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    • 专利摘要:
    Composition comprising garlic extracts, use for the manufacture of a medicament for the treatment of diseases, and obtaining procedure. Use of a composition comprising lyophilized garlic extract and black garlic extract for the manufacture of a medicament for the treatment of diseases selected from the group consisting of sepsis, septic shock, tumor diseases and dermatological diseases. Pharmaceutical and food composition comprising lyophilized garlic extract and black garlic extract. Process for obtaining black garlic extract, comprising extraction, separation and stabilization. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2675282A1
    申请号:ES201730018
    申请日:2017-01-10
    公开日:2018-07-10
    发明作者:Francisco Javier REDONDO CALVO；José Manuel PÉREZ ORTIZ；David PADILLA VALVERDE；Juan Luis SANTIAGO SÁNCHEZ-MATEOS；Ignacio GRACIA FERNÁNDEZ；Juan Francisco Rodríguez Romero；Pedro VILLAREJO CAMPOS；José Ramón MUÑOZ RODRÍGUEZ；Eva María GALÁN MOYA；Luis Antonio GÓMEZ FERNÁNDEZ
    申请人:Sescam Servicio De Salud De Castilla-La Mancha；Sescam Servicio De Salud De Castilla La Mancha；Universidad de Castilla La Mancha；
    IPC主号:
    专利说明:

     DESCRIPTION Composition comprising garlic extracts, use for the manufacture of a medicament for the treatment of diseases, and method of obtaining 5 FIELD OF THE INVENTION The invention relates to medicaments for the treatment of diseases, sepsis, septic shock, tumor diseases and dermatological diseases In particular, the invention relates to a composition comprising lyophilized garlic extract and black garlic extract 10. BACKGROUND OF THE INVENTION Black garlic is the result of the transformation of natural garlic under certain conditions of temperature and humidity. The process takes place in 4 or 5 stages depending on the manufacturer, in which the environmental conditions are varied in ranges of: 60-90ºC; 50-100% relative humidity, and each stage is prolonged between 6- 192 hours. 20 The Maillard reaction is the result of the non-enzymatic glycosylation of proteins, that is, the addition of a carbohydrate to another molecule that is called an acceptor, and which may have a protein or lipid nature. Most of the proteins stored in the rough endoplasmic reticulum undergo glycosylation. The rough endoplasmic reticulum is formed by a series of channels or cisterns that are distributed throughout the cell's cytoplasm, generating flattened sacs in which polypeptide chains are introduced, which form the proteins that undergo non-enzymatic reactions of glycosylation As a result, we have a very complex set of chemical reactions that bring about the production of colored melanoidins ranging from light yellow, toasted colors of varying intensity, and even black as in the case of the transformation of natural garlic to black garlic . In the reactions, a reducing sugar (ketose or aldose) and a free amino group are necessary, from an amino acid or a protein, and depending on the conditions in which they are carried out and the nature of the substrate, 35they can be derived in the transformation of aromatic compounds such as flavonoids and polyphenols as in the case of garlic. Sepsis is defined as an infection (proven or suspected) plus a systemic inflammatory response syndrome (fever, tachycardia, tachypnea and leukocytosis). Severe sepsis 5 is defined as a sepsis with organic dysfunction (hypotension, hypoxemia, oliguria, metabolic acidosis, thrombocytopenia, etc.). Septic shock would be a severe sepsis with hypotension, despite adequate fluid replacement. Septic shock and multiorgan failure are the most frequent cause of death in patients with sepsis. Mortality associated with severe sepsis and septic shock ranges between 25-30% and 40-70% 10 respectively. Sepsis is the final culmination of complex interactions between the infecting microorganism and the immune, inflammatory and coagulation response of the host. The state of the art describes that allicin, the main active component contained in the lyophilized extract, is capable of reacting with free thiol groups and penetrating the biological membranes. However, the mechanisms by which allicin affects cellular systems are not entirely clear. It has been suggested that the antiproliferative effect of allicin is due to the ability of allicin to transiently deplete intracellular glutathione deposits. With respect to black garlic, there are works that indicate its potential anti-tumor effect by inhibiting the cellular proliferation of human tumor lines of gastric cancer. 25 The physical and functional integrity of the skin, the largest organ, is crucial in the survival of mammals to avoid dehydration and allow life in a terrestrial environment. The main function of the skin is to act as a permeability barrier, separating the predominantly aqueous biological environment from the surrounding air environment. 30 The compromise of the epidermal barrier function is a crucial event in the pathophysiology of many chronic inflammatory dermatoses (atopic dermatitis, irritant dermatitis, psoriasis, etc.), since it creates a pro-inflammatory state in the skin that prolongs activity outbreaks and shortens the periods of remission. 35DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a composition comprising lyophilized garlic extract and black garlic extract. Another embodiment is the composition according to the first aspect, where the proportion of lyophilized garlic extract to black garlic extract is between 1: 1 and 1: 5. In a second aspect, the composition according to the first aspect is a pharmaceutical composition. Another embodiment is the pharmaceutical composition according to the second aspect, which comprises antineoplastic drugs selected from the group consisting of cytostatics, cytotoxic agents, biological drugs and immunotherapy compounds. Another embodiment is the pharmaceutical composition according to the second aspect, wherein said antineoplastic drug is oxaliplatin. Another embodiment is the pharmaceutical composition according to the second aspect, which comprises pharmaceutically acceptable excipients. 20 The term “excipients” refers to compounds that stabilize and favor the absorption of active ingredients, dyes, sweeteners, flavorings, protectors against air and / or moisture, binders, etc. The pharmaceutical composition may be formulated with pharmaceutically acceptable excipients, as well as with any other type of pharmaceutically acceptable carriers or diluents, in accordance with conventional techniques in pharmaceutical practice. The pharmaceutical composition can be administered alone or in combination with other active ingredients. The pharmaceutical composition can be administered in single or multiple doses. The pharmaceutical composition of the second aspect of the invention can be administered by any route of administration (eg, systemic, oral, sublingual, perioral,parenteral, urethral, vaginal, rectal, intraperitoneal, intramuscular, intranasal, intravenous, intraarterial, transdermal, subcutaneous, topical, etc.) for which said composition will be formulated in the appropriate pharmaceutical form to the route of administration chosen. The pharmaceutical composition may be formulated to provide controlled release of the active ingredient such as sustained or prolonged release according to methods that are well known in the art. In a third aspect, the composition according to the first aspect is a food composition. Another embodiment is the food composition according to the third aspect, which comprises food additives. In a fourth aspect, the present invention provides the use of a composition according to the first and second aspects for the manufacture of a medicament for the treatment of diseases selected from the group consisting of sepsis, septic shock, tumor diseases and dermatological diseases. The composition comprises a stable allicin-rich extract, from lyophilized extract, and stable s-allyl-cysteine, derived from black garlic. This provides controlled concentrations of these two active compounds. The s-allyl-cysteine acts synergistically with allicin and the other thiosulphinates and compounds present in the lyophilized extract. 25 Black garlic extract is a polar extract of black garlic. With this extract, a water-soluble extract and thermal stability of S-allyl-L-cysteine (SAC) and the enhancement of the activity of both extracts are achieved. The composition provides stabilization for thiosulphinates, polysulfides, prostaglandin E1 (PGE1), vitamin E, inulin and ajoene, compounds derived from garlic. The invention makes it possible to obtain standardized, formulable and therapeutically applicable products on an industrial scale with the temporary stability guarantees set forth above. The compounds obtained from garlic extracts are stable for periodsof time that allow its application in the laboratory, in animals and in humans, with total guarantees of efficacy in relation to its composition. In sepsis, the composition acts in multiple steps of the process, both in the early inflammatory response and in the procoagulant response. Up to now, the therapeutic options raised have been directed at a specific point in the process, which may have been able to contribute to unsatisfactory results. The composition interacts at several key points and thereby decreases the early inflammatory response and the later procoagulant response. Another embodiment is the use according to the fourth aspect of the invention, wherein said tumor diseases are selected from the group consisting of colon cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, ovarian cancer, cancer of testis, pancreatic cancer, kidney cancer, liver cancer, cervical cancer, gastric cancer, head and neck cancer, bone cancer, cancers of the central nervous system, neuroendocrine tumors, thyroid carcinoma, sarcomas, leukemia, lymphomas, Myelomas, melanomas. In dermatological diseases, considering the variations corresponding to each cutaneous pathological process (wound by loss of substance or inflammatory process with or without affecting the epidermal barrier function), the invention provides a composition comprising lyophilized garlic extract and black garlic extract for topical use on the bed of skin wounds due to loss of substance, chronic inflammatory dermatosis or apparently unscathed skin with basic alterations in the barrier function. Topical route 25 is easily accessible, non-invasive, assuming an increase in the bioavailability and therapeutic efficacy of garlic extracts with less adverse effects than other routes of administration. Similarly, the systemic use of the composition comprising lyophilized garlic extract and black garlic extract has a favorable effect on the healing of cutaneous wounds, chronic inflammatory dermatoses or apparently unscathed skin with 30 basis alterations in the barrier function. Therefore, another embodiment is the use according to the fourth aspect of the invention, wherein said dermatological diseases are selected from the group consisting of skin wound healing, epidermal barrier function modulator, production inducer.and secretion of epidermal antimicrobial peptides and inflammatory dermatosis, irritative dermatitis, atopic eczema, irritative eczema and allergic contact eczema. In tumor diseases, making the relevant specific variations according to the nature of the neoplastic process (tumor type), the invention provides a therapeutic option in which lyophilized garlic extract and black garlic extract are combined with antineoplastic drugs selected from the group composed of cytostatics, cytotoxics, biological drugs and immunotherapy compounds. This combination maintains the desirable cytotoxic effect on tumor cells, allowing to reduce the dose of the antitumor drugs / drugs commonly used in the clinic and reducing the adverse (toxic) effects of routine administration of said drugs to standard concentrations. In a fifth aspect, the present invention provides a method of obtaining black garlic extract according to the first, second and third aspects, comprising the following steps: (a) solid-liquid extraction with an organic and / or inorganic solvent a a pressure between 0.1 and 400 atm and at a temperature between -70ºC and 150ºC with an extraction time of more than 1 minute, 20 (b) physical separation of the extracted matter and the leachate obtained, (c) solvent separation or recovery and the concentration of the leachate or extract obtained in the previous stages and (d) chemical-physical stabilization by lyophilization or cold maintenance. Another embodiment is the process according to the fifth aspect, where the physical separation of step (b) is carried out by centrifugation, filtration or decantation. BRIEF DESCRIPTION OF THE FIGURES 30 Figure 1. Weight monitoring for 14 days. The graph shows how in group II and III after infection there is a similar drop in weight. However, after the first 24 hours, the allicin group (group III) begins to gain weight, while group II does so at 48 hours. The lower left panel represents the weight loss in group II and III on day 9, 24 hours after infection. The lower right panel is a graphic representation showing that group III begins on day 10 with aweight recovery that is not observed in group II (statistically significant difference p <0.01) Figure 2. Graphical representation of the number of rats that present, in each of the groups (II and III), the different signs of suffering and stress assessed: ocular secretions, nasal secretions, mustache position, lack of grooming, piloerection, lethargy / hypoactivity and diarrhea. Figure 3. Determination of IL-1, IL-6 and TNF-alpha in each of the groups at different time intervals. 10 Figure 4. Most representative images of each of the variables studied. Figure 5. Resulting cell morphology after treatment of HT-29 colon cancer cells with different concentrations of allicin expressed in µg / mL. 15 Figure 6. RN assay on Caco-2 cells treated with oxaliplatin (OX), lyophilized garlic (represented by allicin; Ali), black garlic (represented by SAC; N), and combinations thereof. The graphs show the cell viability detected with the different treatments, depending on the concentration of each of them. The average value (n = 6) ± SD is displayed. 20 Figure 7. RN assay on HT-29 cells treated with oxaliplatin (OX), lyophilized garlic (represented by allicin; Ali), black garlic (represented by SAC; N), and combinations thereof. The graphs show the cell viability detected after 24 hours with the different treatments, depending on the concentration of each of them. The average value 25 (n = 6) ± SD is shown. Figure 8. LDH assay on HT-29 cells treated with oxaliplatin (OX), lyophilized garlic (represented by allicin; Ali), black garlic (represented by SAC; N), and combinations thereof. The graphs show the LDH released after 24 hours with the different treatments, depending on the concentration of each of them. The average value (n = 6) ± SD is displayed. Figure 9. Closure of the wound by second intention at 24 h (2 topical administrations) of lyophilized garlic extract at a concentration of 50 µg / mL (the two mice on the right) with respect to the control group (the two animals on the left ). The treatment group only 35He received topical treatment with lyophilized garlic extract in the right flank wound, while the contralateral wound was treated with vehicle (V). Figure 10. Closure of the wound by second intention on day 8 of the experiment. Note the difference between the animal in the group treated with lyophilized garlic extract at a concentration of 50 5 µg / mL on the right flank (located on the right) with respect to the animal in the group treated with lyophilized garlic extract at a concentration of 1 µg / mL in the right flank wound (located on the left). Below is a detail of the dermatoscopic image of the wounds of both flanks. 10 Figure 11. Reduction of the vertical axis of the scar (not subjected to Langer tension lines) on day 10 of the pilot experiment to determine the therapeutic concentration of lyophilized garlic extract as a procicatrizer. Note the detail of the dermatoscopic image of the wounds of the right flank in a subject of the treatment groups at a concentration of 1 µg / mL and 50 µg / mL. 15 Figure 12. Histological image of the re-epithelized scar (Masson's stain). Note the decrease in the diameter of the scar in the skin biopsy of the re-epithelized scar of the mouse treated with lyophilized garlic extract 50 µg / mL with respect to the re-epithelized wound of a mouse of the control group on day 14 of the experiment. Below you can see in detail the density of fibroblasts and the appearance of new collagen fibers in the scar. Figure 13. Histological image of the re-epithelized scar (Masson's stain) on day 14 of the experiment. In the upper part, the scar of an animal of the control group appears, below are two histological sections of the two wounds made in the same animal of the group treated with lyophilized garlic extract at a concentration of 50 µg / mL: 1) Bottom image corresponds to the wound treated with garlic extract and 2) the intermediate image is a biopsy of the contralateral wound, which only receives topical treatment with the vehicle. Note the graduation in the decrease in the diameter of the scar in the three images from top to bottom, demonstrating a direct pro-healing effect by topical administration 30 of lyophilized garlic extract at a concentration of 50 µg / mL (bottom image) and a less intense effect, systemic, on the contralateral side scar that only received topical vehicle treatment (intermediate image). Both scars, on the treated side and on the untreated side with lyophilized garlic extract in the same animal, are smaller than the scar of an animal of the control group on day 14 of the experiment. 35Figure 14. Histological image comparing the density and distribution of dermal collagen bundles (orcein staining) in the biopsy of the re-epithelized scar of a mouse from the group treated with lyophilized garlic extract 50 µg / mL with respect to the biopsy of the scar of a mouse from the control group on day 14 of the experiment. 5 Figure 15. Topical application of the freeze-dried extract of Allium sativum (50 µg / mL) accelerated the healing process (** p <0.05). Figure 16. Dermatoscopic details of the healing process in vivo every 2 days until day 10, where the progress of the re-epithelialization tongue (black vertical line) 10 in the treatment group (lyophilized garlic extract 50 µg /) mL) with respect to the control group. Figure 17. Histopathologically, wounds (arrow) treated with lyophilized garlic extract (50 µg / mL) showed an increase in the number of fibroblasts, collagen fiber density and complete re-epithelialization of the wound. 15 Figure 18. Topical application of the fermented extract of Allium sativum (10 µg / mL) accelerated the healing process (** p <0.05). Figure 19. Dermatoscopic details of the healing process in vivo every 2 days until 20 day 10, where the progress of the re-epithelialization tongue (black vertical line) can be seen in the treatment group (black garlic extract 10 µg / mL) with respect to the control group. Figure 20. Histopathologically, wounds (arrow) treated with black garlic extract (10 µg / mL) showed an increase in the number of dermal fibroblasts (25 granulation tissue), collagen fiber density and complete re-epithelialization of the wound. Figure 21. The wound healing model (dashed linear wound edges) with cell cultures of keratinocytes (HaCaT) and fibroblasts (CRL-2072) showed a significant increase (** p <0.05) in proliferation and migration rates under treatment with lyophilized garlic extract with respect to control. Figure 22. The results obtained from proliferation tests using the violet crystal technique, showed that the fermented garlic extract (black garlic) has a dose-dependent effect on the proliferation in both keratinocytes (HaCaT) and 35 fibroblasts (CRL) -2072), producing a significant increase for both cell types. Figures 23 and 24. The combination, especially at low doses in a 1: 5 ratio, of the lyophilized garlic extract with the fermented derivative (black garlic) is capable of inducing an increase in proliferation greater than 80% and 60% in keratinocytes (HaCaT, Figure 23) and fibroblasts (CRL-2072, Figure 24), respectively. These results corroborate that both garlic extract 5 and the fermented derivative thereof have a positive effect on the proliferation of the main components of the dermis and epidermis and point to a synergistic effect of the combination of both. * <0.05 ** <0.01 10 *** <0.001 DESCRIPTION OF EMBODIMENTS Example 1. Obtaining lyophilized garlic extract used in the present invention 15 The lyophilized garlic extract (Aliben®, Patent: ES 2 306 597 B1) used in the present investigations has been used reconstituted in medium aqueous. Allicin has shown activity for more than 10 months if it is maintained at 6 ° C, showing a chemical stability well above what is established in other works for this chemical species. In addition, the lyophilisate obtained shows a stability of not less than 18 months when stored in an opaque and hermetic environment. Procedure for obtaining lyophilized extract from Allium sativum, using ethanol in the extraction 25 Several garlic heads of the purple variety and belonging to the Protected Geographical Indication "Ajo Morado de Las Pedroñeras" (Castilla-La Mancha, Spain) were taken . The outer shirts were removed from these heads and their teeth separated into separate units. The total mass of garlic used in this experiment was 850 g. 30 The teeth were crushed in a domestic crusher to an average particle size of 1 mm. The crushed product was contacted with 2 liters of ethanol (96% v / v) in a cylindrical tank perfectly mixed by means of a mechanical stirrer of propellers / vanes (375 rpm) for solid-liquid extraction. The system was kept thermostated at 33 ° C for 90 minutes. The mixed liquor was filtered under vacuum by means of a Buschtner-Kitasato to which a filter with a 0.5 mm mesh light had been coupled. HeFiltrate was processed on a rotary evaporator for solvent removal in vacuo. The temperature of the rotary evaporator bath was 55 ° C. The product thus obtained was placed in a freeze dryer tray. It was frozen at -37 ° C and a vacuum of 0.2 torrs was applied. The temperature of the condenser was set at -55 ° C and that of the chamber at + 10 ° C. The process was extended for 28 h after which 60 g of lyophilized extract were collected. The lyophilized extract was stored at room temperature in opaque and airtight containers to perform pharmacotherapeutic characterization tests at different times. Obtaining a concentrated extract 10 The ethanol extraction operating procedure described above was followed, which included the stages of conditioning, ethanol extraction, garlic head filtering and solvent removal in the rotary evaporator. The concentrated extract thus obtained was stored in a refrigerator at 6 ° C in opaque and airtight containers. Example 2. Obtaining aqueous extract of black garlic used in the present invention Commercial black garlic (Aurum black®), fruit of the transformation of natural garlic, was used. The process takes place in 4 or 5 stages, in which the environmental conditions are varied in 20 ranges of: 60-90ºC temperature; 50-100% relative humidity, and each stage is prolonged between 6-192 hours of time. The product undergoes an evolution caused by the so-called Maillard reaction. The Maillard reaction is the result of the non-enzymatic glycosylation of proteins, that is, the addition of a carbohydrate to another molecule that is called an acceptor, and that can have a protein or lipid nature. Most of the 25 proteins stored in the rough endoplasmic reticulum undergo glycosylation. The following procedure was followed: i) 133.5 g of black garlic were weighed on teeth without skin. 30 ii) They were crushed together with 120.1 ml of physiological serum (0.9% NaCl), until a homogeneous paste mixture was obtained. iii) An additional 100 ml of physiological serum was added and extraction was performed at 50 ° C for 50 minutes at a stirring speed of 80 rpm in a stirred tank-type extractor of 2 L. 35 iv) Centrifugation was performed in 10 ml vials at 4,000 rpm for 15 minutes.v) The supernatant was collected by pipetting. The solid phase remained as a residue at the bottom of the vials. vi) The extract was filtered on 15 mm x 0.45 µm plastic filters at positive pressure. vii) The obtained black garlic extract, 61.3 ml, was stored at 4 ° C until it was used and formulated for biological tests. 5 viii) A 3 ml aliquot was used for SAC chromatographic analysis, according to the HPLC conditions described in UI-Seong Black-Garlic Farming Association. Source: Biotechnology Center of Korea University. The following Table shows the composition of the black garlic extract obtained with the procedure described above: Concentration Compound (ppm) Concentration Compound (ppm) S-allyl-L-cysteine (SAC) 123.20 Cycloalanine NC α-glucose NC Alanine NC β-glucose NC Lysine NC Fructose NC Arginine NC Citric Acid NC Valine NC Acetic Acid NC Leucine NC Formic Acid NC Isoleucine NC Pyroglutamic Acid NC Threonine NC Lactic Acid NC Choline NC Glutamine NC 5-hydroxymethylfurfural NC NC.- Not quantified. Qualitative analysis by 1 H-NMR (3). Example 3. Sepsis and septic shock An experimental study (therapeutic trial) of intra-abdominal infection has been developed where the experimental animal Sprague-Dawley® rat, Harlan Laboratories Models SL, male, 5 weeks, and 100-125 g has been used . It was carried out at the facilities of the Translational Research Unit (ITU) of the General University Hospital 20 of Ciudad Real.It was done at the same time, to avoid the possible influence of the circadian cycle on the results of the work. All animals were healthy and did not receive prior treatment. The rats were maintained with food and water ad libitum, with a cycle of 12 h of light and 12 h of darkness, and an ambient temperature of 22ºC ± 2ºC with a relative air humidity of 5 50% -70% and with 15-20 renewals / hour without air recirculation. They were established according to RD 53/2013. The animals were kept in these environmental conditions for at least 1 week, to allow acclimatization, prior to the study. The location of 10 animals and their housing was carried out in the animal of the Translational Research Unit of the General University Hospital of Ciudad Real. Conventional, biological wastes were removed and disposed of regularly, safely and in accordance with institutional recommendations for occupational hazards. 15 A model was made where a known concentration of allicin was administered. Randomization was carried out in three groups of rats, from now on we will refer to Group I –group with physiological serum–, Group II –group with ceftriaxone–, Group III –group with ceftriaxone + allicin–. Inclusion of 6 rats in control group and 9 rats in Groups II and 20 III. A peritonitis model was generated in all Groups. For the creation of the peritonitis model, subxiphoid laparotomy was performed prior anesthesia of the rat with ketamine / xylazine 75/10 mg / kg intraperitoneal, about 3 cm in length for the implantation of sublethal dose of E Coli ATCC 25922, obtained after control of colony forming units (1010 CFU / ml) (after extension of 10 µl of E Coli in MacConkey agar and after control and reading after 18-24 h at 37 ° C). The treatment to be administered to each of the groups was carried out as follows: 1) control group – Group I-: 6 animals receiving 4.4 ml of SF 0.9% intraperitoneal 30 2) experimental group II: 9 animals receiving 100 mg / kg of intraperitoneal ceftriaxone (IP) 3) experimental group III: 9 animals receiving 100 mg / kg of ceftriaxone IP + allicin 0.5 mg / kg IP 35 The animals weighed approximately 280 g. Dose calculation 100 mg / kg x 0.28 kg = 28 mg of ceftriaxone IP 0.5 mg / kg x 0.28 kg = 0.14 mg of allicin IP. The duration of the intervention was 7 days if the rats survived the infection. At 5 that day, the rats were expelled, once anesthetized with ketamine / xylazine 75/10 mg / kg intraperitoneal plus lethal doses of Thiopental 100-120 mg / kg, and after performing total peritonectomy with radical evisceration for microbiological study of the peritoneal and posterior histological fluid. The loss of blood volume with this surgical treatment exanguinated the rat, being incompatible with life. 10 The following variables were studied: CLINICAL PARAMETERS 15  Weight in grams each day.  Signs of stress and suffering.  Survival time in 24-hour intervals. BIOCHEMISTRY 20  Blood determination at 3 and 7 days (exitus) for serological study of inflammatory response variables, IL-1, IL-6, TNF.  BLOOD microbiological study (blood cultures) at 7 days. 25 HISTOLOGICAL  Microbiological study of peritoneal fluid.  Liver and peritoneum samples were taken for a histopathological evaluation. 30 The results indicated that GROUP III (ceftriaxone + allicin) is the group with the best clinical response (Figures 1 and 2), finding that this group begins weight recovery 24 hours before GROUP II. It should be noted that all the rats of GROUP I (Control group, which is not treated with either an antibiotic or with allicin, only physiological serum) die after infection at 3-4 h of the same, regardless of the weight they left. In relation to the rest of the clinical parameter (Figure 4), we found clear differences in favoragain from the allicin treatment group, with less signs of suffering and stress (less ocular and nasal secretions, less mustache position, less diarrhea and less lethargy and more active). Based on the biochemical parameters, we found that the rats in the control group at the time of death (Figure 3) had high values of all the proinflammatory factors studied (IL-6, IL-1, TNF), in line with the generalized sepsis that present and that presumably leads to death. Similarly, both the microbiological analysis in the blood cultures and the culture of peritoneal fluid 10 showed growth of E Coli ACCT 25922 (Table 1). Table 1. Blood cultures, peritoneal fluid culture and antibiogram in each of the study groups. 15 CROP HEMOCULTIVES L. PERITONEAL ANTIBIOGRAM CONTROL_1 POSITIVE E.COLI. ATCC 25922 POSITIVE E.COLI ATCC 25922 MULTISENSIBLE CONTROL_2 POSITIVE E.COLI ATCC 25922 POSITIVE E.COLI ATCC 25922 MULTISENSIBLE CONTROL_3 POSITIVE E.COLI ATCC 25922 POSITIVE E.COLI ATCC 25922 MULTISENSIBLE Tensile Grach - Non-Adjustable Group. GROUP II_2 Enterococcus faecalis 100 cfu / ml E. Coli <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. MULTISENSIBLE GROUP II_3 Enterococcus faecalis 200 cfu / ml Enterococcus faecalis Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP II_4 First success 24 h First success 24 h GROUP II_5 E. Coli <100 cfu / ml Compatible ATCC 25922 MULTISENSIBLE NEGATIVE GROUP II_6 Enterococcus faecalis 1300-3000 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP II_7 Enterococcus faecalis <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP II_8 Enterococcus faecalis 800-2000 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP II_9 Enterococcus faecalis 100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_1 NEGATIVE NEGATIVE GROUP III_2 Enterococcus faecalis 200 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_3 Enterococcus faecalis 80000 cfu / ml Enterococcus faecalis Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_4 Enterococcus faecalis 1100-2000 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine.GROUP III_5 Enterococcus faecalis <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_6 Enterococcus faecalis <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_7 Enterococcus faecalis <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_8 Enterococcus faecalis <100 cfu / ml NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. GROUP III_9 Enterococcus faecalis <100 cfu / ml1 NEGATIVE Sensitive to: Ampicillin, Vancomycin Teicoplamine. It should be noted that less amount of IL was detected globally on the third day in the group that added allicin. In both group II and III on the seventh day, no type of IL was detected (which speaks in favor of all the rats having survived sepsis, also reflected by other clinical parameters such as weight gain similar to the state 5 prior to infection). Attending to the histological study, we also found a difference in favor of the allicin group (Table 2 and Figure 4), with less bacterial and polymorphonuclear infiltration in the peritoneum and liver. 10 Table 2. Histopathological alterations found in peritoneum and liver in each of the study groups (congestion in the liver, polymorphonuclear –PMN– in hepatic sinusoids, PMN peritoneal surface, PMN in liver surface, colonization of liver surface bacteria, colonization of bacteria peritoneal surface ). Comparison between group II and 15 group III. Histopathological variable χ2 p value Hepatic congestion 2.22 0.898 PMN sinusoids hep. 5.71 0.456 PMN serosa hep. 4,59 0,597 Bacterial collection hep. 5.71 0.456 Vacuolization hep. 5.3 0.505 PMN perit surface. 4.29 0.83 Perit bacterial collection. 3.99 0.678 After the sacrifice blood cultures were performed in all rats, all of them were negative, except in group II where an ampicillin, vancomycin and teicoplanin-sensitive Enterococcus faecium 5 was found in one of the rats. Also peritoneal fluid culture, in it only in a rat of group II we find that growth of the E. coli bacteria still persists. However, in both groups we found an overgrowth of Enterococcus faecium, possibly due to prolonged treatment with ceftriaxone for seven consecutive days that makes the GRAM - bacilli disappear from the intestinal flora and enterococcal overgrowth occurs (Table 1). As indicated previously, all control rats died at 3-4 h after infection. All the rats of group III survived until the sacrifice on the seventh day and one rat died in Group II of ceftriaxone surviving the remaining eight. Again the 15 group of allicin is the one that best survives. Globally we can establish that the GROUP to which allicin is added has better evolution for all the variables studied. In our particular case, by having a stable composition rich in allicin, from the lyophilized extract, with stable s-allyl-cysteine from the black garlic extract, it allows us to affirm that the resulting mixture can act together or in a dual or synergistic with other thiosulphinates, as well as with other compounds present in the lyophilized extract such as polysulfides, vinyl dithiines and the isomeric mixture EZ-25 ajoene. Example 4. Use of lyophilized garlic extract and black garlic extract in adjuvant therapy with antitumor drugs in various neoplasms Experimental studies (in vitro tests) have been developed. These studies have been based on the treatment of colon cancer tumor cells, Caco-2 and HT-29 cell lines, with garlic extracts, where the antiproliferative effect was determined. These cell lines were used since compounds capable of inhibiting the proliferation of tumor cells may be useful as chemopreventive and chemotherapeutic agents. 10 In addition, in the study, combined treatments of garlic extracts with chemotherapy were used for clinical use to determine if there is a more pronounced effect than that produced by each of them in isolation. The cytotoxic and antiproliferative effects produced by the lyophilized extract containing allicin, the black garlic extract containing SAC, and the oxaliplatin chemotherapeutic agent, in isolation and in combination, on colorectal tumor cells are shown. For this, methods were carried out that provide information on the affectation of cell viability, proliferation and cytotoxicity after the different treatments. These were: Neutral Red Test (RN) and Lactate 20 Dehydrogenase (LDH) Test. Results of cell proliferation, viability and cytotoxicity tests. Determinations based on the examination of different cellular parameters were carried out, by means of which to extract information on the cytotoxic or antiproliferative effect produced by garlic extracts and a cytotoxic, both administered individually and in combination, on cancer cancer cells of colon In Figure 5, the microscope can be seen visually as, as the concentration of allicin increases in treatments applied to HT-29 colon cancer cells, cell morphology is changing. It is observed that for low concentrations of allicin the cells do not appear to be affected, maintaining a spherical shape, joined in cell clusters and well attached to the support used. On the other hand, for higher concentrations, the observed are cells or cell fragments lacking morphological similarity with respect to the control cells and completely detached from the cell support. 35Treatments with garlic extracts in RN trials The analysis of the effect of the different extracts and combinations on the loss of cell viability was performed to evaluate the most effective ones. For this, the cells were treated with 1 concentration of oxaliplatin (500 µM), 2 concentrations of allicin (10 and 50 µg / ml), 2 5 different concentrations of SAC (5 and 25 µg / ml), 4 combinations of garlic extract lyophilisate + black garlic extract (N5 + Alic10, N5 + Alic50, N25 + Alic10 and N25 + Alic50), combinations of black garlic extract + oxaliplatin (N5 + Oxaliplat and N25 + Oxaliplat), and lyophilized garlic extract + extract black garlic + oxaliplatin (N5 + Alic10 + Oxaliplat, N5 + Alic50 + Oxaliplat, N25 + Alic10 + Oxaliplat and N25 + Alic50 + Oxaliplat), and the effect 10 produced by the RN test was evaluated. Cancer-surviving cancer cells will have functional lysosomes that will be able to incorporate and fix the neutral red dye. Therefore, the greater the color observed, the greater the amount of living cells. In Figures 6 and 7, the cell viability observed through the use of spectrophotometry is represented in 15 percent values, taking the absorbance obtained in the cells that have not received treatment as 100% viability, that is, the control cells. When examining these data, it is observed how when applying individualized treatments of the 20 garlic extracts to tumor cells of colon cancer, a dose-dependent cellular affectation occurs, being only the treatment with black garlic 25 µg / ml more effective than the 500 µM oxaliplatin. In fact, cells that show a higher cell replication rate, that is, Caco-2, are more affected. Despite this more pronounced effect on Caco-2, a loss of cell viability also occurs for HT-29. In Caco-2, and with respect to the combination of lyophilized garlic extract + black garlic extract, certain combinations are more cytotoxic than their separate components (N5 + Alic10 and N5 + Alic50), and also a combination of lyophilized garlic extract + 30 black garlic extract + oxaliplatin (N5 + Ali50 + Oxaliplat) further increases the effect of oxaliplatin alone (Figure 6). In HT-29, and with respect to the combination of lyophilized garlic extract + black garlic extract, there is greater involvement with N5 + Alic50, and also the combination of extract of 35Freeze-dried garlic + black garlic extract + oxaliplatin (N5 + Ali50 + Oxaliplat) further increases the effect of oxaliplatin alone (Figure 7). Treatments with garlic extracts in LDH assays 5 LDH is an enzyme belonging to the cellular cytosol, and in the case of being detected outside the cell, it would be a clear indication of cell lysis. Both the presence of the LDH released in the culture medium, and the LDH that remains inside the cells, can be determined by colorimetric quantification of a metabolite resulting from the conversion of a tetrazolium salt into a colored formazan product red, whose production 10 is due to the enzymatic effect of LDH. The quantification of the percentage of LDH released with respect to the total can be used as an index of cellular toxicity, since if there is LDH outside the cell, it is because damage or breakage of the cell membrane must have occurred. This determination was made by means of the LDH release assay of the CytoTox 96® Assay kit (Promega). In Figure 8, carried out in HT-29 cells, it can be seen how the combinations of black garlic (N) and lyophilized garlic (allicin) significantly increase the individual effects, and that the combination of lyophilized garlic extract + black garlic extract + oxaliplatin 20 further increases the effect of oxaliplatin alone. These results support the use of lyophilized garlic, black garlic and its combination as a therapeutic option combined with different antineoplastic drugs for the treatment of different cancer processes. These combinations would maintain or increase the desirable cytotoxic effect on tumor cells, allowing to reduce the dose of antineoplastic commonly used in the clinic and, theoretically, reducing the adverse side effects of the application of said antineoplastic to their standard concentrations. Example 5. Topical or systemic use of lyophilized garlic extract and black garlic extract 30 in healing Murine model of wound healing with loss of substanceThe experimental animals that were used are Skh1 (Charles River Laboratories ©), naked, albino, microbiologically conventional (non SPF), euthymic and immunocompetent mice. Two circular biopsies of 5 mm in diameter were performed on two flanks of each mouse 5 of each of the treatment groups by the same experimenter and consecutively (so as not to have variations between different operators or variations in the technique of the same operator at different times of a day). The biopsy removed complete skin to the level of the muscular fascia, which must be left intact to prevent bruising. In this way, each animal presented two wounds of equal diameter, made at the same time 10 and located in the same anatomical situation of the skin of the back. No topical antibiotics or other topical or systemic products were used that interfered with the healing of each individual except the solution with garlic extract at different concentrations (depending on the treatment group) and the vehicle in the wound-control. 15 ● Histopathological studies Histological samples were collected from each surgical wound and 0.5 cm around the scar, making 3 longitudinal and parallel cuts in the sample: 1) in the center of the scar; 20 2) in the vicinity of the edge of the scar; and 3) on the skin located in the 0.5 cm margin around the scar. After dividing the biopsy into these three parts and properly orienting the cuts in the microtome, the following histological stains were performed: 25 1) Hematoxylin-eosin 2) Masson's trichrome 3) Acetic orcein staining Determination of the therapeutic concentration of lyophilized extract of Topical garlic 30 A study was carried out with 8 male Skh1 mice> 1 year old, in which the healing protocol mentioned above was carried out and separated into 3 groups of 2 animals each: 1) extract of lyophilized garlic at a concentration of 50 µg / mL; 2) lyophilized garlic extract at a concentration of 10 µg / mL; 3) lyophilized garlic extract at a concentration of 1 µg / mL; and 4) control group. In each animal of the same group, the woundSurgical right side is the treatment area, while the contralateral wound acts as a control (the vehicle is applied). Thus, each animal acts as a treatment individual and its own control within each of the three treatment groups, being able to compare them with the control group, in which both wounds are treated with a vehicle. 5 During the follow-up of the experiment (2 weeks), from the realization of the surgical wounds until its closure was observed in all the animals of the experiment, the group treated with lyophilized garlic extract at a concentration of 50 µg / mL showed a faster closure than the group treated with garlic extract at a concentration of 10 µg / mL and both groups had 10 better results in terms of wound closure speed than the group treated at a concentration of 1 µg / mL and the wounds of the control group (in fact, there were no differences between the group treated with 1 µg / mL concentration and the control group). This pro-healing response was objectified after 24 hours of treatment (Figure 9). Therefore, a dose-dependent effect was observed, which is clear for the groups treated with concentrations of 15 50 and 10 µg / mL (right side) with respect to the group treated with 1 µg / mL. In the group treated with lyophilized garlic extract at a concentration of 1 µg / mL, no differences were observed in the treated wound (right side) with respect to the contralateral wound (left side), which showed a torpid evolution towards closure by second intention during The 14 days of the experiment. However, the animals in the garlic extract treatment groups at 20 concentrations 50 and 10 µg / mL had a greater clinical response in the treated wound (right side), than in the contralateral treated with vehicle only (Figure 10). However, all individuals in the garlic extract treatment groups at a concentration of 50 and 10 µg / mL also had a good response on the left side wound, which demonstrates a systemic effect of topical application on the skin wound. on the right side of the lyophilized garlic extract at these concentrations (Figures 10 and 11). ● Histopathological studies Histological stains showed an increase in epidermal thickness (acanthosis) and a greater number of dermal fibroblasts in wounds in the group treated with lyophilized garlic extract at a concentration of 50 µg / mL with respect to the control group treated with vehicle (Figure 12 ). The systemic effect of the topical use of the concentration of 50 µg / mL was manifested at the histological level, with a reduction in the scar of the vehicle treated flank in the animals of the group treated with lyophilized garlic extract 50 µg / mL in the right flank 35 (Figure 13). Also noteworthy was the increase in the production of collagen in the dermis,which was well organized in dense beams with respect to what happens in the control group (Figure 14). Response to topical use of lyophilized garlic extract in the murine healing model 5 A study was conducted with 10 male Skh1 mice of 8 weeks of age, in which the healing protocol mentioned above was carried out and separated into 2 groups of 5 animals each: 1) lyophilized garlic extract at a concentration of 50 µg / mL; 2) control group treated with the lyophilized garlic extract dissolution vehicle. 10 During the follow-up of the experiment (2 weeks), from the realization of the surgical wounds until the closure was observed in the first animals (2 weeks), the group treated with lyophilized garlic extract at a concentration of 50 µg / mL showed a faster closure than the control group. This pro-healing response was objectified by measuring the major and minor diameters of each wound in the healing process to calculate the wound area (Figure 15 15). It was also evidenced through the sequence of dermoscopic photographs taken at 2-day intervals (Figure 16). ● Histopathological studies Histological stains showed a greater number of dermal fibroblasts in wounds 20 of the group treated with lyophilized garlic extract at a concentration of 50 µg / mL with respect to the control group treated with vehicle (Figure 17). Also noteworthy was the increase in the production of collagen in the dermis, which was well organized in dense beams compared to what occurs in the control group (Figure 17). 25 Response to the topical use of black garlic extract in the murine healing model A study was developed with 9 Skh1 male mice of 8 weeks of age, in which the healing protocol mentioned above was carried out and separated into 2 groups of 5 and 4 animals each: 1) black garlic extract at a concentration of 10 µg / mL (n 30 = 5); 2) control group treated with the lyophilized garlic extract dissolution vehicle (n = 4). During the follow-up of the experiment (2 weeks), from the realization of the surgical wounds until the closure was observed in the first animals (2 weeks), the group treated with black garlic extract at a concentration of 10 µg / mL showed a faster closure that hecontrol group. This pro-healing response was objectified by measuring the major and minor diameters of each wound in the healing process to calculate the wound area (Figure 18). It was also evidenced by the sequence of dermoscopic photographs taken at 2-day intervals (Figure 19). 5 ● Histopathological studies Histological stains showed a greater number of dermal fibroblasts in the wounds of the group treated with black garlic extract at a concentration of 10 µg / mL with respect to the control group treated with vehicle (Figure 20). Also noteworthy was the increase in the production of collagen in the dermis, which was well organized in dense bundles with respect to what occurs in the control group (Figure 20). In vitro model (wound healing) in cultures of human dermal keratinocytes and fibroblasts 15 In keratinocyte cultures (HaCaT), a wound healing model was developed to measure its proliferation and migration in response to the freeze-dried extract of Allium sativum, garlic extract black, and the combination of both extracts. It was also studied for its effects in vitro on the proliferation of dermal human fibroblasts (CRL-2072). 20 The results for lyophilized garlic extract in the in vitro model of wound healing showed an increase in the migration and proliferation of human keratinocytes and in the proliferation of dermal human fibroblasts (Figure 21). There was also evidence of increased proliferation of human dermal keratinocytes and fibroblasts in proliferation studies with the violet crystal technique (Figure 22). Finally, the combination of both extracts of Allium sativum, lyophilized garlic and black garlic, in different concentrations, especially 1: 5 (lyophilized garlic / black garlic), showed an increase in the proliferation of both human keratinocytes and dermal fibroblasts 30 (Figure 23). 
    权利要求:
    Claims (13)
    [1]
    CLAIMS 1. A composition comprising lyophilized garlic extract and black garlic extract.
    [2]
    Composition according to claim 1, characterized in that the ratio of freeze-dried garlic extract to black garlic extract is between 1: 1 and 1: 5.
    [3]
    Composition according to one of claims 1 or 2, characterized in that it is a pharmaceutical composition.
    [4]
    Composition according to claim 3, characterized in that it comprises antineoplastic drugs selected from the group consisting of cytostatics, cytotoxics, biological drugs and immunotherapy compounds.
    [5]
    5. Composition according to claim 4, characterized in that said antineoplastic drug is oxaliplatin.
    [6]
    Composition according to any of claims 3 to 5, characterized in that it comprises pharmaceutically acceptable excipients.
    [7]
    Composition according to claim 1, characterized in that it is a food composition. fifteen
    [8]
    Composition according to claim 7, characterized in that it comprises food additives.
    [9]
    9. Use of a composition according to any of claims 1 to 6 for the manufacture of a medicament for the treatment of diseases selected from the group consisting of sepsis, septic shock, tumor diseases and dermatological diseases.
    [10]
    Use according to claim 9, characterized in that said tumor diseases are selected from the group consisting of colon cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, ovarian cancer, testicular cancer, cancer of pancreas, kidney cancer, liver cancer, cervical cancer, gastric cancer, head and neck cancer, bone cancer, central nervous system cancers, neuroendocrine tumors, thyroid carcinoma, sarcomas, leukemias, lymphomas, myelomas, melanomas.
    [11]
    Use according to claim 9 or 10, characterized in that said dermatological diseases are selected from the group consisting of cutaneous wound healing, modulator of the epidermal barrier function, inducer of the production and secretion of epidermal antimicrobial peptides and inflammatory dermatosis, dermatitis irritative and atopic eczema, irritative eczema and allergic contact eczema.
    [12]
    12. Process for obtaining black garlic extract according to any of claims 1 to 8, characterized in that it comprises or may comprise the following steps:(a) solid-liquid extraction with an organic and / or inorganic solvent at a pressure between 0.1 and 400 atm and at a temperature between -70ºC and 150ºC with an extraction time greater than 1 minute, (b) physical separation of the extracted matter and the leachate obtained, (c) separation or recovery of the solvent and the concentration of the leachate or extract obtained in the previous steps and (d) chemical-physical stabilization by freeze-drying or keeping it cold.
    [13]
    Process according to claim 13, characterized in that the physical separation of step (b) is carried out by centrifugation, filtering or decantation.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    KR20110042607A|2009-10-19|2011-04-27|부산대학교 산학협력단|Anti-inflammatory composition comprising extracts from black aged garlic|
    KR20110057012A|2009-11-23|2011-05-31|부산대학교 산학협력단|Composition for suppressing tumor metastasis|
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