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Journal of Ethnopharmacology 109 (2007) 104–112

Evaluation of the wound healing effect of some Jordanian traditionalmedicinal plants formulated in Pluronic F127 using

mice (Mus musculus)

Enam A. Khalil a,∗, Fatma U. Afifi a, Maysa Al-Hussaini b

a Faculty of Pharmacy, University of Jordan, Amman, Jordanb Faculty of Medicine, University of Jordan, Amman, Jordan

Received 6 March 2006; received in revised form 15 June 2006; accepted 6 July 2006Available online 14 July 2006

bstract

The wound healing effect of the aqueous extracts of Inula viscosa, Ajuga chia, Rubia taenifolia and Parieteria diffusa, and the oil of Laurusobilis, dispersed in water, were examined. The 10% (w/w) Pluronic F127 (PF127) was added to the applied preparations, in order to modify thequeous extracts viscosity, and to stabilize the oil dispersion. A full thickness wound was made in the dorsal area of the mice. The wounds werereated with the different preparations with 12 h intervals for four times in two successive days. For 16 days, the wounds were visually observed,

hotographically documented and the wound area was measured. After day 16, the animals were sacrificed and the histology of the wound areaas examined. The best wound healing activity was observed with the extract of Inula viscosa, followed by Parieteria diffusa, Laurus nobilis,juga chia and the least active extract was that of Rubia taenifolia.2006 Elsevier Ireland Ltd. All rights reserved.

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eywords: Wound healing; Inula viscosa; Parieteria diffusa; Laurus nobilis; A

. Introduction

Since mediaeval times plants played an important role in theife of human, as the major source of food, as well as for the

aintenance and improvement of health and for the elimina-ion of the enemies. The World Health Organization (WHO)stimated that about 80% of the world’s population still reliesn plant-based medicines for their primary health care. This inact is a clear indication for the role of medicinal plants in theaintenance of health and treatment of diseases as therapeutic

lternatives throughout the world, still in the late 20th and early1st century (WHO, 2002).

Wound care can be traced back to early civilizations, andany of these treatments were based on the use of herbal reme-

ies. Approximately one-third of all traditional medicines in usere for the treatment of wounds and skin disorders, compared tonly 1–3% of modern drugs (Mantle et al., 2001).

∗ Corresponding author. Tel.: +962 6 5355000x2660; fax: +962 6 5339649.E-mail addresses: ekayoub@ju.edu.jo, enamkhalil@yahoo.com

E.A. Khalil).

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378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2006.07.010

hia; Rubia taenifolia; Pluronic F127

A survey of the ethnobotanical studies, carried out in Jor-an and Palestine, indicated the use of tens of plant speciesy the inhabitants of the area, especially by those habiting theural areas for wound healing purposes (Dafni et al., 1984; Al-halil, 1995; Oran and Al-Eisawi, 1998; Abu-Irmaileh and Afifi,000; Ali-Shtayeh et al., 2000). Some of these plants are usedn the management of fresh wounds, while others for the treat-

ent of chronic wounds. The common ways of the treatmentre either direct application of crushed fresh/dried plant on theound or repeated wash of the wound with a plant decoction.espite the wide spread use of local plants in wound healing,nly few of them has been investigated for their potential use-ulness using excision and incision wound models by rats (Abul-Basal, 2001; Rashed et al., 2003).Reports about medicinal plants affecting various phases of

he wound healing process, such as coagulation, inflammation,broplasia, collagenation, epithelization and wound contraction

re abundant in the scientific literature (Ulubelen et al., 1995;emmati and Mohammadian, 2000; Choi et al., 2001; Bairy,002). Still, one should keep in mind that plants have not onlyeneficial effects in promoting the healing process of wounds

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E.A. Khalil et al. / Journal of Eth

nd burns or protecting the skin from fungal and bacterial infec-ion or anti-tumor activity against skin cancer, they can benvolved in different allergic, photoallergic and irritant skin reac-ions (Mantle et al., 2001). Many traditional remedies are basedn systematic observations and methodologies and have beenime-tested but for many of them, scientific evidence is lacking.here are only few prospective randomized controlled trials thatave proved the clinical efficacy of these traditional wound heal-ng agents. The present study was designed to test the in vivoound healing activity of the aqueous extracts of four selectededicinal plants, namely; Ajuga chia Schreber (Labiatae), Inula

iscosa (L.) Ait. (syn. Dittrichia viscosa) (Compositae), Pari-teria diffusa Mert. et Koch (Urticaceae) and Rubia taenifolia’Urv. (Rubiaceae). In addition, the isolated oily fraction ofaurus nobilis L. (Lauraceae) is included in the experiments.

. Materials and methods

.1. Plant material

Ajuga chia, Inula viscosa, Parieteria diffusa and Rubia taeni-olia were collected from northern parts of the country duringarly summer 2004. Laurus nobilis seeds were collected fromhe campus of the University of Jordan in late summer 2004.he plant materials were identified by Prof. Dr. Barakat Abu-

rmaileh, Faculty of Agriculture, University of Jordan. Herbar-um specimens were kept at the Department of Pharmaceuticalciences, University of Jordan. All plant materials with thexception of Parieteria diffusa, used in the present study wereried prior submitting them to the extraction process.

.2. Plant extract preparation

About 10% (w/v) aqueous extracts from the dried aerial partsf Ajuga chia, Inula viscosa, and Rubia taenifolia were preparedy refluxing the fine powdered plants with tap water for half anour, keeping the decoction overnight at room temperature andubsequent filtration.

Parieteria diffusa aqueous extract was prepared using fresheaves following the same extraction process. Then solid PF127as dissolved in the filtrate in order to obtain the final 10% (w/w)F127 in the liquid extract to be applied on the created wound.

.3. Bay seed oil preparation

Bay seed oil was prepared by diethylether extraction of theoarsely powdered Laurus nobilis seeds and evaporation of theolvent at temperatures below 40 ◦C until a thick oily materialas obtained. To this oil, PF127 in water solution was added inrder to produce an aqueous dispersion containing 2% and 10%w/w) bay seed oil and PF127, respectively.

Pluronics are synthetic, non-ionic surfactants composedf poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene

xide) or (PEO–PPO–PEO) tri-block polymer, widely used inhe pharmaceutical formulations. In the present study, Pluronics used as a vehicle mainly due to its thermogelling propertyhere the vehicle viscosity increases after application to the

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rmacology 109 (2007) 104–112 105

ound area and preventing its loss by flowing. Pluronic, as aurfactant, also improved the dispersion of the plant extracts/oiln water.

.4. Thin layer chromatography (TLC) screening

Plant extracts were applied to pre-coated TLC silica gellates (silica gel 60F254, Fluka, Buchs., Switzerland), devel-ped in appropriate solvent systems and visualized (Wagnernd Bladt, 1996). Chromatograms were examined before andfter spraying under UV and daylight to detect the presencef alkaloids, anthraquinones, phenolics/flavonoids, coumarins,erpenoids and volatile oils.

.5. Animals

This study was conducted in the Experimental Animal Lab-ratory of the Faculty of Medicine, University of Jordan. Allnimals were housed, fed and treated in accordance with the in-ouse guidelines for animal protection. Male and female albinowiss mice (Mus musculus) of 2–3 months of age weighing of0–25 g were used. Animals were kept for 2 weeks to be accli-atized prior to the investigation. During this time they were

iven standard pellet diet and water ad libitum. Throughout thexperimentation period animals were kept single in cages.

.6. Wound induction

Twenty four hours before the beginning of the wound healingxperiments, the dorsal skin of the mice were shaved. In theext day and under chloralhydrate anesthesia (0.25 ml of 3.5%hloralhydrate solution was administered i.p.), a 7 mm × 7 mmull-thickness excision wound was created.

.7. Experimental protocol

After wound creation the mice were divided randomly intoeven groups, consisting of eight mice per group. Six groupsere treated immediately with either the vehicle (10%, w/wF127 in water) or the test solution/dispersion, while group 7as the untreated control group. Using insulin syringes, 0.1 mlf the test solution/dispersion or the vehicle was applied slowlyn the wound area, extending slightly outside the wound areao ensure inclusion of the wound edges. The presence of theluronic in the formula lead to the formation of a thin film of

he liquid extract covering the wound area. The wounds werereated with the different preparations with 12 h intervals forour times in two successive days. The first application was doneirectly after the wound induction. Throughout the experimen-ation period, the mice were observed twice daily and the woundimensions were measured on days 5, 9 and 12 and on day 16fter the last application. Also, optical photographs were takenrom the wound area using a professional Nikon 8700 cam-

ra at 15 cm distance from the wound and right angle to theound surface. At day 16 the experiment was terminated and

he wound area was removed from the surviving animals for his-ological examination. The excisional skin biopsies were fixed

106 E.A. Khalil et al. / Journal of Ethnopharmacology 109 (2007) 104–112

Table 1Wound areas measured after the last application of plant preparation

Wound area relative to the control ± S.D. (n)

5 days 9 days 12 days 16 days

Control 1.00 ± 0.15 (6) 1.00 ± 0.23 (6) 1.00 ± 0.35 (6) 1.00 ± 0.43 (6)Vehicle 1.06 ± 0.28 (7) 1.05 ± 0.35 (7) 0.92 ± 0.44 (6) 1.01 ± 0.57 (6)Inula viscosa 1.11 ± 0.18 (6) 1.08 ± 0.33 (6) 1.27 ± 0.15 (5) 0.54 ± 0.12 (5)Laurus nobilis 1.22 ± 0.33 (7) 1.04 ± 0.23 (7) 1.19 ± 0.37 (7) 0.56 ± 0.30 (7)Parieteria diffusa 1.05 ± 0.38 (7) 1.08 ± 0.41 (7) 1.23 ± 0.51 (6) 0.68 ± 0.25 (6)Ajuga chia 0.89 ± 0.21 (6) 1.07 ± 0.18 (5) 1.12 ± 0.52 (4) 1.20 ± 0.70 (4)Rubia taenifolia 1.30 ± 0.39 (6) 1.38 ± 0.47 (6) 1.40 ± 0.64 (6) 1.52 ± 0.71 (6)

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he relative wound area is calculated by dividing each wound area by the avera

n 4% neutral buffered formaldehyde solution for 24 h. Duringhe experimentation period, two animals treated with Ajuga chiand two animals from the vehicle group died.

Cross sections through the longitudinal aspect of the scarredreas were made. The tissue was processed in the routine wayor histological evaluation and embedded in paraffin. Five-icrometer thick sections were stained with hematoxylin and

osin, the routine stain used in the histopathology, and rec-mmended as a general survey stain. Additional sections weretained with Masson Trichrome for the assessment of collagenontent and maturation within the dermis.

Tissue samples were evaluated for the following histologicalriteria; the extent of re-epithelization, the maturation and orga-ization of the epidermal squamous cells, the thickness of theranular cell layer, the degree of granulation tissue formation,nd for collagenisation and scar formation in the dermis. For thepidermal changes, the adjacent non-traumatized skin serveds the internal control for the assessment of epidermal matu-ation. For the dermis; the presence of disorganized collagenbers, prominent vascularity and inflammation considered as an

ndication for immature granulation tissue. Contrary, the pres-nce of well formed and horizontally-oriented collagen fibers,s evident by Masson Trichrome stain, along with scarce inflam-atory cells and inconspicuous blood vessels were considered

s an indication for the maturity of the scar.The different animal groups were assessed blindly by the

athologist and the results were compared with the vehicle andontrol groups.

.8. Statistical analysis

The relative wound area results were compared using one-ay analysis of variance (ANOVA). The results were considered

ignificantly different at P < 0.05. All tests were conducted usingigmaStat Version 3.10 (SPSS Inc., Chicago, IL).

. Results

.1. Morphological evaluation

During the wound healing period and at the preset time inter-als, the wound area was traced manually and was photographed.

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ntrol wound area measured at the same day.

he wound area was calculated using Motic Image plus version.0 software (Motic China Group Co. Ltd.). The relative woundrea was calculated for each animal by dividing the woundrea for that animal by the average area of the control. Thisalculation was repeated for each plant at each measurementay. The average wound areas together with the standard devi-tions for each plant are listed in Table 1. Representative sets ofhotographs taken on days 5 and 16 are shown in Fig. 1a and.

.2. Histological evaluation

Histological evaluation was carried out for the treated andntreated samples. Comparison between control and somereated animals is shown in Figs. 2 and 3. The best resultsere obtained with Inula viscosa (Fig. 2C and D), when

ompared to the other groups as well as to the control andhe vehicle (Fig. 2A and B). There was full thickness re-pithelization, in which the epidermis was thin and well orga-ized, comparable to the adjacent normal skin not involved inhe wound generation and the healing process. The granularayer was well-formed and one cell in thickness. The keratinayer was thin, composed of orthokeratin. The underlying der-is was occupied by hypocellular collagen, with horizontally-

riented fibers in the absence of inflammation or prominentascularity. The animals treated with Ajuga chia (Fig. 2E and), Parieteria diffusa, and Laurus nobilis showed comparableesults when compared to one another and when the differ-nt groups were compared with the control and the vehicle.here was full thickness epidermal regeneration which cov-red completely the wound area. The epidermis was thicknd disorganized, especially when compared with the adja-ent normal skin. The keratin layer was thick with focalarakeratosis. In all three groups, the granular layer was wellormed but of 2–3 cells in thickness. The basal layer wasell formed in the different groups as it was in the con-

rol and the vehicle groups. In the dermis, maturation andrganization of the collagen fibers as judged by the Mas-

on Trichrome stain was similar. The dermis was cellular,ith proliferation of fibroblasts, laying down disorganized

nd poorly oriented collagen fibers. Prominent capillary-sizedlood vessels were seen. Scattered collections of inflamma-

E.A. Khalil et al. / Journal of Ethnopharmacology 109 (2007) 104–112 107

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ory cells, consisting mainly of macrophages and few neu-rophils were also present through out the whole thickness of theermis.

Animals treated with the plant Rubia taenifolia (Fig. 3),howed the least favorable results. In two animals, the area ofhe wound persisted with minimal re-epithelization. In one ani-

al failure of re-epithelization with persistence of the wound

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rea was seen. In the dermis, the granulation tissue was imma-ure with few haphazardly-oriented collagen fibers, as detectedy Masson Trichrome stain. Inflammatory cells, predominantly

eutrophils, were still detected within the granulation tissue inhe absence of signs of infection around the wound area. Thelood vessels within the granulation tissue area were prominentnd dilated.

108 E.A. Khalil et al. / Journal of Ethnopharmacology 109 (2007) 104–112

Fig. 2. Histological evaluation of control, vehicle, Inula viscosa and Ajuga chia treated animals after sacrifying on the last day of the experiment: (A) control (H&E400×) and (B) vehicle (H&E 400×) showing well formed but thick granular cell layer, the underlying dermis contains deposited collagen fibers with minimali (Masf (E) As he co

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nflammation. (C) Animals treated with Inula viscosa (H&E 400×) and (D)ormation in the dermis and no inflammatory cells in a well organized dermis.howing maturation of the epidermis and the dermis comparable with those of t

.3. Phytochemical evaluation

Table 2 shows the results of the TLC screening of the aque-us extracts of Ajuga chia, Inula viscosa, Parieteria diffusa andubia tinctorum and volatile oil fraction of Laurus nobilis.

. Discussion

.1. Ethnopharmacological and phytochemical evaluation

Ajuga chia is one of the three species of the genus Ajuga foundn Jordan. Despite the traditional use of Ajuga chia in wound

ealing process in Jordan, there are no reported data available inhe literature. Ethnopharmacological studies indicate the woundealing potential of three other Ajuga species, Ajuga iva, Ajugaseudo-iva and Ajuga orientalis (Boulos, 1983; Ali-Shtayeh et

Esav

son Trichrome 400×) showing thin well-formed epidermis with hair folliclenimals treated with Ajuga chia (H&E 400×); (F) (Masson Trichrome 400×)

ntrol and the vehicle groups.

l., 2000). Studies designed to test the biological activities ofhese species revealed hypoglycemic, anti-bacterial and anti-eedant activities for the water and acetone extracts and for somesolated pure compounds such as steroids (makisterone A, cyas-erone, ecdysterone), iridoids (harpagide, 8-O-acetylharpagide),avonoids (naringin, apigenin-7-O-neohesperidoside) and cin-amic acid derivatives (Ghedira et al., 1991; Chaari et al., 2000;annet et al., 2000; Chaari et al., 2002; Hilaly and Lyoussi, 2002).

Inula viscosa, a widely distributed herbaceous plant of Jordannd Palestine is used for the treatment of respiratory, infectiousnd skin diseases and for wound healing properties according toeveral ethnobotanical surveys (Dafni et al., 1984; Oran and Al-

isawi, 1998; Ali-Shtayeh et al., 2000). Lentini (2000) reportedimilar uses for Inula viscosa in Italy. Anti-inflammatory andnti-fungal activities of some isolated substances from Inulaiscosa were tested by several researchers using mice and

E.A. Khalil et al. / Journal of Ethnopharmacology 109 (2007) 104–112 109

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ig. 3. Histological evaluation of Rubia taenifolia treated animals after sacrifyihowing spongiosis of the epidermis with persistence of inflammatory cells inrust overlying the area of the wound, with thickened and immature epidermis.

icrosporum canis, respectively (Manez et al., 1999; Maozt al., 1999; Hernandez et al., 2001). Phytochemical investi-ations resulted in identification of flavonoids, sesquiterpenoidubstances and essential oils (Chiappini et al., 1982; Grande etl., 1985; Benayache et al., 1991; Sanz et al., 1991; Abu Zargat al., 1998; Abu Zarga et al., 2003).

The genus Parieteria is represented in Jordan by threepecies, Parieteria diffusa, Parieteria alsinifolia and Parieteriaucitanica. Commonly it is applied in the traditional medicine asresh plant directly on the wound for healing purposes. Topicalse of Parieteria diffusa is reported by Dafni et al. (1984) and byentini (2000). To imitate the traditional use of this plant in theresent study the fresh collected aerial parts, composed mainlyf leaves was submitted to aqueous extraction and used for its

ound healing activity. There are no published data availableeither for the phytochemical composition nor for the biologi-al activity of Parieteria diffusa. Lecomte et al. (1996) stated aase report for the use of Parieteria in curing of kidney problems.

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able 2LC screening of the aqueous extracts from four plants and the oily fraction of Lauru

lant name Constituents

Phenolics/flavonoids Terpenoids An

nula viscosa ++ + −juga chia ++ − −ubia taenifolia − − ++arieteria diffusa + − −aurus nobilis + + −−) not detected; (+) positive; (++) strongly positive.

the last day of the experiment: (A) H&E 400×; (B) Masson Trichrome 400×ermis; (C) H&E 400× and (D) H&E 600× showing persistent ulceration and

Many phytochemical and biological studies have been carriedut with Rubia tinctorum which occurs only rare in Jordan andsed as an aphrodisiac, anti-diarrhoic, diuretic and for the treat-ent of wounds and ulcers (Hagiwara et al., 1997; El-Emary andackheet, 1998; Oran and Al-Eisawi, 1998; Tanaka et al., 2000;ackheet et al., 2001; Marec et al., 2001; Puchalska et al., 2003).he common species of the genus Rubia encountered in Jordan isubia taenifolia. Both species are known to the local populationith the same common Arabic name “Fuwwah”, therefore either

pecies available is used in the traditional medicine for woundealing purposes. In the present study the widely distributedubia taenifolia has been collected, taxonomically identifiednd used.

Laurus nobilis commonly known as Bay, Sweet Bay, True

aurel or Roman Laurel is well known as a culinary spice. It isidely distributed in the Mediterranean area and Europe. Bay

eaves are recognized as folk remedies in many countries andhe volatile oils obtained from the leaves and fruits are used in

s nobilis

thraquinones Alkaloids Coumarins Volatile oils

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he perfume and soap industries (Duke, 1989). The composi-ion of the laurel volatile oil is reported in several publicationsLin et al., 1990; Polat and Otles, 1999; Bouzouita et al., 2001;raun et al., 2001; Uchiyama et al., 2002; Kilic et al., 2004).he oil of Laurus nobilis showed potential anti-fungal capac-

ty against several common fungi causing spoilage of bakeryroducts (Guynot et al., 2003). Matsuda et al. (2000) foundhat the methanolic extract of Laurus nobilis leaves potentlynhibited blood ethanol evaluation in ethanol-loaded rats andydrochloric acid-ethanol induced gastric lesions in rats. In vivoastroprotective and anti-ulcerogenic activities of the oily frac-ion in rats were studied and reported by Afifi et al. (1997a,b)nd of the sesquiterpenes from the leaves by Yoshikawa (2001).nsulin-like biological activity, using rat epididymal adipocytessay, is reported for the aqueous leaf extract of Laurus nobilisBroadhurst et al., 2000). The potential use of Laurus nobilis inkin diseases and wound healing is found in the ethnobotanicalurveys carried out by Dafni et al. (1984) and by Ali-Shtayeh etl. (2000).

.2. Morphological evaluation

Results of wound area measurements, as shown in Table 1, didot indicate a healing potential for the test extracts. Statistically,rea measurements showed that there is no significant differenceetween the different groups. The wound healing potential forarieteria diffusa, Inula viscosa and Laurus nobilis was evidentn day 16 (Table 1), this potential was further confirmed in theistological evaluation (see Section 4.3). No healing effect wasbserved with Rubia taenifolia (Table 1 and Fig. 1). In previoustudies, Pluronics were investigated for their potential for woundealing (Hokett et al., 2000; Fowler et al., 2002; Lin et al., 2003).n this study; animals treated with the Pluronic vehicle alonehowed results similar to the untreated control animals, withoutmproving the wound healing process (Fig. 1 and Table 1).

.3. Histological evaluation

Wounds are referred to as disruption of the normal anatomictructure and function (Gerald et al., 1994). Skin wounds couldappen through several causes like physical injuries resultingn opening and breaking of the skin. The most common symp-oms of wounds are bleeding, loss of feeling or function belowhe wound site, heat and redness around the wound, painful orhrobbing sensation, swelling of the tissues in the area and pus-ike drainage (Rashed et al., 2003).

Wound healing is a very complex, multifactor sequence ofvents involving several cellular and biochemical processes. Theims in these processes are to regenerate and reconstruct the dis-upted anatomical continuity and functional status of the skinPhillips et al., 1991). Healing process, a natural body reactiono injury, initiates immediately after wounding and occurs inour stages. The first phase is coagulation, which controls exces-

ive blood loss from the damaged vessels. The next stage of theealing process is inflammation and debridment of the woundollowed by re-epithelialization, which includes proliferation,igration and differentiation of squamous epithelial cells of the

A

rmacology 109 (2007) 104–112

pidermis. In the final stage of the healing process collagen depo-ition and remodeling occurs within the dermis. (Chettibi anderguson, 1997; Hj Baie and Sheikh, 2000).

The results in this study are in support that the wound healingnd repair is accelerated by applying Inula viscosa which wasigh-lighted by the full thickness coverage of the wound areay an organized epidermis in the presence of mature scar tissuen the dermis. This ability was especially obvious when the dataere compared with the other plants. The enhanced capacityf wound healing with the Inula viscosa could be explained onhe basis of the anti-inflammatory effects of the plants that areell documented in the literature (Hernandez et al., 2001). Thisutcome was expected according to the wide use of this plant byhe inhabitants of Jordan for enhancement of the healing process.owever, histological evaluation showed that wound healing

apacity was not that clear with the other plants. Moreover, inhe case of Rubia taenifolia interference with the normal healingrocess was observed. Probably this observation is due to theuppression of the proliferative ability of the cells within thepidermis, which has resulted in persistence of the wound areaven after about 3 weeks of induction of the wound. The clearifference in the measured wound area between Rubia taenifoliand the control is supported by the above mentioned histologicalbservations and by the visually observed difference betweenhe wound morphology of Rubia taenifolia and Laurus nobiliss documented in Fig. 1.

In conclusion, while plant based traditional medicine haseen used throughout generations, the efficacy of such treat-ents requires experimental backup and scientific verification.

n this study, the plant species presented were selected based onthnopharmacological information, provided by the local com-unities. However, the plants’ actual therapeutic efficacies were

onsiderably different to those anticipated by the inhabitants ofordan.

cknowledgements

This work was supported by a grant from the Deanshipf Academic Research, University of Jordan. The authors arehankful to Miss Ihsan Omari, Miss Haifa Hattab, Mr. Ismailbaza and Mr. Jamal Alameer for their technical help.

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