL zinc acetate solution and 2g of activated carbon powder, mixing completely after each addition, transferring into a 250mL volumetric flask, adjusting the pH of the extracting solution to 3-4 with oxalic acid, adding water to constant volume, and filtering to obtain colorless clear solution.

In the extraction process, hydrochloric acid-L-cysteine and sodium metabisulfite are further added to ensure the activity of vitamin C in the extraction process, wherein the content of hydrochloric acid-L-cysteine is 0.1 percent, and the content of sodium metabisulfite is 0.2 percent.

1.2 determination of nitrate

the content of nitrite and nitrate in the spinach extract is measured by spectrophotometry, and the result shows that the content of nitrite in the spinach extract is 5.017 × 10^-3mg/ml; the nitrate content was 0.236 mg/ml.

the content of vitamin C in spinach extract is measured by spectrophotometry, and the result shows that the content of vitamin C in spinach extract is 1.21 multiplied by 10^-2mg/ml。

Example 2 preparation of microencapsulated spinach extract

Spinach extracts prepared in example 1 were concentrated to prepare zein based granules of spinach extracts by spray drying a solution consisting of spinach extract, L-cysteine hydrochloride and sodium metabisulphite (vitamin C stabilizer), zein and a volatile solvent, which contained 10% by weight of nitrite. Zein is a proline-rich protein obtained from corn and used as a coating and encapsulating matrix for processing food and pharmaceutical products. It is classified as Generally Recognized As Safe (GRAS) by the U.S. Food and Drug Administration (FDA). This solution was 10% zein (Flo Chemicals,29 buffer St., ashburn ham, MA 01430(Lot F40000111C6)) dispersed in a mixture of ethanol: water (90: 10). The solution also contained stabilizers, L-cysteine hydrochloride with a content of 0.1% and sodium metabisulphite with a content of 0.2%, a vitamin C stabilizer, and was dispersed in a dryer using a rotary disc atomizer, in such a way that particles with a diameter ranging between 10 and 100 microns were formed, these particles comprising a zein matrix in which the spinach extract was dispersed. Zein is insoluble in water, and when the microparticles are exposed to water, the water slowly diffuses into the zein matrix dissolving the sodium nitrite and vitamin C in the spinach extract, and the solution containing sodium nitrite slowly diffuses out of the granules, resulting in a sustained release of sodium nitrite over a longer period of time.

Example 3 release of NO in solution of microencapsulated spinach extract and acidic gel

A100 ml (100ml) aqueous solution was prepared containing 5.6g of citric acid and 0.3g of PE9010 (a preservative, manufactured by Sch and Mayr, 30 Two Bridges Road Suite 225, Fairfield, NJ 07004, USA). 40ml (40ml) of this solution was placed in a beaker and the NO concentration in the solution was measured with an InNO-T nitric oxide measurement system (Innovative Instruments, Inc., Tampa, FL 33637) equipped with an amiNO-700 probe. 10mg (10mg) of the zein microencapsulated spinach extract containing microparticles prepared in example 2 were added to the solution at the indicated time zero (0), and the NO content of the solution was recorded, the production of NO arising from the addition of the microparticles, about 20 minutes the production of NO formed from the liquid, then the NO gradually increased, peaked about 1 hour and then gradually decreased. The total release time of NO from the sodium nitrite emanating from the microencapsulated particles and the acid in solution was maintained for 5 hours according to the reactions of equations (1) - (3) as described above. The release process of NO is shown in FIG. 2.

Example 4 release of NO from microencapsulated spinach extract in solution

10mg (10mg) of the fine particles prepared in example 2 were placed in a container. The mixture was added to a beaker containing 40ml of deionized water and stirred well.

The concentration of NO in the solution was measured using an InNO-T nitric oxide measurement system (Innovative Instruments, Inc., Tampa, FL 33637) equipped with an amiNO-700 probe, to which the probe tip of the amiNO-700 was added at the specified time zero (0), the NO content of the solution was recorded, followed by recording the NO signal, and weak NO was detected during the first three hours of monitoring.

Example 5 release of NO from microencapsulated spinach extract and acidic gel in a paste

This example is intended to simulate the release of NO when equal amounts of microencapsulated spinach extract and acidic gel are applied directly to the body surface of a patient.

10mg of the microparticles prepared in example 2 were placed on a small-wrinkled weighing paper. The probe tip of the amino-700 was inserted and completely covered with the powder mixture. The same amount of the acidic gel prepared in example 3 was mixed with the microencapsulated spinach extract and the NO signal was recorded, optionally with the addition of a small amount of deionized water at the right time depending on the dry state of the paste system, during the whole NO release, from the start of the recording, the NO release reached a relatively high level of about 1 hour and the whole release reached 10 hours. While the level of NO is always in a steady and steady state during the process. The release process of NO is shown in FIG. 3.

EXAMPLE 6 Release of NO from microencapsulated sodium nitrite and acidic gel in a paste

Particles of ethylcellulose-based sodium nitrite (sodium nitrite weight ratio 10%) were prepared by spray-drying a solution consisting of sodium nitrite, ethylcellulose and a volatile solvent according to the microcapsule preparation method of example 2.

10mg of the microencapsulated sodium nitrite microparticles prepared above were placed on a weighing paper with small wrinkles. The probe tip of the amino-700 was inserted and completely covered with the powder mixture. The same amount of the acid gel prepared in example 3 was mixed with the microencapsulated sodium nitrite uniformly, and then the NO signal was recorded, during which trace amount of deionized water was added at the right time according to the dry state of the paste system, and during the whole NO release, from the beginning of recording, about 1 hour the NO release reached a relatively high level, and the whole release reached 8 hours. While the level of NO is always in a steady and steady state during the process. The whole NO release process is shown in FIG. 4.

Example 7 release of NO from microencapsulated spinach extract and acidic gel in a paste, the acidic gel also containing a reducing agent

This example is intended to simulate the release of NO when equal amounts of microencapsulated spinach extract and acidic gel are applied directly to the body surface of a patient. A reducing agent vitamin C is additionally added into the prepared acidic gel.

100ml (100ml) of an aqueous solution containing 5.6g of citric acid, 2.2g of ascorbic acid and 0.3g of PE9010 (a preservative manufactured by Sch and Mayr, 30 Two Bridges Road Suite 225, Fairfield, NJ 07004, USA) was prepared.

10mg of the particles were placed on a small wrinkled weighing paper. The probe tip of the amino-700 was inserted and completely covered with the powder mixture. The same amount of the acidic gel containing additional reducing agent prepared above was mixed well with the microencapsulated spinach extract, and then the NO signal was recorded, during which trace amounts of deionized water were added at the right time depending on the dry state of the paste system, and during the whole NO release, from the start of the recording, the NO release reached a relatively high level in about 40 minutes, and the whole release reached 10 hours. While the level of NO is always in a steady and steady state during the process. The whole NO release process is shown in FIG. 5.

The above experimental results show that the microencapsulated spinach extract of the invention can have a longer NO release time than microencapsulated nitrite (which can sustain NO release for 8 hours), and can sustain the release of NO at a physiologically effective concentration for up to 10 hours without adding a reductant component to the system, and the NO release rate is constant during this process.

Analyzing the above reasons, it is possible that the specific morphology of nitrite and vitamin C in spinach extracts or the formation of complex structures with the presence of other substances maintains the long-term NO release process.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A delayed release system of nitric oxide comprising a microencapsulated spinach extract and an acidic gel having sufficient acidity to convert the spinach extract to nitric oxide; the spinach extract contains vitamin C derived from spinach, and the pH of the spinach extract is adjusted to 3-4 in the extraction process;

the spinach extract has nitrite content of 5.017 × 10^-3mg/ml, nitrate content of 0.236mg/ml, vitamin C content of 1.21 × 10^-2mg/ml, containing 10% by weight, calculated as nitrite, spinach extract, and preparing particles of a zein-based spinach extract by spray drying a solution consisting of spinach extract, L-cysteine hydrochloride and sodium metabisulphite, zein and a volatile solvent, said solution containing 10% zein, 0.1% L-cysteine hydrochloride, 0.2% sodium metabisulphite;

the acid gel is prepared by mixing 5.6g of citric acid and 0.3g of PE9010 in 100ml of water;

the spinach extract and the acidic gel are used in equal proportion.

2. A kit for providing a patient with a delayed release of therapeutic nitric oxide, comprising a microencapsulated spinach extract and an acidic gel having sufficient acidity to convert the spinach extract to nitric oxide; the microencapsulated spinach extract and the acidic gel are placed separately; the spinach extract contains vitamin C derived from spinach, and the pH of the spinach extract is adjusted to 3-4 in the extraction process;

the spinach extract has nitrite content of 5.017 × 10^-3mg/ml, nitrate content of 0.236mg/ml, vitamin C content of 1.21 × 10^-2mg/ml, the microencapsulated spinach extract containsSpinach extract at 10% by weight, calculated as nitrite, and preparing particles of a zein-based spinach extract by spray drying a solution consisting of spinach extract, L-cysteine hydrochloride and sodium metabisulphite, zein and a volatile solvent, said solution containing 10% zein, 0.1% L-cysteine hydrochloride, 0.2% sodium metabisulphite;

the spinach extract and the acidic gel are used in equal proportion.

3. The kit of claim 2, wherein the microencapsulated spinach extract is placed on a wound dressing or bandage and the acidic gel is placed in a separate container.

4. The kit of claim 2, wherein the microencapsulated spinach extract is coated on the inner surface of the condom and the acidic gel is placed in a separate container.

5. Use of a kit according to any of claims 2 to 3 in the manufacture of a medicament for promoting wound healing.

6. Use of a kit according to any of claims 2 to 3 for the manufacture of a medicament for promoting the regeneration of hair loss.

7. Use of a kit according to claim 2 or 4 for the manufacture of a medicament for overcoming sexual dysfunction.