Wound healing potential of Sambucus ebulus L. leaves and isolation ...

Journal of Ethnopharmacology 129 (2010) 106–114

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Wound healing potential of Sambucus ebulus L. leaves and isolation of an active component, quercetin 3-O-glucoside Ipek Pes¸in Süntar a , Esra Küpeli Akkol a,∗ , Funda Nuray Yalçın b , Ufuk Koca a , Hikmet Keles¸ c , Erdem Yesilada d a

Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Sıhhiye 06100, Ankara, Turkey Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200 Afyonkarahisar, Turkey d Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Atasehir 34755, Istanbul, Turkey b c

a r t i c l e

i n f o

Article history: Received 7 November 2009 Received in revised form 25 January 2010 Accepted 25 January 2010 Available online 2 February 2010 Keywords: Caprifoliaceae Circular excision Flavonoids Linear Incision Sambucus ebulus Tensiometer Wound healing

a b s t r a c t Ethnopharmacological relevance: The leaves of Sambucus ebulus L. are used in Turkish folk medicine for treatment of high fever, rheumatic pains, snake bites and wounds. Aim of the study: Four different solvent extracts by using n-hexane, diethyl ether, ethyl acetate and methanol were prepared from the leaves of Sambucus ebulus in order to investigate the claimed wound healing activity in vivo and isolation of active component(s) from the active extract through the bioassayguided fractionation procedures. Materials and methods: Linear incision and circular excision wound models on rats and mice were employed. The methanol extract was fractionated by successive chromatographic techniques. Wound healing activity of each fraction was investigated following the bioassay-guided fractionation procedures. Moreover, the tissue samples were examined histopathologically. Results: Significant wound healing activity was observed for the ointment prepared with methanol extract at 1% concentration. The methanol extract treated groups of animals showed 84.3% contraction in circular excision model, which was close to contraction value of the reference drug Madecassol® (100%). On the other hand, a significant increase (43.7%) in the wound tensile strength was determined with the same extract on incision wound model. Subfractions showed significant but reduced wound healing activity on both in vivo wound models. A flavonoid derivative “quercetin 3-O-glucoside” was isolated and determined as one of the active component of active final subfraction. The results of histopathological examination supported the outcome of linear incision and circular excision wound models. Conclusion: The experimental data revealed that the methanolic extract of Sambucus ebulus leaves displayed remarkable wound healing activity. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction There are two Sambucus species (Caprifoliaceae) in Turkey (Sambucus ebulus L. and Sambucus nigra L.) and the aerial parts, leaves or fruits of both have been used to treat various ailments in Turkey (Tuzlacı and Tolon, 2000; Kultur, 2007). A survey of the literatures indicated that both plants have been used to treat hemorrhoids, rheumatic pain, common cold, high fever, snake bites and wounds in Turkish folk medicine (Fujita et al., 1995; Yesilada et al., 1995; Sezik et al., 1997). However, possibly due to the widespread of distribution, Sambucus ebulus has been referred more frequently in Turkish folk medicine. The fresh leaves of Sambucus ebulus are

∗ Corresponding author. Tel.: +90 312 2023185; fax: +90 312 2235018. E-mail address: [email protected] (E.K. Akkol). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.01.051

collected before the fruits become mature and cooked with milk, about 20 min into a thick poultice form and this poultice is then applied externally on wounds for rapid recovery (Sezik et al., 1992). Moreover, leaves of Sambucus ebulus are wrapped in a cloth, or poultice made out of leaves and they are applied externally to treat burns, infectious wounds, edema, eczema, urticaria, rheumatism and inflammations (Tuzlacı and Tolon, 2000; Kultur, 2007). Fruits are used as purgative, but consumption in higher doses may induce vomitory intoxications particularly in children (Asımgil, 1993; Baytop, 1999). Several pharmacological effects have been previously reported for Sambucus species, such as antiinflammatory (Yesilada, 1997; Ahmadiani et al., 1998; Ebrahimzadeh et al., 2006), antiHelicobacter pylori (Yesilada et al., 1999), antiviral (Martinez et al., 2005), antibacterial (Neto et al., 2002) and radical scavenging (Dawidowicz et al., 2006) activities. Yesilada et al. (1997) also reported the significant inhibitory effects of the plant extracts on

I.P. Süntar et al. / Journal of Ethnopharmacology 129 (2010) 106–114

interleukins-1␣ and 1␤ and tumour necrosis factor-␣. However, wound healing potential of Sambucus species has not been investigated so far. Therefore, the aim of the present study was to evaluate the in vivo wound healing effect of the leaves of Sambucus ebulus and isolation of active component(s) by using bioassay-guided fractionation procedures. 2. Materials and methods 2.1. Plant material Leaves of Sambucus ebulus L. were collected from Abant, BoluTurkey, in May 2007. A voucher specimen was identified by the staff of the Department of Botany, Faculty of Art and Science, Gazi University and it was deposited in the Herbarium of Faculty of Pharmacy, Gazi University (GUE-2602).

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before the next injection was necessary in order to equilibrate the column. 2.5. Phytochemical study The structure of the compound I isolated from the active fraction was identified as “quercetin 3-O-glucoside” by its physical and spectroscopic (1 H NMR, 13 C NMR, DEPT, 2D NMR and MS) data which is also compared with the data obtained from those published in related literature. The subfractions Frs. D2e –D2f of Fr. D2 which showed significant wound healing activities (40.2% and 52.5% respectively) and “quercetin 3-O-glucoside” were analysed by HPLC (Fig. 1). Except for “quercetin 3-O-glucoside” contents, HPLC chromatograms of Fraction D2e and Fraction D2f were similar. “Quercetin 3-Oglucoside” was existed in both fractions. But in Fraction D2f , that was the major compound.

2.2. Preparation of the plant extracts The air-dried and powdered leaves of Sambucus ebulus (250 g) were extracted successively with n-hexane, diethyl ether, ethyl acetate and methanol (4× 1 l, 3 h each) at room temperature. Extracts were filtered, then evaporated at 40 ◦ C (Buchi, Switzerland) to dryness under reduced pressure. Yields of each extracts were 4.12% for n-hexane extract, 3.68% for diethyl ether extract, 4.56% for ethyl acetate extract and 8.32% for methanol extract. 2.3. Fractionation, isolation and structure elucidation The MeOH extract (20 g) was first submitted to a polyamide column and eluted with a solvent gradient of MeOH–H2 O (0:100 → 100:0) to afford five main fractions (Frs. A–E, 200 ml each). The Fraction D (800 mg) was rechromatographed by RP-MPLC (LiChroprep RP-18); eluting with MeOH–H2 O (0:100 → 100:0) mixtures to give six fractions (Frs. D1–6 ). Fraction D2 (250 mg) was subjected to semiquantitative TLC analysis. The plates were exposed to Vanilin:H2 SO4 , a yellow colored major spot (possibly a flavonoid) and several minor pink/purple small spots (possibly terpenic compounds) were detected. This fraction was further fractionated by Sephadex LH-20 column chromatography with methanol, followed by silica gel column chromatography eluted with a gradient of CHCl3 –MeOH–H2 O (80:20:2 → 60:40:4), to yield 1 (10 mg). Its structure was elucidated as “quercetin 3-O-glucoside” by spectroscopic methods (UV, 1 H NMR, 13 C NMR and 2D NMR and mass spectrometry).

2.6. Wound healing activity tests 2.6.1. Animals Male, Sprague–Dawley rats (160–180 g) and Swiss albino mice (20–25 g) were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health (Ankara, Turkey). The animals were left for 3 days at room conditions for acclimatization. They were maintained on standard pellet diet and water ad libitum throughout the experiment. A minimum of six animals were used in each group, otherwise described in procedure. The study was permitted by the Institutional Animal Ethics Committee (Gazi University Ethical Council Project Number: G.U.ET-08.037) and was performed according to the international rules considering the animal experiments and biodiversity right. 2.6.2. Preparation of test samples for bioassay Incision and excision wound models were used to evaluate the wound healing activity of the extracts. For the in vivo wound models, test samples were prepared in an ointment base (vehicle) consisting of glycol stearate, 1,2 propylene glycol, liquid paraffin (3:6:1) in 1% concentration. Each test ointment (0.5 g) was applied topically on the wounded site immediately after wound was created by a surgical blade. Animals of the vehicle group were treated with the ointment base only, whereas animals of the reference drug group were treated with 0.5 g of Madecassol® ointment (Bayer, 00001199) only. Madecassol® contains 1% extract of Centella asiatica.

2.4. Instrument and chromatographic procedure The reference compound was dissolved in methanol (0.2 mg/ml), the fractions were dissolved in methanol (1 mg/ml) filtered through a membrane filter (0.45 ␮m) and injected (20 ␮l). Spectra were generated on a Dionex system HPLC consisting of Dionex P6 80 HPLC pump, Dionex ASI-100-Automated Sample Injector, Dionex Thermostatted Column Compartment TCC-100, Dionex DAD. The chromatographic separation of the fractions was performed using a gradient elution program. Mobile phase, solvent A: HPLC grade water (H2 O) adjusted to pH 3.7 with sulphuric acid (H2 SO4 ); solvent B: acetonitrile (CH3 CN); mode: gradient, increasing the organic phase (CH3 CN) from 25 to 50% over 10 min; flow rate: 1 ml/min; injected volume: 20 ␮l. Detection wavelength of 200 nm on Channel A, 250 nm on Channel B, 300 nm on Channel C and 350 nm on Channel D. The wavelength scanning range was 200–600 nm at 2 nm/step. After the end of each run, a delay time of 10 min then modifying B to 25% and a second delay for 15 min

2.6.3. Circular excision wound model This wound model was used to monitor wound contraction and closure time. Each group of animals (six animals placed in each group) was initially anaesthetized by 0.01 cm3 Ketalar® . The back hairs of the mice were depilated by careful shaving. The circular wound was created with a 5 mm biopsy punch by excising only the skin on the dorsal interscapular region of each animal; following, wounds were left open (Tramontina et al., 2002). The extracts/fractions, the reference drug Madecassol® and the vehicle ointments were applied topically once a day till the wound was completely healed. The progressive changes in wound area were monitored by a camera (Fuji, S20 Pro, Japan) every other day. At the end of the treatment schedule, wound area was evaluated by using AutoCAD program. Wound contraction was calculated as percentage of the reduction in wounded area. A specimen sample of tissue removed from the healed skin of all the mice were utilized in the experiment, in order to be analyzed by histopathological examination (Sadaf et al., 2006).

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Fig. 1. HPLC chromatogram of Fr. D2e - Fr. D2f and “quercetin 3-O-glucoside.

Table 1 Wound healing processes and healing phases in the Sambucus ebulus L. extracts and Madecassol® administered animalsa . Groups

Vehicle Negative control Methanol extract Fr. D Fr. D2 Fr. D2f Madecassol®

Wound healing processes

Healing phases

S

U

RE

FP

CD

MNC

PMN

NV

MC

I

P

R

++/+++ ++/+++ ++/+++ ++ ++/+++ ++ +/++

++ + − −/+ − − −

−/+ −/+ ++ + +/++ +/++ ++

++ +/++ +/++ +/++ +/++ +/++ ++

+/++ +/++ +/++ +/++ ++ +/++ +/++

+/++ + + +/++ ++ +/++ +

+ + −/+ + + + −/+

+/++ +/++ −/+ + +/++ + +

+/++ +/++ + +/++ +/++ ++ +

+/++ ++ + +/++ +/++ +/++ +

++ +/++ + +/++ +/++ +/++ +

−/+ −/+ ++ + + + ++

a HE, VG and TB stained sections were scored as mild (+), moderate (++) and severe (+++) for epidermal and/or dermal re-modeling. S: scab, U: ulcus, RE: re-epithelization, FP: fibroblast proliferation, CD: collagen depositions, MNC: mononuclear cells, PMN: polymorphonuclear cells, NV: neovascularization, MC: mast cells, I: inflammation phase, P: proliferation phase, and R: re-modeling phase.

2.6.4. Linear incision wound model All the animals were anaesthetized with 0.15 cm3 Ketalar® and the back hair of the rats were shaved with an electrical shaving machine. Five centimeters long, two linear-paravertebral incisions were made with a sterile surgical blade through the full thickness of the skin at a distance of 1.5 cm from midline of each side of the vertebral column (Ehrlich and Hunt, 1969). The wounds were closed with three surgical interrupted sutures of 1 cm apart. The extracts/fractions, the reference drug (Madecassol® ) and the vehicle were topically applied once in a day throughout 9 days. Animals of the negative control group, were not treated with a material. All the sutures were removed on the 9th post-wound day. On day 10, all the animals were killed under anesthesia. One linear-paravertebral incised skin was analyzed using tensiometer (Zwick/Roell Z0.5, Germany) to measure its tensile strength, the other incised skin was sent for histopathological examination (Suguna et al., 2002; Lodhi et al., 2006). Tensiometer measures the breaking strength in N (Newton), which is called tensile strength. %Tensile strength (TS) of extract =

TSextract − TSvehicle × 100 TSvehicle

TSreference − TSvehicle × 100 %Tensile strength of reference = TSvehicle %Tensile strength of vehicle =

TSvehicle − TSnegative TSnegative

× 100

2.6.5. Histopathology The cross-sectional full thickness of skin specimens from each group were collected at the end of the experiment for evaluation of the histopathological alterations. Samples were fixed in 10%

buffered formalin, processed and blocked with paraffin and then sectioned into 5 micrometer sections and stained with hematoxylin & eosin (HE), Van Gieson (VG) and toluidine blue (TB) stains. The tissues sections were examined and scored as mild (+), moderate (++) and severe (+++) for epidermal or dermal re-modeling. Re-epithelization or ulcus in epidermis; fibroblast proliferation, mononuclear and/or polymorphonuclear cells, neovascularization and collagen depositions in dermis were analyzed to score the epidermal or dermal re-modeling. Van Gieson stained sections were examined for collagen deposition, in addition, toluidine blue stained sections checked for metachromatic staining of mast cells. At the end of the examination, all the wound healing processes were combined and staged for wound healing phases as inflammation, proliferation, and re-modeling in all groups. 2.6.6. Statistical analysis of the data The data on percentage wound healing was statistically analyzed using one-way analysis of variance (ANOVA). The values of p ≤ 0.001 were considered statistically significant. Histopathological data were considered to be nonparametric; therefore, no statistical tests were performed. 3. Results and discussion Wound healing is a spontaneous process, includes phases of hemostasis, inflammation, proliferation, and re-modeling. Each step involves a series of interactions between variety of components in a tissue. When a wound occurs, it is thoroughly exposed to the infections and complications. Consequently, the aim of the wound management is to heal the wound as quick as possible with minimal pain and scar formation (Clark, 1991). The agents, which shorten the healing process are always required in order to contribute for a rapid and better healing of wounds without scar.


109

Fig. 2. Histopathological view of wound healing and epidermal/dermal re-modeling in the extracts of Sambucus ebulus L and Madecassol® administered animals. Hematoxylin & eosin (HE) stained epidermis and dermis in rats (A) and mice (B), and the dermis stained with Van Gieson (VG) and toluidine blue (TB) in rats in C and D respectively. The original magnification was ×100 and the scale bars represent 75 ␮m for figures in A and B, and the original magnification was ×400 and the scale bars represent 25 ␮m for both B and C. 1) The vehicle group, 10 day old wound tissue treated with the vehicle only 2) The negative control group (untreated), 10 day old wound tissue 3) Fraction D group, 10 day old wound tissue treated with the fraction D 4) Fraction D2 group, 10 day old wound tissue treated with the fraction D2 5) Fraction D2f group, 10 day old wound tissue treated with the fraction D2f 6) The methanol extract group, 10 day old wound tissue treated with methanol extract 7) Madecassol® group, 10 day old wound tissue treated with Madecassol® .

S.E.M.: standard error meaning. Percentage of the contraction values: the vehicle group was compared to the negative control group. The extracts and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

± ± ± ± ± ± ± 19.10 9.02 6.13 4.85 0.64 0.57 0.00 1.84 1.75 (33.1) 0.67 (21.4) 1.32 (47.6)* 0.51 (85.1)*** 0.19 (85.4)*** 0.18 (84.3)*** ± ± ± ± ± ± ± 18.77 9.13 8.43 6.21 1.21 0.48 0.34 0.46 1.56 (21.8) 1.14 (15.0) 1.45 (38.9) 0.64 (32.1) 0.46 (47.4)* 0.29 (46.5)* ± ± ± ± ± ± ± 20.36 10.68 9.12 7.23 5.51 1.73 1.16 1.49 2.19 (22.1) 1.59 (26.5) 1.98 (29.5) 1.15 (23.8) 0.56 (13.1) 0.50 (17.9) ± ± ± ± ± ± ± 17.66 10.64 7.89 8.35 6.19 2.86 1.78 1.68 2.82 (30.2) 1.64 (38.8) 0.98 (15.7) 1.77 (9.5) 0.81 (34.7) 0.73 (14.3) ± ± ± ± ± ± ± 18.92 9.53 6.57 9.99 7.35 2.15 1.86 2.16 1.94 1.78 2.16 1.95 1.52 0.98 ± ± ± ± ± ± ± 0 2 4 6 8 10 12

18.24 13.65 10.73 11.85 8.12 3.29 2.17

± ± ± ± ± ± ±

1.73 1.88 (18.8) 1.54 (38.9) 1.52 (16.9) 1.12 (31.9) 0.61 (41.9) 0.54 (28.4)

19.73 16.81 17.55 14.26 11.92 5.66 3.03

Methanol extract Ethyl acetate extract Diethyl ether extract n-Hexane extract Negative control Vehicle

Wound area (mm2 ) ± S.E.M. (% contraction) Day

Table 2 Effects of the extracts from Sambucus ebulus L. on circular excision wound model.

1.56 1.48 (33.9) 1.41 (42.9)* 2.31 (67.5)** 0.45 (92.1)*** 0.27 (82.6)*** 0.00 (100.0)***


Madecassol®

110

In order to verify the claimed traditional usage of Sambucus ebulus in Turkish folk medicine, leaves of the plant were extracted successively with n-hexane, diethyl ether, ethyl acetate and finally with methanol. The wound healing activity of these extracts was investigated by pharmacological methods, i.e. excision and linear incision models in mice and in rats. The former model was used for the assessment of the test samples on wound contraction and epithelialization capacity. Rapid healing is characterized with faster contraction. Alternatively, promotion of the tensile strength of a treated wound is an indication of wound healing, possibly due to increase in collagen content and stabilization of the fibers. This may be well monitored by measurement of the tensile strength in the linear incision model through a tensiometer (Swamy et al., 2007). In order to confirm the experimental results histopathological examination was also carried out in the tissue sections of the wound area. In histopathological examination, steps of wound healing processes (inflammation, proliferation, and re-modeling) were observed within the experimental groups, especially in the methanolic extract group (Fig. 2 and Table 1). Delayed wound healing processes were observed in the vehicle and negative control groups compared to the other groups. In comparison with the vehicle and negative control groups, faster re-modeling were noticed in the extract/fraction treated groups. Re-modeling, particularly, re-epithelization in the methanolic extract group were equal to Madecassol® group. However, weaker and slower wound healing activity was observed, in the successive fractionations, i.e. in the fraction D and subfractions D2 and D2f compared with the crude methanolic extract group. The size of the wound area and percentage of contraction rates were monitored during the 12-day experimental period to assess the wound healing potential of the plant extracts, reference drug, vehicle and negative control groups in the excision wound model are shown in Table 2. In the animals treated with the methanol extract, significant healing effect was observed starting from day 6 with 47.6% (p < 0.05) and reached to 85.4% (p < 0.001) contraction on day 10 of the experimental period. This extract demonstrated 84.3% (p < 0.001) contraction on day 12, which was close to contraction value of the reference drug Madecassol® (100%). The ethyl acetate extract also showed significant activity with the values of 47.4% (p < 0.05) on day 10 and 46.5% (p < 0.05) on day 12. Tensile strength of the animals (Table 3) treated with the methanol extract demonstrated a remarkable value (43.7%, p < 0.001) on day 10, followed by the ethylacetate extract with the value of 31.1% (p < 0.05). Since methanol extract demonstrated higher wound healing activity in both excision and incision wound models following studies were conducted on

Table 3 Effects of the extracts from Sambucus ebulus L. on linear incision wound model. Material

Statistical mean ± S.E.M. (mm2 )

Vehicle Negative control n-Hexane extract Diethyl ether extract Ethyl acetate extract Methanol extract Madecassol®

19.42 14.50 20.50 21.22 25.46 27.90 30.08

± ± ± ± ± ± ±

1.96 2.12 1.63 2.49 3.05 1.82 1.07

%Tensile strength 25.3 – 5.6 9.3 31.1* 43.7*** 54.9***

S.E.M.: standard error meaning. Percentage of the tensile strength values: the vehicle group was compared to the negative control group. The extracts and the reference material were compared to vehicle group. * p < 0.05. *** p < 0.001.

3.17 2.42 (22.9) 3.80 (32.0) 1.83 (58.1)** 0.78 (66.2)** 0.71 (74.6)*** 0.00 (100.0)*** S.E.M.: standard error meaning. Percentage of the contraction values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

± ± ± ± ± ± ± 19.02 13.87 10.22 4.72 2.25 0.98 0.00 18.31 17.01 13.63 10.55 5.82 3.02 1.98 18.94 13.81 10.81 5.80 2.82 1.22 0.77 ± ± ± ± ± ± ± 19.21 14.55 11.43 6.79 4.68 2.02 0.99

Fr. C

3.51 3.04 (2.6) 2.61 (4.8) 3.23 (8.9) 0.79 (27.5) 0.54 (32.4) 0.11 (44.8) ± ± ± ± ± ± ± 19.81 17.54 14.31 10.26 4.82 2.61 1.59

Fr. B

2.57 1.44 (6.6) 3.22 2.65 2.33 (12.5) 0.71 (41.9) 1.53 (44.4) ± ± ± ± ± ± ± Fr. A

18.76 16.82 15.98 12.77 5.82 2.24 1.60 3.43 2.98 3.37 2.93 2.02 1.72 0.88 ± ± ± ± ± ± ± 19.79 18.23 16.05 12.44 7.46 4.17 3.49

Negative control

2.19 1.88 (1.2) 3.10 (6.4) 2.04 (9.4) 1.97 (10.9) 1.66 (7.4) 0.72 (17.5) ± ± ± ± ± ± ±

Vehicle

19.54 18.01 15.03 11.27 6.65 3.86 2.88 0 2 4 6 8 10 12


Table 4 Effects of the polyamide column fractions from the methanolic extract of Sambucus ebulus L. on circular excision wound model.

2.67 2.09 (19.2) 2.94 (23.9) 1.72 (39.8) 1.32 (29.6) 0.95 (47.7)* 0.31 (65.6)**

Fr. D

± ± ± ± ± ± ±

2.30 2.99 (23.3) 1.09 (28.1) 1.10 (48.5)* 0.21 (57.6)** 0.33 (68.4)** 0.03 (73.3)***

Fr. E

± ± ± ± ± ± ±

3.09 1.84 (5.6) 2.87 (9.3) 3.81 (6.4) 2.07 (12.5) 0.69 (21.8) 0.63 (31.3)

Madecassol®


111

this extract and submitted to bioassay-guided fractionation and isolation procedures in order to determine the active constituent(s). The methanolic extract was subjected to successive chromatographical techniques and the wound healing effect of each fraction was investigated using the same wound models. Among the fractions, which were eluted from polyamide column, C and D showed statistically remarkable contraction (Table 4) and tensile strength (Table 5) values. The wound contracting ability of the fraction D on excision wound model was significantly greater than that of the negative control and the vehicle itself, with the contraction values increasing from 23.3% on 2nd day to 73.3% on 12th day (p < 0.001). As for applied linear incision wound model, the tensile strength value of the wounds treated with fraction D was significantly higher than that of the vehicle and negative control group (Table 5). Fraction D was rechromatographed by RP-MPLC to give six subfractions (Frs. D1–6 ). Among all the fractions, the wounds treated with D2 exhibited significant activity with the contraction values between 7.6% (on the 2nd day) to 65.8% (on the 12th day) (p < 0.01) on excision wound model and tensile strength value of 37.1% (p < 0.01) on linear incision wound models (Tables 6 and 7). Final fractionation of the active subfraction D2 was performed on Sephadex LH-20 column chromatography to give 7 subfractions (Frs. D2a –D2g ). Subfractions D2e and D2f exerted remarkable wound healing activity on excision and linear incision wound models compared to others (Tables 8 and 9). Results expressed that subfractions considerable but step by step reduced wound healing activity on both in vivo wound models. The structure of the compound quercetin 3-O-glucoside isolated from the active fraction was identified by its physical and spectroscopic (1 H NMR, 13 C NMR, DEPT, 2D NMR and MS) data, was obtained from previously published literature (Harborne and Mabry, 1982; Harborne, 1994). A number of studies have been reported on the phytochemical composition of Sambucus ebulus leaves. Recently, six new iridoid glycosides of the Valeriana type were isolated from the leaves of the plant (Pieri et al., 2009). In addition to the secondary metabolites, a new family of acidic type 2 (ebulitins ␣, ␤ and ␥) and basic type 1 (ebulin 1) ribosome-inactivating nontoxic protein derivatives (Girbes et al., 1993; De Benito et al., 1995) and dimeric and mucin binding lectins (Rojo et al., 2003) were isolated from the matured leaves. However, other phytochemicals in the leaves of the plant have not been studied thoroughly.

Table 5 Effects of the polyamide column fractions from the methanolic extract of Sambucus ebulus on linear incision wound model. Material


Vehicle Negative control Fr. A Fr. B Fr. C Fr. D Fr. E Madecassol®

18.88 16.23 21.52 24.97 25.63 26.61 22.95 30.25

± ± ± ± ± ± ± ±

2.67 2.21 1.15 3.18 1.43 2.05 2.73 1.68

%Tensile strength 16.3 – 13.9 32.3 35.8* 40.9** 21.6 60.2***

S.E.M.: standard error meaning. Percentage of the tensile strength values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

S.E.M.: standard error meaning. Percentage of the contraction values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

19.37 16.89 14.12 9.04 6.18 4.28 2.44 21.59 17.62 13.63 8.59 5.27 2.68 1.37 22.61 17.98 13.73 10.24 6.03 4.19 2.50 3.02 2.59 2.01 2.57 (1.4) 1.87 (3.3) 0.45 (8.2) 0.39 (11.5) ± ± ± ± ± ± ± 21.17 18.32 14.88 9.55 6.52 4.36 2.61 0 2 4 6 8 10 12

19.06 16.42 14.28 9.69 6.74 4.75 2.95

± ± ± ± ± ± ±

2.81 2.07 (4.2) 1.89 (9.7) 1.74 (9.5) 2.53 (11.8) 0.82 (11.7) 0.24 (8.4)

19.81 17.14 15.82 10.71 7.64 5.38 3.22

± ± ± ± ± ± ±

3.58 1.79 2.36 2.78 2.04 1.99 0.75

20.11 17.47 14.05 9.03 5.92 4.02 2.38

± ± ± ± ± ± ±

2.54 3.27 2.84 (1.6) 1.71 (6.8) 1.59 (12.2) 0.89 (15.4) 0.54 (19.3)

19.63 15.18 11.64 7.04 4.25 2.07 1.01

± ± ± ± ± ± ±

2.83 2.41 (7.6) 1.72 (18.5) 1.15 (27.3) 1.01 (36.9) 0.87 (56.4)** 0.13 (65.8)**

Fr. D3 Fr. D2 Fr. D1 Negative control Vehicle


Table 6 Effects of the subfractions of fraction D (after rechromatographed by RP-MPLC) on circular excision wound model.

Fr. D4

± ± ± ± ± ± ±

2.41 2.56 2.13 (3.9) 2.69 0.97 (10.5) 0.81 (11.8) 0.14 (15.3)

Fr. D5

± ± ± ± ± ± ±

3.46 2.98 1.87 (4.6) 1.61 (11.4) 1.33 (21.8) 0.95 (43.6) 0.23 (53.6)

Fr. D6

± ± ± ± ± ± ±

3.56 2.44 2.24 (1.1) 1.27 (6.7) 1.41 (8.3) 1.79 (9.9) 0.60 (17.3)

19.44 14.76 10.42 5.78 2.68 0.97 0.00

± ± ± ± ± ± ±

2.43 1.86 (10.1) 1.85 (27.0) 1.62 (40.4)* 0.93 (60.2)** 0.13 (79.6)*** 0.00 (100.0)***


Madecassol®

112

Although wound healing activity of Sambucus ebulus leaves have not been investigated so far, several closely related pharmacological activities have been reported. One of which could be the antiinflammatory activity of the plant. Antiinflammatory agents have also contribution into the wound healing process, as it includes an inflammation phase at the beginning of healing. According to published literatures, plenty of studies were reported on the in vivo and in vitro antiinflammatory activity of Sambucus ebulus leaves (Yesilada, 1997; Yesilada et al., 1997; Ahmadiani et al., 1998; Ebrahimzadeh et al., 2006). Ahmadiani et al. (1998) postulated that the antiinflammatory effect of Sambucus ebulus may be related to flavonoid or steroid ingredients of the plant, while n-hexane extract of leaves were shown to lack of the mentioned activity. Flavonoids are used for their several therapeutic effects such as antioxidant, antiinflammatory, antifungal, and wound healing (Okuda, 2005; Nayak et al., 2009). Inhibition of lipid peroxidation effect by flavonoids, is supposed to increase the viability of collagen fibrils, by activating the DNA synthesis and preventing the cell damage (Getie et al., 2002; Shetty et al., 2008). Flavonoids are also known to promote the rapid wound healing due to their antimicrobial and astringent properties (Tsuchiya et al., 1996). Therefore, wound healing potential of Sambucus ebulus may be attributed to the phytochemicals exist in the leaves, which might be additive effect that accelerates the progress probably during the proliferation phase of wound healing. The present study demonstrated that, the methanolic extract of Sambucus ebulus leaves was found to be remarkably active on in vivo wound models, whereas the other extracts were not as active as the methanol extract. This prompted us to analyze and identify the chemical composition of the methanol extract. According to bioassay-guided fractionation procedures, a flavonoid component, quercetin 3-O-glucoside was isolated from active fraction as one of the active wound healing ingredient. However, the wound healing activity of the final active fraction was somewhat less than the initial methanolic extract; the activity decreased from 85.4 to 52.5% for excision model and from 43.7 to 32.6% for incision model (Tables 2, 3, 8, 9). Therefore, a synergistic interaction between the active ingredient and other phytochemicals in the plant extract should be questioned.

Table 7 Effects of the subfractions of fraction D (after rechromatographed by RP-MPLC) on linear incision model. Material


Vehicle Negative control Fr. D1 Fr. D2 Fr. D3 Fr. D4 Fr. D5 Fr. D6 Madecassol®

16.72 14.23 17.99 22.92 17.68 17.86 19.45 18.13 26.17

± ± ± ± ± ± ± ± ±

1.80 3.17 3.11 1.54 2.15 1.88 2.09 2.32 1.57

%Tensile strength 17.5 – 7.6 37.1** 5.7 6.8 16.3 8.4 56.5***

S.E.M.: standard error meaning. Percentage of the tensile strength values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. ** p < 0.01. *** p < 0.001.

113

± ± ± ± ± ± ±

1.88 1.24 (15.2) 1.56 (28.8) 1.31 (32.2)* 0.45 (46.9)** 0.16 (81.6)*** 0.00 (100.0)***

Table 9 Effects of the subfractions of fraction D2 (after rechromatographed by Sephadex LH-20) on linear incision model.

S.E.M.: standard error meaning. Percentage of the contraction values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

Fr. D2g

19.21 15.09 11.20 6.87 3.21 0.79 0.00 19.65 16.76 14.26 7.99 4.29 2.55 1.31 21.07 17.33 14.85 8.86 5.14 2.80 1.65 1.56 1.89 2.01 1.93 (1.2) 0.59 (2.8) 0.47 (11.7) 0.16 (15.6) ± ± ± ± ± ± ± 20.62 18.17 15.97 10.01 5.87 3.79 2.33

Fr. D2d

2.43 1.82 (2.9) 1.96 (5.1) 2.10 (12.2) 1.45 (13.7) 0.81 (16.8) 0.62 (17.8) ± ± ± ± ± ± ± 19.83 17.26 14.92 8.89 5.21 3.57 2.27

Fr. D2c

2.04 1.82 (5.5) 1.86 (5.6) 1.28 (11.4) 1.69 (7.5) 0.47 (9.8) 0.79 (16.3) ± ± ± ± ± ± ± 20.07 16.81 14.84 8.98 5.59 3.87 2.31

Fr. D2b

2.02 2.14 1.55 (2.1) 1.53 1.78 (1.2) 0.69 (2.8) 0.21 (6.9) ± ± ± ± ± ± ± 19.11 18.09 15.39 11.37 5.97 4.17 2.57 2.39 1.91 1.34 1.27 2.52 1.32 0.65 ± ± ± ± ± ± ± 19.71 18.85 16.67 11.19 6.53 4.74 3.37

Negative control Fr. D2a

2.23 1.98 (5.6) 2.56 (5.7) 1.46 (9.5) 1.63 (7.5) 0.99 (9.5) 0.17 (18.1) ± ± ± ± ± ± ±

Vehicle

20.51 17.79 15.72 10.13 6.04 4.29 2.76 0 2 4 6 8 10 12

Day Wound area (mm2 ) ± S.E.M. (% contraction)

Table 8 Effects of the subfractions of fraction D2 (after rechromatographed by Sephadex LH-20) on circular excision wound model.

Fr. D2e

± ± ± ± ± ± ±

2.60 2.29 (2.6) 1.72 (5.5) 1.59 (12.5) 1.56 (14.9) 0.39 (34.7)* 0.12 (40.2)*

Fr. D2f

± ± ± ± ± ± ±

1.44 20.59 ± 1.36 2.13 (5.8) 18.25 ± 1.79 1.42 (9.3) 15.51 ± 1.72 (1.3) 1.33 (21.1) 8.23 ± 1.54 (9.9) 0.69 (28.9) 5.36 ± 1.17 (11.3) 1.68 (40.6)** 3.41 ± 0.49 (20.5) 0.63 (52.5)** 1.81 ± 0.87 (34.2)

Madecassol®


Material


Vehicle Negative control Fr. D2a Fr. D2b Fr. D2c Fr. D2d Fr. D2e Fr. D2f Fr. D2g Madecassol®

17.87 15.03 18.60 19.79 20.45 21.17 22.98 23.70 22.39 28.43

± ± ± ± ± ± ± ± ± ±

1.51 2.66 2.03 1.06 1.72 1.19 1.84 1.02 1.20 1.01

%Tensile strength 18.9 – 4.1 10.7 14.4 18.5 28.6* 32.6** 25.3 59.1***

S.E.M.: standard error meaning. Percentage of the tensile strength values: the vehicle group was compared to the negative control group. The fractions and the reference material were compared to vehicle group. * p < 0.05. ** p < 0.01. *** p < 0.001.

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