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.     

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.     

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.     

Toxicity and potential anti-trypanosomal activity of ethanolic extract of Azadirachta indica (Maliacea) stem bark: An in vivo and in vitro approach using Trypanosoma brucei

Aim of the study

To determine the toxicity and anti-trypanosomal activity of the ethanolic extract of Azadirachta indica (Maliacea) stem bark, through in vivo and in vitro approach using Trypanosoma brucei brucei.

Materials and methods

Graded concentrations (100, 200, 400, 800, 1600 and 3200 mg/kg) of the crude stem bark ethanolic extract of Azadirachta indica, Hochst ex. A. Dc. (Maliacea) was tested for acute toxicity in 35 out bred Swiss (Wister) adult albino rats of both sexes. Secondly, the in vitro activity in test tubes and in vivo activity of the extract in 30 out bred Swiss (Wister) adult albino rats against Trypanosoma brucei brucei strain NITR/14 (Federe) was evaluated in a graded dose manner.

Results

The calculated intra-peritoneal LD₅₀ of the extract was 870 mg/kg and produced toxicity at high doses (>800 mg/kg). Graded concentrations of the ethanolic extract produced remarkable in vitro activity against Trypanosoma brucei brucei within seconds of inoculation. It also suppressed the establishment of parasitaemia at 100 mg/kg when administered simultaneously with infection in vivo. Similarly, at 200 and 400 mg/kg, the extract administered at the onset of parasitaemia for 4 consecutive days reduced parasitaemia, modulated declined packed volume (PCV) changes by day 48 post-infection in vivo.

Conclusion

The results confirm that the folkloric medicinal application of the extract of Azadirachta indica (Maliacea) has a pharmacological basis. Further investigation is however, needed to optimize the effectiveness of the extract.     

The traditional Hungarian medicinal plant Centaurea sadleriana Janka accelerates wound healing in rats

Ethnopharmacological relevance

The aerial parts of Centaurea sadleriana Janka, a species native to Hungary, have been used for the healing of wounds of livestock in Hungarian folk medicine. This is the first report of the ethnomedicinal use of this plant.

Aim of the study

This study was aimed at investigating the wound-healing efficiency of different extracts of Centaurea sadleriana.

Materials and methods

Experimental wounds inflicted on healthy rats by means of a branding iron were treated topically with different extracts and fractions of extracts of the aerial parts of Centaurea sadleriana. To assess the effectiveness of treatment, an absolute control (no treatment), a vehicle control (Carbomer gel) and a positive control group (1% salicylic acid in Carbomer gel) were applied.

Results

The n-hexane fraction of the methanol extract significantly accelerated the wound-healing process. This effect was rather similar to that of the positive control gel. Other fractions exhibited more moderate activities.

Conclusions

The apolar fraction of the methanol extract of Centaurea sadleriana facilitated wound healing significantly, corroborating the folk medicinal use of this plant.     

The essential oil of Croton zehntneri and trans-anethole improves cutaneous wound healing

Ethnopharmacology relevance

Croton zehntneri is a Euphorbiaceae species native to northeastern Brazil, where teas and beverages made from Croton zehntneri leaves are used as healing agents. To our knowledge, there is no experimental study supporting this claim of pharmacological activity.

Materials and methods

Full-thickness excisional wounds were made in the left and right sides of the dorsum of anesthetised Swiss mice, and a topical pharmaceutical formulation, developed by including essential oil extracted from the leaves of Croton zehntneri (2% and 20% EOCz) in pluronic-127 (PF-127), was administered to mice twice daily for 15 days post-wounding. To evaluate the contribution of trans-anethole, the major constituent of EOCz (85.7%), in the wound healing activity of EOCz, the effect of the topical administration of trans-AT on wound tissue repair was also evaluated and compared to other groups. A macroscopic analysis of swelling and exudates was performed and scored as 0 (missing), 1 (light), 2 (moderate) and 3 (intense). The number of capillaries and leukocytes was counted in hematoxylin and eosin (HE)-stained sections of the injured tissue. For extracellular matrix remodelling analysis, fibroblasts and collagen fibres present in the photomicrography of the Masson’s Trichrome (MT)-stained sections were counted. Each experimental group comprised six mice.

Results

At day 3 post-wounding, it was observed that treatment with 20% EOCz greatly reduced the swelling and exudates with a similar magnitude to the dexamethasone treatment. The inflammatory cell infiltration and angiogenesis were not altered by either the EOCz- or trans-AT treatments. In contrast, an acceleration of the wound closure was observed, with an enhanced number of fibroblasts and collagen fibres in both the 20% EOCz- and trans-AT-treated mice.

Conclusion

Our data indicate that EOCz exerts significant wound healing activity, demonstrating its relevant therapeutic potential.     

Evaluation of the wound healing property of Boesenbergia longiflora rhizomes

Ethnopharmacological relevance

The rhizomes of Boesenbergia longiflora (Wall.) Kuntze (Zingiberaceae) have been traditionally used for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess by decoction with alcohol.

Aim of the study

The rhizomes of Boesenbergia longiflora were carried out to investigate for anti-inflammatory and wound healing activities in order to support the traditional use.

Material and methods

The ethanolic extract of Boesenbergia longiflora and its fractions were tested using relevant in vitro anti-inflammatory and wound healing assays. For the in vitro studies, murine macrophage RAW264.7 cells and mouse fibroblast L929 cells were assessed for anti-inflammatory and fibroblast stimulatory activities, respectively. In vivo anti-inflammatory activity was determined by carrageenan-induced rat paw edema model as well as acute toxicity estimated by the up-and-down method in mice.

Results

The present study has demonstrated that the ethanolic extract of Boesenbergia longiflora rhizomes possesses a potent anti-inflammatory and wound healing activities. Among the isolated fractions, the CHCl₃ fraction showed potent anti-inflammatory effect through nitric oxide inhibitory activity (IC₅₀=5.5 μg/ml) and reduction of carrageenan-induced rat paw edema (ED₅₀=222.7 mg/kg), whereas this fraction exhibited wound healing property via fibroblast migration on both day 1 (77.3%) and day 2 (100%) as well as enhanced collagen production (187.5 μg/ml) at concentration of 3 μg/ml, compared to that of the controls, 39.4% for fibroblast and 60.8 μg/ml for collagen, respectively. The anti-inflammatory mechanism of the CHCl₃ fraction is found to suppress the iNOS and COX-2 mRNA expression.

Conclusion

The scientific investigation of wound healing activity of Boesenbergia longiflora rhizomes support the Thai traditional uses for treatment of inflammatory bowel disease, ulcerative colitis, aphthous ulcer and abscess. The EtOH extract and CHCl₃ fraction exert potential wound healing property through NO inhibition, anti-oxidant effect and stimulation of fibroblast migration and collagen production. The phytochemical screening revealed that the CHCl₃ fraction of Boesenbergia longiflora rhizomes contains diarylheptanoids, flavonoids and terpenes. The isolation of the compounds responsible for the wound healing effect is now in progress.     

Evaluation of wound healing activity of root of Mimosa pudica

Ethno pharmacological relevance

Mimosa pudica, commonly known as touch-me-not, is used in folklore medicine in arresting bleeding and in skin diseases.

Aim

There was no scientific evidence justifying the use of Mimosa pudica, therefore the present study was aimed at evaluation of wound healing activity of the plant.

Materials and methods

In the present study the roots of Mimosa pudica were studied for wound healing activity by incorporating the methanolic and the total aqueous extract in simple ointment base B.P. in concentration of 0.5% (w/w), 1% (w/w) and 2% (w/w). Wound healing activity was studied in three types of model in rats viz. excision, incision and estimation of biochemical parameter. In case of the excision wound model wound contraction and period of epithelization was studied while in incision wound model was evaluated by determining tensile strength and hydroxyproline content in the scab.

Results

Treatment of wound with ointment containing 2% (w/w) the methanolic and 2% (w/w) the total aqueous extract exhibited significant (P < 0.001) wound healing activity. The methanolic and total aqueous extracts were analyzed for total phenols content equivalent to Gallic acid. The content of total phenols was 11% (w/w) and 17% (w/w) in methanolic and total aqueous extract respectively.

Conclusion

The methanolic extract exhibited good wound healing activity probably due to phenols constituents.     

Anti-inflammatory,wound healing and in-vivo antioxidant properties of the leaves of Ficus amplissima Smith

Ethno pharmacological relevance

Ficus amplissima, commonly known as kal-itchchi, being used in folklore medicine, leaves juice is applied externally on old chronic wounds and the latex is applied on the wound.

Aim

There was no scientific evidence justifying the medicinal use of Ficus amplissima, therefore the present study was aimed at evaluation of anti-inflammatory, wound healing and in-vivo antioxidant activity of the plant.

Materials and methods

In the present study the acetone extract of Ficus amplissima leaves were studied for enzymatic antioxidant activity through different assays, anti-inflammatory by using carrageenan and histamine induced rat paw edema models (50 mg, 100 mg acetone extract) and wound healing activity by incorporating the two doses (1% (w/w) and 2% (w/w)) of acetone extract and simple ointment base B.P. in concentration of 0.5% (w/w) using excision and incision wound models in rats. In case of the excision wound model wound contraction and period of epithelization was studied while incision wound model was evaluated by determining tensile strength.

Results

Ficus amplissima leaves expressed the potent anti-inflammatory and in-vivo antioxidant activity where 100 mg acetone extract showed high activity. Treatment of wound with ointment containing 2% (w/w) acetone extract exhibited significant (P<0.001) wound healing activity.

Conclusion

The acetone extract of Ficus amplissima leaves exhibited better anti-inflammatory, wound healing and in-vivo antioxidant activity probably due to phenols constituents.     

A biochemical comparison of the in vivo effects of Bulbine frutescens and Bulbine natalensis on cutaneous wound healing

Aim of the study

In South Africa the local population relies extensively on indigenous plants in the formulation of traditional medicines to treat skin ailments. The scientific merits of many of these plants used to treat wounds and burns are yet to be validated. Bulbine natalensis and Bulbine frutescens of the Asphodelaceae family are indigenous to only southern Africa and are widely used as a skin remedy. This study aimed to explore the scientific value of these plants through investigating the in vivo biochemical effects of Bulbine natalensis and Bulbine frutescens on cutaneous wounds.

Material and methods

Excisional and incisional wounds treated with either B. natalensis or B. frutescens and mirrored control wounds were created on the back of 12 domestic pigs. Wound contraction was recorded daily. The excisional wounds, biopsied at days 2, 4, 7, 10 and 16, were used to analyse the biochemical composition of the wounds by estimating the total amount of protein, DNA, collagen and hexosamine present. The incisional wounds, biopsied at day 16, were used to test the tensile strength of the healed wounds using a tensiometer.

Results

Wound contraction following treatment with Bulbine natalensis on days 2, 4 and 10 (p = .004, 0.007 and 0.03, respectively), and Bulbine frutescens on day 4 (p = 0.004) increased significantly when compared to the corresponding untreated wounds. The tensile strength of the wounds treated with the leaf gels was significantly stronger than that of the untreated wounds. There was also a significant increase in the collagen, protein and DNA content of the Bulbine natalensis- and Bulbine frutescens-treated wounds compared with that of the untreated wounds (collagen content: p = 0.014 and 0.018; protein content: p = 0.03 and 0.04; DNA content p = 0.04 and 0.04; respectively) over the 16-day experimental period. Treatment with both leaf gels followed the same pattern in hexosamine content with a maximum hexosamine content on day 4 followed by a steady decrease to day 16. No significant difference between the hexosamine content of the wounds of animals treated with either Bulbine frutescens or Bulbine natalensis was found.

Conclusions

These findings validate the traditional use of the leaf gel extracts of B. frutescens and B. natalensis in the treatment of wounds and may warrant further investigation towards producing a low-cost effective topical treatment for wounds.     

Topical continuous use of Lippia sidoides Cham. essential oil induces cutaneous inflammatory response,but does not delay wound healing process

Ethnopharmacological relevance

The essential oil of Lippia sidoides (EOLS) has been used in Brazilian folk medicine as a topical antiseptic agent in skin for treatment of wounds and superficial infections of the body. The aim of this study was to investigate the effects of EOLS on intact and damaged skin, including its action on expression of mediators, COX-2 and VEGF, involved in healing full-thickness cutaneous lesions in vivo.

Material and methods

EOLS was analyzed chemically and used at different concentrations to dose-response experiments in skin mice. Skin irritation tests by one-dosage and multiple-dosages and irritation to damaged skin were assessed by macroscopy, morphometry and histological and immunohistochemical analyses. To evaluate the effects of EOLS on wound healing, excision wounds were surgically created on the dorsum of rats, and the ointments at 6% and 12% were applied daily to the wound area. Cutaneous lesions were assessed by planimetric (wound contraction) and macroscopic parameters.

Results

Skin irritation tests showed that topical application of EOLS promoted cutaneous inflammation in varying degrees, which was demonstrated by increase of skin thickness and formation of cutaneous edema and erythema. Topical administration of EOLS in high concentrations presented an irritant response to skin, but this irritation is lighter when low concentrations this oil were used. Histological evaluation supported the outcome of these models, which revealed accentuated presence of inflammatory cells infiltration. In wound healing process, the lesions treated with EOLS showed intense edema and exsudation up to day 5, but there were not significant differences in the wound contraction on days 14 and 21. No immunohistochemical staining was verified to COX-2 and VEGF mediators in skin treated with EOLS 12%.

Conclusion

The continuous application of EOLS in adequate concentrations on cutaneous wounds increases inflammatory response without delay the lesions closure. The association of these results with antimicrobial action previously related to EOLS allows its indication as an alternative therapeutic modality for topical treatment of infected cutaneous wound. Nevertheless, further studies need to be performed to determine the mechanism of action and support its application in clinical practice.     

Evaluation of the wound healing activity of methanol extract of Pedilanthus tithymaloides (L.) Poit leaf and its isolated active constituents in topical formulation

Ethnopharmacological relevance

Pedilanthus tithymaloides leaves are widely used in Indian medicine to heal wounds, burn, mouth ulcers. However, systematic evaluation of these activities is lacking. Thus, the present study aimed to assesses the wound healing activity of Pedilanthus leaves and its isolated constituents in topical ointment formulation.

Materials and Methods

Bioassay-guided chromatographic fractionation of the methanol extract of leaves resulted in the isolation of 2-(3,4-dihydroxy-phenyl)-5,7-dihydroxy-chromen-4-one and 1, 2-tetradecanediol, 1-(hydrogen sulfate), sodium salt. The ointment formulation of methanol extract (2.5%, 5% w/w) and isolated compounds (0.25% w/w) was prepared and evaluated on excision, incision and dead space wound models in rats. The effects of formulations on wound healing were assessed by the rate of wound closure, period of epithelialization, tensile strength, granulation tissue weight, hydroxyproline content and histopathology.

Results

Significant wound healing activity was observed with methanol extract and isolated constituents. Topical application of isolated compound ointments caused faster epithelialization, significant wound contraction (95.41%), and better tensile strength (565.33 g) on 16 post-wounding day, while 5% extract showed wound epithelialization with 95.55% contraction on 18th post-wounding day, better than the control group (76.39% on 22 day). The tensile strength of incision wound was significantly increased in extract and compound treated animals. Moreover, in dead space model the extract significantly increased granuloma tissue weight, tensile strength and hydroxyproline content. The tissue histology of ointment treated groups showed complete epithelialization with increased collagenation, compared to the povidone–iodine group.

Conclusions

The results validated the traditional use of Pedilanthus tithymaloides for cutaneous wound management.     

雄黄致体内外染色体畸变

目的:用仓鼠体内染色体畸变试验和中国仓鼠肺细胞CHL细胞染色体畸变试验评价雄黄的遗传毒性.方法:体内染色体畸变试验用毛足属仓鼠连续ig5 d给予33.25,66.5,133 mg·kg-1剂量雄黄悬浊液,处死前2 h ip秋水仙素.处死后取骨髓细胞制备染色体.油镜下观察每只动物骨髓细胞的有丝分裂指数和100个中期分裂相细胞的畸变类型.CHL细胞染色体畸变试验用雄黄浸出液终质量浓度为0.15,0.3,0.6g.L-1作用于CHL细胞,培养24 h或48 h,终止培养前4h加入秋水仙素.收获细胞,制备染色体,油镜下观察CHL细胞有丝分裂指数和200个中期分裂相细胞的畸变类型.结果:①雄黄265.0 mg· kg -1组和雄黄530.0 mg·kg-1组ig给药和雄黄浸出液(1.2,2.4g·L-1)作用于CHL细胞显示有明显的抑制有丝分裂作用.②与阴性对照组比较,雄黄ig给药仓鼠体内染色体畸变率和雄黄体外给药CHL细胞染色体畸变率均显著升高,差异有极显著意义,且有明显的量效关系.但体内试验的畸变率低于体外试验.③雄黄给药后CHL细胞染色体畸变试验中可见较多的染色体断片,而仓鼠体内染色体畸变试验中未发现,这可能与体内外药物作用的方式不同.结论:①雄黄能致体内外细胞染色体畸变,具有遗传毒性.②仓鼠体内染色体畸变试验可作为中药新药遗传毒性评价试验组合的方法之一.     

In vitro and in vivo antimicrobial activity of Algerian Hoggar Salvadora persica L. extracts against microbial strains from children's oral cavity

Ethnopharmacological relevance

Salvadora persica L. (miswak) is traditionally used to ensure oral hygiene Muslim people in developing countries where it is growing. The antibacterial properties of Salvadora persica L. originating from various geographic areas have already been reported. However, they have never been tested for samples originating from Hoggar, where extreme weather conditions could lead to different properties for this Salvadora persica L. ecotype.

Aim of the study

To assess the in vitro and in vivo antimicrobial activities of methanolic extract of Algerian Hoggar Salvadora persica L. (miswak) on some isolated and identified strains from the oral cavity of school children aged from 6 to 12 with (n=20) and without (n=20) caries.

Materials and methods

After a qualitative and quantitative analysis of dental plaque samples from the selected children, the effect of methanolic extract of Hoggar miswak against oral bacterial and fungal strains isolated from the oral cavity of children with caries was tested by both agar disc diffusion and microdilution methods. The stability and physicochemical parameters of Hoggar Salvadora persica L. mouthwash were also assessed compared. The in vivo antimicrobial effect of Hoggar miswak rinse on dental plaque samples was also tested over a week.

Results

Four bacterial genera (Staphylococcus, Streptococcus, Escherichia and Lactobacillus) were commonly identified in all subjects at different colonization levels. A statistically significant difference in colonization levels between the two groups of children was recorded. Hoggar miswak extract more significantly inhibited the growth of Gram negative bacteria from the dental plaque than Gram positive ones. Hoggar Salvadora persica L. mouthwash was stable at 4 °C and 25 °C over the period of conservation (one week) while a temperature of 40 °C induced variations in the physicochemical parameters and considered not suitable for preservation. The in vivo study revealed a significant reduction in bacteria of the oral cavity using miswak mouthwash as compared to placebo.

Conclusion

Hoggar miswak extract displayed a strong antimicrobial effect both in vitro and in vivo. Its use as a mouthrinse could therefore be recommended as a preventive measure to preserve from tooth decay.     

Comparative assessment of dermal wound healing potentials of various Trifolium L. extracts and determination of their isoflavone contents as potential active ingredients

Ethnopharmacological relevance

Trifolium species are used in Turkish folk medicine as a wound healing agent, expectorant, antiseptic, sedative and to alleviate pain in rheumatism. In the present study, the aqueous methanolic extracts (80%) of 13 Trifolium species (Trifolium ambigum, Trifolium arvense var. arvense, Trifolium campestre, Trifolium canescens, Trifolium hybridum var. anatolicum, Trifolium hybridum var. hybridum, Trifolium pannonicum, Trifolium pratense var. pratense, Trifolium purpureum var. purpureum, Trifolium repens var. repens, Trifolium resupinatum var. microcephalum, Trifolium spadiceum and Trifolium trichocephalum) collected from different regions of Anatolia were evaluated for their in vivo wound healing effects.

Materials and methods

In vivo wound healing activities of the plant aqueous methanolic extracts were evaluated by linear incision and circular excision wound models subsequent to histopathological analysis. Active constituents were determined by a validated high performance liquid chromatographic method. Precision of the method was performed by the evaluation of intra-day and inter-day variations of the each standard at limits of quantification (LOQ) levels.

Results

The aqueous methanolic extracts of Trifolium canescens and Trifolium pretense var. pratense possessed better wound healing activity compared to the other extracts and control groups. The animal groups treated with the Trifolium canescens extract demonstrated increased contraction (48.96%) on excision and a significant increase in wound tensile strength (35.6%) on incision models. The main compounds were detected as genistein and biochanin A for Trifolium canescens.

Conclusion

The results of the present study revealed the wound healing potential of Trifolium canescens. This might be due to the combined effect of the isoflavones genistein, formononetin, daidzein, and biochanin A present in the extract.     

Evaluation of toxicity of Calophyllum brasiliense stem bark extract by in vivo and in vitro assays

Ethnopharmacological relevance

Calophyllum brasiliense Camb., Clusiaceae, is commonly known as “guanandi” and its stem bark is used in Brazilian traditional medicine to treat rheumatism, vein problems, hemorrhoids and gastric ulcers. The aim of this study was to evaluate the toxicity of hexane extract of Calophyllum brasiliense stem bark (HECb) using in vitro and in vivo experimental models.

Materials and methods

In vitro toxicity was evaluated by Alamar Blue cytotoxicity assay and micronucleus test, using Chinese hamster ovary (CHO-k1) epithelial cells. in vivo toxicity was evaluated by oral acute and subchronic toxicity assays. In the oral acute toxicity screening, a single dose of HECb was administered to mice at doses ranging from 250 to 1000 mg/kg. In the subchronic study, HECb was administered orally for 30 days to Wistar rats at doses of 100 mg/kg and 500 mg/kg. Phytochemical analyses were performed by HPLC/UV–vis, secondary metabolites were quantified by spectrophotometric methods.

Results

HECb presented IC₅₀=119.94±4.31 µg/mL after a 24 h cytotoxicity test using CHO-k1 cells, showing low cytotoxicity. However, when the cells were exposed to HECb for 72 h, the IC₅₀ value was 8.39±2.00 µg/mL, showing in this case, a pronounced cytotoxic effect. In the oral acute toxicity studies, doses up to 500 mg/kg of HECb did not cause any changes in both male and female mice. At 1000 mg/kg, male mice showed signs typical of depression and stimulation that were reversed at 72 h. Besides, female mice were more sensitive to the toxic effect of HECb at 1000 mg/kg, which initially presented typical agitation signals, followed by depression signals, leading to death of all the animals at 24 h. In subchronic assay with rats, HECb administered orally at doses of 100 and 500 mg/kg did not cause significant changes in all clinical parameters evaluated. Histopathological analyses showed no deleterious effect in the vital organs of rats. Preliminary phytochemical analysis revealed the presence of phenolic compounds, steroids, and volatile coumarins. Analysis by HPLC showed two major peaks characteristic of chromanones.

Conclusions

In vitro toxicological tests showed that HECb exhibited cytotoxicity especially after 72 h of exposition, and mutagenicity on the highest tested dose. The in vivo studies demonstrated that HECb produced some toxicity signs at the highest dose tested, particularly, in the acute toxicity test but showed no significant signs of toxicity in the subchronic assay. Based on these and previous pharmacological studies, it is possible to say that HECb did not exhibit significant toxicity at its effective dose. This suggests that HECb is relatively safe in humans at its effective dose.     

In vivo wound healing activity and phytochemical screening of the crude extract and various fractions of Kalanchoe petitiana A. Rich (Crassulaceae) leaves in mice

Ethnopharmacological relevance

The leaves of Kalanchoe petitiana A. Rich (Crassulaceae) are used in Ethiopian folk medicine for treatment of evil eye, fractured surface for bone setting and several skin disorders including for the treatment of sores, boils, and malignant wounds.

Aim of the study

In order to scientifically prove the claimed utilization of the plant, the effects of the extracts and the fractions were investigated using in vivo excision, incision and dead space wound models.

Materials and method

Mice were used for wound healing study, while rats and rabbit were used for skin irritation test. For studying healing activity 80% methanolic extract and the fractions were formulated in strength of 5% and 10%, either as ointment (hydroalcoholic extract, aqueous and methanol fractions) or gel (chloroform fraction). Oral administration of the crude extract was used for dead space model. Negative controls were treated either with simple ointment or sodium carboxyl methyl cellulose xerogel, while positive controls with nitrofurazone (0.2 w/v) skin ointment. Negative controls for dead space model were treated with 1% carboxy methyl cellulose. Parameters, including rate of wound contraction, period of complete epithelializtion, hydroxyproline contents and skin breaking strength were evaluated.

Results

Significant wound healing activity was observed with ointment formulated from the crude extract at both 5% and 10% concentration (p<0.01) compared to controls in both excision and incision models. In dead space model, 600 mg/kg (p<0.01) but not 300 mg/kg significantly increased hydroxyproline content. Fractions showed variable effect, with the chloroform fraction lacking any significant effect. Both 5% and 10% formulations of the aqueous and methanolic fractions significantly increased wound contraction, decreased epithelializtion time and increased hydroxyproline content in excision wound model (p<0.05) as compared to controls. These fractions were also endowed with higher skin breaking strength in incision wound model (p<0.01).

Conclusions

The present study provided evidence that the leaves of Kalanchoe petitiana A. Rich possess remarkable wound healing activities supporting the folkloric assertion of the plant. Fractionation revealed that polar or semi polar compound may play vital role, as both aqueous and methanolic fractions were endowed with wound healing activity.     

Evaluation of wound healing properties of Arrabidaea chica Verlot extract

ETHNOPHARMACOLOGICAL RELEVANCE: Arrabidaea chica Verlot. (Bignoniaceae), popularly known as Crajiru, has been traditionally used as wound healing agent. AIM OF THE STUDY: Investigate in vitro and in vivo healing properties of Arrabidaea chica leaves extract (AC). MATERIALS AND METHODS: AC was evaluated in vitro in fibroblast growth stimulation (0.25-250 microg/mL) and collagen production stimulation (250 microg/mL) assays. Allantoin (0.25-250 microg/mL) and vitamin C (25 microg/mL) were used as controls respectively. DPPH and Folin-Ciocalteau assays were used for antioxidant evaluation, using trolox (0.25-250 microg/mL) as reference antioxidant. To study wound healing properties in rats, AC (100mg/mL, 200 microL/wound/day) was topically administered during 10 days and wound area was evaluated every day. Allantoin (100mg/mL, 200 microL/wound/day) was used as standard drug. After treatment, wound sites were removed for histopathological analysis and total collagen determination. RESULTS: AC stimulated fibroblast growth in a concentration dependent way (EC50=30 microg/mL), increased in vitro collagen production and demonstrated moderate antioxidant capacity. In vivo, AC reduced wound size in 96%, whereas saline group showed only 36% wound healing. CONCLUSION: AC efficiency seems to involve fibroblast growing stimulus and collagen synthesis both in vitro and in vivo, beyond moderate scavenging activity, corroborating Crajiru folk use.     

正交试验法优选了哥王中西瑞香素的提取工艺

目的:优选了哥王中西瑞香素的提取工艺.方法:采用单因素及正交试验,以西瑞香素含量为指标,考察超声功率、甲醇用量、提取时间、提取次数4个因素对提取工艺的影响,优选了哥王中西瑞香素的提取条件.结果:了哥王中西瑞香素的最佳提取工艺为16倍量甲醇超声提取2次,每次1.5h,超声功率160 W.结论:提取工艺简单合理可行,可用于了哥王中西瑞香素的提取.