Burn Wound Healing Activity of Lythrum salicaria L. and Hypericum scabrum L.

Objective. Burns are complicated traumatic injuries caused by several physical or chemical factors. Plants with a wide range of secondary metabolites, with valuable properties like antioxidant and anti-inflammatory activities, can be a promising source of wound healing agents. Materials and Methods. Effects of hydromethanolic extracts of Lythrum salicaria and Hypericum scabrum, individually and in combination, were assessed in second-degree burn wounds in rats in comparison to a white oleaginous base (negative control) and silver sulfadiazine (positive control). Histological assessments as well as total thiol molecules, lipid peroxidation, and total antioxidant power were evaluated in skin tissue samples. Total phenol, flavonoids, and tannins along with the antioxidant and antimicrobial activities of the extracts were also assessed. Results. Total phenol, total flavonoid, and total tannin amounts for L. salicaria and H. scabrum were 331 ± 3.7 and 308.1 ± 5.2 µg gallic acid/mg extract, 5.8 ± 0.4 and 4.3 ± 0.3 µg quercetin/mg extract, and 430 ± 2.33 and 13.4 ± 0.5 µg tannic acid/mg extract, respectively. H. scabrum significantly inhibited S. aureus and L. salicaria moderately suppressed Staphylococcus aureus and Candida albicans growth. Wound contraction percentage with L. salicaria and H. scabrum was 89.5 ± 3.7 and 77.6 ± 4.1, respectively. A well-organized epidermal layer and normal appearance in dermis layer were more observable in the L. salicaria group. Moreover, L. salicaria ointment individually displayed better influence on tissue oxidative stress parameters than H. scabrum and the negative control (P < 0.05). Conclusion. Results of this study clearly confirm the effectiveness of L. salicaria topical ointment as a wound healing agent, possibly due to the considerable polyphenolic content and antioxidant properties.

Burns are a kind of tissue injury due to various causes such as extreme heat, flame, radiation, radioactivity, electricity, or contact with chemicals.¹ Burn severity is categorized in 3 degrees: First degree, in which only the epidermis is damaged; second degree, where the epidermis and dermis are disrupted; and third degree, in which all layers of skin tissue are injured.² The wound healing process is divided into 4 phases: hemostasis, inflammation, proliferation, and tissue remodeling, during which neutrophils, cytokines, and the extracellular matrix play an important role.³ There are not many drugs and therapeutic agents for effectively healing burn wounds with fewer complications than the natural process of wound healing. Thus, medicinal plants with a background of anti-inflammatory, antinociceptive, and antimicrobial activity could be a promising source of new products for burn healing.⁴ In Iranian Traditional Medicine several plants including Achillea millefolium, Aloe spp, Blechnum spp, Caesalpinia spp, Cedrus spp, Centaurea spp, Commiphora spp, Lilium spp, Myrtus communis, Malva spp, Rosa spp, Hypericum perforatum, Lawsonia inermis, and Sesamum indicum have been used frequently as burn healing agents. In addition to burn and wound healing, anti-inflammatory, antioxidant, and antibacterial effects of these herbs were prominent as well.^5,6  Various plants such as Galium odoratum,⁷Pistacia atlantica,⁸Punica granatum,⁹Alternanthera brasiliana,¹⁰Fagonia indica,¹¹ and Hypericum perforatum¹² have been investigated by scientists for their healing properties and has shown observable wound healing effects. Hypericum scabrum L. (Hypericaceae) grows widespread in various regions of Europe, Africa, North America, and Iran.¹³ The plant contains various secondary metabolites such as hypericin, anthraquinone derivatives, flavonoids, tannins,¹⁴ phenols, thymol, carvacrol,¹⁵ fatty acids, and volatile oils.¹⁶ Pharmacological and biological activities of all parts of the plant, especially its aerial parts, were evaluated in previous studies.^9,13-16 Antimycobacterium,¹⁷ antibacterial (against Staphylococcus aureus and Bacillus cereus),^18-20 antiseptic, antidiarrhea, antihemorrhoid, antieczema, antipsoriasis, antidepressant, and antihypoxic activities of H. scabrum were demonstrated in a previous report.¹³ In Süntar et al,¹² the wound healing effects of H. perforatum and H. scabrum were compared in an incision wound model. H. perforatum demonstrated remarkable anti-inflammatory and wound healing activities, while H. scabrum did not show any wound healing or anti-inflammatory effects. 

Lythrum salicaria L. (Lythraceae), commonly known as surmankhal, yerpoose, and turbinkwash in Persian, grows throughout the world and in north and northwest Iran.²¹ Previous studies^22-24 have reported this plant contains phenolic acids, flavonoids, coumarins, tannins, anthocyanins, glycosides, triterpenoids, and organic acids. Aerial flowering parts of the plant are used for treatment of diarrhea, dysentery, chronic intestinal catarrh, hemorrhoids, hematuria, leucorrhoea, dysmenorrhea, vaginitis, and eczema. An extract of L. salicaria showed inhibition activity against Clodosporium cucumerinum, S. aureus, Proteus mirabilis, and Micrococcus luteus²⁵ as well as antifungal activity against Candida albicans.²⁶ This plant also has antidiabetic, anti-Helicobacter pylori, hypoglycemic, antioxidant, anti-inflammatory, and antinociceptive activity,²⁷ along with cytotoxic effects mainly towards the human colon carcinoma (HT-29) cell line.²⁸ The properties of L. salicaria and H. scabrum, especially their antimicrobial, antioxidant, and anti-inflammatory effects, are desirable for the burn healing process. In the research presented in this paper, the authors studied the healing effect on burn wounds of each plant individually, as well as in combination, along with histological and biochemical evaluations. In addition, the authors investigated phytochemicals, antioxidant, and antimicrobial activities of the plant extracts.

Plant material
The aerial parts of H. scabrum were obtained in May 2013 from Damavand, Tehran, Iran and were identified by Professor Gholamreza Amin. A voucher specimen of the plant (NO: 6758-TEH) has been deposited in the herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. Aerial parts of L. salicaria were collected from Guilan province (Lahijan city) in May 2013 and deposited in the central herbarium of medicinal plants, Jahade-daneshgahi (ACECR), Karaj, Iran. A voucher specimen was authenticated by Mr. Youssef Ajani (NO: Ajani313).

Chemicals
All necessary solvents — aluminum chloride (AlCl3); trichloroacetic acid (TCA); 2-thiobarbituric acid (TBA); 2, 4, 6-Tripyridyl-s-triazine (TPTZ); 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB); ketamine; and xylazine — were purchased from Merck KGaA (Darmstadt, Germany). Polyvinylpolypyrrolidone (PVPP), Folin-Ciocalteu reagent (FCR), 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), sodium bicarbonate, and butylated hydroxyanisole (BHA) were purchased from Sigma-Aldrich (Taufkirchen, Germany), and silver sulfadiazine (SS) was purchased from Sobhan Darou Co (Tehran, Iran).

Preparation of the extracts
Aerial parts of both plants were cleaned, shade dried, powdered in a mechanical grinder, and stored at room temperature.  Then, 100 g of each plant powder were extracted 3 times with a mixture of water: methanol (20:80) at room temperature and evaporated by rotary evaporator. The extracts were then lyophilized and stored at -20˚C for further investigations.

Total phenol assay
Total phenolic content was evaluated by FCR.²⁹ Several concentrations of the plant extracts were prepared, and 1 mL of each concentration was mixed with 5 mL of FCR (1:10 dilution with distilled water) and kept in the dark for 10 minutes. The mixture was mixed with 4 mL of sodium bicarbonate (75 mg/mL). Distilled water was added to increase the volume to 10 mL. All solutions were kept at room temperature for 30 minutes. The absorbance of the sample was measured at 765 nm and total phenolic contents were expressed as µg gallic acid equivalent (GAE)/mg extract (EXT). The calibration curve was illustrated using different concentrations of gallic acid (50–200 µg/mL). All experiments were done in triplicate.

Total flavonoid assay
A mixture of 2% AlCl3.6H2O reagent was used to measure the flavonoid content.³⁰ It was added to 20 µL of the sample, and the volume was increased to 10 mL with distilled water. The absorbance of the solution was measured after 10 minutes in 425 nm. Total flavonoid content was expressed as µg quercetin equivalent in mg extract (µg QE/mg EXT) using the following formula: 

(a × 0.875)/b

Where a is the absorbance of the test solution at 425 nm and b is the mass of the powdered extract in grams.

Total tannin assay
Total tannins of the extracts were measured using PVPP, which bind to tannin-phenolics making them insoluble.²⁵ Therefore, PVPP precipitated tannin-phenolic to eliminate them from the extracts. Total tannin percentage was calculated as µg tannic acid in mg extract (µg TAE/mg EXT) using this formula:

Y = 0.042 x + 0.077

Where Y is the amount of tannin in the gram of evaluated extract and x is the absorbance.

Antimicrobial activity
The minimum inhibitory concentrations (MIC) of the extracts were determined by the usual agar dilution method³¹ in which the extract was mixed with Muller-Hinton agar, and the bacterial suspensions were inoculated by spot inoculation on the surface of agar (S. aureus ATCC6538, Pseudomonas aeruginosa ATCC9027, and C. albicans ATCC10231). These microorganisms were obtained from the Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences. Different concentrations of the 2 extracts were mixed with 19 mL of Muller-Hinton agar at 50˚C to give the final concentrations of 7, 3.5, 1.7, 0.85, 0.42, 0.21, 0.1, 0.05, and 0.027 mg/mL. The fresh bacteria and C. albicans were prepared in Muller-Hinton agar and Sabouraud dextrose agar, respectively. Suspensions of freshly cultured microorganisms were prepared in normal saline (0.9% NaCl) to reach the concentration of 10⁷ CFU/mL. One µL each of this suspension was added to plates and reached 10⁴ CFU/mL. The lowest concentration of the extracts that inhibit growth of the microorganism was considered as the MIC value.

Animals
In this experiment, 30 male Wister rats approximately of the same weight (200-250 g) were used. Animals were housed under standard vivarium condition (1 rat per cage, 23˚C ± 1˚C, and 12-hour light/dark cycles) with access to food (ie, standard pellet) and water ad libitum.³²  The ethical committee of animal experimentation, Tehran University of Medical Sciences, Tehran, Iran, approved the treatment protocol.

Wound induction
Rats were anaesthetized by injection of ketamine/xylazine (45/10 mg/kg, intraperitoneal injection) and the dorsal areas of their skin were shaved. A second-degree burn measuring 260 mm² was created with 110˚C heat and 1 standard atmospheric pressure for 10 seconds using an electric heater.⁷

Preparation of ointments
Three types of ointments were prepared in a white oleaginous base including 10% of H. scabrum extract (H), 10% L. salicaria extract (L), and 10% H. scabrum extract plus L. salicaria extract (HL). For control and reference standard, white oleaginous base and SS 1% were used, respectively.

Experimental design
The animals were divided into 5 groups of 6. The control group was treated with the base cream, the positive control received 1% SS, and the 3 other groups were treated by L. salicaria, H. scabrum, and a combination of both extracts. Treatment protocol started 24 hours after burn induction and lasted 14 days.  Animals received ointments once a day and wound areas were photographed with a digital camera using a paper ruler as scale. Wound areas were calculated with Adobe Photoshop CS3, and the wound contraction rate was measured using the following formula:

Wound Contraction (%) = 100 × [(Wound Size on Day 0 – Wound Size on Specific Day) / Wound Size on Day 0]

At the end of the experiment (day 14) all rats were euthanized and tissue samples of the wound area were collected for further investigation.

Histological assessment
Skin samples were halved, with one half used for histological studies. The tissues were fixed in 10% buffered formalin and stained using hematoxylin and eosin; protocol and parameters used were prepared and evaluated according to Haghdoost et al.⁸ The microscopic samples were prepared and examined by a pathologist who was blinded to which group each sample was from, and images of histological sections were taken under 400× magnification.

Analyses of antioxidant defense mechanisms
The other half of each skin sample was kept in storage at -80˚C for assessment of antioxidant defense mechanism biomarkers. Three markers of oxidative stress including total thiol molecules (TTM), cellular lipid peroxidation (LPO), and total antioxidant power (TAP) were assessed.^33,35 All experiments were performed in triplicate.

Lipid peroxidation
This marker was detected using the reaction of TBA with lipid peroxides. The homogenate tissue was mixed with TCA (20%), sulfuric acid (0.05 M) and 0.2 % TBA. The tissue was placed in falcon tubes and was incubated via bain-marie method for 30 minutes in a boiling water bath; the absorbance was measured at 532 nm.³⁴

Total antioxidant power
This marker was measured by ferric-reducing antioxidant power (FRAP) test using FRAP reagent (25 mL of 0.3 M) acetate buffer, 2.5 mL of TPTZ solution, and 2.5 mL of FeCl3.6H2O solution).³⁵ This method is based on the creation of a complex between Fe²⁺ and TPTZ and measuring the absorbance of the obtained complex at 593 nm.

Total thiol molecules
A tris-ethylenediaminetetraacetic acid buffer (0.6 mL) was added to homogenate tissue and DTNB in methanol (40 mL) was mixed in to bring the volume up to 4 mL. After centrifuging the test tubes at 3000 g for 10 min, the absorbance of supernatants was measured at 412 nm.³³

Statistical analysis
A one-way analysis of variance (ANOVA) was used to assess the statistical significance. Data were reported as mean ± standard deviation (SD) and P < 0.05 was considered a statistically significant difference.

Phytochemical screening
Results of total phenols, total flavonoids, total tannins, and antioxidant activity of the extracts are shown in Table 1. Extracts of L. salicaria and H. scabrum contained observable total phenolic content (331 ± 3.7 and 308.1 ± 5.2 µg GAE/mg EXT, respectively). Moreover, the respective total flavonoid of the examined extracts of the plants was calculated as 5.8 ± 0.4 and 4.3 ± 0.3 µg QE/mg EXT. The extract of L. salicaria comprised a considerable amount of tannins (430 ± 2.33µgTAE/mg EXT) in comparison to H. scabrum (13.4 ± 0.5 µgTAE/mg EXT). Both extracts of L. salicaria and H. scabrum showed significant radical scavenging activity with IC50 values of 13.5 µg/mL and 16.4 µg/mL, respectively. However, it is obvious that L. salicaria represented better antioxidant activity compared with vitamin E (s: 14.2 µg/mL), while both extracts displayed lower inhibitory potency toward free radicals in comparison to BHA (7.2 µg/mL). 

Antimicrobial activity assay
The MIC values of the extracts against major pathogens of burn wounds are shown in Table 2. The extract of H. scabrum demonstrated significant antimicrobial activity against S. aureus with an MIC value of 27 µg/mL. The L. salicaria extract had an MIC value of 1.7 × 10³ µg/mL against S. aureus and 2 × 10⁴ µg/mL for C. albicans. The test results of L. salicaria extract exhibited average bactericidal and fungicidal effects. In another examination, both of the extracts were combined and their antimicrobial activities were evaluated, but no additive, synergistic, or antagonist effect was observed. 

Assessment of wound healing activity
The percentages of wound size reduction in different groups are shown in Table 3. There was no significant difference between the wound sizes on day 0 between groups    (P > 0.05). The ointment of L. salicaria was the most effective treatment with 89.56% ± 3.7% wound contraction on the last day of the experiment compared with other treatments. The ointment of the individual plant extracts demonstrated better activity than their combination. Significant differences were observed between treatment groups (H. scabrum and L. salicaria) and the negative control group on day 14, but the difference in wound closure for the combination-treated animals (65.34 ± 1.4) was not statistically significant in comparison to the negative control (57.76 ± 5.6). On the other hand, the difference between wound contraction of the animals treated with L. salicaria and H. scabrum (77.68 ± 4.1) and SS (76.93 ± 3.2) was not significant. 

Histological study
The photomicrographs of the positive and negative controls and extract-treated groups are shown in Figure 1A-D, 1E-H. In the positive control group (SS group), numbers of polymorphonuclear (PMN) cells were decreased in comparison to the negative control group and the large numbers of fibroblast as well as better reepithelialization were seen in the tissue sample (Figure 1A). In the negative control group, despite no reepithelialization in the wound center, a decrease in the number of macrophages and PMN cells occurred (Figure 1B). L. salicaria caused well-organized epidermal layer along with a normal appearance in the dermis. Moreover, fibrino-leukocytic exudates and PMN cells were fewer in the L. salicaria-treated group compared with tissue samples of the other groups (Figures 1C and 1D). Reepithelialization occurred around the wounds in the H. scabrum-treated group, but the number of fibroblasts and macrophages were relatively high (Figure 1E and 1F). Additionally, in the HL group, the epidermis layers were organized around the wound, but the appearance of the dermis was abnormal with a high number of macrophage cells and numerous vacuoles (Figures 1G and 1H). 

Analyses of antioxidant defense mechanisms and lipid peroxidation
All tested materials including positive control and the plant extracts individually or in combination caused a decrease in LPO of the tissue significantly compared with the negative control (P < 0.01 for H. scabrum, P < 0.001 for the L and H groups; Figure 2). The results indicated that SS and L. salicaria ointment showed similar activity in decreasing LPO in the tissue of treated animals (P > 0.05; Figure 2). 

Total antioxidant power
Assay of TAP in tissue samples showed significant elevation occurred in TAP in the L group in comparison to negative control group (P < 0.05; Figure 3). In addition, ointment of L. salicaria considerably enhanced TAP level in the tissue samples more than other treatments; however, the level of TAP in H- and HL-treated groups were evaluated the same as the negative control group (P > 0.05; Figure 3). 

Total thiol molecules
The TTM levels in all treatment groups were successfully evaluated, and the obtained results showed that all treatments caused enhancement in TTM levels compared with the negative control group. Furthermore, comparison of TTM levels in L and HL groups with the negative control group demonstrated significant elevation of the thiol molecules in plant-treated tissues (P < 0.01 for L and P < 0.05 for HL; Figure 4). Both L. salicaria and SS ointments caused a similar increase in TTM levels in examined tissues (P > 0.05; Figure 4). 

Burn wound healing is a complicated process with 4 main phases including hemostasis, inflammation, proliferation, and tissue remodeling in which neutrophils, cytokines, and the extracellular matrix play principal roles.³ There aren’t many drugs and therapeutic agents available for healing burn wounds; therefore, medicinal plants could be a promising source of new products for burn healing.⁴ The present study aimed to determine the role of topical treatments with ointments of L. salicaria and H. scabrum individually and in combination in second-degree burn wounds. Quantitative phytochemical tests revealed that both tested extracts contained phenolic compounds, flavonoids, and tannins in different amounts. The test results also demonstrated L. salicaria extract contained a higher amount of tannins with more potent radical scavenging activity compared with H. scabrum extract. Although both extracts showed antimicrobial activities, H. scabrum strongly suppressed S. aureus growth; while L. salicaria was more active against C. albicans. Ointment of L. salicaria extract individually exhibited better wound healing activity compared with SS. Moreover, histological studies demonstrated L. salicaria ointment caused complete reepithelization in the dermis layer. Inflammatory agents including fibrino-leukocytic exudates and PMN cells were fewer in the L. salicaria-treated group compared with other tissue samples. Assay of oxidative damage biomarkers including LPO, TAP, and TTM levels in the L. salicaria group also confirmed these study findings in the animal experiment and histological analysis. Lipid peroxidation has a crucial role in pathogenesis of various diseases, which is harmful for viability of cells and tissues.³⁶ In the present study, L. salicaria decreased LPO in tissue samples similar to the positive control. L. salicaria ointment also improved TAP as well as TTM in skin tissue samples. In addition, in vitro antioxidant activity of L. salicaria was the same as vitamin E against the free radical DPPH. L. salicaria has been applied traditionally for treatment of hemorrhage, heavy menstrual flow, diarrhea, typhoid, and eczema due to the high amount of phenolic compounds, like tannins and flavonoids.^23,24,37 Flavonoids are well-known free-radical scavengers and LPO inhibitors.³⁸ In a previous study,²⁷ methanolic fraction of L. salicaria extract, rich in flavonoids, showed higher antioxidant and anti-inflammatory activity compared with other fractions. Phytochemical investigations of L. salicaria indicated the presence of ellagic acid and triterpenoid derivatives including erythrodiol, usrolic acid, oleanolic acid, and corosolic acid along with beta-sitosterol and its glycosylated form, daucosterol, in the plant extract.²² The results of the present study showed the extract of L. salicaria was rich in tannins, astringent properties, and antimicrobial agents.^39,40 The mechanism of tannins in wound healing is described as their ability to bind and precipitate proteins forming a protective coat^41-43; moreover, they have antioxidant and anti-inflammatory properties.⁴⁴ Triterpenoides also possess antioxidant and anti-inflammatory activity as well as wound healing properties.⁴⁵ Beta-sitosterol, a plant sterol, also has been shown to accelerate epithelialization⁴⁶ and has anti-inflammatory effects.⁴⁷ Therefore, extract of L. salicaria reduced pain and healing duration of the wound through its antioxidant properties, forming a protective coating, and its anti-inflammatory and antinociceptive activities which are attributed to tannins such as ellagic acid derivatives, triterpenoides, beta-sitosterol, and its glycosylated form, daucosterol. Moreover, L. salicaria exhibited in vitro antimicrobial activity, which is important in the protection against infection in wounds as well as in the wound healing process. The results of a previous study²⁷ provided evidence about anti-inflammatory activity of the L. salicaria extract. Although inflammation is a defense mechanism of skin against microbial contamination, continuation of the inflammation phase delays wound healing.^12,48 The beneficial effect of the plant in healing burn wounds can be attributed to its active phytochemicals like tannin, ellagic acid derivatives, triterpenoides, and sterols.

The findings of a previous report¹² indicated ethanol extract of H. scabrum from Turkey showed no remarkable wound healing and anti-inflammatory activity. However, the results of the present study revealed the ointment of H. scabrum provided better activity in comparison to negative control, and that may be due to the presence of a high amount of flavonoids, phenolic compounds, and tannins. The combined treatment of L. salicaria and H. scabrum did not show significant activity in wound healing compared with the negative control. In addition, the ointment of both plants presented weaker wound contraction properties in comparison to the ointment of each plant ointment used individually. According to previous literature,^40,43,44 tannins could interact strongly with some primary and secondary metabolites. Phenolic groups of tannins are excellent hydrogen donors that form strong hydrogen binding with carboxyl groups of proteins. Thus, the dissatisfying effects of combined formulation may have occurred as a result of an increase in the tannin concentration in the extracts’ mixture that interacted with some other active compounds and made the formulation less effective. Further studies are warranted to explore the detailed mechanism of the wound healing effect of the plant. 

This study was part of Dr. Fatemeh Vafi’s PharmD thesis, supported by Tehran University of Medical Sciences.

Affiliations: Department of Pharmacognosy, Faculty of Pharmacy and Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran; Department of Pharmacology and Toxicology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences; Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences; Pharmacology and Applied Medicine, Department of Medicinal Plants Research Center, Institute of Medicinal Plants, Karaj, Iran; Department of Drug and Food Control, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences; Department of Anatomy, Tehran University of Medical Sciences; and Faculty of Land and Food Systems, University of British Columbia, Vancouver, Canada 

Correspondence:
Mahnaz Khanavi, PharmD, PhD
Associate Professor of Pharmacognosy
Faculty of Pharmacy, Tehran University of Medical Sciences
Tehran, Iran
khanavi@mail.ubc.ca or 
khanavim@tums.ac.ir 

Disclosure: The authors disclose no financial or other conflicts of interest.