In vivo activity assessment of some Tanacetum species used as traditional wound healer along with identification of the phytochemical profile by a new validated HPLC method.

OBJECTIVES: Tanacetum species are traditionally used as insecticide, and externally wound healer as well as for anti-inflammatory and antihistaminic properties. The in vivo wound-healing and anti-inflammatory potential of four Tanacetum species, Tanacetum argenteum (Lam.) Willd. subsp. argenteum (TA), Tanacetum heterotomum (Bornm.) Grierson (TH), Tanacetum densum (Lab.) Schultz Bip. subsp. sivasicum (TD), and Tanacetum vulgare L. (TV) was investigated.
MATERIALS AND METHODS: The chloroform (CHCl3) and methanol:water (80:20) extracts were prepared from the aerial parts of each plant. For assessment of the wound-healing activity, linear incision on rats and circular excision on mice wound models were used and histopathological analyses were conducted on the tissues treated with the test materials. For the evaluation of the anti-inflammatory activity, Whittle Method based on the inhibition of the acetic acid-induced increase in capillary permeability was used. In order to elucidate the phytochemical contents of the extracts, HPLC profiles of active fractions were screened and quantitative analysis was conducted within the scope of HPLC analysis.
RESULTS: The CHCl3 extracts of TD, TA and TV were found to have significant wound healing activity (37.1%, 30.8% and 26.1% tensile strength; 88.05%, 72.93% and 44.88% contraction values, respectively) and anti-inflammatory activities (31.5% and 26.6% inhibition values for TD and TA). Parthenolide content of the CHCl3 extracts of TA, TH and TV were found 242.66±1.53, 190.16±5.62 and 177.51±3.73 µg/100 mg plant material, respectively.
CONCLUSION: According to the results, the other secondary metabolites present in the aerial parts of the Tanacetum species possibly exerted synergistic effects on the observed healing of the wounds.

Introduction

The genus Tanacetum L., which belongs to the Asteraceae family and grows in the temperate regions of Europe and West Asia, comprises about 200 species. The species of this genus are traditionally used for anti-inflammatory, antihistaminic, and stomachic properties and as insecticide (1-3). In addition, these species are used against psoriasis, migraine, nausea, vomiting, tinnitus, dizziness, hysteria, neuralgia, asthma, kidney weakness, constipation, gynecological disorders, and emmenagogue as an anthelmintic food additive and are reported to be used externally as a poultice to heal eruptive skin diseases (4-10). The leaves of Tanacetum vulgare L. were reported to be utilized for wound-healing (11) and the infusion prepared from the aerial parts of Tanacetum densum (Labill.) Sch. Bip. subsp. densum is used for the treatment spelling (12, 13). These species contain sterols, essential oil components, sesquiterpene lactones, resins, bitter substances, acetylenes, flavonoids, coumarins and tannic acid (6, 9, 14-17). Several biological activity studies have also been conducted on Tanacetum species revealing their analgesic, antipyretic, antitumor, antioxidant, cardiotonic, spasmolytic, hypoglycemic, diuretic, laxative, acaricidal, antileishmanial, antifungal, antibacterial and herbicidal activities (6-10, 18-24). However, there has been no study on the assessment of the wound-healing activity of Tanacetum species despite their previously reported traditional utilizations.

The aim of the present study is to evaluate the in vivo wound-healing and anti-inflammatory potentials of four Tanacetum species namely Tanacetum argenteum (Lam.) Willd. subsp. argenteum, Tanacetum heterotomum (Bornm.) Grierson, Tanacetum densum subsp. (Lab.) Schultz Bip. sivasicum and T. vulgare. Among them, T. densum subsp. sivasicum and T. argenteum subsp. argenteum are endemic to Turkey. In order to elucidate the phytochemical contents of the extracts, HPLC profiles of these species were screened and quantitative analysis of three flavonoid aglycones; apigenol, quercetin, kaempferol and a sesquiterpene lactone; parthenolide were conducted within the scope of HPLC analysis.

Materials and Methods

Plant material

Tanacetum species were collected from different regions of Anatolia (Table 1). Taxonomic identification of the plants was confirmed by Assistant Professor Mehmet Tekin (Department of Pharmaceutical Botany, Faculty of Pharmacy, Trakya University). Voucher specimens were stored in the Herbarium of Cumhuriyet University, Faculty of Science (CUFH).

Table 1

Locality of the plant samples of different Tanacetum species

Plant species	Locality	Altitude	Date	Herbarium No
T. argenteum subsp. argenteum (TA)	Böğrüdelik village, Sivas	1845 m	2012	M.Tekin 1255
T. heterotomum (TH)	Ziyarettepe, Sivas	1402 m	2012	M.Tekin 1315
T. densum subsp. sivasicum (TD)	Böğrüdelik village, Sivas	1850 m	2012	M.Tekin 1257
T. vulgare (TV)	Karaçayır, Sivas	1440 m	2012	M.Tekin 1313

Dried and powdered aerial parts of each plant (30 g) were extracted with 300 ml CHCl3 during 8 hr for 3 days. Residues were dried and extracted with methanol: water (MeOH:H2O) (80:20) at room temperature during 8 hr for 3 days by continuous stirring. The extracts were filtered and concentrated using evaporator to yield dry crude extracts.

HPLC analysis

Quercetin, kaempferol, apigenin and parthenolide contents of CHCl3 extracts of Tanacetum species were investigated using HPLC analysis. HPLC analyses were carried out using Agilent LC 1200 model chromatograph (Agilent Technologies, California, USA). Separation was carried out by using gradient elution on ACE 5 C18 (250 mm× 4.6 mm; 5 µm) column. 0.2% phosphoric acid in water (A) and acetonitrile (B) were used in gradient elution as the mobile phase. The analysis was started with the ratio of A:B 90:10, v/v. Afterwards, the ratio of A:B was linearly changed to 0:100 in 36 min. During the last 4 min of the analysis, the solvent ratio was isocratic at the rate of A:B 0:100, v/v. The analyses were conducted with the flow rate of 1 ml/min and the injection volume was 10 µl. The column temperature was kept at 40 °C during the analyses. Parthenolide and flavonoid aglycones were analyzed at 214 nm and 330 nm, respectively. Peak areas were integrated automatically by computer using Agilent Software.

Preparation of standard solutions and calibration

The stock solution for the reference compound was prepared at the concentration of 0.1 mg/ml. The compound was weighed and dissolved with methanol in volumetric flask and the final volume was adjusted to 10 ml. The stock solution was diluted to obtain the concentration levels between 0.0005 mg/ml and 0.05 mg/ml. Triplicate analyses were carried out for each concentration level and the calibration curve was obtained using peak areas against concentration.

Optimization of the sample extraction procedure and preparation of samples

Aerial parts of the four different Tanacetum species were used in this experiment. CHCl3 and MeOH:H2O (80:20) mixture were used successively for the extraction of plant samples. For HPLC analysis, CHCl3 extracts were prepared from plant material to obtain total extract. Ten mg of CHCl3 extracts of four species were weighed in 10 ml volumetric flask, dissolved in MeOH and adjusted to the final volume separately, and each extract was filtered through 0.45 µm membrane filter after adjusting to a final volume of 10 ml with same solvent. Triplicate 10 µl injections were performed for plant samples.

Validation procedure-Limit of detection and quantification

The injections were repeated 9 times to verify the values of limit of detection (LOD) and limit of quantification (LOQ).

Biological activity tests

Animals

Male Sprague Dawley rats (160-180 g) and Swiss albino mice (20–25 g) were provided from Laboratory of Experimental Animals, Kobay, Turkey. Each group consisted of six animals. Before the experiments, the animals were left 3 days for acclimatization at room temperature, standard humidity and light-controlled (12 hr light/12 hr dark) conditions. The animals were maintained on standard pellet diet and water ad libitum. Animals were processed according to the suggested European ethical guidelines for the care of laboratory animals. The study was performed according to the international rules considering the animal experiments and biodiversity rights (Gazi University Ethical Council Project Number: G.U.ET-10.027).

Preparation of test samples for bioassay

For the assessment of wound-healing activity, the test samples were prepared in an ointment base (glycol stearate, 1,2 propylene glycol, liquid paraffin, 3:6:1) in 1% concentration and applied 0.5 g onto the wounded sites. Madecassol® (Bayer) was used as the reference ointment. For the evaluation of anti-inflammatory activity, extract suspensions were prepared as described previously (25).

For the anti-inflammatory activity evaluation, test samples were administered orally to the test animals after suspending in a mixture of distilled H2O and 0.5% sodium carboxy methyl cellulose (CMC). The control group animals received vehicle only (26). Indomethacin (10 mg/kg) in 0.5% CMC was used as a reference drug (27).

Wound-healing activity

Linear incision wound model

Linear incision wound model was performed on the rats according to the model, which was previously described by Lodhi et al. 2006 (28), and Suguna et al. 2002 (29) with some modifications (25). Two linear incisions were made on the dorsal part of the rat. Tensile strength of the treated skin was measured with a tensiometer (Zwick/Roell Z 0.5, Germany)

Circular excision wound model

A circular wound was created on the dorsal region of each mouse according to the method described by Süntar et al. 2013 (25). After treatment of the wounds, wound contraction was calculated as percentage of the reduction. A specimen sample of tissue was taken in order to be analyzed histopathologically (30).

Histopathology

The tissues were stained and histopathologically examined (25).

Anti-inflammatory activity

Acetic acid-induced increase in capillary permeability

Inhibitory activity of the test samples on the increased vascular permeability induced by acetic acid in mice was evaluated according to Whittle method (31) with some modifications (32).

Statistical analysis of the data

One-way analysis of variance (ANOVA) and Students-Newman-Keuls post hoc tests were used to analyze the data. The values of P≤ 0.05 were considered statistically significant. No statistical tests were performed for histopathological data, which were considered to be nonparametric.

Results

In the present study, in vivo wound-healing and anti-inflammatory activities of various Tanacetum species were investigated. Among these species, T. densum subsp. sivasicum, T. vulgare and T. argenteum subsp. argenteum were found to have wound-healing activity potential in both wound models. The highest activity was observed for the CHCl3 extract of TD, with the tensile strength value of 37.1% in linear incision wound model (Table 2) and with the contraction value of 88.05% in circular excision wound model (Table 3). Histopathological findings also supported the biological activity results. Phases in wound-healing processes (inflammation, proliferation, and remodeling) were observed within the experimental groups with different degree (Table 4). Comparing the other experimental groups, best remodeling was observed in the reference group and then in the T. densum subsp. sivasicum CHCl3 extract group. Delayed wound-healing processes were observed in the negative control and vehicle groups. Histopathological results are shown in Figure 1, which stained with hematoxylin & eosin (HE) and Van Gieson (VG).

Table 2

Effects of the test materials on linear incision wound model

Material	Extract type	Tensile strength ± SEM	(%Tensile strength)
Vehicle		15.42 ± 2.18	7.2
Negative Control		14.39± 2.21	-
T. argenteum subsp. argenteum	CHCl3	20.17 ± 1.65	30.8**
	MeOH:H2O	18.41 ± 1.79	19.4
T. heterotomum	CHCl3	18.26 ± 1.94	18.4
	MeOH:H2O	17.46 ± 1.78	13.2
T. densum subsp. sivasicum	CHCl3	21.14 ± 1.89	37.1**
	MeOH:H2O	15.32 ± 2.25	-
T. vulgare	CHCl3	19.44 ± 1.86	26.1*
	MeOH:H2O	15.95 ± 2.52	3.3
Madecassol®		23.16 ± 1.50	50.2***

: P<0.05;

: P<0.01;

: P<0.001;

SEM.: Standard error of the mean, percentage of the tensile strength values: The vehicle group was compared to the negative control group; The test materials and the reference material were compared to vehicle group

Table 3

Effects of the test materials on circular excision wound model

Material	Extract type	Wound area (mm2) ± SEM (Contraction%)
		Day 0	Day 2	Day 4	Day 6	Day 8	Day 10
Vehicle		20.25±3.24	18.32±2.45 (2.86)	15.49±2.08 (3.61)	10.24±1.69 (9.70)	6.56±1.89 (7.48)	4.10±0.47 (6.18)
Negative Control		21.16±3.31	18.86±2.78	16.07±2.16	11.34±1.79	7.09±0.91	4.37±0.66
T. argenteum subsp. argenteum	CHCl3	22.24±3.83	15.52±1.68 (15.28)	12.29±1.84 (20.66)	7.71±1.92 (24.71)	3.86±0.69 (41.16)*	1.11±0.55 (72.93)***
	MeOH:H2O	20.75±2.54	16.92±1.95 (7.64)	13.88±1.99 (10.39)	8.91±1.36 (12.98)	5.45±0.99 (16.92)	3.49±0.70 (14.88)
T. heterotomum	CHCl3	21.19±3.04	15.98±1.75 (12.77)	13.13±1.92 (15.24)	8.35±1.86 (18.45)	4.64±0.75 (29.26)	2.81±0.39 (21.71)
	MeOH:H2O	19.96±3.15	18.42±2.25 -	14.04±2.22 (9.36)	8.91±1.74 (12.99)	5.54±0.91 (15.54)	3.03±0.52 (26.09)
T. densum subsp. sivasicum	CHCl3	21.47±2.99	15.56±1.93 (15.07)	12.08±1.79 (22.01)	7.11±1.33 (30.57)*	2.55±0.81 (61.13)**	0.49±0.48 (88.05)***
	MeOH:H2O	20.68±2.90	17.31±2.10 (5.51)	14.50±2.09 (6.39)	8.99±1.50 (12.21)	5.73±0.91 (12.65)	1.76±0.61 (8.29)
T. vulgare	CHCl3	21.55±3.31	17.91±2.07 (2.23)	14.32±2.11 (7.55)	8.45±2.01 (17.48)	4.02±1.10 (38.71)*	2.26±0.43 (44.88)*
	MeOH:H2O	20.52±3.41	15.77±2.17 (13.91)	13.38±2.07 (13.62)	8.49±1.70 (17.08)	4.98±0.98 (24.08)	2.66±0.59 (22.92)
Madecassol®		20.44±2.67	15.07±1.91 (17.74)	11.27±1.71 (27.24)	5.29±1.12 (48.34)*	2.01±0.32 (69.36)**	0.00±0.00 (100.00)***

: P<0.05;

: P<0.01;

: P<0.001;

SEM: Standard error of the mean; percentage of the tensile strength values: The vehicle group was compared to the negative control group; The test materials and the reference material were compared to vehicle group

Table 4

Wound healing processes and healing phases of the experimental groups

Groups	Extract type	Wound Healing Processes								Healing Phases
		S	U	RE	FP	CD	MNC	PMN	NV	I	P	R
Vehicle		+++	+++	-	+++	+++	+++	+++	+++	+++	+++	-
Negative Control		+++	+++	-	+++	+++	+++	+++	+++	+++	+++	-
T. argenteum subsp. argenteum	CHCl3	++/+++	++	-	+++	+++	+++	+++	+++	++/+++	++/+++	-
	MeOH:H2O	++/+++	++	-	++/+++	++/+++	+++	+++	+++	++/+++	++/+++	-
T. heterotomum	CHCl3	++/+++	++	-	+++	+++	+++	+++	+++	++/+++	++/+++	-
	MeOH:H2O	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	-
T. densum subsp. sivasicum	CHCl3	+	-	++	++	++	++	++	++	++	++	++
	MeOH:H2O	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	-
T. vulgare	CHCl3	++	++	-	++/+++	++/+++	+++	++/+++	++/+++	++/+++	++/+++	-
	MeOH:H2O	+++	+++	-	+++	+++	+++	+++	+++	+++	+++	-
Madecassol®		++	-	+/++	++	++	++	++	++	++	++/+++	+/++

Hematoxylin & eosin (HE) and Van Gieson (VG) 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, I: Inflammation phase, P: Proliferation phase, R: Re-modeling phase

Figure 1

Histopathological view of treated tissues; Skin sections show the hematoxylin & eosin (HE) stained epidermis and dermis in A, and the dermis stained with Van Gieson (VG) in B. The original magnification was x 100 and the scale bars represent 120 µm for figures in A, and the original magnification was x 400 and the scale bars represent 40 µm for B. Data are representative of 6 animal per group. 1) Vehicle group; 2) Negative control group (untreated group); 3) T. argenteum subsp. argenteum MeOH:H2O extract group; 4) T. argenteum subsp. argenteum CHCl3 extract group; 5) T. heterotomum MeOH:H2O extract group; 6) T. heterotomum CHCl3 extract group;7) T.densum subsp. sivasicum MeOH:H2O extract group; 8) T.densum subsp. sivasicum CHCl3 extract group; 9) T. vulgare MeOH:H2O extract group; 10) T. vulgare CHCl3 extract group; 11) Reference group (wound tissue treated with Madecassol®); Arrows pointing events during wound healing; s: scab, u: ulcus, re: re-epithelization, f: fibroblast, c: collagen, mnc: mononuclear cells, nv: neovascularization

The results of the anti-inflammatory activity assessment revealed that CHCl3 extracts of T. densum subsp. sivasicum and T. argenteum subsp. argenteum possess anti-inflammatory activity in Whittle Method by displaying 31.5% and 26.6% inhibition values, respectively (Table 5).

Table 5

Inhibitory effect of the test materials on acetic acid-induced increase in capillary permeability

Material	Extract type	Dose (mg/kg)	Evans blue concentration (μg/ml) ± SEM	Inhibition (%)
Control			12.01 ± 2.18	-
T. argenteum subsp. argenteum	CHCl3	100	8.82 ± 0.91	26.6*
	MeOH:H2O	100	10.91± 1.35	9.2
T. heterotomum	CHCl3	100	11.58 ± 1.23	3.6
	MeOH:H2O	100	10.94 ± 0.97	8.9
T.densum subsp. sivasicum	CHCl3	100	8.23 ± 0.79	31.5**
	MeOH:H2O	100	9.59 ± 1.44	20.2
T. vulgare	CHCl3	100	10.39 ± 1.46	13.5
	MeOH:H2O	100	9.65 ± 1.81	19.7
Indomethacin		10	5.97 ± 0.51	50.3***

SEM: Standard error of the mean;

: P<0.05.

: P<0.01.

: P<0.001 significant from the control

In the present study, the quantification of three flavonoid aglycones and a sesquiterpene was conducted on the same Tanacetum species by using HPLC method. Selected HPLC method was determined by comparing the chromatographic profile and data obtained from the standards and samples, considering the following parameters; retention time, analyzing time for samples, separation, peak shapes and maximum UV absorption of the standards. In order to optimize the suitable chromatographic separation of four compounds in four different Tanacetum species, many different isocratic and gradient elution were investigated. Phosphoric acid was also used as modifier. Furthermore, different temperatures (30°C, 35°C and 40°C) and different flow rates (1.2 ml/min, 1 ml/min, 0.8 ml/min) were tested. Finally good separations for the extracts of Tanacetum species have been achieved under 40°C temperature, 1 ml/min flow rate and gradient elution of water (containing 0.2% phosphoric acid) (A), and acetonitrile (B). The chromatograms were obtained at 214 nm and 330 nm for standards and samples. Identification of the peaks was confirmed by comparison of the retention times and UV absorption spectra with acquired standards (Figure 2).

Figure 2

HPLC chromatogram of T. argenteum subsp. Argenteum (214 nm)

The highest content of the parthenolide was detected in T. argenteum subsp. argenteum CHCl3 extract. The parthenolide contents of the CHCl3 extracts of T. argenteum subsp. argenteum, T. vulgare and T. heterotomum were found as 242.66±1.53 µg/100 mg; 177.51±3.73 µg/100 mg and 190.16±5.62 µg/100 mg, respectively. Parthenolide was not detected in T. densum subsp. sivasicum CHCl3 extract. Furthermore, quercetin, kaempferol and apigenin have also been investigated in CHCl3 extracts. Quercetin and kaempferol has not been detected even at LOD levels. However, T. heterotomum was determined as the only species containing apigenin in quite small amount. According to the HPLC chromatograms and UV absorbances of the peaks, investigated Tanacetum species have flavonoids in varying amounts together with other compounds. LOD and LOQ levels of parthenolide were determined as 0.4139 µg/ml and 1.3793 µg/ml, respectively.

Discussion

Tanacetum species are popular among the people living in rural areas evidenced by the previously published ethnomedicinal data (1-10). Among Tanacetum species, Tanacetum parthenium (L.) Schultz-Bip. (Feverfew) is popular for its use in migraine prophylaxis (5, 9). The activity potential was confirmed by several clinical trials, which demonstrated its lowering effect on the intensity and frequency of headache, visual disturbance, nausea and vomiting induced by migraine (33-35). The secondary metabolites present in the leaves have been shown to exert the activity synergistically and the whole leaf extract has been suggested to be used for the prevention of migraine (5, 36). In vivo and in vitro studies revealed that parthenolide-depleted feverfew extract possessed antioxidant activity. According to the in vitro studies, the extract decreased cigarette smoke-mediated damage, UV-induced hydrogen peroxide and pro-inflammatory cytokine release (37). Wound-healing and anti-inflammatory activity results exhibited herein could be related to potential antioxidant activity of the tested Tanacetum species. The phytochemical studies yielded the chemical constituents of T. parthenium as volatile oil components, sesquiterpene lactones, coumarin derivatives and flavonoids (mainly kaempferol, quercetin, apigenin and luteolin derivatives) (9, 14). Besides T. parthenium, another species such as T. vulgare is also a well-known folk remedy that is externally used as poultice to heal some eruptive skin diseases, sprains, gout, contusions and scabies, or to kill lice, and fleas (4). Previous study by Brown et al. (1997) demonstrated that T. vulgare, Tanacetum ptarmiciflorum (Webb & Berth.) Schultz. Bip. and Tanacetum niveum (Lagasca) Schultz-Bip. contain parthenolide and these species exhibit in vitro anti-inflammatory activity. However, lower parthenolide contents of T. vulgare and T. ptarmiciflorum proved that parthenolide was not the only constituent responsible for such activity (4). T. vulgare was further reported to have antibacterial and antihelmintic compounds as well as polysaccharides (6-8, 10). The oil obtained from T. vulgare is used by applying on skin as repellent against insects and the common tick Ixodes ricinus (1, 15). T. vulgare comprises of sesquiterpenes and sesquiterpene lactones, flavonoid derivatives, hydroxycoumarins, sterols, tannic acid, resins and essential oil components (6, 15-17).

Anti-inflammatory activity studies have been conducted on Tanacetum microphyllum DC., which is traditionally used for inflammatory conditions and rheumatic diseases. In vivo trials showed that flavonoids; 5,7,3’-trihydroxy-3,6,4’-trimethoxyflavone (centaureidin) and 5,3’-dihydroxy-4’-methoxy-7-carbomethoxyflavonol as well as a sesquiterpene lactone, and hydroxyachillin isolated from the aerial parts of this species also exhibited these activities (38, 39). Anti-inflammatory activity of the plant was also confirmed by in vivo studies. The flavonoid derivatives; ermanin and 5,3’-dihydroxy-4’-methoxy-7-methoxycarbonylflavonol isolated from T. microphyllum were reported to inhibit inducible nitric oxide synthase and cyclooxygenase-2, which were assumed to be the mechanisms of their anti-inflammatory activity (40). Tanacetum larvatum (Griseb. ex Pant.) Kanitz is another species that was reported to exhibit anti-inflammatory and antiulcerogenic activity. The mechanism of such activity was supposed to be due to the inhibition of DNA binding of the transcription factor NF-κB (21). In the present study, T. densum subsp. sivasicum and T. argenteum subsp. argenteum demonstrated significant anti-inflammatory activity.

The essential oil of Tanacetum santolinoides (DC.) Feinbr. and Fertig was found to possess antimicrobial activity on Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Candida albicans (19). According to the other records, significant antimicrobial effects of Tanacetum balsamita L., Tanacetum aucherianum (DC.) Schultz. Bip. and Tanacetum chiliophyllum (Fisch. Et Mey.) Schultz. Bip. var. chiliophyllum were demonstrated (22, 41). Antimicrobial activity tests were conducted with the aerial part extract of T. densum subsp. sivasicum and the extract was found to be active against B. subtilis and Klebsiella pneumoniae (42). Evaluation of antioxidant activities of T. densum subsp. sivasicum, T. densum subsp. eginense and T. densum subsp. amani revealed that the most active subspecies was T. densum subsp. sivasicum in compliance with its higher phenolic content (43). The wound-healing activity of T. densum subsp. sivasicum demonstrated in the present study, could be related to its both antimicrobial and antioxidant effects, which were previously reported.

In previous studies, the secondary metabolites present in the leaves of T. parthenium have been shown to exert the analgesic activity synergistically and thus the whole leaf extract has been suggested to be used for the prevention of migraine (5, 36).

Tanacetum species were found to contain sterols, essential oil components, sesquiterpene lactones, resins, bitter substances, acetylenes, flavonoids, coumarins and tannic acid (6,9, 14-17). According to the current results, wound-healing activity does not seem to be in accordance with parthenolide content. In this case, flavonoid aglycones and terpenic compounds that may be found in CHCl3 fraction are supposed to be responsible for such an activity.

In addition, studies on T. vulgare and T. ptarmiciflorum have shown that parthenolide was not the only constituent responsible for anti-inflammatory activity (4).

Conclusion

Parthenolide appears not to be the only principle compound responsible for the wound-healing activity but other secondary metabolites present in the aerial parts of the Tanacetum species studied possibly exerted synergistic effects on the observed healing of the wounds.