RO128713B1 - Ointment with complex effects for treating burns and wounds, made exclusively of natural products - Google Patents

Description

The present invention relates to a composition of ointment for the treatment of burns and wounds.

Burns and, in general, the different types of skin wounds are an important public health problem, with multiple and serious medical, economic, family, social implications.

Wound healing involves a complex, dynamic process that re-establishes the continuity and functions of the skin [1], and involves interactions between the extracellular matrix mediators and different cells, such as fibroblasts and keratinocytes. In the healing process, reactive oxygen species are also generated, and their presence can cause cellular damage through different mechanisms, such as membrane lipid peroxidation [2, 3]. This phenomenon suggests that the presence of antioxidants in the treatment of wounds and burns may be favorable for healing [3].

Numerous experimental researches have shown the beneficial effects that the preparations of plant origin have in improving (and even healing) the wounds of different etiologies. At present, numerous researches are being carried out worldwide to discover new natural products and new pharmaceutical formulations dedicated to the treatment of wounds.

Most of the solutions offered by the pharmaceutical industry are based on synthetic substances and can produce various side effects. For example, silver sulfadiazine cream, the current topical gold-standard drug in the treatment of burns, delays wound healing, slows the rate of epithelialization, and in prolonged treatment, leads to the formation of hypertrophic or atrophic scars, may have local cytotoxicity (on keratinocytes and fibroblasts) and can generate systemic disorders, such as: neutropenia, erythema multiforme, renal toxicity and methemoglobinemia [4,5].

An alternative to synthetic products is provided by medicinal plants. These have a long use in the history of mankind for the treatment of various skin conditions, including wounds and burns. Numerous current scientific studies demonstrate the healing ability of wounds with plant-based preparations [6-13], and highlight the many benefits, such as low cost, effectiveness, ease of application, safety at operation, and relatively low side effects (especially local hypersensitization).

Involvement in multiple mechanisms of wound repair and tissue healing of traditional remedies requires validation through scientific studies. Of the bioactive plant compounds, polyphenols represent a significant class. Gallic acid exhibits antimicrobial, antifungal and antioxidant activity [14, 15], and caffeic acid and its esters have anti-inflammatory [2] and antioxidant [16] action, accelerate the healing of skin wounds [2], and stimulate collagen synthesis [3]. Ferulic acid promotes skin healing by accelerating epithelialization [17,18] and acting as an antioxidant by inhibiting lipid peroxidation [17], Antioxidant activity is a common feature of flavonoids; The protective action of quercetin and routine on dermal cutaneous neurovascular cells undergoing oxidative stress has been demonstrated [19]. The presence of routine in dermatological formulations stimulated wound healing by slowing down lipid peroxidation, increasing catalase activity and decreasing protein carboxylation [20].

The object of the present invention is to introduce a new preparation, with a complex therapeutic action, in the range of medicinal preparations for burns and wounds. The invention proposes a complex formula, based on a mixture of 9 medicinal plants, conditioned in the form of ointment, to stimulate an adequate healing of the wounds, which has been called "Dermaplant".

The ointment presented is made exclusively from natural components and herbs. The formula contains an extract in olive oil of a mixture of medicinal herbs, along with other components with demonstrated action of wound healing, such as sea buckthorn oil (Oleum Hippophae), lavender essential oil (Lavandulae Aetheroleum), visco-growth agents (coconut oil - Coconut Oleum), beeswax (Cera Flava) and coniferous resin (Pina Resin). The active ingredient is represented by the oily extract of the mixture of plants, and the conditioned form is represented by an ointment.

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The Romanian medicinal plants used to make the oily extract are: 1 Calendula officinalis L. (Asteraceae), Matricaria chamomilla L. (Asteraceae), Symphytum officinale L. (Boraginaceae), Hypericum perforatum L. (Hypericaceae), Achillea millefolium L. 3 (Asteraceae ), Arctium lappa L. (Asteraceae), Plantago major L. (Plantaginaceae), Althaea officinalis L. (Malvaceae), Quercus roburL. - bark (Fabaceae). 5

The ointment contains the components in the following mass relation:

Marigold Calendula officinalis ........ ...... 15 ... 25 parts by weight 7 rattle comfrey Hypericum perforatum ....... Symphytum officinale ....... ...... 15 ... 25 parts by weight ...... 15 ... 25 parts by weight 9 Burdock hollyhock Arctium lappa ............. Althea officinalis ........... ...... 15 ... 25 parts by weight ...... 15 ... 25 parts by weight 11 Chamomile plantain Chamomilla Matricaria ....... Plantago major ............ ...... 20 ... 40 parts by weight ...... 20 ... 40 parts by weight 13 Yarrow Oak bark Achillea millefolium ......... Quercus cortex ............ ...... 10 ... 20 parts by weight ...... 10 ... 20 parts by weight 15 Olive oil Coconut oil Olivarum oleum ............ Coconut oleum .............. ........ 800 parts by weight ........ 100 parts by weight 17 Seabuckthorn oil Bee wax Hippophae oleum ......... Cera fi ava ................ ........ 100 parts by weight ..... 50 ... 150 parts by weight 19 Coniferous resin Volatile Lavender Oil Resin (fir, pine) ......... Lavandulae aetheroleum ..... ..... 50 ... 150 parts by weight ....... 5 ... 10 parts by weight 21

Medicinal plants in the composition of the ointment have multiple, synergistic properties: epithelizing, healing, antimicrobial, antiseptic, soothing, anti-allergic, immunomodulatory, 23 anti-inflammatory, anti-allergic, anti-purifying etc. [21-30]. The active principles of the plants are extracted in olive oil in a water bath, under the boiling water temperature, under optimum conditions, 25 non-destructive, which ensures the therapeutic bioavailability of the active principles and their maximum concentration. 27

Olive oil is a support base for the other ingredients, conferring the extraction of their active principles. Containing glycerides, fatty acids, flavonoids, vitamins A, 29 E and K, this oil stimulates cell regeneration, collagen and elastin, and provides good tissue hydration. Alternatively, Helianthi oil can be used in the same amount instead of olive oil, having a similar composition, but olive oil is preferable due to its specific anti-inflammatory action [31-33]. In fact, 33 it has been used frequently over time for skin and hair care.

Coconut oil is useful due to its particular biochemical structure; unlike 35 other vegetable oils and animal fats, it is mainly made up of medium chain saturated fatty acids (lauric, capric, caprylic, myristic and palmitic acids). Among other things, 37 this property prevents the oxidation and rinsing of the oil, making it suitable for the preservation of medicinal plants and for the treatment of wounds, along with the other 39 biologically active minor components of its composition [34-35].

Chestnut oil contains many essential fatty acids, carotene, lycopene, tocopherols, 41 phytosterols, etc. and has antioxidant, epithelializing, anti-inflammatory, anti-allergic properties [36-38].

The oily extract of the coniferous resin has antiseptic, comforting, anti-inflammatory, 43 healing, healing, astringent, antioxidant, immunomodulatory [39-41]

Beeswax has antioxidant, anti-inflammatory, antiseptic, and is a good 45 solidifying agent with both the protection of the wound against the external environment and the conditioning of the ointment. Wax esters and flavonoids stimulate tissue regeneration, 47 contribute to tissue hydration and have anti-inflammatory effect [42-43].

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The volatile lavender oil provides a pleasant aroma (especially beneficial in burn patients) and has antiseptic, antimicrobial, local anesthetic and anti-allergic action [44-48].

The proposed ointment, based on vegetable oils, medicinal plants, beeswax and coniferous resin, provides a humid microclimate favorable to epithelial regeneration, in conjunction with the intrinsic therapeutic actions, particular of the ingredients used.

Wet wound management represents the therapeutic concept of the future in the treatment of lesions of different etiologies, in order to obtain a much faster healing, of a better quality and with increased psychic comfort for the patient [49-54],

Compared with a dry environment, maintaining a moist environment at the level of the wounds favors the healing process and their epithelialization through multiple therapeutic properties:

- reduces the loss of bodily fluids;

- prevents tissue dehydration and cell death;

- isolates the plague from potentially harmful environmental factors;

- accelerates angiogenesis;

- improves local microcirculation and thus recovers the semi-viable cells;

- increases the elimination of dead tissue and fibrin;

- significantly reduces pain;

- promotes healing by reducing scars and acceptable aesthetic results. The proposed ointment promotes wound healing through multiple actions:

- provides a local microclimate favorable for healing by isolating (sealing) the wound against the environment, by stimulating epithelial cells, keratinocytes and endothelial cells, by restoring the skin's lipid barrier and stimulating collagen and elastin production;

- promotes epithelialization even in the case of deep wounds, by stimulating the reserves of residual epithelial cells (from the hair follicles, sebaceous glands and sweat glands);

- anti-inflammatory effect and absorption of excess exudate allow for early debridement and easy dressing removal;

- the antiseptic and antimicrobial action of the different ingredients;

- acceleration of neoformation vascularization;

- increasing patient comfort by reducing pain and local lubrication, thus allowing early mobilization;

- favoring the development of a good quality epithelium;

- dressing removal is easy and atraumatic, due to the non-adherence to the tissues;

- ease of application even in the case of wounds in difficult anatomical areas (natural skin folds, cutaneous mucosal junctions, deep, brittle wounds).

The ingredients used enhance each other's actions and give the proposed ointment beneficial properties and multiple therapeutic actions. It can be used to treat various skin lesions, as a natural adjuvant with healing, epithelizing, antiseptic, antimicrobial, anti-inflammatory, anti-allergic, immunomodulatory, anti-inflammatory etc.

In the following, a brief presentation will be made, for comparison, of preparations of natural origin with indication in skin lesions, from US patents (http://patft.uspto.gov/):

US 3943249 of 1976, inventor Max Shulman: method of treatment of burns by preparations containing rosin. The author proposes the treatment of burns and other skin lesions through a preparation containing 25 ... 60 parts by weight rosin (abietic acid, resin) and alpha tocopherol, dissolved in an oily environment (vegetable oil, fish oil or lanolin ).

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US 4725438 of 1988, inventor Billie Leazer: aloe vera gel ointment for 1 treatment of skin conditions. The ointment contains 5 ... 20 parts by weight of aloe vera gel and

80 .. .95 parts by weight base (approximately equal parts mixture of mineral oil, petroleum jelly, 3 lanolin and paraffin).

US 4857328 of 1989, inventor Theodore Trenzeluk: therapeutic mixture with extract 5 of aloe vera, for the treatment of skin disorders. The mixture contains 3 ... 50 parts by weight aqueous extract of aloe vera leaves as a therapeutic agent, 5 ... 20 parts by weight sulfatiazole straight 7 preservative, 5 ... 16 parts by weight zinc oxide as white inert pigment and 14 ... 87 parts by weight petroleum jelly (petrolatum) as a soluble oil base. 9

US 5266318 from 1993, inventor Darlene Taylor-McCord: therapeutic mix with

... 98% aloe vera gel extract, 0.1 ... 8% allantoin, 0.01 ... 2% lavender essential oil, additionally 11 topical base, cosmetic preservative and surfactant.

US 5785972 from 1998, inventor KathleenTyler: antiseptic composition with 500 ml 13 colloidal silver (6 ... 100 ppm), 0.94 ml essential oil of Helichrysum angustifolium or Helichrysum italicum, 1.25 ml raw honey, emulsified with 0.35 water soluble lecithin. 15

US 5766614 of 1998, inventor Liu Yong: a composition for the treatment of burns, prepared from Chinese rhubarb, calcium hydroxide, rhizome of sanguisorba officinalis, camphor, 17 rhizome of coptis chinensis, bark of phellodendron amurense and oldenlandia diffusa.

US 5997876 of 1999, inventors Nino Shikhashvili, Zurab Darsavelidze (Tbilisi, 19 Georgia): ointment with Chelidonium majus 15 ... 25 g, Plantago major 15 ... 25 g, Matricaria chamomilla 15 ... 25 g, Achillea millefolium 15 ... 25 g, Calendula officinalis 15 ... 25 g, Hypericum 21 perforatum 15 ... 25 g, Eucalyptus globulus 15 ... 25 g, Oleum olivarum 1000 g and Cera flava

80 .. .130 g. The active principles of the plants are extracted in olive oil by boiling in the bath of 23 water for 6 hours, then the filtrate obtained (700 ... 900 g) is boiled again in the water bath and mix with minced wax for 30 min. 25

US 6099866 of 2000, inventor K. Slimak,: composition for topical application of fresh beeswax and oil, with and without water, with a volume ratio from 1: 1/10: 0 to 1:20:20 27

US 6200570 of 2001, inventors Prakash Diwan et al. (Hyderabad, India): herbal composition, useful in therapeutic and cosmetic applications for the treatment of 29 general skin problems. The composition comprises plant extracts: Gymnena sylvestrae

3 .. .6 parts by weight, Tridaxprocumbens3 ... Q parts by weight, Alliumsativum oil 1 ... 3 parts by weight, dry juice of aloe vera 2 ... 6 parts by weight, Gum Olibanum powder in natural form 4 ... 7 parts by weight, Gum Olibanum resin extract in organic solvent 3 ... 8 parts by 33 weight, Gum Olibanum mass without resins 5 ... 10 parts by weight, optional, any drug with anti-inflammatory properties of the Diclofenac group sodium 1 ... 3 parts by weight, salicylic acid 35

1 .. .4 parts by weight, Piroxicam 1 ... 2 parts by weight, turmeric powder 0.1 ... 1 parts by weight, a base containing an aqueous cream or a gel containing carbopoly 1 ... 4 parts by weight 37, an emulsifying ointment 20 ... 40 parts by weight, preservatives 0.05 ... 0.3 parts by weight, moisturizing 1 ... 4 parts by weight and the rest of the water, to make 100 parts in weight. 39

US 6482442 from 2002, inventor Suleiman Dado from Austria: composition for topical applications in skin conditions, prepared from honey, olive oil, propolis, sage, optional beeswax 41, chamomile, thyme or lavender.

US 6576269 of 2003, inventor Alexander Korneyev: treatment of open skin lesions 43 using (oily) sea buckthorn extract. The composition is prepared from 5 ... 95 parts by weight oily extract of seafood, 0 ... 95 parts by weight oily extracts from herbs: mulberries, 45 ringworms (Hypericum perforatum), rockfish (Stellaria media), patlagina (Plantago major), yellow flowers (Calendula officinalis), 0 ... 10 parts by weight an essential oil, 0 ... 15 parts by weight 47 antioxidant, 0 ... 80 parts by weight diluent (a vegetable oil).

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US 6699512 of 2004, inventor Quintanilla Almagro et al., From the Biotechnology and Investigacion Company, Alicante, Spain: stable lipid extract from Styrofoam (Hypericum perforatum), which can be used in pharmaceutical preparations, such as water-soluble gels, as a healing agent.

US 6956144 of 2005 and RE42,755 of 2011, inventor Peter Molan of New Zealand: honey-based compositions for the treatment of wounds, such as foldable and flexible ointments, gels or foils.

US 7141252 from 2006, inventor Eliana Soubhie from Canada: a composition for the treatment of burns, prepared from olive oil, beeswax, lemon juice and boric acid.

US 2007/0141168 A1, inventor Wafa Alkazemi of Kuwait: a composition for the treatment of burns prepared from almond oil, purified honey, beeswax, pollen, propolis, lavender oil and water.

US 7985431 of 2011, inventor Rey-Yuh Wu from Taiwan: a pharmaceutical composition for the treatment of wounds, including in diabetic patients, containing extracts from Chinese medicinal plants (Plectranthus amboinicus and Centella asiatica).

Among the patents registered with OSIM, which refer to preparations containing some ingredients of natural origin, recommended in skin disorders, the following can be noted:

RO 106503 B1 from 1992, inventor Gheorghe Neagoe: ointment for wound healing and other skin lesions, consisting of nitroglycerin, dermatol, mafenide, acetate hydrocortisone, Balsam of Peru, vitamin E, vitamin F, vitamin A, incorporated in a base of simple ointment.

RO 107087 B1 from 1992, inventor Loan Mânzatu et al .: therapeutic ointment for the treatment of wounds and burns (commercial preparation ARMON), consisting of processed conifer resin, seaweed oil, propolis or soft extract of poplar buds, soluble collagen, extract oil of yellows, rattles and root of brust, lavender oil, incorporated in a base of ointment composed of beeswax, cetyl alcohol, cholesterol, anesthesia.

RO 108643 B1 from 1994, inventors Stoica Felician Titus, Bratu Ion Tiberiu, Lupuliasa Dumitru: ointment for the treatment of burns, consisting of white beeswax, rosin, incense, subgalic bismuth powder and sunflower oil.

R0120530 B1 of 2002, inventors Andrei Constantin and Andrei Diana Cristina: ointment based on herbal extracts, for the treatment of psoriasis, prepared from extracts from Saponaria officinalis, Glycyrrhiza glabra, Chelidonium majus, Hypericumperforatum, long-lived free radicals and / or precursors. these (as sterically hindered amines), salicylic acid, vitamins A, E and D and pharmaceutically acceptable excipients.

RO 121672 B1 of 2002, inventor Mititelu Magdalena: ointment for the regeneration and healing of wounds and burns, consisting of yellow beeswax, propolis, stearin, lanolin, volatile lemon oil, shark liver oil and calcium hydroxide solution 0, 15%.

R0122334 B1 from 2007, inventors Văduva Constantin et al: composition of ointment intended for the restoration of traumatized teguments and process of obtaining, consisting of 5 ... 12% sack, 10 ... 16% glue of resinous tree, 10 ... 16 % paraffin, 10 ... 20%, beeswax, 40 ... 50% edible oil and 2 ... 6% propolis granules.

RO 122475 B1 from 2006, inventor Miron Ghiorghi et al: anti-inflammatory, antirheumatic and antiseptic ointment, consisting of 22 ... 47 parts of coniferous resin extract in sunflower oil, 42 ... 65 parts of flower oil - sun food, 12 ... 26 parts yellow beeswax, 0.5 ... 3 parts lavender oil, 4.1 ... 7.5 parts propolis extract in food sunflower oil or tincture of sunflower propolis, and 10 ... 18 parts distilled water.

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RO 122836 B1 from 2007, inventors Butnariu Monica et al: pharmaceutical ointment and 1 process for obtaining yellow flowers grown on zinc-enriched soils, prepared from 3 to 4 parts dry extract and purified by Calendula officinalis, together with a formula 3 based on ointment, consisting of 5 ... 6 parts cetyl alcohol, 5 ... 6 parts cocoa butter, 1 ... 2 parts lauryl sodium sulfate, 25 ... 30 parts petroleum jelly, 1 ... 2 parts cetacean and 50 ... 60 parts preserved solution. 5 RO 122893 B1 of 2005, inventor Dumbrava Delia et al .: pharmaceutical ointment based on carotenoid extract from Zea mays, for the treatment of skin conditions, prepared from 7 0.7 ... 1 parts carotenoid compounds made of beta-carotene, alpha-carotene, zeaxanthin and lutein, together with 8.6 ... 10 parts lanolin, 88.8 ... 90 parts vaseline and 0.2 ... 0.7 parts nipagin. 9 RO 122952 B1 from 2007, inventors Butnariu Monica et al: pharmaceutical ointment and method for obtaining mouse tail inflorescences, grown on 11 zinc-enriched soils, with anti-inflammatory, healing and antibiotic action, prepared from 3 ... 4 parts dried and purified extract of Alchillea millefolium, together with a base of ointment made up of 13

5 ... 6 parts cetyl alcohol, 5 ... 6 parts cocoa butter, 1 ... 2 parts lauryl sodium sulphate, 25 ... 30 parts petroleum jelly, 1 ... 2 parts cetacean and 50 ... 60 parts preserved solution. 15

RO 123440 B1 from 2007, inventor Pârvuloiu Constantin: ointment destined to regenerate the skin traumatized by burns, prepared from sheepskin, beeswax, sunflower oil 17 and egg white.

Also, on the Romanian market there is the Carpicon brand - the Carpicon ointment, 19 designed by the lady doctor Viorica Vintilă, made from oily extract from coniferous resin and beeswax (company SC Romdan SRL), as well as Carpicon Plant cream with vegetable extracts 21 and conifer resin (Elzin Plant company), made from lavender oil, basil and coniferous oil resin, pine essential oil, castor oil and beeswax. 2. 3

As can be seen from the foregoing patents, a large number of compositions have been proposed for the treatment of burns, starting from substances of natural origin, but generally containing ingredients and pharmacological substances that are expensive and not always accessible. In addition, the topical application of some of them may require 27 careful and uninterrupted medical treatment, and may cause adverse reactions to some non-naturally occurring ingredients. In view of these things, as well as the increasing importance nowadays given to preparations of natural origin throughout the world, the present invention provides a topical composition for the treatment of burns and other skin lesions of 31 which is relatively inexpensive, easy to obtain, applied. and used, safe, stable, and can be administered not only under strict medical supervision. This eliminates or reduces the problems mentioned above, the limitations or disadvantages associated with conventional topical preparations, in addition, it has a complex therapeutic action, which combines the therapeutic effects of the 35 oil extracts from a wide variety of medicinal plants with a lipophilic ointment base, consisting of vegetable oils (with intrinsic therapeutic action), beeswax, coniferous resin and lavender 37 volatile oil (which, among other things, gives a pleasant, comforting, refreshing, physical and mental scent, especially for burn patients). Also, the ointment presented is made 39 exclusively from medicinal plants that are found in the flora of Romania, easy to obtain, with a real value and therapeutic importance, which have been confirmed by international studies. 41 Comparative analysis regarding the fulfillment of the conditions of novelty and of inventive activity in relation to the European patent EP 0952 839 B1, published in Bulletin 2002/03, regarding 43 to the ointment that was named Green Ointment (Gruene Salbe, Baume Vert) and which contains : rattlesnake (Chelidonium majus) 15 ... 25 g, grasshoppers (Plantago major) 15 ... 25 g, camomile 45 (Matricaria chamomilla) 15 ... 25 g, tail (Achillea millefolium ') 15 ... 25 g, yellowfin (Calendula officinalis) 15 ... 25 g, St. John's wort (Hypericum perforatum) 15 ... 25 g, eucalyptus 47 (Eucalyptus globulus) 15 ... 25 g, olive oil (Oleum olivarum) 1000 g and wax of bees (Cera flava) 80 ... 130 g. 49

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Both the product of this invention and the Green Ointment contain extracts from medicinal plants in olive oil, but the composition according to the invention is richer in quantity both in terms of medicinal plants and in supplementation with coconut oil, castor oil and resin. of conifers (the equivalent of eucalyptus would be, in this case, the volatile oil of lavender), each having its biochemical composition and its specific effects, which were also mentioned in the bibliography related to this work, which gives additional properties and the innovative character of the product.

The process of preparing the ointment according to the invention is carried out in the following way: in a bowl, the olive oil is mixed with the powders of medicinal plants, heated to a temperature of 7O ... 9O ° C, for 4 ... 6 h (on a water bath, under boiling water, for gentle, progressive extraction of active principles from plants), and then filter. The hot oil obtained (with the extracts from herbs) is mixed first with the beeswax and the coniferous resin until dissolved, then with the coconut and sea buckthorn oil (as the temperature of the preparation decreases) and finally , add the volatile lavender oil, stirring continuously until it is completely cooled to room temperature. The vessel in which the ointment is prepared can be optionally provided with a stirring, timing and temperature measuring element.

So far, we have made two series of determinations on the product.

The first researches were carried out together with a team from the Faculty of Chemistry, the University of Bucharest (Claudia-Valentina Popa), the Institute of Organic Chemistry CD Nenițescu (Liliana Lungu, Victorița Tecuceanu), the National Research and Development Institute for Biological Sciences, Bucharest Valentina Alexandru, Rodica Tatia), which materialized by publishing an article in the journal Romanian Biotechnological Letters, vol. 21 (2) of 2016, entitled: "An innovative ointment made of natural ingredients with increased wound healing activity, in which we examined the ointment from multiple points of view: biochemical characterization by LC-MS analysis of ethanolic extracts, total phenol determinations and flavonoids, DPPH free radical scavenging activity, fibroblast cytotoxicity test, stability test, as well as highlighting the effectiveness of the ointment by presenting suggestive clinical cases.

The following researches were carried out with a team from the National Institute of Chemical Research and Development in Bucharest, under the coordination of Dr. biochim. Radu Albulescu, President of the Scientific Council of the National Institute of Chemical-Pharmaceutical Development, Chief of the Pharmaceutical Biotechnology Department, and were the subject of a research project won by competition, carried out by the financing contract with no. 108CI of 25/07/2017 (PN-III-P2-2.1 -CI-2017-0487), concluded with the Executive Unit for the Financing of Higher Education, Research, Development and Innovation (UEFISCDI).

Test methodology and results published in the journal "Romanian Biotechnological Letters"

Preparation of extracts

Alcoholic extracts were obtained in 70% ethanol, using the following methods: maceration at room temperature, with occasional stirring for 7 days and continuous stirring for 3 hours; reflux for 3 h and 30 min respectively. The mixture of plants was ground, then every 10 g of powder was extracted and the ratio of plant material / solvent was 1:10 (g / v). The extracts were filtered through filter paper.

To prepare the oily extract, 30 g of vegetable powder mixture and 90 g of olive oil were kept for 6 h at 60 ° C in a thermostatic water bath, left overnight at room temperature and then filtered. through sterile gauze. The test sample a

EN 128713 Β1 of the oily extract was prepared by alcoholic re-extraction in a separating funnel (20 ml 1 oil extract and 60 ml 70% ethanol, v / v). After vigorous stirring of the funnel for 15 min, the resulting emulsion was centrifuged at 3500 rpm for 7 min. The supernatants were collected 3 and filtered through filter paper.

The sample analysis of the ointment was prepared as follows: a weighed portion of the total amount of ointment 5 was extracted with 70% (v / v) ethanol by ultrasonic assisted extraction (15 min); the ointment / solvent ratio was 1:20 (g / v). The resulting extract was centrifuged and filtered 7 under the same conditions as above. All extracts were stored at 4 ° C until further analysis. 9

1. Evaluation of action on fibroblasts ("fibroblast bioassay")

Cells in the mouse fibroblast cell line NCTC clone 929 (European 11 cell culture collection, Sigma-AIdrich, USA) were seeded into 24-well plates to form a subconfluent monolayer at a density of 2 x 10 ⁴ cells per cell. well 13 in the Minimal Essential Environment (MEM) Eagle, containing 10% fetal calf serum (FCS) and 1% antibiotics (penicillin / neomycin / streptomycin), and maintained at 37 ° C in an incubator with humidity of 5 % CO ₂ . After 24 h, the culture medium was removed by aspiration.

Dilutions of two tested plant extracts were made in MEM medium to give a range of final concentrations in wells from 0.1 pg / ml to 50 pg / ml. The analysis was performed in triplicate for each sample concentration. The control cultures received normal environment without 19 herbal extracts. Cells were incubated and tested for cell growth at 24 h, 48 h and 72 h. The test samples were ethanol extract and oil extract which was prepared for in vitro analysis by ethanol re-extraction. These two extracts were initially concentrated under rotary vacuum evaporator at 40 ° C, solubilized in water and diluted in MEM 23 medium to obtain a concentration of 50 µg / ml, and filtered through Millipore sterile 0.2 µm filter before adding to cells. 25

The effect of plant extracts on fibroblast cell growth was evaluated according to the Red Neutral (RN) method described by Borenfreund and Purner (1984), with some 27 modifications. RN analysis is used to measure cell viability, as an indicator of cytotoxicity in fibroblast cultures and on other cell lines. This simple and sensitive procedure is based on morphological and spectrophotometric criteria. Both assays were performed on the same cell culture in a 24-well microtiter test plate. 31 After the appropriate time interval, the plates were transferred to an inverted microscope for any visible signs of morphological change (growth inhibition, vacuolization, rounding, 33 detachment and lysis). The quantitative spectrophotometric procedure is based on the incorporation of RN, a supravital dye, into viable cell lysosomes. The medium was removed by suction and the 35 cells were washed with phosphate buffered saline, 0.5 ml of freshly prepared neutral red (RN) solution, 5 µl NR (50 μΙ / ml) in 495 μΙ MEM medium, and were added to each 37 well and the cells were incubated at 37 ° C for 3 h. The RN solution was removed and the cells were washed rapidly with 1% CH ₂ O / 1% CaCl ₂ to remove unincorporated RN. To extract the dye from the lysosomes, 1% (v / v) acetic acid and 50% (v / v) acetic acid were added to each well, and the plates were stirred for 10 ... 15 min. The plates were transferred to a 41 microplate reader and the optical density measured at 540 nm. Cell culture experiments were performed in triplicate for each sample and data were reported as mean ± SD, calculated 43 with Excel for Windows. Pairwise comparison between control and each sample was performed by t-test. Significant statistical differences were considered p <0.05. 45

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The morphological changes of the fibroblast culture treated with ethanol extract and oil reextract were examined microscopically for all concentrations and at all selected time intervals. No sign of modification was observed for any of the samples.

The results of the spectrophotometric determinations are shown in the figure. The absorbance is proportional to the number of intact membrane cells. Figure 1a and 1b show the viability of fibroblasts in the presence of ethanolic and oily extracts. The results of the associated t-student tests showed that there were no significant differences (p> 0.05) between control and each concentration of extracts, which shows that the two extracts are not cytotoxic.

2. Biochemical characterization of ethanol extracts by LC-MS analysis

The characterization of the polyphenols in the extracts was performed by liquid chromatography coupled with mass spectrometry (LC-MS). Chemical characterization was performed using an Alltech C18 5U column (100 x 3.2 mm, particle size of 5 µm). The mobile phase was bidistilled methanokap = 70:30 (v / v) and flow rate of 0.6 ml / min.

As standard, gallic acid (AG), caffeic acid (AC), chlorogenic acid (ACh), ferulic acid (AF), salicin (S), (+) -catenin (C), quercetin (Q) and routine were used (R). 1 ml of each methanol solution of 0,1 mg / ml of the said standards were mixed and brought to 25 ml with methanol in a volumetric flask. Thus, the concentration for each standard in the mixture was 4 pg / ml.

The concentration of the standard in the extract was calculated by the formula (1):

A IC = A IC (1Ί '' standard ⁷ '-'standard''sample ⁷ '-'probate \ ¹ / where: A _standard is the integrated peak area for each standard; C _standard is the concentration known by the standard; The _sample is integrated peak area for each extract; C _sample is the standard _sample concentration determined in the extract. Each sample was performed in triplicate and SD values were given. The results were expressed in standard mg / 100 g su

Total determinations of phenols and flavonoids

The total phenol content (TF) was determined by the Folin-Ciocâlteu method. The absorbance (A) was measured at the wavelength of 761 nm. The equations r ² / n (correlation coefficients / number of determinations) and the linearity fields for the calibration curves were:

A = 0.0037 x caffeic acid concentration - 0.0626 (2) r ² / n = 0.9933 / 11 (linearity range = 30 ... 200 mg / l);

A = 0.0018 x chlorogenic acid concentration - 0.0438 (3) i ^ / n = 0.9956 / 10 (linearity range = 40 ... 200 mg / l).

The total flavonoid content of the extracts was measured colorimetrically, using the aluminum chloride method, and the absorbance was recorded at λ = 425 nm. The equation, the correlation coefficient (r2) and the linearity domain obtained for the absorption of the calibration curve as a function of the routine concentration were:

A = 0.0029 x routine concentrations + 0.0103 (4) i ^ / n = 0.9933 / 11 (linearity range = 30 ... 200 mg / l).

The sample concentrations were determined from the equations of the calibration curves versus the linear range, and the final results were expressed in standard mg / 100 g dry weight (su) for all extracts. All determinations were made in triplicate and SD values were given.

The LC-MS method allowed the identification and quantification of the main phenolic compounds from the extracts of plant mixtures. The compounds in the extracts were identified by detailed studies of the MS-MS spectral data by comparing their spectra with those of the standards and with the literature data. The results are presented in table 1:

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Table 1

Quantitative LC-MS analysis of alcohol extracts and extracts

Sample Determined compounds mg / 100 g s.u. AG AF NEEDLE ACh Q R Macerated 7 days 0.339 ± 0.10 0.122 ± 0.006 0.347 ± 0.063 5.95 ± 0.31 1.20 ± 0.22 2.73 ± 0.11 Macerated 3 h 0.336 ± 0.041 0.161 ± 0.059 0.416 ± 0.089 11.9 ± 1.40 1.13 ± 0.15 3.57 ± 1.00 Reflux 3 h 0.252 ± 0.031 0.142 ± 0.010 0.412 ± 0.014 3.62 ± 0.43 3.56 ± 0.93 2.76 ± 0.31 Reflux 30 min 0.278 ± 0.042 0.118 ± 0.017 0.433 ± 0.20 5.26 ± 0.26 2.99 ± 0.26 2.55 ± 0.27 Re-extract (pg / g) Reextract oils 2.43 ± 0.44 0.561 ± 0.16 1.26 ± 0.11 12.2 ± 2.3 1.42 ± 0.59 8.23 ± 2.7 Oil reextract through US 6.98 ± 0.33 0.832 ± 0.047 1.76 ± 0.26 5.49 ± 0.11 - 7.29 ± 0.49

Of all the standards used, the extracts analyzed were identified and quantified: gallic acid (AG), caffeic acid (AC), chlorogenic acid (ACh), ferulic acid (AF), quercetin (Q) and routine (R). The results of the LC-MS analysis are presented in Table 2. Of the total measured compounds, chlorogenic acid was found in larger quantities, followed by routine and quercetin, and, in smaller quantities, caffeic acid, gallic acid and ferulic acid in ethanolic extracts. As for re-extracts, the values for polyphenols and flavonoids are lower, because smaller amounts of substances are extracted into the oil. The values for some compounds are higher for the cream re-extract, and this is probably due to the presence of the other ingredients in the cream.

Table 2

The total content of phenols, flavonoids and antioxidant activity of the extracts from the mixture of plants and from the extracts from the oil and cream extracts

Sample TF FL DPPH EAC (g / 100 g dw) EACh (g / 100 g dw) ER (g / 100 gdw) ET (mM) Macerat 7z 1.77 ± 0.14 3.26 ± 0.28 1.54 ± 0.038 12.1 ± 1.3 Macerat3h 2.51 ± 0.027 4.76 ± 0.055 1.53 ± 0.14 12.4 ± 1.7 Reflux 3h 3.63 ± 0.32 6.65 ± 0.65 0.91 ± 0.088 10.9 ± 1.2 Reflux 30 min 4.55 ± 0.36 8.55 ± 0.74 50.92 ± 0.086 13.5 ± 0.90 TE extract (μΜ) Reextract oils 0.728 ± 0.11 13.5 ± 0.23 4.90 ± 0.53 1.10 ± 0.059 Oil reextract through US 2.77 ± 1.7 52 ± 3.4 41.6 ± 4.7 -

CAE - caffeic acid equivalents, ChAE - chlorogenic acid equivalents, RE - rutin equivalents, TE - Trolox equivalents.

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The amount of polyphenols is twice as high when expressed as chlorogenic acid equivalents compared to caffeic acid. The results showed that the extracts contain significant amounts of polyphenols, such as caffeic, chlorogenic, gallic and ferulic acids, as well as quercetin and routine, compounds reported in the literature as having a good wound healing activity. These quantities determine and confirm the ability of the ointment to promote epithelialization and wound healing.

DPPH free radical scavenging activity

500 µl of standard / sample were mixed with 500 µl of DPPH solution (0.135 mM in 70% v / v ethanol) and the mixture was kept in the dark for 30 min. The absorbance was measured at the wavelength of 525 nm. The percentage inhibition of the calibration curve (% I) relative to the Trolox concentration was extracted. The equation curve, the correlation coefficients (r ² ) and the obtained linearity domains were:

% I = 0.9455 Trolox concentration + 47.5 (5) ^r2 / n = 0.9902 / 7 (linearity range = 2.5 ... 25 pmol / L trolox).

From the equation of the calibration curve the capacity of the antioxidant compounds from samples for the elimination of the free radical DPPH was determined. The antioxidant capacity of the samples was expressed as an equivalent sample mmol trolox (TE) / L. All determinations were made in triplicate and SD values were given.

The antioxidant activity of the extracts evaluated by the DPPH free radical scavenging assay DPPH is illustrated in Table 2. The increased DPPH radical scavenging activity for the alcoholic extracts indicates a high antioxidant activity of the extracts and also confirms the potential of the wound healing ointment. Moreover, the antioxidant capacity of the various ingredients in the ointment has been highlighted by numerous data in the literature. For oil re-extracts, the DPPH free radical scavenging ability has very low values and cannot be detected in the case of cream re-extract.

The stability test of the ointment. The centrifugation test g of ointment was centrifuged for 15 min at 3000 rpm at room temperature 24 h after preparation. The ointment was then homogenized by vortexing for 30 s and centrifuged again for another 15 min. The separation step was evaluated before the ointment was homogenized at the vortex and after the second centrifugation.

Phase separation after centrifugation of 30 (2x15) min was not observed.

Aspect. The samples were semi-solid, pale green, leaving a homogeneous trace on the glass plate. No phase separation was observed within 3 months of storage at different temperatures (5 ° C, 22 ° C).

Determination of the pH of the ointment

The pH values of the ointment were obtained by direct potentiometric method and according to Bucur. Thus, 6 ml of distilled water heated to 80 ° C was poured over 1 g of ointment and stirred vigorously for 1 min, then filtered. The filtrate was brought to room temperature and the pH was measured.

The pH value of the ointment, obtained by the direct potentiometric method for the cream at different temperatures (5 °, 27 ° C and 45 ° C), was 4.2. When the Bucur method was applied, the pH value obtained for the filtrate was 4.3. According to literature data, local acidification of the wound (with the pH of the ointment around 4, as in the case of honey, for example) promotes tissue healing by preventing the appearance of the non-ionized histotoxic form of ammonia, formed under the action of urea (from microorganisms). urea production) on urea in extracellular fluid. In an acidic environment, ammonia (NH ₃ ) is converted to the non-toxic ammonium ion (NH ⁴⁺ ). In addition, acidification of the wound increases the contribution of oxygen and pO ₂ to the surface of the wound by increasing the oxyhemoglobin-hemoglobin dissociation, due to a corresponding shift of the oxyhemoglobin-hemoglobin dissociation curve (Bohr effect).

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Case presentations 1

The efficacy of the ointment was clinically evidenced by a series of cases, both with acute wounds (mainly burns) and chronic wounds, after obtaining informed consent from 3 patients.

The first case presented is a hot liquid burn IIA-IIB degree on the dorsal side of 5 left hands. The characteristics of the wound were: moist, pinkish-white, intermediate depth lesions, moderate and serous secretions, bright portions indicating plasmoragia (deeper 7 zones). After painless treatment by applying ointment, the patient achieved significant improvements after only 4 days, involving two dressing changes, with 9 beginning of the epithelialization process and blurring of the glossy areas. The wound was almost completely epithelized after only 10 days. 11

The second case is a chronic wound - a calf ulcer located in the distal portion of the right leg, with arteriovenous venous etiology, dry staircase and small amount 13 secretion. Chest ulcer was 1 year old and was refractory to other common treatments. After only 12 days, with the change of dressings at the interval of 1 ... 2 days, a remarkable evolution was observed, with clear wound contraction and progressive marginal epithelialization.

Other cases with favorable evolution and accelerated epithelialization are represented by the application of the ointment on the donor areas of skin grafts, both in the case of burns and in the case of other reconstructive surgeries. 19

The test methodology and the results obtained by the 21 research project no 108CI, concluded with UEFISCDI

The research project aimed to obtain experimental data that would allow 23 preclinical pharmacological evaluation of the product, tested both as an extract and as a conditioned product (ointment), in order to establish the basic aspects regarding the safety and efficacy of the product. 25 In vitro and in vivo experimental models were approached to estimate the activity:

- product safety tests: 27

- reconstructed dermal culture, in vitro experimental model, for the evaluation of irritant or corrosive activity on the epidermis;

- in vitro pyrogenicity test \ testing the presence of bacterial endotoxins (LAL);

- microbial load test.31

- effectiveness tests:

- testing the sensitizing potential is kept alive) ', 33

- in vitro wound repair test ',

- healing of thermal wounds keep alive) .35

The testing of this product is done for the category of non-invasive medical devices that come in contact with damaged skin, of class 11b, in accordance with Law 176 37 of 2000 and with Directive 93/42 / EEC of 1993 on medical devices.

Test methodology and results39

1. Evaluation of dermal toxicity

Test protocol41

It was worked in accordance with the testing protocol provided by the kit supplier, Mattek from Bratislava, distributor of in vitro kits produced by the American company 43 Mattek (Protocol: in vitro EpiDerm ™ Skin Irritation Test (EPI-200-SIT)) .

Material and methods45

Test kit (human reconstituted dermis), delivered as viable cultures, 24 pieces reconstructed human dermal inserts, in 24-well plate, inserted in agarose gels and 47 supplemented with culture medium.

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Culture medium - DMEM F12 with 10% serfetal.

Positive control: sodium dodecyl sulfate (SDS), 0.1 g; it is recommended to dissolve it in saline phosphate buffer, for administration, achieving a working concentration of 1%.

6-well culture plates, 24-well and 96-well culture plates, for carrying out various experimental stages.

a) Tissue precultivation

The tissues are received in the laboratory on the day of arrival (usually the second day of the week).

Day 1. After examining the contents of the package, which must contain the 24-well tissue plate, culture medium (fully formulated), MTT reagent, positive control (SDS), phosphate buffered saline (for dissolving MTT reagent), and solvent for dissolution of formazan resulting from the reduction reaction of MTT, the culture plates with 6 and 24 wells required for the experiments), the MTT and SDS reagent are stored properly.

Each of the tissue inserts is then examined to verify their integrity. The culture medium is distributed, each 1 ml in each well of 6-well plates; 8 such 6-well plates, which are pre-incubated in the CO ₂ incubator for about 30 minutes, will be prepared to preheat at room temperature. It is similar to the plaque containing the pieces of reconstituted tissue. After bringing and stabilizing at 37 ° C, the plates and tissue are removed from the incubator. Each tissue insert is taken, a TPS wash is performed, to remove traces of agarose, and one piece of tissue is placed in three of the 6 wells of a plate. It is similar to all 24 wells.

Incubate overnight (12 h, 37 ° C, 5% CO ₂ ).

b) Preparation of the samples to be treated

SDS (positive control) is dissolved in phosphate buffered saline at the recommended concentration in the protocol. The sample does not require weighing, being delivered in the quantity that reconstitutes the concentration applied to the test.

The plant extract samples are administered as an aqueous solution, at a concentration of 10% after sterilizing filtration, through a 0.2 µm filter. The 10% concentration exceeds the usual concentrations of orthopedic or skin care products (dermacosmetics). The sample of ointment is administered by spatula, uniformly stretching 0.2 g on each piece of tissue.

c) Sample administration and incubation.

Day 2. Remove the culture medium from the refrigerator and preheat to 37 ° C. The plates with 6 necessary wells containing tissue inserts are removed from the incubator. For all independent replicates, 3 inserts will be used:

- for positive control;

- negative control;

- try.

Transfer the tissue pieces from the top row of wells to the bottom row of wells. It is re-examined for integrity. Absorb any trace of the environment on the surface of the insert, and wipe with a sterile cotton swab.

Using an automatic pipette, a 20 µl sample volume (SDS, medium or sample extract) is accurately applied to the upper surface of the dermis. As some operations will require about 1 min to handle the pieces, the samples will be applied at 1 minute intervals. Prior to administration, the type of sample applied to each of the plates will be marked.

After all the samples have been applied, the tissue pieces are moved back to the incubator, where exactly 35 minutes are kept. After 35 minutes, the tissue pieces are removed with the laminar flow hood, where a further 25 minutes are maintained.

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d) removal of the substances to be treated and replacement of the environment 1

Each of the tissue pieces was washed repeatedly (20 washes with 0.3 ml each) with phosphate buffered saline. Finally, each sample is immersed in the GST, to ensure that the lower face 3 is cleaned of any contaminants. Repeated washing takes about 1 minute (including re-insert). The samples were re-inserted into the upper well. 5

After the treatment of all the pieces is completed, they are reintroduced to the incubator at 37 ° C, 5% CO ₂ . Incubate again for 18 h, under standard conditions. The MTT reagent is brought to working temperature 7. Mark a plate with 24 wells, which will be incubated with MTT reagent. The treated cultures are removed from the incubator. The culture medium is extracted from each well with 9 pieces; this medium can be stored for testing secretions of pro-inflammatory cytokines such as IL-1 beta and TNF alpha. 11

Each of the pieces is carefully removed from the environmental traces, by buffering on sterile filter paper, and placed in a well of the plate of 24. Incubate the pieces at 37 ° C, 5% CO ₂ , 13 for 3 h. During this time, depending on the existence of viable cells, the MTT reagent will be reduced to an insoluble formazan, blue. 15

The pieces are extracted from the incubator, and 1 ml of acidified isopropyl alcohol is added over the culture medium, the plate is protected from light and incubated for 2 h on the rotary shaker, 17 at room temperature, for the solubilization of the colored formazan. Finally, it is possible to drain the entire amount of liquid from the insert, for this purpose proceeding with the needle perforation of 19 of each piece of tissue for the complete drainage of the formazan solution.

From each well, 200 µl of supernatant is taken 2 times and 21 is transferred to a 96-well plate, in order to read the optical density.

The optical density measurement is performed on a microplate reader, at a wavelength of 23 570 nm, against a blank represented by the acidified isopropyl alcohol. For each of the samples, the average of the 2 readings is calculated. A similar procedure is performed for the other 2 independent replicates.

For each of the samples,% viability is calculated using the formula: 27

Relative viability of TS (%) = [ODts / Average OD _NC ] x 100

Relative viability NC (%) = [ODnc / Mean OD _CN] x 10029

Relative viability of PC (%) = [ODPC / _NC OD Media] x 100, where: 31

TS = test substance;

OD _TS = optical density of the test substance; 33

OD _nc = optical density of negative control;

OD _PC = positive control optical density.35

For each type of tested substance, the positive or negative control sample, the average values of relative viability for three individual tissues are calculated and used for 37 classification according to the Predictive Model.

Data interpretation procedure (Predictive model) .39

According to the EU and GHS classification (category R38 / Category 2 or without marking), an irritant is predicted if the average tissue viability of three individual tissues exposed to the test substance is 41 below 50% compared to the relative viability of the negative controls.

In vitro result In vivo prediction Average tissue viability <50% Irritating (I), (R38 or GHS category 2) Average tissue viability> 50% Neiritant (NI)

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The results obtained when testing the irritant-corrosive action on a reconstructed dermal model.

Viability tests were performed, by applying the EPI-SKIN model, on the Active Product - Dermaplant Extract, Therapeutic Product - Dermaplant Ointment, in three sets of independent replicates, of 3 sets of determinations in each independent replicate.

The data recorded after the test were:

The product Average DO STDEV p ⁽²⁾ Relative viability PBS, 1 1887 .2703 - 100% SDS ** 0.031 0.0175 - 0 Extract Dermaplant * 1413 .4634 0.035 69.88% Dermaplant ointment 1328 .6436 0.035 74.46%

'The average of three independent replicates.

“A positive witness; 1: negative control; 2: Student's t test, 2 queues, homoscedatic distributions, versus positive control.

According to the classification criteria, it is concluded that Dermaplant Active Product and Dermaplant Therapeutic Ointment products are non-irritating (NI), and can therefore be safely used in accordance with the instructions for use.

2. Evaluation of loading with endotoxins - LAL method - gel-clot technique.

Bacterial endotoxin (BET) assay is used to detect endotoxins from gram-negative bacteria using amoebocyte lysate from Limulus polyphemus or Tachypleus tridentatus. The technique used in the microbiology laboratory for the detection of bacterial endotoxins is LAL "LONZA gel-clot. The gel-clot method is based on the opacification and gelation reaction of LAL extract in the presence of bacterial endotoxins. Bacterial endotoxin control (LAL test - gel-clot method) provides an in vitro alternative to the pyrogenicity assay performed in rabbits.

The LAL reagent used in the assay is a lyophilized product obtained from amoebocyte lysate from Limulus polyphemus. The LAL reagent is reconstituted extemporaneously with LAL water, by gentle and gentle spins.

LAL water is used for the reconstitution of the Standard Control Endotoxin (CSE). Standard Reference Endotoxin (CSR) is a reference preparation with a predetermined endotoxin content, specified in the Certificate of Analysis. In the case of the use of CSR endotoxin, before the start of the test, the certificate must be accessed and verified on the manufacturer's site, for the expiration date, as it is not mentioned in the analysis certificate.

Standard Control Endotoxin (CSE) is a substitute for the standard reference endotoxin (CSR) and has an endotoxin content expressed in EU / ng. The potential of bacterial endotoxins to induce a pyrogenic effect is expressed in EU / ng (ml). The potential of CSE is determined by the ratio between CSR / CSE, tested at a certain sensitivity of the LAL reagent.

Both the Standard Reference Endotoxin (CSR) and the Standard Control Endotoxin (CSE) are reconstituted with LAL water, according to the instructions specified by the manufacturer in the certificate: CSR or CSE is used in routine testing; after reconstitution, the standard series is valid for a fixed period of time, if stored at 2 ... 8 ° C.

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The pH buffer is used only when the pH value of the test solution is within 1 outside the normal admissibility values between 6 ... 8. It is used according to the manufacturer's specification and must be free of detectable endotoxins and 3 interference factors.

Confirmation of sensitivity of LAL reagent 5

Standard Reference Endotoxin (CSR) or Standard Control Endotoxin (CSE) is a reference preparation with a predetermined endotoxin content and which is reconstituted with a quantity of LAL water specified in the working procedure specified by the manufacturer of the LAL kit. The solution resulting from reconstitution of endotoxin with LAL water is the stock solution and has a concentration of endotoxin determined and expressed in lU / ml or EU / ml. Serial dilutions are made so that, from the predetermined endotoxin content of the stock solution, concentrations of 1 of 1 EU / ml, respectively 2, Ă, Ă / 2, Ă / 4, respectively, are reached, where λ = reactive sensitivity LAL used. 13

Confirmation of sensitivity of LAL reagent and standard series (CSR / CSE):

- there are at least 4 replicates; 15

- is performed before the beginning of the test cycle;

- is made when a new stock solution is reconstituted; 17

- is performed when using a new batch of reagent.

Each replicate consists of 0.1 ml LAL reagent, reconstituted extemporaneously 19 according to the working procedure specified by the kit manufacturer, added over 0.1 ml of the concentrations 2λ, λ, Ă / 2, λ / 4 of CSR / CSE . 21

The mixture will be incubated according to the manufacturer's specification at 37 ± 1 ° C for 60 + 2 min.23

The standard series result is not valid if:

- one of the replicates at the lowest concentration of the standard series A / 4 induces a 25 positive result represented by a firm gel that remains stable upon the return of the tube at 180 °;

- one of the replicates at concentration 2 X induces a negative result.27

Testing the endotoxin (L) limit for a sample involves:

- dilution of the sample (if necessary), 29

- positive control standard series (C +);

- negative control (C -); 31

- positive control sample (PPC).

The endotoxin concentration in the product to be analyzed must be lower than the permitted endotoxin limit of 33. In order to test the endotoxin limit, it is necessary to prepare dilutions by dissolving or diluting in LAL water the active substances or medicinal products.

Control tests: 37

- negative control test (C-) consisting of 0.1 ml LAL water, over which 0.1 ml LAL reagent is added;

- positive control test (C +) consisting of 0.1 ml CSE dilution at twice the sensitivity concentration, over which 0.1 ml LAL reagent is added;

- the positive control test (PPC) test is performed by combining 0.05 ml dilution PPC sample, to which 0.05 ml endotoxin is added, with 0.1 ml LAL reagent, 43

- the test of the sample consists of adding over 0.1 ml of the dilution of the sample of 0.1 ml reagent

LAL.45

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The reagent distribution in all the test tubes is done in maximum 2 minutes. After the reagent has been distributed, the tubes are gently shaken to homogenize the contents, then the tube stand is incubated in the thermostatic bath at 37 ± 1 ° C for 60 + 2 min. After the incubation period, the results are read.

Conformity of the result

The test result is as follows:

- In both replicates with the sample (or dilution) to be analyzed, the result is negative (the endotoxin concentration in the analyzed sample is below the maximum allowed level), ie the reaction product is not a gel or a viscous substance is formed which is not kept in the inside of the test tube after its rotation at 180 °.

The product is considered to conform if a negative result is found in both replicates of the sample to be analyzed.

The product is considered non-compliant, if in both replicates the result is positive (the endotoxin concentration in the product to be analyzed is greater than or equal to the maximum permissible limit).

Results obtained when evaluating endotoxin loading

The load with endotoxins was estimated by the LAL test for Dermaplant Extract, as the ointment product may possibly be contaminated with endotoxins from the plants used in the manufacturing process. The ointment has characteristics that make it impossible to apply the test for endotoxins.

According to the reported data, Dermaplant - Extract was found to be within the recommended endotoxin content limits for topical products and type II medical devices, respectively below 0.5 EU / ml.

3. Method of testing microbial contamination

Equipment and glassware: 90 mm diameter, single-use, Petri dishes; sterile graduated pipettes of volume 10 ml; sterile test tubes; sterile Erlenmayer cup; automatic pipettor; analytical balance; laminar airflow (LAF) biological cabinet; thermostats set at 25, 35 and 44 ° C.

Culture media used (according to the FE in force): medium A (Casein soy bean digest broth); medium B (Casein soy bean digest agar); medium C (Sabouraud-glucose agar with chloramphenicol); peptonate water buffer solution with sodium chloride, pH = 7; medium E (Enterobacteria enrichment broth-Mossel); medium D (Lactose monohydrate broth); medium F (Crystal violefneutral red, bile agar with glucose); medium N (Cetrimid agar); medium O (Baird-Parker agar); medium I (Tetrationat bile brilliant green broth); medium K (XLD agar).

Test methodology total number viable aerobic microorganisms - deep inoculation method

The samples are processed under aseptic conditions, at the working point of class A. Prepare the dilution 1/10: 10 g or 10 ml of the product, dissolve in 90 ml buffer solution and homogenize. Subsequently, serial dilutions (1 / 100,1 / 1000) are prepared, using the same solvent. Each 1 ml of the sample dilution is divided into 2 sterile Petri dishes and each 15 ... 20 ml medium culture medium B, melted and cooled to a temperature of 45 ° C is added.

Cover each Petri dish with the lid, gently rotate on a flat surface and allow to solidify at room temperature. The plates are thermostated with culture medium B at a temperature of 3O ... 35 ° C for 5 days.

Results

After the incubation period expires, Petri dishes with microbial growth are selected, but the microbial colonies are counted only from plates with a maximum of 250 colony-forming units (ufc). The total number of viable aerobic microorganisms is

EN 128713 Β1 is equal to the arithmetic mean of the number of ufc recovered from culture medium B. If no microbial recovery is obtained, the result is expressed as being lower than the detection limit (ie the lowest dilution tested), ie <10 ufc / g or ml.3

Methodology of testing the total number of yeasts and molds - deep inoculation method5

Sample processing is carried out as for the total number of viable aerobic microorganisms: serial dilutions (1/10, 1/100) are prepared, using buffer solution and 1 ml 7 of the sample dilution are divided into 2 sterile Petri dishes. To each plate is added 15-20 ml of culture medium C (Sabouraud glucose agar with chloramphenicol), melted and cooled 9 to a temperature of 45 ° C.

Cover each Petri dish with the lid, gently rotate on a flat surface and allow to solidify at room temperature. The plates are thermostated with the culture medium C at a temperature of 2O ... 25 ° C for 5 days. 13

Results

After the incubation period has expired, the Petri dishes with 15 microbial growth are selected, but the microbial colonies are counted only from the plates with a maximum of 50 cfu. The total number of yeasts and molds is equal to the arithmetic mean of the number of ufc 17 recovered from culture medium C. If no microbial recovery is obtained, the result is expressed as being lower than the detection limit (ie the lowest dilution tested). ), and 19 ie <10 cfu / g or ml.

Test methodology Enterobacteria and other Gram negative bacteria 21 in 100 ml of culture medium D add 10 g / ml of product (dilution 1/10), homogenize and incubate at 2O ... 25 ° C for 2 h. incubation is transferred 1 ml (from 1/10 dilution) into 23 100 ml culture medium E which is thermostated at 3O ... 35 ° C for 18 ... 48 h. Subcultures are made from medium E by surface inoculation of two plates of culture medium F 25 (1 ml each in each plate) and incubated at 3O ... 35 ° C for 18 ... 24 h.

Results 27

After the incubation period has expired, the Petri dishes that present the microbial growth are selected. Occurrence on culture medium F of red bacterial colonies, with a reddish area around 29 may be the indication of the presence of enterobacteria.

Test methodology Pseudomonas aeruginosa 31 in 100 ml culture medium 10 ml (equivalent to 1 g / ml product) is added from the dilution 1: 10a of the sample prepared as total viable aerobic microorganisms, homogenized and 33 incubated at 3O. ..35 ° C for 18 ... 48 h. Sub-cultures are made by surface inoculation of two plates of culture medium N (1 ml in each plate) and incubated at 3O ... 35 ° C 35 for 18 ... 72 h.

Results 37

After the incubation period has expired, Petri dishes are selected that show microbial growth. Occurrence on culture N medium of yellow-green bacterial colonies may be an indication of P. aeruginosa presence.

Test Methodology Staphylococcus aureus 41 in 100 ml culture medium 10 ml (equivalent to 1 g / ml product) from dilution 1: 10a of the sample prepared as a total number of viable aerobic microorganisms is added, homogenized and 43 incubated at 3O. ..35 ° C for 18 ... 48 h. Subcultures are then made by surface inoculation of two plates of culture medium O (1 ml each in each plate) and incubated 45 at 3O ... 35 ° C for 18 ... 72 h.

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Results

After the incubation period has expired, Petri dishes are selected that show microbial growth. Occurrence on culture medium O of some yellow or white bacterial colonies and surrounded by a yellow area may be an indication of the presence of S. aureus.

Test methodology for detecting pathogenic microorganisms of the genus Salmonella in 100 ml culture medium. 10 g / ml sample is added, homogenized and incubated at 3O ... 35 ° C for 18 ... 48 h. After incubation, transfer 0.1 ml (from 1/10 dilution) in 10 ml culture medium I which is thermostated at 43 ... 45 ° C for 18 ... 48 h. Sub-cultures are then made by inoculating two plates on the surface of culture medium K (1 ml in each plate) and incubated at 3O ... 35 ° C for 18 ... 72 h.

Results

After the incubation period has expired, Petri dishes are selected that show microbial growth. The appearance on culture medium of some reddish bacterial colonies may be the indication of the presence of Salmonella sp.

Highlighting the microorganism Escherichia coli in 100 ml culture medium 10 ml (equivalent to 1 g / ml product) from the dilution 1: 10a of the sample prepared as total viable aerobic microorganisms is added, homogenized and incubated at 3 O ... 35 ° C for 18 ... 24 h. After incubation, transfer 1 ml into 100 ml culture medium G which is thermostated at 43 ... 45 ° C for 24 ... 48 h. Subcultures are made by inoculation to surface of 1 ml on two plates of culture medium H and incubated at 3O ... 35 ° C for 18 ... 72 h.

Results

After the incubation period has expired, Petri dishes are selected that show microbial growth. The appearance on culture medium H of some bacterial colonies may be the indication of the present E. coli.

Results obtained in the evaluation of microbial contamination

The evaluation used the Dermaplant Extract and Dermaplant Ointment for testing, in the conditions of the determination of bacterial, fungal and yeast contaminants, according to the protocol described.

Following the evaluation, no contaminants from the category of pathogenic microorganisms were discovered.

at the same time, although it has a microbial load represented by non-pathogenic species, the product in its present form is not yet recommended for medical devices that come into contact with the damaged tissues, for which a new sterilization operation is recommended, most probably by irradiation, which would ensure the recommended sterility level for products intended for the treatment of burns.

4. Tests for thermal burn repair

The mouse model

The model uses young mice (6-8 weeks old) who are anesthetized with ketamine and xylazine or other intraperitoneally administered anesthetics. In some cases, 1 ml of subcutaneous saline is administered along the spine to protect the spinal cord from any injury. Subsequently, the hair on the dorsal side is removed. The dorsal region is the area of choice, because the animal does not have access and no additional scratching lesions appear in the area of the thermal injury caused. The mouse is then fixed with the epilated region in a template with a window that provides predetermined skin exposure. The exposed surface of the mouse in the template is then immersed in a water bath at 60 ... 100 ° C for 8 ... 12 s to cause a total burn. The animals are then frequently observed for signs of pain or discomfort and are given analgesics.

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In mice, thermal injury is limited by the size of the animals, as they can only tolerate a burn of 30% of the total body surface area. Some authors mention that the hypermetabolism phase is not fully activated except by thermal injuries greater than 40% of the 3 total body surface area. Thus, although the burn pattern of the mouse is simple to achieve, it loses relevance when studying the post-burn hypermetabolic status. 5

The model on the rat

The rat burn model is performed in the same way as the 7 mouse model, with some minor differences, such as temperature and duration of exposure to heated water. Rats, being larger animals, can withstand a burn of up to 60% of 9 total body surface area, using the aforementioned model for the dorsal region and additionally another thermal injury in the abdominal region. The provocation of a wound by thermal burn 11 with more than 60% of the total body surface area in rats is followed by a low degree of survival and is not sustainable for experimentation. There is a need for a thermal lesion model 13 large enough to cause the hypermetabolism observed in human burns on large surfaces. Because in the initial stages of post-burn in humans, glucose extraction of the affected tissue occurs, it induces hyperglycemia and hyperlactatemia.

Therefore, the rat burn model is superior to the mouse model with 17 increased ability to recover hypermetabolism, which becomes deficient when attempting to include the risk of infection to achieve post-burn sepsis observed in 19 human patients with extensive burns above 60 %.

The results obtained when testing the ability to favor the repair of burned wounds 21

Testing was performed using the model using rats. We worked on male Wistar rats, 180 ... 200 g, provided by the bio-base of the Cantacuzino institute. Two variants of the test model were applied, namely the conventional model, in which the lesion is carried on both sides of the animal, and a modified model, in which 25 the thermal lesion is performed on a single flank, and a batch of animals is used. "Control (in which the lesion is not treated) and one batch" treated, in which the test preparation is administered. 27 in both cases, the wounds were obtained by applying an exposure of the epilated dorsal tegument at a temperature of 90 ° C for 10 s, on animals anesthetized with ketamine 29 (50 mg / kg, ip) After this exposure, the animals were left to stand for 24 hours, after which the treatment was applied. 31

There were the following batches, which were treated every 48 hours, for 6 days:

Nr. lot Lot name Remarks 1 Treated lot Bilateral thermal injury 2 Witness batch Unilateral thermal injury 3 Lot 2 treated Unilateral thermal injury

Experiment results - visual observations of tissue regeneration:

Nr. lot Lot name Remarks Result (% lesion area) 1 Treated lot Bilateral thermal injury - control area 52% Bilateral thermal injury - treated area 41% 2 Witness batch Unilateral thermal injury 31% 3 Lot 2 treated Unilateral thermal injury 56%

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From the analysis of the results, it can be deduced that in the case of the animals injured bilaterally, there is probably an influence (probably mediated by the production of cytokines and lympholines) that generates an improved repair also on the untreated flank. The “unilateral” model shows a clearer stimulation of the reparative process in the treated animals, compared to the animals in the control group.

In order to deepen these issues, further investigations are proposed, using multiplex assay kits to monitor the effect of the product on the production of cytokines and lymphokines involved in the inflammatory process and then wound repair.

5. Determination of cytokine levels

The levels of cytokines - IL1 beta, TNFalfa and IL6 were determined in culture supernatants collected from the experiment on the ToxDerm Kit, using commercial ELISA kits, according to the manufacturer's instructions (ab214025, ab46087 and ab46042), in triplicate. There was a significant reduction in the level of pro-inflammatory cytokines IL1 beta and TNF alpha, as well as a maintenance level comparable to the control for IL6.

A more advanced reepithelialization of the treated animals compared to the control group was observed. The treatment consisted of daily application of the preparation as an ointment on the wound surface. The wounds were made by applying, on day 1 of the experiment, a device heated to 80 ° C, and placing it in contact with the dermis for 10 s, on the previously epilated surface of the animals. After the lesions occurred, the treatment was applied. No occlusive dressing was used.

The treatment was continued for a period of 14 days, on days 2 ... 4, 7 ... 10, 13, 14. During the experiment, the animals were fed and received water ad libitum. No adverse effects due to the application of the treatment were observed. Although recovery of the control group was slower, no serious adverse effects or mortality were reported in this case.

Conclusions and proposals

Due to the growing interest in natural preparations, the ointment offers a composition for the treatment of burns and wounds, which is efficient, stable, pleasant, relatively cheap, easy to administer, applied and used safely.

The favorable therapeutic properties result from adding the therapeutic effects of the oily extracts from a variety of medicinal plants to those of a lipophilic ointment base, consisting of vegetable oils, beeswax, coniferous resin and lavender essential oil, all of which have a certain value and therapeutic importance, which have been confirmed by a wealth of data from the literature.

The work done on the products Active product - Dermaplant extract and Therapeutic product - Dermaplant ointment demonstrated the following:

- the preparations do not exhibit dermal toxicity, as demonstrated by using the EpiDerm ™ Skin Irritation Test (EPI-200-SIT) in vitro test model;

- Dermaplant Extract preparation, tested in vitro on 3T3 fibroblasts, demonstrated a remarkable ability to stimulate proliferation (+ 18% increase in proliferation rate);

- safety tests have shown that the product is free of endotoxin-type contaminants, and therefore safe for application on damaged skin;

- the presence of pathogenic germs was not highlighted, and the microbial load was within the limits imposed by the Pharmacopoeia for topical preparations;

- the evaluation of the in vivo reparative effect revealed a stimulation of the repair of the damaged dermal tissue, on a model of unilateral thermal injury in the rat, the repair process at 6 days being accelerated by 69%;

- the study on the effect on the production of signaling molecules, by applying a model to evaluate the level of pro- and anti-inflammatory cytokines, chemokines and lymphokines in the treated animals, showed a significant decrease in the level of pro-inflammatory cytokines IL1 beta and TNF alpha, as well as maintaining a level comparable to the control for IL6.

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Claims (1)

Claim 1 Composition of ointment for the treatment of burns and wounds, characterized in that 3 is made up of oily extracts of powders of yellow calendula officulais 15 ... 25 parts by weight, sounding Hypericum perforatum 15 ... 25 parts by weight, dried 5 Symphylum officinale 15 ... 25 parts by weight, Arctium lappa brusture 15 ... 25 parts by weight, root of Althea officinalis 15 ... 25 parts by weight, chamomile Matricaria chamomilla 1 20 .. .40 parts by weight, Plantago major platter 20 ... 40 parts by weight, Achillea millefolium rat tail 10 ... 20 parts by weight, Quercus coertex oak bark 10 ... 20 parts by 9 weight, from Vegetable oils: Helianthioleum olive oil 800 parts by weight, coconut oil Cocos oleum 100 parts by weight and Hippophae oleum custard oil 100 parts by weight, from 11 beeswax Cera flava 50 ... 150 parts by weight, resin of coniferous resins albies, pines 50 .. .150 parts by weight, and from volatile lavender oil Lavandulae aetheroleum 5 ... 10 parts by 13 weight.