WO2022025852A1

WO2022025852A1 - Gel formulation prepared with okra fruit extract and use of the gel formulation as a wound healer

Info

Publication number: WO2022025852A1
Application number: PCT/TR2021/050745
Authority: WO
Inventors: Hande SIPAHI; Ahmet Aydin; Erdem Yesilada; Rengin REIS; Duygu ORAK; Kübra YALMAN; Derya ALGÜL KURÇEREN
Original assignee: Yeditepe Universitesi
Current assignee: Yeditepe Universitesi
Priority date: 2020-07-27
Filing date: 2021-07-27
Publication date: 2022-02-03
Anticipated expiration: 2023-01-27
Also published as: TR202011889A1

Abstract

The present invention relates to obtaining formulas, which are used for wound healing purposes, from aqueous or alcoholic extracts of okra ( Abelmoschus esculentus ) fruit. Wound healing properties of the okra fruit grown in our country were investigated based on the use thereof as a folk medicine in the treatment of abscesses in the skin and subcutaneous tissue. As a result of the in vitro and in vivo studies conducted within this context, the formulation prepared with the extract of okra from Kilis region is found to be very effective in wound healing. In the histopathological analyses, it was observed that collagenisation and granulation tissue maturation were better in the reference and the 5% ethanolic extract groups. Furthermore, the present gel formulation was found to be non- irritant as a result of in vitro EpidermTM reconstituted skin irritation test (SIT) conducted within the scope of the biocompatibility tests.

Description

GEL FORMULATION PREPARED WITH OKRA FRUIT EXTRACT AND USE OF THE GEL FORMULATION AS A WOUND HEALER

Field of the Invention

The present invention relates to obtaining formulas, which are used for wound healing purposes, from aqueous or alcoholic extracts of okra ( Abelmoschus esculentus) fruit.

Background of the Invention

Okra (. Abelmoschus esculentus ) plant is a plant species that belongs to the Malvaceae family and can be grown in warm climates. Its fruit, which is in the form of a long, thin pyramid and contains round, oval and green seeds, is consumed as food. Due to its high fiber and polysaccharide content, it is a type of food that allows proper functioning of the stomach and intestines and supports the immune system thanks to its rich mineral content and can be consumed dry or wet. A raw okra fruit has 90% water, 2% protein and 7% carbohydrate content by weight, and is rich in fiber content, vitamin C, vitamin K, as well as thiamine, folic acid and magnesium, which a person should intake daily.

The fruits of Abelmoschus esculentus (Okra) are consumed as food not only in Turkey but also in many parts of the world. The fruits with abundant mucilage have been used for many years in traditional medicine to treat gastric lesions and inflammatory diseases. In the study conducted, the total antioxidant capacity in aqueous and ethanolic extracts of okra fruit differs according to the literature. These different effects are thought to be due to the diverse antioxidant potentials of the aqueous and ethanolic extracts. Previously different parts of the okra plant, such as the fruit or the seed, have also been reported to possess various activities. In the present study, it is aimed to get higher activity potential, and therefore whole fruit, including the seeds, are extracted together. The literature has previously stated that the variations, particularly in the polyphenolic and flavonoid contents, may be due to the cultivation conditions, harvesting time of the plant or the climatic conditions of the concerned region. In our study, it is shown that the ethanolic extract of Kilis origin fruits has a very high phenolic content. It is known that geographical location is also an influencing factor in terms of lipid, fatty acid and amino acid compositions. Furthermore, it has been reported that the protein level in okra in Turkey is higher compared to the okras from other countries.

In our country, porridge prepared with okra fruits in the Southeastern Anatolia Region is used as a folk medicine, as a wound healing remedy in the treatment of abscesses in the skin and subcutaneous tissue, and also as an antispasmodic in gastrointestinal system disorders. In the literature, antioxidant and antiproliferative activities of okra species have been shown as well as antibacterial activities thereof, but no detailed scientific study has been conducted on its effect on wound healing which is the subject matter of the present invention. Considering its use as a folk medicine, it is predicted that okra may yield a good wound healing agent. There is no wound healing formulation produced with okra grown in Turkey. For this reason, a gel formulation that is easier to use and less costly than the reference ointments in the market has been developed by using okra grown in Turkey as a local source. In addition, the fact that the developed formulation has a gel structure provides an advantage in terms of ease of use, as it is a factor that increases patient compliance.

The study entitled "EVALUATION OF WOUND HEALING ACTIVITY OF ABELMOSCHUS ESCULENTUS (LINN) IN ALBINO WISTAR RATS" conducted by Farooqui M. B. et ak, one of the known applications in the art, discloses an evaluation of wound healing activity of Abelmoschus esculentus (okra). Within the scope of this study, the wound healing potential of the aqueous extract of Abelmoschus esculentus (okra) fruit as a result of being applied to the wound in ointment form (200 mg/kg/day) is examined upon comparing it with Framycetin (1% w/w). In that study, wound healing was examined only on the percentage of wound closure, and the details of this examination were not specified. In the present study, the effect of aqueous and alcoholic extracts of Abelmoschus esculentus (okra) on wound healing has been demonstrated by evaluating the percentage of wound closure, as well as histopathological features such as vascularization, fibroblastic activity, presence of inflammatory cells, collagenisation, and many other parameters in serum and tissue samples taken from animals. It was distinctly seen with the 5% alcoholic extract that with the anti-inflammatory activity, epithelization was provided in the scar tissue, and the granulation tissue matured. It is clearly seen that our study is more reliable for the intended effect, as the reference substance used to compare wound healing and the activity is confirmed by different parameters. In addition, no studies have been conducted on the characterization of the ointment formulation produced in the said study. Only a mixture prepared to be tested in in vivo experiments was used, and when the said study is examined, it will be seen that there is no formulation development.

The Chinese patent application document no. CN107362283, an application known in the art, discloses a traditional Chinese medicine for treating fractures and traumatic injuries. One of the ingredients of the Chinese medicine material of the said invention is okra.

The Chinese Patent application document no CN106074868, an application known in state of the art, discloses selenopolysaccharides immune enhancers. It is disclosed that the selenopolysaccharides immune enhancer of the said invention is prepared from 120-150 parts of selenopolysaccharides, 15-20 parts of okra extract, 20-35 parts of oil tea seed oil extract and 10-30 parts of propolis extract. It is stated that the said immune enhancer has various other effects, including enhancing wound healing. The study titled "Solid-Liquid Extraction Kinetics of Flavonoids from Okra {Abelmoschus esculentus L. Moench) Pods with Applicability" conducted by Zhou CS et al., one of the known applications in the art discloses the performance of the liquid-solid extraction kinetics of the flavonoids obtained from the pods of okra (. Abelmoschus esculentus L. Moench). In the preliminary explanation of the article, it is stated that okra is known to be used as a folk medicine to accelerate wound healing as well as its use as food.

Summary of the Invention

The objective of the present invention is to use the biocompatible formulation produced by extraction of the Okra ( Abelmoschus esculentus) fruit for wound healing purposes.

The objective of the present invention is to use the formulation produced by extraction of the Okra ( Abelmoschus esculentus) fruit which has antibacterial, antioxidant and antiproliferative activities, which are essential factors for wound healing purposes. Within this scope, it has been shown that the gel formulation prepared from aqueous and ethanolic extracts of okra fruit from Kilis region is effective in wound healing.

Detailed Description of the Invention

Within the scope of the present invention, the formulation produced by extraction of Okra (. Abelmoschus esculentus) fruit which has antibacterial, antioxidant and antiproliferative activities, is aimed to be used for wound healing purposes and the activity thereof is determined within the novel invention. Within the scope of the invention, the extract of okra fruit is discussed, and it is ensured to develop gel formulations from it and its wound healing activity. The gel formulation, which is especially considered within the scope of the invention, contains a solvent containing 5-10% by weight (g/g) of okra extract, and preferably water or ethanol is used as this solvent. In addition, since okra has antibacterial, antioxidant and antiproliferative activities, the gel formulation produced within the scope of the invention is used to have antibacterial, antioxidant, antiproliferative activities on the wound.

For wound healing purposes, during the development of the invention, the in vitro antioxidant activities of 2 different extracts prepared from okra fruits were determined and their phytochemical contents (phenolic, flavonoid and proanthocyanidin) were determined and clarified and also their antimicrobial activity were investigated. By means of the in vitro cell culture study, the cytotoxic effects of different concentrations of both extracts on HDF human skin fibroblast cell and RAW 264 macrophage cell line were investigated, and the effects of okra on both skin and immune system were analysed using both cell lines.

Within the scope of the invention, it was aimed to formulate a product in the form of a topical gel having 1% carbapol content with okra aqueous and ethanolic extract, and the activity of these formulations that were prepared was studied by comparing it with a reference product, which is used in the pharmaceutical market and all phase studies of which have been completed, and comparing in vivo wound healing accelerating activity thereof.

The formulation was prepared with okra aqueous and ethanolic extracts. 0.005 g/g benzalkonium chloride was used as the preservative, and the removal of bubbles in the gel was achieved via a sonicator. pH, viscosity, spreadability, gel tension and bioadhesion tests were carried out to ensure optimum gel structure and to investigate physicochemical properties. Table 1. Gel groups selected for the formulations

KS: Kilis Okra aqueous extract, KET : Kilis Okra ethanolic extract

According to this formulation, gels prepared from 5% okra aqueous and alcoholic extracts were applied to groups of experimental animals (rats) once a day at a dose of 0.5 g/day to cover the wounds as a thin layer. The wound healing process was monitored every day via a camera.

In addition to inflammation markers in serum samples, expressions of proinflammatory cytokines such as TNF-a, ILl-b, TGF-b and expression of EGF, which is an important marker in wound healing, in skin samples were evaluated, and the wound healing efficacy studies were evaluated through many different biochemical and molecular mechanisms. Wound healing was also evaluated histologically in tissues. In addition, in vitro Epiderm TM reconstituted skin irritation test (SIT) was performed on the developed formulation product within the scope of advanced preclinical trials.

The experimental studies conducted within the scope of the invention and the histopathological analyses showed that collagenisation and granulation tissue maturation were better in the reference and the 5% ethanolic extract groups. In accordance with the biochemical, histopathological findings and gene expression analysis results obtained from in vivo wound healing model, the okra ethanolic extract was found to be highly effective on wound healing. It was determined that the ethanolic extract at a concentration of 30 mg/ml was effective in 15 of the 18 strains that were studied. There are many wound healing products used in the market today. It is known that many of these products are insufficient, especially in healing deep wounds such as bedsores. There is no gel carrier system that is widely used as a wound healer today. The commercially available products used for wound healing are mostly products developed with conventional carrier systems such as creams and ointments. Among these products, an ointment with herbal content was chosen as a reference product in our experimental animal studies. Lack of moisture in the wound bed adversely affects the wound healing process. Our expectation from a good topical wound healing product is that it stays on the wound for a longer time and acts as a wound dressing, and at the same time releases the active ingredients that it contains to the wound bed. A natural gel is formed using the mucilage content in the fruits of the okra plant, which are used among the people in Turkey by swelling it in hot water. Thanks to this gel-like, bioadhesive structure and the wound healing ingredients therein, it covers the wound bed for a long time, clings to it and releases these ingredients to the wound environment. Within the scope of this invention, the formulation was designed by evaluating all these beneficial effects and natural structure of okra. Instead of ointment or other carrier systems, a gel carrier system suitable for the natural content of okra has been developed. Topical gel carrier systems are frequently preferred as wound care products. These systems preserve the moisture of the wound bed, allow oxygen permeation, fill the gap formed in the wound and can trap the excess formed in the wound bed. It also has advantages such as being bioadhesive and easy to apply by patients. Carbapol is used as a drug carrier system in many transdermal applications due to its advantages such as high viscosity, high compatibility with drugs, good thermal stability and tissue compatibility. Due to these properties, carbapol has been added to the formulation as an excipient.

Claims

1. Gel formulation used for wound healing purposes and comprises extraction of 5-10% by weight (g/g) of okra (Abelmoschus esculentus) fruit.

2. A gel formulation according to Claim 1, comprising water or ethanol as the solvent in extraction of the okra fruit.

3. A gel formulation according to Claim 1, comprising 1% carbapol.

4. A gel formulation according to Claim 1, comprising 0.5% Triethylamine.

5. A gel formulation according to Claim 1, comprising 0.005% preservative.

6. A gel formulation according to Claim 1, comprising benzalkonium chloride as the preservative.

7. A gel formulation according to Claim 1 comprising a sonicator for removing the gel bubbles.

8. A gel formulation according to Claim 1, which used to have antibacterial, antioxidant, antiproliferative activities on the wound.

9. A gel formulation according to Claim 1, which is topically used for the treatment of wounds.

10. A gel formulation according to any one of the preceding claims applied once a day to cover the wound as a thin layer.