Esra Küpeli Akkol

Investigations on the in vivo wound healing potential of Hypericum perforatum L.

ETHNOPHARMACOLOGICAL RELEVANCE Olive oil extract of the flowering aerial parts of Hypericum perforatum L. (Hypericaceae) is a popular folk remedy for the treatment of wounds in Turkey. AIM OF THE STUDY In order to prove the claimed utilization of the plant, the effects of the extracts and the fractions were investigated by using bioassay-guided procedures. For the wound healing activity assessment, in vivo excision and incision wound models were applied. For the anti-inflammatory activity, an in vivo model, based on the inhibition of acetic acid-induced increase in capillary permeability was used as well. Moreover, a parallel study was run on Hypericum scabrum L., which is a widespread species of the gender but not known as a folk remedy for wound healing, to provide a preliminary data to compare and emphasize the selection of correct plant species. RESULTS Initial investigations proved that the olive oil extract of Hypericum perforatum has a significant wound healing effect on excision (5.1-82.6% inhibition) and circular incision (20.2-100.0% inhibition) wound models. In order to determine the active wound healing ingredient(s), aerial parts of the plant was extracted with ethanol, noteworthy wound healing activity profile was observed with the wound models; between 18.3% and 95.6% in excision model and from 13.9% to 100.0% inhibitions in incision model were determined. The ethanolic extract was then submitted to successive solvent extractions with n-hexane, chloroform and ethyl acetate (EtOAc). Each solvent extract was also applied on the same wound models, consequently, EtOAc subextract was found to be the most active one by inhibiting wounds between 17.9% and 100.0% in excision model, subsequently between 9.4% and 100.0% in incision model. However, all subfractions obtained from the EtOAc subextract using Sephadex LH-20 column chromatography showed wound healing activity not more than the whole EtOAc subextract, which revealed that a possible synergistic activity that might be questioned. Among the active Sephadex fractions, Fr. A further yielded hyperoside, isoquercitrin, rutin and (-)-epicatechin and Fr. B yielded hypericin as the major components. Moreover, a dose-dependent anti-inflammatory activity was found for the ethanol extract, EtOAc subextract and Sephadex fractions of Hypericum perforatum. These results suggest that anti-inflammatory activity of the active fractions might have a contributory role in the wound healing effect of the plant. CONCLUSION Results of the present study have proved that aerial parts of Hypericum perforatum possess remarkable wound healing and anti-inflammatory activities supporting the folkloric assertion of the plant in Turkish folk medicine. Flavonoids [hyperoside, isoquercitrin, rutin and (-)-epicatechin] and naphthoquinones (hypericins) were found as the active components of Hypericum perforatum. On the other hand, ethanol extract of Hypericum scabrum showed neither remarkable wound healing nor anti-inflammatory activity demonstrating the importance of correct plant species selection in therapeutic applications.

Phenolic composition and biological activities of Salvia halophila and Salvia virgata from Turkey

The aerial parts of Salvia halophila and Salvia virgata were subjected to Soxhlet extraction with different solvents such as n-hexane, ethyl acetate, methanol, and aqueous methanol (50%). Plants were also extracted with water under reflux. The effects of the extracts were studied in p-benzoquinone-induced abdominal constriction test for the assessment of antinociceptive activity and carrageenan-induced hind paw edema and 12-O-tetradecanoyl-13-acetate (TPA)-induced ear edema models in mice for the anti-inflammatory activity. The extracts were analysed using a HPLC-PDA method. Results showed that methanol extract of S. virgata significantly inhibited carrageenan-induced paw edema and p-benzoquinone-induced abdominal constriction at 100mg/kg dose, while it showed no effect in the TPA-induced ear edema. On the other hand, the other extracts did not show any inhibitory antinociceptive and anti-inflammatory activities in these in vivo models. Rosmarinic acid was found as main constituent in the extracts, while caffeic acid and luteolin derivatives were also detected.

Exploring the wound healing activity of Arnebia densiflora (Nordm.) Ledeb. by in vivo models

ETHNOPHARMACOLOGICAL RELEVANCE The roots of Arnebia densiflora (Nordm.) Ledeb. (Boraginaceae), which is an endemic plant for Turkey, have been used for healing of wounds in folk medicine. The extract of the roots in olive oil and mostly the root barks was applied onto open wounds for rapid healing. AIM OF THE STUDY To evaluate the wound healing activity of the roots, extracts were prepared with different solvents; hexane, chloroform, ethyl acetate and methanol, respectively from the roots of Arnebia densiflora. MATERIALS AND METHODS Incision by using tensiometer and excision models were employed on mice and rats. RESULTS Significant wound healing activity was observed with the ointment formulation prepared by using hexane extract at 1% concentration on the mentioned models. The results of histopathological examination supported the outcome of both incision and excision wound models. The wound healing effect was comparatively evaluated with a reference ointment Madecassol. CONCLUSION The experimental data demonstrated that Arnebia densiflora displayed remarkable wound healing activity.

A novel wound healing ointment: a formulation of Hypericum perforatum oil and sage and oregano essential oils based on traditional Turkish knowledge.

AIM OF THE STUDY Hypericum perforatum L. (Hypericaceae), olive oil (Oleaceae), Origanum Tourn ex L. and Salvia L. species (Lamiaceae) are used against inflammatory disorders and for healing of skin wounds in traditional Turkish medicine. A new ointment formulation was developed to provide more efficient wound healing activity. The content of the formulation was as follows; olive oil extract of flowering aerial parts of Hypericum perforatum L., olive oil, an equivalent mixture of Origanum majorana L. and Origanum minutiflorum Schwrd. et Davis essential oils (Origani aetheroleum), Salvia triloba L. essential oil. The aim of the present study is to assess the wound healing potential of this new formulation by using in vivo and in vitro models as well as histopathological methods. MATERIALS AND METHODS For the evaluation of wound healing potential of this formulation (HPP crème mit Rotöl ointment) in vivo wound healing experimental models were employed on rats and mice and the efficiency was comparatively assessed against a reference ointment Madecassol(®). Tissue sections were also evaluated histopathologically. Furthermore, the wound healing activity of each component was also investigated individually to determine the improvement in the healing capacity of the formulation. RESULTS The ointments of HPP crème mit Rotöl and Hypericum perforatum L. demonstrated the highest activities on both wound models when compared to reference ointment Madecassol(®), while the other ingredients did not show any remarkable wound healing effect. However, the efficacy of the formulation was remarkably higher than the Hypericum L. ointment alone which was also confirmed by histopathological evaluation. On the other hand, the formulation did not reduce elastase activity in vitro, but inhibited the collagenase activity. HPP crème mit Rotöl also exerted bactericidal and candicidal activities. CONCLUSIONS The experimental studies revealed that HPP crème mit Rotöl formulation displays remarkable wound healing activity. To be acting on the different stages of wound healing process could be considered as a beneficial effect of the formulation for the treatment of wounds.

Bioassay-guided isolation of anti-inflammatory and antinociceptive principles from a folk remedy, Rhododendron ponticum L. leaves

ETHNOPHARMACOLOGICAL RELEVANCE Rhododendron ponticum L. (Ericaceae) is used for the treatment of inflammatory diseases and to alleviate rheumatic pain and against toothache in Turkish traditional medicine. AIM OF THE STUDY To evaluate the anti-inflammatory and antinociceptive effects of Rhododendron ponticum leaves using in vivo models, and isolation and chemical characterization of the biologically active constituents through bioassay-guided fractionation procedures. MATERIAL AND METHODS Carrageenan-induced hind paw edema model was used for anti-inflammatory activity and p-benzoquinone induced abdominal contractions model for the antinociceptive activity assessment. RESULTS The ethylacetate fraction displayed marked anti-inflammatory (28.4-40.7% inhibition) and antinociceptive (50.7% inhibition) effects as compared to reference compounds. Through bioassay-guided fractionation and isolation procedures flavonol glycosides [a mixture of hyperoside and isoquercitrin (1) and quercitrin (2)] along with one flavanone glycoside [6-C-glycosylnaringenin (3)] were isolated as the active ingredients of ethylacetate extract against carrageenan-induced edema and p-benzoquinone-induced writhes and their structures were elucidated by spectral techniques. 1 and 2 also showed a significant anti-inflammatory activity against 12-O-tetradecanoyl-13-acetate (TPA)- induced mouse ear edema model. CONCLUSION Results of the present study supported the utilization of the plant in Turkish folk medicine and revealed that flavones are the major anti-inflammatory and antinociceptive principles of the leaves.

Beneficial effects of Aesculus hippocastanum L. seed extract on the body's own antioxidant defense system on subacute administration.

AIM OF THE STUDY Seeds of Aesculus hippocastanum L. have long been used in European phytotherapy to treat inflammatory and vascular problems. In Turkish folk medicine, tea prepared from the crushed seeds was used to pass kidney stone and against stomach ache, while a fraction of seed was swallowed to alleviate hemorrhoids symptoms. In order to evaluate the in vivo effects of escin mixture from Aesculus hippocastanum seed on the blood and tissue antioxidant defense systems in standard pellet diet (SPD) and in high-fat diet (HFD) consumed male mice. MATERIALS AND METHODS Escin mixture was obtained from the ethanol extract of seeds. Escin mixture was administered orally to male mice fed either standard pellet diet (SPD) or high-fat diet (HFD) at 100mg/kg doses daily for 5 weeks and the tissue (liver, kidney and heart) and blood samples were collected at the end of experimental period. The effect of escin mixture on the plasma antioxidant activity; blood and tissue malondialdehyde (MDA) and reduced glutathione (GSH) levels; erythrocyte and tissue superoxide dismutase (SOD) and catalase activity (CAT) in SPD and HFD consumed animals were experimentally studied. RESULTS Escin mixture prohibited the adverse effects of oxidative stress and showed a protective effect on the liver architecture both in SPD and HFD consumed male mice. Escin mixture prohibited the adverse effects of oxidative stress and showed a protective effect on the liver architecture both in SPD and HFD consumed male mice. Combined administration of high-fat diet with escin mixture decreased blood (p<0.01), liver (p<0.01), kidney (p<0.05), and heart (p<0.05) of MDA, liver SOD (p<0.01) and CAT (p<0.05) levels and increased blood (p<0.01) and liver GSH (p<0.001) levels in mice. CONCLUSION The present results indicate that Aesculus hippocastanum increase the antioxidative defense system of the body and prevent HFD-induced lipid peroxidation in male mice.

Efficacy of Daphne oleoides subsp. kurdica used for wound healing: identification of active compounds through bioassay guided isolation technique.

ETHNOPHARMACOLOGICAL RELEVANCE In Turkish traditional medicine, the aerial parts of Daphne oleoides Schreber subsp. kurdica (DOK) have been used to treat malaria, rheumatism and for wound healing. The aim was to evaluate the ethnopharmacological usage of the plant using in vivo and in vitro pharmacological experimental models, and to perform bioassay-guided fractionation of the 85% methanolic extract of DOK for the isolation and identification of active wound-healing component(s) and to elucidate possible mechanism of the wound-healing activity. MATERIALS AND METHODS In vivo wound-healing activity was evaluated by the linear incision and the circular excision wound models. Anti-inflammatory and antioxidant activities, which are known to support the wound healing process, were also assessed by the Whittle method and the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assays, respectively. The total phenolic content of the extract and subextracts was estimated to establish any correlation between the phenolic content and the antioxidant activity. The methanolic extract of DOK was subjected to various chromatographic separation techniques leading to the isolation and identification of the active component(s). Furthermore, in vitro hyaluronidase, collagenase and elastase enzymes inhibitory activity assays were conducted on the active components to explore the activity pathways of the remedy. RESULTS After confirmation of the wound-healing activity, the methanolic extract was subjected to successive solvent partitioning using solvents of increasing polarity creating five subextracts. Each subextract was tested on the same biological activity model and the ethyl acetate (EtOAc) subextract had the highest activity. The EtOAc subextract was subjected to further chromatographic separation for the isolation of components 1, 2 and 3. The structures of these compounds were elucidated as daphnetin (1), demethyldaphnoretin 7-O-glucoside (2) and luteolin-7-O-glucoside (3). Further in vivo testing revealed that luteolin-7-O-glucoside was responsible for the wound-healing activity of the aerial parts. It was also found to exert significant anti-inflammatory, antioxidant, anti-hyaluronidase and anti-collagenase activities. CONCLUSION The present study explored the wound-healing potential of Daphne oleoides subsp. kurdica. Through bioassay-guided fractionation and isolation techniques, luteolin-7-O-glucoside was determined as the main active component of the aerial parts. This compound exerts its activity through inhibition of hyaluronidase and collagenase enzymes activity as well as interfering with the inflammatory stage.

Bioassay-guided isolation of anti-inflammatory, antinociceptive and wound healer glycosides from the flowers of Verbascum mucronatum Lam.

ETHNOPHARMACOLOGICAL RELEVANCE The leaves, flowers and whole aerial parts of Verbascum L. species have been used to treat respiratory problems, haemorrhoids and other types of inflammatory conditions in traditional Turkish medicine. AIM OF THE STUDY In order to evaluate this traditional information, the anti-inflammatory, antinociceptive and wound healing activities of Verbascum mucronatum Lam. which is used as haemostatic in Turkish folk medicine were investigated. MATERIALS AND METHODS In vivo inhibitory effect of the extracts on the carrageenan-induced hind paw edema model in mice was studied for the assessment of anti-inflammatory activity. Moreover, the wound healing potential of the plant were evaluated by using in vivo wound healing experimental models, i.e. incision and excision models on mice and rats, were comparatively assessed with a reference ointment Madecassol(®). Skin samples were also evaluated histopathologically. RESULTS The results of these experimental studies exhibited that Verbascum mucronatum displays anti-inflammatory, antinociceptive and wound healing activities. Through bioassay-guided fractionation and isolation procedures four iridoid glucosides, ajugol (1), aucubin (2), lasianthoside I (3), catalpol (4), two saponins, ilwensisaponin A (5) and C (6) and a phenylethanoid glycoside, verbascoside (7) were isolated and their structures were elucidated by spectral techniques. Verbascoside (7) was found to possess significant wound healing activity as well as antinociceptive and anti-inflammatory potentials, per os without inducing any apparent acute toxicity or gastric damage. CONCLUSION The experimental study revealed that Verbascum mucronatum displays remarkable antinociceptive, anti-inflammatory and wound healing activities.

Wound Healing Activity of Rubus sanctus Schreber (Rosaceae): Preclinical Study in Animal Models

Young shoots of Rubus species have been used for healing of wounds, infected insect bites and pimples in folk medicine for ages. In order to evaluate the wound healing activity of Rubus sanctus, four different extracts were prepared from the whole aerial parts of the plant by using n-hexane, chloroform, ethyl acetate and methanol, respectively. Incision wound healing model by using tensiometer on rats and excision model on mice were employed to assess the activity. Remarkable wound healing activity was observed with the ointment formulation of the methanol extract at 1% concentration on the mentioned models. The results of histopathological examination also supported the outcome of both incision and excision wound models. The wound healing effect was comparatively evaluated with a reference ointment Madecassol. The experimental data confirmed the ethnobotanical usage of R. sanctus.

In vivo anti-inflammatory and wound healing activities of Centaurea iberica Trev. ex Spreng

AIM OF THE STUDY Several Centaurea species (Asteraceae) are used in Turkish folk medicine to alleviate pain and inflammatory symptoms in rheumatoid arthritis, high fever, head ache and for wound healing. Particularly, the aerial part of Centaurea iberica Trev. ex Spreng. has been practiced on wounds for healing. In order to evaluate the anti-inflammatory and wound healing activities of the plant, extracts were prepared with variety of solvents: hexane, chloroform, ethyl acetate and aqueous methanol (85%) from the aerial parts of Centaurea iberica. MATERIALS AND METHODS The incision by using tensiometer and excision models were used in order to assess the effect of the plant extracts on wound healing in mice and rats. Results were also evaluated histopathologically. In vivo inhibitory effect of the extracts on acetic acid-induced increase in capillary permeability was studied for the assessment of anti-inflammatory activity. RESULTS The wound healing effect was comparatively evaluated with a reference ointment Madecassol. Noteworthy wound healing activity was observed for the ointment formulation prepared with 1% methanol extract. The results of histopathological evaluation supported the outcome of both incision and excision wound models. Moreover, the methanol extract exerted remarkable wound healing activity and also demonstrated a significant and dose-dependent anti-inflammatory activity. CONCLUSION The experimental study revealed that Centaurea iberica displays remarkable wound healing and anti-inflammatory activity.