WO2015009260A2

WO2015009260A2 - A wound healing herbal product

Info

Publication number: WO2015009260A2
Application number: PCT/TR2014/000273
Authority: WO
Inventors: Fikrettin Sahin; Selami DEMIRCI; Aysegul DOGAN; Serpil DEMIRCI
Original assignee: Yeditepe Universitesi
Priority date: 2013-07-16
Filing date: 2014-07-16
Publication date: 2015-01-22
Also published as: WO2015009260A3; WO2015009260A9

Abstract

In the embodiment of the invention, a product which enables wound healing and accelerates cell division and veining is obtained by using the plant extract obtained from Verbascum speciosum leaves via extraction. The obtained product can be applied to the cells of L929 (Rat Fibroplast Cell Line), HF (Human Dermal Fibroplast Cell Line) and HUVEC (Human Umbilical Vein Endothelial Cells) which is human endothelial cell line.

Description

DESCRIPTION

A WOUND HEALING HERBAL PRODUCT

Field of the Invention

The present invention relates to an herbal product which enables wound healing and accelerates cell division and veining.

Background of the Invention

Wound healing is the response given by the body after loss of soft or hard tissue integrity. It is generally comprised of five steps, namely inflammation, neovascularization, granulation, re-epithelialization and remodeling, which are interdependent and overlapping. Although wound healing normally does not require special care, it poses serious problems in chronic wounds observed in 45 out of every 10000 people (Sasidharan et al., 2010). Even though important drugs have been discovered in pharmaceutical drug industry for wound healing, their clinical applications have not reached the desired level yet due to their high costs, low accessibility and side effects. Thus, drugs developed from herbal products have become an area of attraction for scientists since they are reliable, clinically effective, inexpensive and easily tolerable by the patients (Balekar et al, 2012).

Plants and formulations obtained from plants have been used for treatment of wounds, cuts and burns since antique ages even though their activity mechanisms were unknown. It was determined that the biologically active molecules (tannins, triterpenoids and alkaloids) in the plant extracts affect at least one of the steps of wound healing (Kumar et al., 2007; Nayak and Pereira, 2006). Thus, isolating these components in the plant extracts and making them into formulations have become a requirement of modern medicine. In this sense, Verbascum species are one of the plants that are widely studied. 196 out of 232 Verbascum species in the world are grown only in the flora of Turkey. (Kozan et al., 20 1). It was determined that the said plant species includes molecules important for the pharmaceutical industry such as flavonoids, phenylethanoids, neolignan glycosides, and saponins (Tatli et al., 2011). Different parts, such as roots, leaves and flowers of Verbascum species have been used for treatment of many diseases. It was stated that Verbascum thapsus, the most famous member of the said family, can be used for treatment of cancer (Zhao et al., 2011), viral infection (Escobar et al., 2012), flue and dysentery (Ali et al., 2012). In addition, it was claimed that other species of this family can be used for treatment of hemorrhoid, rheumatic pains, fungal infections (Tatli et al., 2011). No wound healing property of Verbascum speciosum has been disclosed before. It was stated that it can be used for treatment of mycodermatitis (Altundag and Ozturk, 2011).

The International patent document no. WO2012059926 discloses one or more plant extracts for treating burns and soft tissue wounds.

Summary of the Invention

An objective of the present invention is to provide an herbal product which enables to accelerate wound healing.

Another objective of the present invention is to provide an herbal product which enables to eliminate the poor living conditions and the pain resulting from prolongation of wound treatment process.

A further objective of the present invention is to provide an herbal product which enables to increase angiogenesis which is one of the steps of wound healing. Another objective of the present invention is to provide an herbal product which enables to increase fibroplast proliferation.

A further objective of the present invention is to provide an herbal product which enables to increase the level of extracellular matrix and cytoskeleton proteins such as collagen and vimentin.

Detailed Description of the Invention "A wound healing herbal product" developed to fulfill the objectives of the present invention is illustrated in the accompanying figures wherein

Figure 1 is the view of the results of cell viability after application of the plant extract (A) L929, B) HUVEC, C) HF (nc: negative control).

Figure 2 is the view of in vitro Matrigel tube formation (A) formation of tubelike structures after a 7 hour incubation (B) total number of branching (Magnifying scale: ΙΟΟμπι, C, Control; E: Plant extract *p < 0.05). Figure 3 is the view of wound healing after application of the plant extract and the levels of apoptic genes in L929 cells. When increasing (A) collagen type I gene expression, it caused decrease in the levels of (B) caspase 3, (C) p53 gene (C: Control; E: Plant extract, *p < 0.05). Experimental Study

In the embodiment of the invention, Verbascum speciosum leaves were used for obtaining the plant extract. These leaves were collected from Trabzon (Akcaabat) region in Turkey in autumn 2011. The collected leaves were washed and dried at room temperature in a sunless area. The dried leaves were crushed by using a grinder. Extraction of 15 grams of ground leaves was performed by soxhlet extractor (ISOPAD, Heidelberg, Germany) using 500 ml of methanol. Extraction was performed at room temperature for 48 hours. The resulting extract was filtered with a filter paper. Then, 80% of methanol was evaporated at 40 °C by using a rotary evaporator. The remaining part was added distilled water and was lyophilized by being frozen to -80 °C. This way the product of the present invention was obtained. The obtained product was experimentally applied to the cells of L929 (Rat Fibroplast Cell Line), HF (Human Dermal Fibroplast Cell Line) and HUVEC (Human Umbilical Vein Endothelial Cells) which is human endothelial cell line.

In the experimental studies that were conducted, different concentrations of V. speciosum methanol extract varying between 10μg/ml and 2mg/ml were tested on L929, HF and HUVEC cells. It was determined that it was effective at concentrations up to 400 pg/ml.

Characterization Studies

Cell Viability Test

The cells of L929 (Rat Fibroplast Cell Line), HF (Human Dermal Fibroplast Cell Line) and HUVEC (Human Umbilical Vein Endothelial Cells), which is human endothelial cell line, were cultured at 37°C in Dulbecco's Modified Essential Medium (DMEM) comprising 10% Fetal Bovine Serum (FBS) and 1% penicillin- streptomycin-amphotericin (PSA) in a humid medium containing 5% C0₂. For cytotoxic analyses, the cells were seeded into 96-well cell culture plates such that there will be 5000 cells per well. 24 hours following the seeding, different concentrations (10μg/ml-2mg ml) of the plant extract prepared in the culture medium were applied on the cells and the 3-day effect of the plant extract on cell viability was analyzed by using MTS method.

In Vitro Scratch Wound Model

L929 and HF cells were seeded into 12-well cell culture plates such that there will be 100,000 cells per well. The cells were cultured at 37 °C for 24 hours in a humid medium containing 5% C0₂ and cell adhesion was attained. A scratch was formed on the single layer cell culture by using the tip of a 200μ1 sterile micropipette. The medium was immediately replaced with the fresh medium containing 200μg/ml V. speciosum plant extract and thereby the removed cells were prevented from adhering back. Cicatrization rates were analyzed for different groups under microscope using NIH image J software at 0th, 12th and 24^th hours. In Vitro Tube Formation Method

Tube formation method was performed in matrigel coated 24-well cell culture plates as mentioned before in the literature (Yalvac et al., 2009). Generally, after the cell culture plates were coated with matrigel dissolved on ice, they were incubated at 37 °C for 30 minutes in order to enable polymerization of matrigel. HUVEC cells were added to matrigel coated wells in the presence of 200μ^ηι1 of plant extract such that it will be 10⁶ cells/ml. The branchings at five regions randomly selected at the end of 7 hours of incubation at 37 C in a humid medium containing 5% C0₂.were counted and analyzed under the microscope.

Real Time PZR analysis The primers required for PZR analysis of Collagen Type I, caspase3 and p53 genes were designed by using Primer-BLAST software of the Biotechnology National Center (Table 1). β-actin sequence was used as it was previously synthesized in the literature (Wilkins et al., 2009). Total RNA isolation from the cells treated with V. speciosum extract was performed by using High Pure RNA-isolation kit (Roche, Indianapolis, IN). cDNA synthesis was performed by using High Fidelity cDNA- synthesis kit (Roche, Indianapolis, IN). The real time PZR method, performed by using SYBR Green method, was used for detecting mRNA levels of the mentioned genes.

Table 1 : Forward and reverse primers used in the study

Primer Forward (5'-3') Reverse (5'-3') bp

GAGGCGGTTGTAGAAGAGTTTCG

caspase3 TGGGGGAAGAGGCAGGTGCA 177

TG

CCATTGCTTGGGACGGCAAGG

p53 ACGCTTCCCTGGATTGGCAGCC 166

G

Collagen ATTGGTGGGATGTCTTCGTCTG

CCACGCATGAGCGGACGCTAA 150 Type I G

β-actin GACAGGATGCAGAAGGAGATTACT TGATCCACATCTGCTGGAAGGT 141

Statistical Analysis

The statistical differences were determined by using One Way Variance Analysis (ANOVA) on the data obtained in the experiments. p<0.05 values were accepted to be statistically significant.

Experimental Results The cell viability analyses, which were carried out for 3 days in order to determine the toxic effect of V. speciosum methanol extract on L929, HF and HUVEC cells at different concentrations varying between l(^g/ml and 2mg/ml, were determined by MTS method. While no cytotoxic effect was observed at concentrations up to 40(^g/ml, cytotoxic effect was observed at concentrations of 400μ§/ιη1 or above (Figure 1).

HUVEC cells were cultured on matrigel and the tubelike structures formed in the presence of the plant extract were counted with the purpose of finding out whether the V. speciosum methanol extract triggered angiogenesis which is an important step in wound healing. It was observed that 20(^g/ml of extract caused a significant increase in the number of the tubelike structures (Figure 2).

Collagen type I gene level having a significant role in wound healing and caspase3 and p53 gene levels showing apoptic state of the cells were analyzed by real time PZR. It was determined that 20(^g/ml of methanol extract significantly increased collagen type I gene level while significantly decreasing the caspase3 and p53 gene levels (Figure 3). Application of the Invention

To the herbal product composition of the present invention may be added separately or in combination antimicrobial, astringent, anesthetic, analgesic, vasoconstrictor, preservative, anti-inflammatory, antihistaminic, keratolytic, anticholinergic agent(s) or other wound healing agent(s). Activity of the invention can be developed and enhanced with the said agents.

The herbal product can be applied for partial or full-thickness burn wounds, sunburns, gangrene, ulcer wounds, cut wounds occurring as a result of surgical operations, traumatic or purulent wounds, varicosis, pathological cleft or hemorrhoids, bedsores and diabetic wounds.

The product of the invention can be produced in the form of a cream, ointment, solution, spray, foam, suppository, medicated pad, pastille, bandage, powder, suspension, film, strip, hard or soft gelatin capsule or tablet.

The invention of the present invention may contain herbal content at a therapeutically effective dose.

Claims

1. A product obtained by the extracts of wound healing plant leaves, comprising the steps of

- collecting the plant leaves,

- drying the collected leaves at room temperature in a sunless area,

- crushing the leaves in a grinder,

- extracting with methanol for 48 hours,

- filtering the plant extract with a filter paper,

- removing 80% of methanol at 40 °C by using a rotary evaporator,

- adding distilled water to the remaining part and lyophilizing by freezing it to -80 °C,

- obtaining the herbal product.

The herbal product according to Claim 1, characterized in that the plant leaves are the leaves of Verbascum speciosum plant comprising active ingredient.

The herbal product according to Claim 2, wherein antimicrobial, astringent, anesthetic, analgesic, vasoconstrictor, preservative, anti-inflammatory, antihistaminic, keratolytic, anticholinergic agent(s) or other wound healing agent(s) may be added separately or in combination in order to enhance activity.

The herbal product according to Claim 3, which may be in the form of a cream, ointment, solution, spray, foam, suppository, medicated pad, pastille, bandage, powder, suspension, film, strip, hard or soft gelatin capsule or tablet. The herbal product according to Claim 4, which can be applied for partial or full-thickness burn wounds, sunburns, gangrene, ulcer wounds, cut wounds occurring as a result of surgical operations, traumatic or purulent wounds, varicosis, pathological cleft or hemorrhoids, bedsores and diabetic wounds.

The herbal product according to Claim 5, which contains plant content at a therapeutically effective dose.

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