WO2019150266A1 - Preparation for treatment of wounds - Google Patents

Preparation for treatment of wounds Download PDF

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Publication number
WO2019150266A1
WO2019150266A1 PCT/IB2019/050731 IB2019050731W WO2019150266A1 WO 2019150266 A1 WO2019150266 A1 WO 2019150266A1 IB 2019050731 W IB2019050731 W IB 2019050731W WO 2019150266 A1 WO2019150266 A1 WO 2019150266A1
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Prior art keywords
treatment
composition
preparation
hsar
gangrene
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PCT/IB2019/050731
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French (fr)
Inventor
Humaid Saeed Mohammad Sultan ALARYANI
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Alaryani Humaid Saeed Mohammad Sultan
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Application filed by Alaryani Humaid Saeed Mohammad Sultan filed Critical Alaryani Humaid Saeed Mohammad Sultan
Priority to EP19747189.9A priority Critical patent/EP3573634A4/en
Priority to CN201980003785.9A priority patent/CN111065402A/en
Publication of WO2019150266A1 publication Critical patent/WO2019150266A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/889—Arecaceae, Palmae or Palmaceae (Palm family), e.g. date or coconut palm or palmetto
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

  • the invention relates to the use of a preparation for treatment of chronic infected wounds
  • the wounds include diabetes foot ulcers and gangrene in addition to an antiviral activity.
  • Wound healing is a complex biological process that aims to repair damaged tissue (Korblatt et al. 2016). More than 6.5 million Americans suffer from chronic, non-healing wounds, generating annual healthcare costs of more than $25 billion (Sen et al. 2009). These numbers are expected to increase with the growth of high-risk populations, including the increase in number of patients suffering from diabetes and obesity, as well as the elderly. The significant disability and costs to society associated with chronic wounds highlight the inadequacy of our current therapeutic armamentarium (Whittam et al. 2016).
  • the composition comprises a mixture of Allium sativum, Ferula assa-foetida, Curcuma longa, Acacia nilotica, Calligonum comosum, Phoenix dactylifera with sodium chloride mixed in honey, and then burnt to form a powdery composition.
  • the powdery composition is then mixed with at least one of water, honey, cream, oil or a gel. In one aspect, the preparation has been burnt and afterwards suspended in water.
  • the method for the preparation comprises mixing the Allium sativum, Ferula an.na-foetida, Curcuma longa, Acacia nilotica, Calligonum comosum, Phoenix dactylifera with sodium chloride and honey and subsequently burning the mixture to produce a powdery composition.
  • the preparation may be used for the treatment of gangrene, skin ulcers, or eczema.
  • Figure 1 shows the antibacterial and antifungal activity of the HSAR preparation.
  • Figure 2 shows the antiviral activity of the aqueous extract.
  • Figures 3A & 3B show a goat udder with deep injury.
  • Figure 4A shows a foot with gangrene before treatment.
  • Figure 4B shows the foot after treatment.
  • Figure 5 shows a diabetic foot ulcer with gangrene after treatment.
  • Figure 6 shows the fingers after treatment.
  • Figure 7 A shows a varicose vein with ulcer before treatment.
  • Figure 7B shows the varicose vein with ulcer during treatment.
  • Figures 7C and 7D show the varicose vein with ulcer after treatment.
  • Figure 8A shows a hand with eczema before treatment
  • Figure 8B shows the hand after treatment with the FIS AR preparation.
  • HSAR Preparation The following herbs were cleaned, ground and mixed together in equal proportions: Allium sativum /Garlic/, Ferula assa-foetida (type of dried latex), Curcuma longa (Turmeric), seeds of Acacia nilotica (gum Arabic tree), Aerial parts of Calligonum comosum, Phoenix dactylifera (Dates) together with Salt (NaCl) (lg per kg of the ground herb) were mixed properly with honey (250ml per kg) to form a thick paste.
  • the honey as an Acacia honey, but this is not limiting of the invention.
  • the paste was then heated over an open fire in air atmosphere (because of the undesired smell of Ferula and garlic) for almost three hours until the paste burned completely and turned to black charcoal.
  • the black charcoal was then powdered (called HSAR) and ready to be used.
  • the HSAR preparation forms the paste as an emulsion when suspended in honey, oil, cream or a gel.
  • the oils can be saturated vegetable oils or animal fats.
  • the cream is typically one used as the base of a moisturizing cream.
  • the HSAR preparation may be applied as a powder especially in case of wet wounds. All ingredients were purchased from local market in Abu Dhabi.
  • Luria-Birtani broth was prepared as follows: Tryptone 10 g /L (HiMedia, India), Yeast extract 5 g /L (HiMedia, India), and sodium chloride 10 g /L (HiMedia, India) at pH 7.2. This medium was used in all the protocols. In case of Luria-Birtani agar (LA) 14 g /L of agar bacteriology No.1 (HiMedia, Mumbai, India) was used for culture maintenance. Bacterial dilutions from 18 h LB cultures grown at 37°C were carried out in phosphate buffered saline (PBS, Oxoid, UK).
  • PBS phosphate buffered saline
  • the‘soft layer agar’ used was LB prepared in Lambda- buffer [6 mmol /L Tris pH 7.2; 10 mmol/L Mg(S04)2.7H20; 50 mg/L gelatin (Oxoid, UK)] and supplemented with 4 g /L agar bacteriology No. 1 (HiMedia, India). Potato Dextrose Broth and Agar (Fluka, USA) were used to maintain Candida.
  • Pseudomonas aeruginosa ATCC 25668 Staphylococcus aureus ATCC 6538, Escherichia coli NTCC 12900 and Candida albicans (Patient isolates from UAE hospitals) were used to evaluate the antimicrobial activity of the composition.
  • Pseudomonas aeruginosa ATCC 25668 was used throughout the antibacterial activity as well as antiviral activity investigations. Cultures were stored at -20°C in 15% glycerol. Prior to investigation, a stock culture of the microorganism was maintained on an LA plate.
  • Antibacterial and antifungal activity Antimicrobial activity of 10% of the HSAR composition in distilled water (w/v) was evaluated using strains of Pseudomonas aeruginosa ATCC 25668, Staphylococcus aureus ATCC 6538, Escherichia coli NTCC 12900 and Candida albicans. Aliquots of 18 hours broth of each microorganism was mixed with 10% HSAR composition suspension, PBS was used for control, all tubes were incubated for 24 hours at 37 C, Candida albicans was incubated in 30 C. The antimicrobial activities were quantitatively investigated using Miles and Misra method (Miles and Misra, 1938).
  • Figure 1 shows the antibacterial and antifungal activity of the HSAR preparation. It can be seen that the HSAR preparation was very effective against P. aeruginosa ATCC 25668 with more than six log reduction, Escherichia coli NTCC 12900 with two log reduction and C. albicans with one log reduction, while did not show any activity against S. aureus ATCC 6538 and C. albicans. This is probably due to differences in the cell walls.
  • Bacteriophages The virus that was used in this study was lytic Pseudomonas phage ATCC 14209-B1.
  • the phage stocks were prepared on the host strain, P. aeruginosa ATCC 25668, by a plate lysis procedure (Davis and Sinsheimer, 1963; Vidaver, 1973; Sambrook, et al. 1989 ; Ausubel, et al. 1991). Briefly, an aliquot (100 ml) of the phage sample (10-fold serially diluted with Lambda-buffer) was mixed with 100 ml of an overnight LB culture of P.
  • aeruginosa ATCC 25668 in a sterile 1.5 ml Eppendorf micro-centrifuge tube (Greiner bio-one) and incubated for 15 min at 37°C to facilitate attachment of the bacteriophage to the host cells.
  • the mixture was transferred from the Eppendorf micro-centrifuge tube to a 5 ml Bijou bottle and then 2 ml of soft layer agar was added which had been melted and cooled to 40°C in a water bath. The contents of each bottle were then gently mixed by swirling, poured over the surface of a plate of LA and allowed to set for 15 min at room temperature.
  • the plates were incubated for 18 h at 37°C, and a plate showing almost confluent plaques was used to prepare a concentrated phage suspension by overlaying with 5ml of Lambda buffer [titer 10 11 plaque-forming units per ml (pfu /ml)].
  • the final purification process used chloroform to separate the bacteriophage from the bacterial cells (Ausubel, et al. 1991).
  • the phage stocks, at a titer of 10 11 pfu/ml, were maintained in Lambda- buffer at 4°C.
  • phage inactivation was performed as previously described (Adams, 1959). Briefly, an aliquot (100 m ⁇ ) of phage suspension containing 10 10 pfu was added to 900 m ⁇ of 10% HS AR in distilled water (w/v) in an Eppendorf micro-centrifuge tube, the control contained 900 m ⁇ of lambda buffer, all in triplicate. Tubes were kept at room temperature for 18 hours. The number of the survived bacteriophages was measured by enumerating the pfu count.
  • Eigure 2 shows the antiviral activity of the aqueous extract, which is determined by enumerating the pfu count of survived phages.
  • the HSAR 10% suspension induced 2 log reductions in phage titer (From 10 9 to 10 7 PFU/ml) in 18 hours.
  • Such a reduction in the titer of Pseudomonas bacteriophage which is known for its resistance to alcohols, organic acids and alkalines (Jones, 1991) makes the present aqueous extract an absolutely promising antiviral extract.
  • Farm animal model Figures 3 A & 3B show a goat udder with deep injury dropping milk
  • Figure 3A illustrates the goat udder deep injury before treatment and Figure 3B shows the goat udder after treatment with the HSAR
  • FIG. 4A shows the foot with the gangrene before treatment with the HSAR preparation.
  • Figure 4B shows the foot after treatment with the HSAR preparation.
  • the wound was totally covered by the HSAR preparation in powder form and which sticks to the wet wound. The powder then gradually falls off when the powder become dry and will be replaced by new powder once a day at night for approximately one month until the wound was found to be cured and new healthy skin is generated.
  • a second case is gangrene in which amputation was also recommended.
  • the patient was male, 53 years, and had a diabetic foot ulcer.
  • the patient refused amputation and used the HSAR composition.
  • Figure 5 shows the diabetic foot ulcer with gangrene after treatment once a day with the HSAR composition for about one month.
  • Figures 7A, 7B, 7C & 7D The patient was a male, 60 years, and did not respond to the provided medical interventions. After one and half year of suffering the patient applied the HSAR preparation.
  • Figure 7A shows the varicose vein with ulcer before treatment.
  • Figure 7B shows the varicose vein with ulcer during treatment with the HSRA preparation and figures 7C and 7D show the varicose vein with ulcer after two weeks of treatment with the HSAR preparation.
  • FIGs 8A & 8B The application to the treatment of eczema is illustrated in Figures 8A & 8B.
  • the patient was female, 12 years old, and had had eczema for the last three years on her hand. Treatment once a day was made by the HSAR preparation.
  • Figure 8 A shows the hand with eczema before treatment with the HSAR preparation and
  • Figure 8B shows the hand after one week of treatment with the HSAR preparation.
  • Bacteriophage f6 a lipid-containing virus of Pseudomonas phaseolicola. J. Virol. 11 :799-805

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Abstract

A method is disclosed for the preparation of a composition comprising mixing Allium sativum, Ferula assa-foetida, Curcuma longa, Acacia nilotica, Calligonum comosum, Phoenix dactylifera with sodium chloride and burning the mixture to produce a powdery composition. The composition is used for the treatment of gangrene, skin ulcers, or eczema.

Description

Title: Preparation for Treatment of Wounds
Summary of the Invention [0001] The invention relates to the use of a preparation for treatment of chronic infected wounds
(such as but not limited to bacterial and fungal infections). The wounds include diabetes foot ulcers and gangrene in addition to an antiviral activity.
Background to the Invention
[0002] Chronic Infected Wound Healing. Wound healing is a complex biological process that aims to repair damaged tissue (Korblatt et al. 2016). More than 6.5 million Americans suffer from chronic, non-healing wounds, generating annual healthcare costs of more than $25 billion (Sen et al. 2009). These numbers are expected to increase with the growth of high-risk populations, including the increase in number of patients suffering from diabetes and obesity, as well as the elderly. The significant disability and costs to society associated with chronic wounds highlight the inadequacy of our current therapeutic armamentarium (Whittam et al. 2016).
[0003] In addition to the economic cost, there is a significant deterioration in the quality of life for patients suffering from chronic wounds (Sen et al. 2009). A physical injury to the human skin may leave a footprint in the form of a scar which can range in appearance from barely visible to extensively disfiguring. Cutaneous scars commonly form after surgical operations, trauma, burns and infections (Al-Shaqsi and Al-Bulushi, 2016). It is estimated that approximately 100 million people develop scars after trauma and elective surgery in middle-income countries every year and 15% of this population will require surgical intervention for their scars due to aesthetic considerations or functional impairment (Gold et al. 2014). In conclusion, there is an unmet clinical need for effective solutions to skin scarring (Sidgwick etal. 2015).
[0004] Diabetic Foot Ulcers and Gangrene. The association between gangrene of the foot and diabetes was discovered 160 years ago. Although the treatment of diabetic ulcers has developed greatly over the past one and a half centuries, the treatment of chronic diabetic ulcers remains a significant problem in which the treatment options are very limited. Patients with chronic diabetic ulcers must deal with pain, infection, hospital stays, and amputations. This leads to a poor quality of life for the patients and families as well as large healthcare expenditures for the community (Naves, 2014). The major consequence of a non-healing ulcer of the foot in a diabetic patient is amputation (Akkurt et al. 2017). About 50% of patients with diabetic foot infections who have foot amputations die within five years (Schaper et al. 2003)
[0005] Alternative and complementary medicine interventions are used across the world and are either the mainstay of health care delivery or serve as a complement to health care delivery (Sebastian and Thampan, 2007; Buffoni, 1996). Alternative and complementary medicine interventions date back basically to the beginning of the humanity (Cragg, et al. 1997; Farnsworth, et al. 1985).
Summary of the Invention
[0006] It is an object of the present invention to provide an alternative composition which can be used to treat any infected wounds including chronic infected wounds, diabetic ulcer foot and gangrene in addition to its use as an antibacterial, antifungal and antiviral agent. It is also an object of the present invention to provide a method of preparation of this composition.
[0007] The composition comprises a mixture of Allium sativum, Ferula assa-foetida, Curcuma longa, Acacia nilotica, Calligonum comosum, Phoenix dactylifera with sodium chloride mixed in honey, and then burnt to form a powdery composition. The powdery composition is then mixed with at least one of water, honey, cream, oil or a gel. In one aspect, the preparation has been burnt and afterwards suspended in water.
[0008] The method for the preparation comprises mixing the Allium sativum, Ferula an.na-foetida, Curcuma longa, Acacia nilotica, Calligonum comosum, Phoenix dactylifera with sodium chloride and honey and subsequently burning the mixture to produce a powdery composition.
[0009] The preparation may be used for the treatment of gangrene, skin ulcers, or eczema.
Description of the Figures [0010] Figure 1 shows the antibacterial and antifungal activity of the HSAR preparation.
[0011] Figure 2 shows the antiviral activity of the aqueous extract. [0012] Figures 3A & 3B show a goat udder with deep injury.
[0013] Figure 4A shows a foot with gangrene before treatment.
[0014] Figure 4B shows the foot after treatment.
[0015] Figure 5 shows a diabetic foot ulcer with gangrene after treatment.
[0016] Figure 6 shows the fingers after treatment.
[0017] Figure 7 A shows a varicose vein with ulcer before treatment.
[0018] Figure 7B shows the varicose vein with ulcer during treatment.
[0019] Figures 7C and 7D show the varicose vein with ulcer after treatment.
[0020] Figure 8A shows a hand with eczema before treatment
[0021 ] Figure 8B shows the hand after treatment with the FIS AR preparation.
Detailed Description of the Invention
[0022] HSAR Preparation The following herbs were cleaned, ground and mixed together in equal proportions: Allium sativum /Garlic/, Ferula assa-foetida (type of dried latex), Curcuma longa (Turmeric), seeds of Acacia nilotica (gum Arabic tree), Aerial parts of Calligonum comosum, Phoenix dactylifera (Dates) together with Salt (NaCl) (lg per kg of the ground herb) were mixed properly with honey (250ml per kg) to form a thick paste. In one aspect, the honey as an Acacia honey, but this is not limiting of the invention. The paste was then heated over an open fire in air atmosphere (because of the undesired smell of Ferula and garlic) for almost three hours until the paste burned completely and turned to black charcoal. The black charcoal was then powdered (called HSAR) and ready to be used. The black powder (HSAR) is slightly soluble in water with pH= 9 ± 0.2.
[0023] The HSAR preparation forms the paste as an emulsion when suspended in honey, oil, cream or a gel. The oils can be saturated vegetable oils or animal fats. The cream is typically one used as the base of a moisturizing cream. The HSAR preparation may be applied as a powder especially in case of wet wounds. All ingredients were purchased from local market in Abu Dhabi.
[0024] Media. Luria-Birtani broth (LB) was prepared as follows: Tryptone 10 g /L (HiMedia, India), Yeast extract 5 g /L (HiMedia, India), and sodium chloride 10 g /L (HiMedia, India) at pH 7.2. This medium was used in all the protocols. In case of Luria-Birtani agar (LA) 14 g /L of agar bacteriology No.1 (HiMedia, Mumbai, India) was used for culture maintenance. Bacterial dilutions from 18 h LB cultures grown at 37°C were carried out in phosphate buffered saline (PBS, Oxoid, UK). For performing the plaque assay, the‘soft layer agar’ used was LB prepared in Lambda- buffer [6 mmol /L Tris pH 7.2; 10 mmol/L Mg(S04)2.7H20; 50 mg/L gelatin (Oxoid, UK)] and supplemented with 4 g /L agar bacteriology No. 1 (HiMedia, India). Potato Dextrose Broth and Agar (Fluka, USA) were used to maintain Candida.
[0025] Microbial Strains. Pseudomonas aeruginosa ATCC 25668, Staphylococcus aureus ATCC 6538, Escherichia coli NTCC 12900 and Candida albicans (Patient isolates from UAE hospitals) were used to evaluate the antimicrobial activity of the composition. Pseudomonas aeruginosa ATCC 25668 was used throughout the antibacterial activity as well as antiviral activity investigations. Cultures were stored at -20°C in 15% glycerol. Prior to investigation, a stock culture of the microorganism was maintained on an LA plate. One loopful of each microorganism was inoculated into a 15 ml sterile centrifuge tube with flat cup (ExtraGene, USA) containing 10 ml of LB and incubated for 18 h at 37°C and 70 rpm in an incubator shaker (Innova 4000, New Brunswick Scientific). Appropriate serial dilutions were made in LB and PBS. 2% Tween 80 (BDH, UK) was used as a neutralizer.
[0026] Antibacterial and antifungal activity. Antimicrobial activity of 10% of the HSAR composition in distilled water (w/v) was evaluated using strains of Pseudomonas aeruginosa ATCC 25668, Staphylococcus aureus ATCC 6538, Escherichia coli NTCC 12900 and Candida albicans. Aliquots of 18 hours broth of each microorganism was mixed with 10% HSAR composition suspension, PBS was used for control, all tubes were incubated for 24 hours at 37 C, Candida albicans was incubated in 30 C. The antimicrobial activities were quantitatively investigated using Miles and Misra method (Miles and Misra, 1938).
[0027] Figure 1 shows the antibacterial and antifungal activity of the HSAR preparation. It can be seen that the HSAR preparation was very effective against P. aeruginosa ATCC 25668 with more than six log reduction, Escherichia coli NTCC 12900 with two log reduction and C. albicans with one log reduction, while did not show any activity against S. aureus ATCC 6538 and C. albicans. This is probably due to differences in the cell walls.
[0028] Bacteriophages. The virus that was used in this study was lytic Pseudomonas phage ATCC 14209-B1. The phage stocks were prepared on the host strain, P. aeruginosa ATCC 25668, by a plate lysis procedure (Davis and Sinsheimer, 1963; Vidaver, 1973; Sambrook, et al. 1989 ; Ausubel, et al. 1991). Briefly, an aliquot (100 ml) of the phage sample (10-fold serially diluted with Lambda-buffer) was mixed with 100 ml of an overnight LB culture of P. aeruginosa ATCC 25668 in a sterile 1.5 ml Eppendorf micro-centrifuge tube (Greiner bio-one) and incubated for 15 min at 37°C to facilitate attachment of the bacteriophage to the host cells. The mixture was transferred from the Eppendorf micro-centrifuge tube to a 5 ml Bijou bottle and then 2 ml of soft layer agar was added which had been melted and cooled to 40°C in a water bath. The contents of each bottle were then gently mixed by swirling, poured over the surface of a plate of LA and allowed to set for 15 min at room temperature. The plates were incubated for 18 h at 37°C, and a plate showing almost confluent plaques was used to prepare a concentrated phage suspension by overlaying with 5ml of Lambda buffer [titer 1011 plaque-forming units per ml (pfu /ml)]. The final purification process used chloroform to separate the bacteriophage from the bacterial cells (Ausubel, et al. 1991). The phage stocks, at a titer of 1011 pfu/ml, were maintained in Lambda- buffer at 4°C.
[0029] Anti-viral activity. The phage inactivation was performed as previously described (Adams, 1959). Briefly, an aliquot (100 mΐ) of phage suspension containing 1010 pfu was added to 900 mΐ of 10% HS AR in distilled water (w/v) in an Eppendorf micro-centrifuge tube, the control contained 900 mΐ of lambda buffer, all in triplicate. Tubes were kept at room temperature for 18 hours. The number of the survived bacteriophages was measured by enumerating the pfu count.
[0030] Eigure 2 shows the antiviral activity of the aqueous extract, which is determined by enumerating the pfu count of survived phages. The HSAR 10% suspension induced 2 log reductions in phage titer (From 109 to 107 PFU/ml) in 18 hours. Such a reduction in the titer of Pseudomonas bacteriophage which is known for its resistance to alcohols, organic acids and alkalines (Jones, 1991) makes the present aqueous extract an absolutely promising antiviral extract.
[0031] Animal model. Male adult Sprague-Dawley rats (200-250 g) from Zayed Herbal Center. The rats were housed under standard conditions and were supplied with drinking water and food. All animal experiments were performed in accordance to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications number 85-23, revised 1996).
[0032] In Vivo Wound healing. Male ones of the rats were shaved 25 cm2, within the shaved area, 5 cm cut was done using surgical blades. Test animals were divided into three groups; group 1 (control) did not receive anything, group 2 was treated topically by Mebo, Group 3 was treated topically with the HSAR composition. The wounds in the HSAR-treated group 3 became completely dry on day three, therefore, we were not able to apply the HSAR preparation as a dry powder again anymore.
[0033] Farm animal model: Figures 3 A & 3B show a goat udder with deep injury dropping milk,
HSAR was applied and the wound was cured. Figure 3A illustrates the goat udder deep injury before treatment and Figure 3B shows the goat udder after treatment with the HSAR
composition.
[0034] Human cases: The HSAR composition has been tested on many severe chronic infected wounds (Wet gangrene, flesh eating disease, allergy), it was successful in all cases, only few patients allowed us to use their pictures and only one of them provided a hospital report, which confirms very clearly that he was infected with gangrene and amputation was recommended.
[0035] In a first case, gangrene was found, and amputation of the foot was recommended, as shown in Figures 4A & 4B. The patient was male, 67 years old and a diabetic. Figure 4A shows the foot with the gangrene before treatment with the HSAR preparation. Figure 4B shows the foot after treatment with the HSAR preparation. The wound was totally covered by the HSAR preparation in powder form and which sticks to the wet wound. The powder then gradually falls off when the powder become dry and will be replaced by new powder once a day at night for approximately one month until the wound was found to be cured and new healthy skin is generated.
[0036] A second case is gangrene in which amputation was also recommended. The patient was male, 53 years, and had a diabetic foot ulcer. The patient refused amputation and used the HSAR composition. Figure 5 shows the diabetic foot ulcer with gangrene after treatment once a day with the HSAR composition for about one month.
[0037] A third case of gangrene in which amputation was also recommended is shown in figure
6. The patient was a male, 38 years, and a diabetic. Three fingers of the right hand; Index, middle and ring finger, had developed gangrene. The patient refused amputation of the three fingers and used the HSAR composition. Figure 6 shows the fingers after treatment with the HSAR composition.
[0038] The use of the HSAR composition to treat a varicose vein with an ulcer is shown in
Figures 7A, 7B, 7C & 7D). The patient was a male, 60 years, and did not respond to the provided medical interventions. After one and half year of suffering the patient applied the HSAR preparation. Figure 7A shows the varicose vein with ulcer before treatment. Figure 7B shows the varicose vein with ulcer during treatment with the HSRA preparation and figures 7C and 7D show the varicose vein with ulcer after two weeks of treatment with the HSAR preparation.
[0039] The application to the treatment of eczema is illustrated in Figures 8A & 8B. The patient was female, 12 years old, and had had eczema for the last three years on her hand. Treatment once a day was made by the HSAR preparation. Figure 8 A shows the hand with eczema before treatment with the HSAR preparation and Figure 8B shows the hand after one week of treatment with the HSAR preparation.
References
Kornblatt AP, Nicoletti VG, Travaglia A. 2016. The neglected role of copper ions in wound healing . J Inorg Biochem. pii: S0l62-0l34(l6)30038-l . Sen CK., Gordillo GM, Roy S, et al. 2009. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Re gen 17:763-771
Whittam AJ, Maan ZN, Dushe D et al. 2016. Challenges and Opportunities in Drug Delivery for Wound Healing. Adv Wound Care (New Rochelle) 5(2): 79-88.
Al-Shaqsi S and Al-Bulushi T. 2016. Cutaneous Scar Prevention and Management. Sultan Qaboos UnivMed J e3-e8.
Gold MH, Berman B, Clementoni MT, et al. 2014. Updated international clinical
recommendations on scar management: Part 1 - Evaluating the evidence. Dermatol Surg.
40:817-24.
Sidgwick GP, McGeorge D, Bayat A, 2015. A comprehensive evidence-based review on the role of topicals and dressings in the management of skin scarring. Arch Dermatol Res 307(6): 461- 477.
Naves CCLM, 2014. The Diabetic Foot: A Historical Overview and Gaps in Current Treatment. Advances in Wound Care 5 (5): 191 -197.
Akkurt MO, Demirkale I, Oznur A, 2017. Partial calcanectomy and Ilizarov external fixation may reduce amputation need in severe diabetic calcaneal ulcers. Diabet Foot Ankle 8(1):
1264699.
Schaper NC, Apelqvist J, Bakker K, 2003. The international consensus and practical guidelines on the management and prevention of the diabetic foot. Curr Diab Rep. 3:475-9. doi:
10.1007/s 11892-003-0010-4 Sebastian, K.S. and Thampan, R.V. 2007. Chem Biol Interact. 17, 135-43 Buffoni, F. 1996. Acta Phytotherapeutica. 2, 3-6 Cragg, G.M. et al, 1997. JNat Prod. 60:52-60 Farnsworth, N.R. et al., 1985. Bull. WHO. 63:965-981
Miles, A. A. & Misra, S.S. 1938. The estimation of the bactericidal power of the blood. Journal of Hygiene, 38, 732-749
Davis, J. E., and R. L. Sinsheimer. 1963. The replication of bacteriophage. MS2. J. Mol. Biol. 6:203-207
Vidaver, A. K., R. K. Koski, and J. L. Van Etten. 1973. Bacteriophage f6: a lipid-containing virus of Pseudomonas phaseolicola. J. Virol. 11 :799-805
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed., p. 2.65. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Ausubel, F.M. et al. 1991. Wiley Interscience . 1.12.1-1.12.3
Adams, M. H. Bacteriophages, p. 450-456. Interscience Publishers Inc.1959, New York
Jones, M.V. et al, 1991. J Hosp Infect. l7(4):279-85

Claims

Claims
1. A method for the preparation of a composition comprising:
mixing Allium sativum, Ferula assa-foetida, Curcuma longa, Acacia nilotica,
Calligonum comosum, Phoenix dactylifera with sodium chloride; and
burning the mixture to produce a powdery composition.
2. The method of claim 1, further comprising mixing the powdery composition with honey.
3. The method of claim 1 or 2, further comprising mixing the powdery composition in at least one of water, honey, cream, oil or a gel.
4. The use of the composition prepared by the method of claims 1 to 3 for the treatment of gangrene, skin ulcers, or eczema.
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GB2586252A (en) * 2019-08-14 2021-02-17 Saeed Mohammad Sultan Alaryani Humaid Anti-viral Preparation

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US5401504A (en) * 1993-12-28 1995-03-28 University Of Mississippi Medical Center Use of turmeric in wound healing
US6126950A (en) * 1997-06-24 2000-10-03 Council Of Scientific And Industrial Research Composition useful for healing and protecting skin
WO2002089826A1 (en) * 2001-05-04 2002-11-14 Morepen Laboratories Ltd. Method of preparing garlic ointment and garlic ointment composition for topical use in skin infections
WO2004087179A1 (en) * 2003-04-04 2004-10-14 Indena S.P.A. A process for the preparation of ferutinine from ferula genus plants
FR2883183A1 (en) * 2005-03-16 2006-09-22 Soc Extraction Principes Actif Use of an extract of date seeds as an antioxidant and/or anti-radicalizing agent, optionally in combination with at least another active agent, in or for the preparation of a cosmetic and/or pharmaceutical composition
US20130109748A1 (en) * 2011-08-16 2013-05-02 King Abdul Aziz City For Science And Technology Method for obtaining extract of calligonum species and extract thereof

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US7357950B2 (en) * 2003-03-21 2008-04-15 Elizabeth Anne Mazzio Topical treatment for dyshidrosis (pompholyx) and dry skin disorders
CN104225504A (en) * 2014-10-11 2014-12-24 朱珍玉 Traditional Chinese medicine unguent for treating diabetic foot gangrene and preparation method thereof
CN104740106A (en) * 2015-04-07 2015-07-01 万小耀 External-use pharmaceutical composition for treating scalds, external-use medicinal oil for treating scalds and preparation method for external-use medicinal oil
CN105233148A (en) * 2015-09-29 2016-01-13 李文远 Externally-used ointment for treating sores and ulcers and diminishing swelling and inflammation and application of externally-used ointment

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US5401504A (en) * 1993-12-28 1995-03-28 University Of Mississippi Medical Center Use of turmeric in wound healing
US5401504B1 (en) * 1993-12-28 1998-04-21 Univ Mississippi Medical Cente Use of tumeric in wound healing
US6126950A (en) * 1997-06-24 2000-10-03 Council Of Scientific And Industrial Research Composition useful for healing and protecting skin
WO2002089826A1 (en) * 2001-05-04 2002-11-14 Morepen Laboratories Ltd. Method of preparing garlic ointment and garlic ointment composition for topical use in skin infections
WO2004087179A1 (en) * 2003-04-04 2004-10-14 Indena S.P.A. A process for the preparation of ferutinine from ferula genus plants
FR2883183A1 (en) * 2005-03-16 2006-09-22 Soc Extraction Principes Actif Use of an extract of date seeds as an antioxidant and/or anti-radicalizing agent, optionally in combination with at least another active agent, in or for the preparation of a cosmetic and/or pharmaceutical composition
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