Composition obtainable by passing singlet oxygen through extra virgin olive oil

Abstract

The present invention relates to a novel composition and methods of preparation thereof. More specifically, the compositions are useful as personal care products, as wound healing compositions, as a disinfectant and also for nosocomial infections.

Claims

1. A composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanedioate, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate, wherein the composition is obtained by passing singlet oxygen through extra virgin olive oil for a period of 325 to 385 hours.

2. A personal care product comprising the composition as claimed in claim 1, and a pharmaceutically acceptable excipient.

3. The personal care product as claimed in claim 2 in the form of a solid, a liquid, or a semi-solid.

4. The personal care product as claimed in claim 3 in the form of a cream, a suppository, a soap, a body scrub, a body oil, a body wash, a shampoo, a hair conditioner, a moisturizer, a body oil, a balm, a baby product or a solution.

5. The personal care product as claimed in claim 4 for use in treatment of acne, dandruff and dry skin.

6. The composition as claimed in claim 1 for use in wound healing, nosocomial infection or disinfection.

7. A process for preparing a composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octyl cyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate, the process comprising the steps of: (a) preparing singlet oxygen; (b) passing the singlet oxygen through extra virgin olive oil for a period of 325 to 385 hours; and (c) obtaining the composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanedioate, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octyl cyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Preparation of Incision to dressing.

(2) FIG. 2: Application process of the drug test.

(3) FIG. 3: Macroscopical results of the wound after 7 days.

(4) FIG. 4: Microscopical results of the wound after 7 days.

DETAILED DESCRIPTION OF THE INVENTION

(5) While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings, graphs and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims. Before the present methods and the products are described, it is to be understood that this invention is not limited to particular method, product and experimental conditions described; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

(6) The tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

(7) The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

(8) The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by “comprises . . . a” does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.

(9) The terms, “alone or in combination” or any other variations thereof, are intended to described and/or cover a non-exclusive inclusion, wherein the molecules or the oligonucleotides exist individually or together with any one or all of the other oligonucleotides.

Definitions

(10) In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

(11) It must be noted that, as used in the specification/description and the appended claims and examples, the singular forms “a”, “an” and “the” may include plural referents unless the context clearly dictates otherwise.

(12) An aspect of the present invention relates to a composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxy octanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octyl cyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate.

(13) In yet another aspect of the present invention, it relates to a composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate obtained by passing singlet oxygen through extra virgin olive oil.

(14) An aspect of the present invention relates to a personal care product comprising the composition and a pharmaceutically acceptable excipient useful for personal care products.

(15) An another aspect of the present invention relates to a product, wherein the personal care products are in the form of solid, liquid or semi-solid.

(16) An another aspect of the present invention relates to personal care products, wherein the personal care products are in the form of cream, suppository, soap, body scrub, body oil, body wash, shampoo, hair conditioners, moisturizer, body oil, balm, baby products or solution.

(17) Another aspect of the present invention relates to personal care products which are as and when used in the treatment of acne, dandruff and dry skin.

(18) Another aspect of the present invention relates to composition for use is wound healing, nosocomial infection or disinfectant.

(19) Another aspect of the present invention relates to a process for preparing a composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate, the process comprising the steps of: (a) preparing singlet oxygen, (b) passing the singlet oxygen through extra virgin olive oil, and (c) obtaining the composition comprising methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate.

(20) Another aspect of the present invention relates to a process wherein the singlet oxygen is passed through the extra virgin olive oil for a period of 325 to 385 hours.

(21) Another aspect of the present invention relates to a process wherein the singlet oxygen is passed through the extra virgin olive oil for a period of about 360 hours.

(22) In another aspect of the present invention, the wound may be caused by injury or any other source.

(23) In another aspect of the present invention, the composition of the present invention results in perfect epithelialization, sufficient fibroplasia, and a lot of vascularization and no scars of the wound.

(24) TABLE-US-00001 Proportion of all Ester in Real lipid acid No. Time Name Structure (%) 1 1.86 Methyl Pelargonate C.sub.10H.sub.20O.sub.2 11.9 2 2.15 Metyl Caprate C.sub.11H.sub.22O.sub.2 0.2 3 2.67 Methyl Azelaal- dehydate C.sub.10H.sub.18O.sub.3 0.7 4 2.72 Dimethyl Suberate C.sub.10H.sub.18O.sub.4 0.3 5 3.05 Methyl 6,6,6- Trimethoxy Hexanoate C.sub.11H.sub.22O.sub.4 0.2 6 3.45 Dimethyl Azelate C.sub.11H.sub.20O.sub.4 8.9 7 3.91 Methyl 6,6 Dimethoxy Octanoate 4.5 8 5.51 Methyl Myristate C.sub.15H.sub.30O.sub.2 0.1 9 5.89 Dimethyl Un- decanediote C.sub.12H.sub.24O.sub.2 0.4 10 10.02 Methyl Palmitate C.sub.17H.sub.34O.sub.2 0 16.4 11 15.19 Methyl Linoleate C.sub.9H.sub.34O.sub.2 3.1 12 15.43 Methyl Oleate C.sub.19H.sub.36O.sub.2 21.7 13 15.54 Metil 8- Octadecenoate C.sub.19H.sub.36O.sub.2 1 14 16.15 Methyl Stearate C.sub.19H.sub.38O.sub.2 27.8 15 19.61 Methyl 2- OctylCyclo- propane- octanoate C.sub.20H.sub.38O.sub.2 1.1 16 19.98 Methyl 11- Eicosenoate C.sub.21H.sub.40O.sub.2 0.3 17 20.54 Methyl Eicosanoate C.sub.21H.sub.42O.sub.2 1 18 24.20 Methyl Docosanoate C.sub.23H.sub.46O.sub.2 0.5

EXAMPLES

(25) The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration to the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention various changes to the described embodiments may be made in the functions and arrangement of the elements described without departing from the scope of the invention.

Example 1: Analysis Study Gas Chromatography, Free Fatty Acid, Acid, Iodine, Saponification, and Peroxide Value

(26) Research Protocol: Analysis Study Gas Chromatography, Free fatty Acid, Acid, Iodine, Saponification, dan Peroxide value on the composition

(27) Tested Parameters: Oleic Acid, Acid value, Iodine Value, Saponification Value and Peroxide value

(28) Result

(29) TABLE-US-00002 Parameter Result Method Free fatty acid as Oleic Acid  .sup. [19.84 ± 0.3] % Titration Acid Value [45.17 ± 1.1] Titration Iodine Value  [7.4 ± 0.8] Volumetric Saponification Value .sup. [247 ± 1.5] Volumetric Peroxide Value [745.4 ± 1.4] Titration
II. Lipid Gas Chromatography Profile
Conclusion

(30) The laboratory tests consisting of Oleic Acid, Acid value, Iodine Value, Saponification Value and Peroxide value, have been performed. As the result of the GC test on, lipid profiles are indicated in the proportion of ester lipid acid (>10%): Methyl Pelargonate (11.9), Methyl Palmitate (16.4), Methyl Oleate (21.7) and Methyl Stearate (27.8).

Example 2: Effectivity Study on Microbes which Cause Nosocomial Infection

(31) Backgrounds: Nosocomial infection may occur on patient or medical-staff in hospitals. It is epidemically occurred in developing-countries in Asia, Africa, and South-America (10-40%), but rarely found in developed-countries in North-America, Europe (1%). In general, 10% of the infections are found in patients who are treated at hospital. Mortality may increase on patients who have ever received surgery, get immunocompromised, or have been treated in the hospital for a long time.

(32) The infection can get worse when a patient is contaminated by the germs found in hospital, known as Multi Drug Resistance Organisms (MDROs). The types of MDRO usually found at hospital are:

(33) 1. Methicillin Resistant Staph aureus (MRSA)

(34) 2. E-coli ESBL (Extended Spectrum Beta-Lactamases)

(35) 3. K. Pneumoniae ESBL (Extended Spectrum Beta-Lactamases)

(36) 4. CRE (Carbapenem Resistant Enterobacteriaceae) a.1 KPC (Klebsiella pneumoniae carbapenemases)

(37) 5. Candida fungi

(38) Those pathogens are commonly found from patient's clinical isolate, that the scope for the therapy becomes tight and limited, and an antibiotics-injection is required.

(39) Material

(40) 1. Composition in liquid form

(41) 2. Liquid bacterial growth medium (Mueller Hinton Broth)

(42) 3. Bacteria's Clinical Isolate

(43) 1. Methicillin Resistant Staph aureus (MRSA)

(44) 2. E coli ESBL (Extended Spectrum Beta-Lactamases)

(45) 3. K. Pneumoniae ESBL (Extended Spectrum Beta-Lactamases)

(46) 4. CRE (Carbapenem Resistant Enterobacteriaceae) a.1 KPC (Klebsiella pneumoniae carbapenemases) 5. Candida fungi

(47) No Batch WIP 030616 (110 CPS)

(48) No Batch 010616 (560 CPS)

(49) No Batch 010616 (580 CPS)

(50) Method

(51) The method of dilution sensitivity test (Tube Test), following these steps:

(52) 1. 1 ml bacteria suspension is added to tube I, 1 ml with 100% concentration

(53) 2. 1 ml bacteria suspension is added to tube II, 1 ml with 50% concentration

(54) 3. 1 ml bacteria suspension is added to tube III, 1 ml with 25% concentration

(55) 4. 1 ml bacteria suspension is added to tube IV, 1 ml with 12.5% concentration

(56) 5. 1 ml bacteria suspension is added to tube V, 1 ml with 6.25% concentration

(57) 6. 1 ml bacteria suspension is added to tube VI, 1 ml with 3.125% concentration

(58) 7. Make 1 ml positive control solution and add 1 ml aquadest

(59) 8. Incubate at the incubator for 24 hours.

(60) 9. Observe the results by comparing with control solution, clearer tube indicates the minimum inhibitory concentration (MIC)

(61) 10. Do the streaking for two tubes before and after MIC on a plate, or pour it on the plate to see minimum bactericidal concentration (MBC). Incubate for 24 hours.

(62) Results

(63) 1. MRSA Bacteria

(64) TABLE-US-00003 TABLE 1a Using WIP 030616 (110CPS) Strain MRSA 100% 50% 25% 12.5% 6.25% 3.125% 6 + + + + + + 10 + + + + + + 11 + + + + + + 12 + + + + + + 13 + + + + + +
Explanation: +cloudy pathogen's growth on the plate is visible

(65) TABLE-US-00004 TABLE 1b Using 010616 (560 CPS) Strain MRSA 100% 50% 25% 12.5% 6.25% 3.125% 6 0 0 + + + + 10 0 0 + + + + 11 0 0 + + + + 12 0 0 + + + + 13 0 0 + + + +
Explanation: 0 Absence of bacteria's growth
+min minimum pathogen's growth is visible
+cloudy and there are pathogen's growth on the plate is visible
2. Other MDROs Bacteria

(66) TABLE-US-00005 TABLE 2 using 010616 (580 CPS) Strain 100% 50% 25% 12.5% 6.25% 3.125% E coli 0 0 + + + + ESBL K pneu 0 0 + + + + ESBL KPC 0 0 + + + +
Explanation: 0 Absence of bacteria's growth
+min minimum pathogen's growth is visible
+cloudy and there are pathogen's growth on the plate is visible
3. Candida Fungi

(67) TABLE-US-00006 TABLE 3 Using 010616 (580 CPS) Strain Candida 100% 50% 25% 12.5% 6.25% 3.125% Albicans 0 0 + + + + Kruseii 0 0 0 0 + + tropicalis 0 0 +min + + + Glabrata 0 0 0 + + +
Explanation: 0 Absence of bacterias growth
+min t minimum bacteria growth is exist
+cloudy and there are pathogen's growth on the plate is visible
Conclusion: After doing sensitivity test on microbes that cause nosocomial infection, the conclusions are:

(68) 1. Other MDRO (ESBL and KPC) can be inhibited and terminated at 50% concentration

(69) 2. Candida fungi can be inhibited and terminated at 50% concentration

Example 3: Acute Toxicity Study on the Incised Wound Healing Rattus Norvegicus: In Vivo Research on Animals

(70) Research Protocol: Acute Toxicity Study on the Incised Wound healing Rattus Norvegicus In Vivo research on animals

(71) Background: Wound is a loss of tissue continuity caused by injury or any other source. Wound can occur in almost all part of the body and will be perfectly healed physiologically. The wound Healing Processes occur in a few steps:

(72) 1. Hemostatic

(73) 2. Inflammation

(74) 3. Proliferation (Fibroplasia)

(75) 4. Maturation and Remodelling.

(76) In general, all kind of wounds will undertake these processes. Some factors may interfere in speeding up and helping the wound healing, either assisting by the application of any medical substances or none.

(77) Material

(78) 1. Composition comprising a mixture composed of methyl pelargonate, methyl caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate.

(79) 2. Vaseline

(80) 3. Sibro (from Mebo)

(81) 4. Gentamicin ointment (generic)

(82) Methods

(83) 1. Preparation of animals:

(84) 1. Inject the animals with anesthesia drug containing ketamine hydrochloride, 0.1-0.2 ml as illustrated in 101 of FIG. 1.

(85) 2. Place them back into the cage while waiting them fully unconscious as the effect of the drug.

(86) 3. Shave the back hair of the animals with razor blade.

(87) 4. Give antiseptics, i.e. povidone iodine and alcohol. Wait for three minutes as illustrated in 102 of FIG. 1.

(88) 5. Make a 2-cm long incision on the animal's back as illustrated in 103 of FIG. 1. Should any bleeding occurred, wipe with cotton.

(89) 6. Apply cotton bud ton the incision spot.

(90) 7. Cover the spot with gauze bandage and stick it with plaster as illustrated in 104 of FIG. 1.

(91) 8. Place the animal back in to the cage.

(92) 2. Fixation Tissue Protocol

(93) The skin organs from treatment and control group are taken after the incision, then put them to the bottles containing fixation solution, and give labels. The intensions of the fixation are:

(94) 1. To maintain cell structure and component.

(95) 2. To prevent autolysis on post mortem

(96) 3. To prevent decomposition and bacteria/fungi growth

(97) There are various solutions used for the fixation process, but this research used neutral buffer formalin, which contains:

(98) Formaldehyde 40%: 100 cc

(99) Aquadestilata: 900 cc

(100) Sodium hydrogen posphate monobasic (NaH.sub.2PO.sub.4): 4 gr

(101) Sodium hydrogen posphate dibasic (Na.sub.2HPO.sub.4): 6.5 gr

(102) Minimum time of fixation is 12-18 hour before the next process.

(103) 3. Tissue Processing Protocol/Paraffin Block

(104) The tissue's processing is started after the fixation completed. The steps of this process are dehydration, clearing, and impregnation/embedding. Dehydration is taken by de-hydrating the tissue using ethanol in the gradually increased concentrate until the absolute concentrate is reached. Afterward, do the clearing by place the tissue into xylol. Then, do the impregnation/embedding by using paraffin. The detail of this process is as follows:
After the fixation is completed, the tissues are placed into the following solution, consecutively.
1. Alcohol 70%: -
2. Alcohol 80% 1 hour
3. Alcohol 95%: 2 hour
4. Alcohol 95%: 1 hour
5. Alcohol 100%: 1 hour
6. Alcohol 100%: 1 hour
7. Alcohol 100%: 1 hour
8. Xylol: 1 hour
9. Xylol: 1 hour
10. Xylol: 2 hour
11. Paraffin (56-58° C.): 2 hour
12. Paraffin (56-58° C.) 2 hour
13. Paraffin (56-58° C.) 2 hour

(105) After this process, block the tissue into paraffin with this steps; first, prepare paraffin block printer, place it to a flat surface, pour liquid paraffin (56-60° C.) into the printer and take the tissue from impregnation process, put it into the printer that contain liquid paraffin, give label, wait for a few minutes until the paraffin is frozen, then take it out from the printer. These steps called Embedding.

(106) 4. Tissue Slicing and Attaching Process on object glass Protocol.

(107) The next process is to slice the tissue and attach it on object glass.

(108) 1. Tissue that already inside the paraffin block is sliced with microtome for 4 micron thick

(109) 2. Ribbon slice is taken into water bath which has temperature between 45-55° C., with object glass that has been applied with albumin glue.

(110) 3. The ribbon slice is attached into object glass and let it dry on room temperature

(111) 4. To make tissue attached into object glass, heat it into the oven at 56-58° C. for

(112) 3-4 hour,

(113) 5. Remove from the oven and live it in room temperature, then the tissue is ready to dye.

(114) 5. Hematoxilin Eosin Dyeing Protocol

(115) The steps of HE dyeing method are:

(116) Dip the object glass which contains tissue into solution:

(117) 1. Xylol 1,2,3

(118) 2. Absolute Alcohol 1×

(119) 3. Alcohol 96% 2×, wash under running water, if use zenker clip in to lugol for 1 minutes, iodine alcohol for 10-15 minutes, wash sodium thiosulfate (hypo), wash with water.

(120) 4. Harris hematoxilin 10-15 minutes

(121) 5. Wash under running water

(122) 6. Acid alcohol, 3-10 dip to suit the color.

(123) 7. Wash under running water

(124) 8. Ammoniac water 1-5 dips, check under microscope, the nucleus is blue

(125) 9. Wash under running water

(126) 10. Eosin 1-5 minutes

(127) 11. Alcohol 95 2×1-5 minutes

(128) 12. Absolute alcohol 2×1-5 minutes

(129) 13. Xylol 3×5 minutes

(130) 14. Mounting with entellan and cover glass

(131) 6. Observe Results

(132) 1. The animals were managed to survive after being applied the substances to their incised wound.

(133) 2. The wound of the animals were increasingly healed macroscopically

(134) 3. The wound of the animals were increasingly healed microscopically

(135) Results of the Research

(136) 1. The animals were manage to survive after being applied the substances to their incise wound

(137) This research use 24 Rats divided into 4 groups of treatment; they are Vaseline, Sibro, Gentamicin and Test. Composition. Each group has 6 rats.

(138) On the second day, 1 rat died from Vaseline group.

(139) TABLE-US-00007 TABLE 1 Animals that survived after being applied the substance on day −7 Amount Group of Rats Dead Rats Survival on day −7 Vaselin 6 1 5/6 Sibro 6 0 6/6 Gentamisin 6 0 6/6 Test Composition 6 0 6/6
2. The Wound of the Animals were Increasingly Healed Macroscopically
In this research, a 2-cm long incision was made on the animal's back using razor blade after they were anesthetized with ketamine injection on the tight. After then stopped the bleeding, applied Vaseline, Sibro, Gentamicin and Test Composition to each group.
Application of Vaseline (201), Sibro (202), Gentamicin (203) and Test Composition (204) are illustrated in FIG. 2.

(140) TABLE-US-00008 TABLE 2 Incised wound macroscopic condition Amount Survival Group of Rats day −7 Wound condition on day −7 Vaselin 6 5/6 Closing Sibro 6 6/6 Closing (with 1 granulation) Gentamisin 6 6/6 Closing (with 1 granulation) Test Composition 6 6/6 Closing (with 2 granulation)

(141) Macroscopic condition of the wound with Vaseline (301), Sibro (302), Gentamicin (303) and Test Composition (304) after 7 days are illustrated in FIG. 3.

(142) 3. The Wound of Animals were Increasingly Healed Microscopically

(143) There are 4 parameters in observing incised wound microscopically, they are Epithelization, Fibroplasia, Neovascularization, and scar.

(144) TABLE-US-00009 Neo Group Epitheliazation Fibroplasia vascularization Scar Vaselin + + + + Sibro + ++ sufficient + + Gentamisin + + ++ + Test Composition + ++ sufficient ++ 0 Ket: 0 absence, + visible, ++ visible, a lot or thick

(145) Microscopical results of the wound with Vaseline (401), Sibro (402), Gentamicin (403) and Test Composition (404) are illustrated in FIG. 4.

(146) Conclusion

(147) 1. Based on this research, the applying of composition on animals was non-toxic on the animals, and they were managing to survive until day-7.

(148) 2. The applying of the composition had increasingly healed their wounds macroscopically on day 7.

(149) 3. The applying of the composition had increasingly healed their wounds microscopically, indicated by the perfect epithelization, sufficient fibroplasia, and a lot of vascularization so there are no scars on day 7.

Example 4

(150) Effectivity Study on Infected Incised Wound of Rattus Norvegicus Experimental Research on Animal Testing

(151) Background: Wound is a continuity loss of tissue as the result of an injury or any other cause.

(152) Wound can occur in almost all part of the body and will be perfectly healed physiologically.

(153) The wound healing processes occur in the following stages:

(154) 1. Hemostatic.

(155) 2. Intimation.

(156) 3. Proliferation (Fibroplasia).

(157) 4. Maturation and Remodelling.

(158) When a wound got infected, it can affect a serious problem in the healing process.

(159) The infection is usually caused by the microbes from patient's body or from the environment. Certain microbes that caused infection can be resistant on anti-microbes that called MDROs (Multiple Drugs Resistance Organisms). Here are the MDRO usually found at the hospital:
1. Methicillin Resistant Staph aureus (MRSA)
2. E coli ESBL (Extended Spectrum Beta-Lactamases)
3. K. Pneumoniae ESBL (Extended Spectrum Beta-Lactamases)
4. CRE (Carbapenem Resistant Enterobacteriaceae) a.1 KPC (Klebsiella pneumoniae carbapenemases)
5. Candida Fungus

(160) Those microbes are usually found from the patient's clinical isolates, so the therapy spectrum became more narrow and limited, and an antibiotic-injection is required.

(161) Material

(162) 1. Composition comprising a mixture composed of methyl pelargonate, caprate, methyl azelaaldehydate, dimethyl suberate, methyl 6,6,6-trimethoxy hexanoate, dimethyl azelate, methyl 6,6 dimethoxyoctanoate, methyl myristate, dimethyl undecanediote, methyl palmitate, methyl linoleate, methyl oleate, methyl 8-octadecenoate, methyl stearate, methyl 2-octylcyclopropaneoctanoate, methyl 11-eicosenoate, methyl eicosanoate, and methyl docosanoate.

(163) 2. Bacteria's Growth Media solution (Mueller Hinton Broth)

(164) 3. Vaseline

(165) 4. Mupirocin (for MRSA)

(166) 5. Clinical isolate of bacteria, such as:

(167) 1. Staphylococcus aureus

(168) 2. Staphylococcus epidermidis

(169) 3. Streptococcus pyogenes

(170) 4. Pseudomonas aeruginosa

(171) 5. Acinetobacter baumanii

(172) 6. Methicillin Resistant Staph aureus (MRSA)

(173) 7. E coli ESBL (Extended Spectrum Beta-Lactamases)

(174) 8. K. Pneumoniae ESBL (Extended Spectrum Beta-Lactamases)

(175) 9. CRE (Carbapenem Resistant Enterobacteriaceae) a.1 KPC (Klebsiella pneumoniae carbapenemases)

(176) 10. Candida Fungus

(177) Method: Solution Preparation Protocol for Infected Incised Wound

(178) Preparing the solution with these following steps:

(179) 1. Bacteria from the infected patient clinical isolate is planted on the media by streaking at for quadrant

(180) 2. The isolated bacteria in separated colonies are dissolved in the tube that contain pz liquid

(181) 3. In order to analyze the cloudness level of the solution on the tube, nephelometer is used to figure the cloudness level to 0.50 McFarland.

(182) 4. 0.5 McFarland is equal to 1.5×108 CFU bacteria.

(183) Infected Incised Wound Preparation Protocol on Animals

(184) 1. Preparation of animals:

(185) 1. Inject the animals with anesthesia drug containing ketamine hydrochloride, 0.1-0.2 ml

(186) 2. Place them back into the cage while waiting them to be fully unconscious as the effect of the drug.

(187) 3. Shave the back hair of the animals with razor blade.

(188) 4. Give antiseptics, i.e. povidone iodine and alcohol. Wait for three minutes.

(189) 5. Make a 2-cm long incision on the animal's back. Should any bleeding occurred, wipe with cotton.

(190) 6. Swipe the incised spot with the bacteria solution using a cotton bud.

(191) 7. Cover the spot with gauze bandage and stick it with plaster.

(192) 8. Place the animal back in to the cage.

(193) 9. If there is any requirement for adding test material, the material should be shortly added up on the wound before it is bandaged.

(194) Protocols for Making Histology Preparate and Dyeing Technique of Hematoxilin Eosin

(195) 1. Protocol for Fixation Tissue

(196) The skin organs from treatment and control group are taken after the incision then put them to the bottles containing fixation solution, and give labels. The intentions of the fixation are:

(197) 1. To maintain cell structure and component.

(198) 2. To prevent autolysis on post mortem

(199) 3. To prevent decomposition and bacteria/fungi growth

(200) There are various solutions used for the fixation process, but this research used neutral buffer formalin, which contains:

(201) Formaldehyde 40%: 100 cc

(202) Aquadestilata: 900 cc

(203) Sodium hydrogen posphate monobasic (NaH.sub.2PO.sub.4): 4 gr

(204) Sodium hydrogen posphate dibasic (Na.sub.2HPO.sub.4): 6.5 gr

(205) Minimum time of fixation is 12-18 hour before the next process.

(206) 2. Protocol for Tissue Processing/Paraffin Block

(207) The tissue's processing is started after the fixation completed. The steps of this process are dehydration, clearing, and impregnation/embedding. Dehydration is taken by de-hydrating the tissue using ethanol in the gradually increased concentrate until the absolute concentrate is reached. Afterward, do the clearing by place the tissue into xylol. Then, do the impregnation/embedding by using paraffin. The detail of this process is as follows:
After the fixation is completed, the tissues are placed into the following solution, consecutively.
1. Alcohol 70%: -
2. Alcohol 80%: 1 hour
3. Alcohol 95%: 2 hour
4. Alcohol 95%: 1 hour
5. Alcohol 100%: 1 hour
6. Alcohol 100%: 1 hour
7. Alcohol 100%: 1 hour
8. Xylol: 1 hour
9. Xylol: 1 hour
10. Xylol: 2 hour
11. Paraffin (56-58° C.): 2 hour
12. Paraffin (56-58° C.) 2 hour
13. Paraffin (56-58° C.) 2 hour
After this process, block the tissue into paraffin with this steps; first, prepare paraffin block printer, place it to a flat surface, pour liquid paraffin (56-60° C.) into the printer and take the tissue from impregnation process, put it into the printer that contain liquid paraffin, give label, wait for a few minutes until the paraffin is frozen, then take it out from the printer. This steps called Embedding.
3. Protocol for Tissue Slicing and Attaching Process on object glass.
The next process is to slice the tissue and attach it on object glass.
1. Tissue that already inside the paraffin block is sliced using microtome for 4 micron thick
2. Ribbon slice is taken into water bath which has temperature between 45-55° C., with object glass that have been applied with albumin glue.
3. The ribbon slice is attached into object glass and let it dry on room temperature
4. To make tissue attached into object glass, heat it into the oven at 56-58° C. for 3-4 hour,
5. Remove from the oven and leave it in room temperature, then the tissue is ready to dye.
4. Hematoxilin Eosin Dyeing Protocol
The steps of HE dyeing method are:
Dip the object glass which contains tissue into solution:
1. Xylol 1,2,3
2. Absolute Alcohol 1×
3. Alcohol 96% 2×, wash under running water, if use zenker dip in to lugol for 1 minutes, iodine alcohol for 10-15 minutes, wash sodium thiosulfate (hypo), wash with water.
4. Harris hematoxilin 10-15 minutes
5. Wash under running water
6. Acid alcohol, 3-10 dip to suit the color.
7. Wash under running water
8. Ammoniac water 1-5 dips, check under microscope, the nucleus is blue
9. Wash under running water
10. Eosin 1-5 minutes
11. Alcohol 95 2×1-5 minutes
12. Absolute alcohol 2×1-5 minutes
13. Xylol 3×5 minutes
14. Mounting with entellan and glass cover
Observation Results
4. The animals were managed to survive after being applied the substances to their incised wound.
5. The wound of the animals were increasingly healed macroscopically
6. The wound of the animals were increasingly healed microscopically
Result of the Research
4. Survival after adding another substances to their incise wounds
This research use more than 1-Rats divided into 3 groups which is nosocomial, MDRO, and fungus. Each group has sub group with 6 rats.
On the second day, 1 rat died from Acinobacter bumanii, and Streptococcus pyogenes group.

(208) TABLE-US-00010 Number of rats Dead Rats Survival Group I Nosocomial (n = 60) Staph aureus + Vaseline 6 0 6/6 Staph aureus + Medcare 6 0 6/6 Staph epidermidis + Vaseline 6 0 6/6 Staph epidermidis + Medcare 6 0 6/6 Streptococcus pyogenes + Vaseline 6 1 5/6 Streptococcus pyogenes + Medcare 6 0 6/6 Pseudomonas aeruginosa + Vaseline 6 0 6/6 Pseudomonas aeruginosa + Medcare 6 0 6/6 Acinetobacter baumanii + Vaseline 6 1 5/6 Acinetobacter baumanii + Medcare 6 0 6/6 Group II MDRO (n = 72) MRSA 10 vaseline 6 0 6 MRSA10 mupirocine 6 0 6 MRSA 10 medcare 6 0 6 MRSA 6 mupirocine 6 0 6 MRSA 6 mupirocine 6 0 6 MRSA 6 medcare 6 0 6 E coli ESBL vaseline 6 0 6 E coli ESBL medcare 6 0 6 K pneumonia ESBL vaseline 6 0 6 K pneumonia ESBL medcare 6 0 6 KPC Vaseline 6 0 6 KPC medcare 6 0 6 Group III Fungi (n = 48) Candida albican Vaseline 6 0 6 Candida albican medcare 6 0 6 Candida krusei Vaseline 6 0 6 Candida krusei medcare 6 0 6 Candida glabrata Vaseline 6 0 6

(209) While the invention has been described and illustrated with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various adaptations, changes, modifications, substitutions, deletions, or additions of procedures and protocols may be made without departing from the spirit and scope of the invention. For example, effective dosages other than the particular dosages as set forth herein above may be applicable as a consequence of variations in responsiveness of the mammal being treated for any of the indications with the compounds of the invention indicated above. The specific pharmacological responses observed may vary according to and depending upon the particular active compounds selected or whether there are present pharmaceutical carriers, as well as the type of formulation and mode of administration employed, and such expected variations or differences in the res tills arc contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be defined by the scope of the claims which follow and that such claims be interpreted as broadly as is reasonable.