An in vitro ethnopharmacological study on Prangos ferulacea: a wound healing agent

Introduction

The Prangos genus belongs to the family Apiaceae. This genus includes about fifty species distributed in the Mediterranean and Middle-East regions including Iran. Fifteen species of this genus are growing wildly in many regions of Iran.¹

The plants of this genus have been used as emollient, carminative, tonic, anti-flatulent and anthelmintic in the traditional medicine of Iran.² According to the literature and also our observations from local people, the freshly crushed root oily exudate of Prangos ferulacea has been used (as topical ointment) as an effective wound healing agent especially for pus-filled wounds both in human and stocks in the northern west of Iran.² Fig. 1 depicts the oil secreted from fresh plant’s root by local people of Toujaleh and Mishadeh villages, Piranshahr, West Azarbayjan province, Iran. In this study, we aimed to evaluate the potential antimicrobial and wound healing activity of the essential oil of P. ferulacea roots obtained from the western north region of Iran. Additionally, by using GC-MS, we could identify the components of the essential oil, to which the antimicrobial and healing effects could be attributed.

Fig. 1. The fresh oil exudes from the roots of Prangos ferulacea traditionally used as a wound healing agent by local people of western north of Iran (Mishadeh and Tujaleh, Piranshahr, Iran).

× The fresh oil exudes from the roots of Prangos ferulacea traditionally used as a wound healing agent by local people of western north of Iran (Mishadeh and Tujaleh, Piranshahr, Iran).

Fig. 1.

Essential oils of Apiaceae taxa are well-known for their variability with respect to the growing site and time of harvest.³ There are several studies investigating the chemical composition of the essential oils of different parts of P. ferulacea such as aerial parts, fruits, seeds, flowers, stems and leaves from different locations. Monoterpene compounds have been well established to be the major component in the essential oil of leaves, stems, flowers and fruits of P. ferulacea,^4,5 and reported to possess antibacterial activity.⁶

Sajjadi et al, for the first time, carried out the GC/MS analysis of the essential oil of the roots of P. ferulacea from centre of Iran and reported the oil to be consist of monoterpene hydrocarbons (78.4%), oxygenated monoterpenes (9.4%), sesquiterpene hydrocarbons (5.3%) and three oxygenated sesquiterpenes.⁷ In addition, a phytochemical screening of the root extract of P. ferulacea resulted in the identification of coumarin compounds, including osthole, isoimperatiorin, oxypeucedanin, psoralen and gosferol, some of which with reported anti-HSV and cytotoxic effects. Moreover, the compound osthole showed anti-inflammatory property⁸ and in vivo antispasmodic^9,10 and antitumor^11,12 activities.

There are some reports evaluating the antimicrobial property of the essential oil from different parts of P. ferulacea. In spite of significant antibacterial activity reported for the fruits, leaves, stems and flowers of P. ferulacea essential oil against both gram-positive and gram-negative bacteria, it has been demonstrated that methanol, ethanol and n-hexane extracts of aerial parts of P. ferulacea showed only slight antimicrobial activity against some gram-positive bacteria.^4,5,13 Razavi et al. reported high antibacterial effect of the fruits and umbels of P. ferulacea essential oil especially against gram-positive bacteria Bacillus cereus.¹⁴ Basically, regarding its traditional use as a wound healing agent, this study was undertaken to evaluate the wound healing activity and antimicrobial effect of the essential oil of P. ferulacea roots from the western north of Iran. For the first time, we aimed to analyze its chemical composition to which these biological activities could be correlated. However, the traditional use of this plant by local people as a wound healing agent is the basis of this study.

Materials and Methods

Results

Effect of PFE on viability of L929 fibroblast cells

The effect of treatment with PFE on fibroblast cells, at a concentration rage of 1-132 µg/mL, was studied by MTT assay. According to the results, as shown in Fig. 2, PFE not only showed no toxic effect on L929 fibroblast cells, but also demonstrated a viability increasing effect at a range of 4-16 µg/mL. Accordingly, for the next step, 4 and 16 µg/mL doses were selected to be evaluated in scratch test for their wound healing potential.

Fig. 2. The effect of the essential oil of P. ferulacea roots (PFE) on the viability of L929 fibroblasts. PFE at different concentratioons (1- 132 μg/mL) was added to fibroblast cells and the viability of cells after treatment was calculated using MTT assay. At doses 4-16 μg/mL, PFE treatment led to a moderate increase in the viability of fibroblast cells.

× The effect of the essential oil of P. ferulacea roots (PFE) on the viability of L929 fibroblasts. PFE at different concentratioons (1- 132 μg/mL) was added to fibroblast cells and the viability of cells after treatment was calculated using MTT assay. At doses 4-16 μg/mL, PFE treatment led to a moderate increase in the viability of fibroblast cells.

Fig. 2.

The effect of PFE on in vitro scratch test

Scratch test, as a standard in vitro method for wound healing studies, was performed to investigate the impact of PFE on the migration of L929 cells to the scratched area, so-called the wounded section. According to the quantitative results listed in Table 3, PFE, at doses of 4 and 16 µg/mL, was able to increase substantially (P < 0.001) the migration of fibroblast cells into the wounded site by 15.4-57.05%. Fig. 3 illustrates the results of scratch assay.

Table 3. The effect of the essential oil of Prangos ferulacea roots (PFE) on the migration of fibroblast cells in the in vitro scratch test
Day	Group	Length between scratch (µm)	Migration Rate (%)
Control	0	195±3.6	-
	1	175.5±2.3	10±0.36
	2	136±5.2	30.2±1.3
PFE (4 µg/mL)	0	208±4.5	-
	1	155±2.8a	25.4±1.1a
	2	76±4.1a	63.5±0. 8a
PFE (16 µg/mL)	0	200.8±1.5	-
	1	92.9±1.7a	53.7±1.3a
	2	26±5.2a	87.05±2.4a
Values are mean ± SD (n = 4). aP<0.001 as compared with normal control group of same day using one way ANOVA with Student-Newman-Keuls post hoc test.

Fig. 3. The effect of the essential oil of P. ferulacea roots (PFE) in scratch wound healing model using L929 fibroblasts. The migration rate of cells into the scratched site evaluated in groups including Control (no treatment) and PFE groups (treated with 4 and 16 μg/mL) at days 0, 1 and 2 after incubation. Photographs were taken at a 4x magnification. Scale bar indicates 0.1 inches.

× The effect of the essential oil of P. ferulacea roots (PFE) in scratch wound healing model using L929 fibroblasts. The migration rate of cells into the scratched site evaluated in groups including Control (no treatment) and PFE groups (treated with 4 and 16 μg/mL) at days 0, 1 and 2 after incubation. Photographs were taken at a 4x magnification. Scale bar indicates 0.1 inches.

Fig. 3.

The effect of PFE on the production of collagen by L929 fibroblast cells

To realize the effect of PFE on the total amount of type I collagen produced by L929 fibroblast cells, the standard collagen assay kit was used as described earlier. As shown in Fig. 4, PFE increased significantly (P < 0.001) the total concentration of type I collagen in a dose-dependent manner.

Fig. 4. The effect of the essential oil of P. ferulacea roots (PFE) on the production of collagen by L929 fibroblast cells. Values are mean±S.D. (n = 4). ^aP<0.01 and ^bP<0.001 as compared with normal control group using one way ANOVA with Student-Newman-Keuls post hoc test.

× The effect of the essential oil of P. ferulacea roots (PFE) on the production of collagen by L929 fibroblast cells. Values are mean±S.D. (n = 4). ^aP<0.01 and ^bP<0.001 as compared with normal control group using one way ANOVA with Student-Newman-Keuls post hoc test.

Fig. 4.

Discussion

Wound healing process is an intricate procedure which concerns many events including angiogenesis, reepithelialization, granulation tissue formation, and remodeling of extracellular matrix.²⁴ Fibroblasts’ role, as the cornerstone cells of the architecture of tissue repair, is of utmost importance. In the earlier days of healing process, fibroblasts proliferate and migrate to the wounded site. Then, from third day after wounding, fibroblasts obtain a new responsibility, collagen production, which is continued until about three weeks.^25,26 Collagen is another important factor during healing process and is an imperative element for events like angiogenesis and extracellular matrix remodeling. The total amount of collagen produced by fibroblasts in a wound is directly associated with the final tensile strength and integrity of newly formed blood vessels.²² Hence, using fibroblast cells in the in vitro wound models, like scratch assay, and evaluating collagen production by these cells is of significant value in studying the wound healing potential of natural products. On the other hand, assessment of proliferative activity or in contrast, toxicity of natural products against fibroblast cells, could be a valuable examination which somehow shows the potency and the safety of the tested product.

In this study, even after treating with high concentrations of PFE (up to 132 µg/mL), the viability of L929 fibroblast cells was remained close to that of normal control cells (Fig. 2). Besides, within the concentration range of 4-16 µg/mL, an increase in the viability of L929 cells was observed; therefore, we tested these doses in scratch assay. Our results showed that PFE at 4 and 16 µg/mL was able to increase significantly (P < 0.001) the population of L929 fibroblast cells in scratched area which could be due to immigration of fibroblasts or proliferation of migrated cells. Furthermore, collagen production by fibroblasts which was assessed after treatment with different concentrations of PFE, increased markedly (P < 0.001) in a dose-dependent manner. This finding can be considered as a consequent event of fibroblast proliferation induced by PFE and confirms the wound healing activity of PFE.

Additionally, monoterpenes were shown to be major constituent of PFE. There are some controversial studies on the impact of monoterpenes in different types of cells. In some studies, they act as antiproliferative,^27,28 in contrast, there are some other reports showing their proliferative effect.²⁹ It is highly important to note that, depending on the cell type, the same compound can elicit different actions. However, we propose that the increased proliferation and migration rate of fibroblast cells by PFE seen in this study could be attributed to its high monoterpene content. On the other hand, non-toxicity of PFE was a valuable finding demonstrating its safety for topical application.

The chemical compositions of the essential oil of different parts of P. ferulacea have been previously investigated. In almost all parts of the plant, monoterpene hydrocarbons have been found to be the most abundant compounds of which 2, 3, 6-trimethyl benzaldehyde, δ-3-carene, α-pinene, sabinene, limonene and phellandrene were the main constituents of the essential oils.^4,5,14 In the present study, PFE was characterized by a high content of the monoterpene hydrocarbons. Thus, this result is consistent with the previous studies. We found that monoterpene hydrocarbons predominated in the oil of P. ferulacea roots (67.3%) with β-Phellandrene (32.1%), m-Tolualdehyde (26.2%), and δ-3-carene (25.8%) and α-Pinene (4.7%) as the major components.

Some hurdles can appear in the wound healing process path. Wound contamination by different categories of microorganisms is a serious and undesirable problem which can result in wound infection and as a consequent, complicating the wound healing process. The situation is more deteriorating because of the growing emerges of multidrug-resistant strains. To combat the shortcomings of the commonly used synthetic antibiotics, there has been an upward global trend in development and application of naturally originating antimicrobial agents since the past few decades. Natural remedies have been widely used in traditional medicine in Iran and it backs to more than 1000 years ago.² The oil extracted from the roots of P. ferulacea has been used as a potent wound healing remedy especially for pus-filled both in human and stocks in the western north of Iran where its effectiveness practically has been proven. Therefore, we hypothesized that essential oil of P. ferulacea roots could have strong antimicrobial activity especially against the strains involving in wound infections so this way, it helps wound healing process.

The normal indigenous skin microbiota such as staphylococci, micrococci and propionibacteria are the very first sources by which the wound is colonized. In addition, exogenous microorganisms originated from the environment predominantly pseudomonas spp. have a high probability to contaminate wounds and worsen the patient's treatment situation.³⁰ Among the contaminant bacteria sources, S. aureus and P. aeruginosa are the most mutant to multidrug-resistant forms.³¹ According to Tammelin et al, 12.5% of S. aureus isolates and 21.7% of Pseudomonas species were found to be resistant to a clinically relevant synthetic antibiotic.³² In addition, strains from Entrobacteriaceae, especially E. coli, have the potential to contaminate wounds. For example, the mostfrequent pathogens isolated from the post-operation infected wounds of 676 surgery patients were S. aureus (28.2%), P. aeruginosa (25.2%), E. coli (7.8%), and S. epidermidis (7.1%).³³

Now, care professionals unanimously believe in that aerobic or facultative pathogens such as S. aureus, P. aeruginosa, and beta-hemolytic streptococci are the primary causes of delayed healing and infection in both acute and chronic wounds.³⁰ According to our antimicrobial results, the essential oil of P. ferulacea roots remarkably suppressed the growth of S. aureus and P. aeruginosa in agar disc diffusion assay with MIC values of 20 µg/mL (Table 2) reflecting its significant role in wound healing activity.

The antibacterial activity of essential oils rich in monoterpenes has been frequently reported.^34-39 For instance, the essential oil of Melaleuca alternifolia (tea tree oil), which consists largely of cyclic monoterpenes, exhibited a broad-spectrum antimicrobial activity against S. aureus, E. coli and C. albicans.³⁶ Therefore, the high monoterpene content, which was revealed in the essential oil of this plant to be as 67.3% of the total oil, and the probability of synergistic effect of the individual monoterpene constituents, is proposed to be responsible for its strong antimicrobial activity.

Conclusion

Taken all together, the present study demonstrated the promising wound healing property of the essential oil of P. ferulacea roots from western north of Iran, as evidenced by its potent antibacterial activity along with its ability to induce proliferation, migration and collagen production of fibroblasts and these findings corroborate its traditional use as a wound healing agent. The observed effects could be attributed to the high monoterpene content of the essential oil. Lastly, we suggest that further phytochemical and in vivo studies are needed to elucidate the components responsible for its healing activity and to clarify the actual effect of the oil on skin after its topical application.

Ethical approval

There is no ethical issue to be considered.

Competing interests

The authors declare that they have no conflict of interest.

Acknowledgment

Authors are thankful to Dr. Marco Leonti (Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia, Università di Cagliari, Cagliari, Italy) for revising the manuscript and also to Mr. Ahmad Yousefi (from Piranshahr) and Mr. Esmaeil Padash (villager from Mishadeh) for their help in collecting the plant. Financial support of this study by the Research Vice-Chancellor of Tabriz University of Medical Sciences is faithfully acknowledged.

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