Attenuation of dermal wounds via downregulating oxidative stress and inflammatory markers by protocatechuic acid rich n-butanol fraction of Trianthema portulacastrum Linn. in wistar albino rats

Biomedicine & Pharmacotherapy 96 (2017) 86–97

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Original article

Attenuation of dermal wounds via downregulating oxidative stress and inflammatory markers by protocatechuic acid rich n-butanol fraction of Trianthema portulacastrum Linn. in wistar albino rats ⁎

MARK

⁎

Ekta Yadav, Deepika Singh, Pankajkumar Yadav , Amita Verma

Bioorganic & Medicinal Chemistry Research Laboratory, Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom University of Agriculture, Technology & Sciences (SHUATS), Allahabad 211007, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Trianthema portulcastrum Wound Epithelialization Hydroxyproline content Collagenation

Oxidative stress and inflammation contribute as a key factor for retarding the process of dermal wound healing. Trianthema portulcastrum Linn. (TP) leaves reported to possess antioxidant, antifungal, anti-inflammatory and antibacterial properties, which could make TP a promising wound healing agent. The current study was aimed to estimate the antioxidant potential of the fractionated hydroethanolic extract of TP leaves and evaluate wound healing activity by excision and incision wound models along with the assessment of possible underlying mechanism. Ethyl acetate, chloroform and n-butanol fractions of the hydroethanolic extract of TP leaves were examined for in vitro antioxidant ability by DPPH method. Strongest antioxidant activity bearing n-butanol fraction (nBuTP) was further analyzed quantitatively by High Performance Liquid Chromatography coupled with Diode Array Detector (HPLC-DAD). Wound healing potential of nBUTP using excision and incision wound model was studied. Wistar albino rats were randomly divided into four groups, containing six animals in each group; group I served as control treated with simple ointment base, group II was standard group, treated with povidoneiodine ointment USP (5%), group III treated with nBuTP 5% w/w ointment, and group IV treated with nBuTP 10%w/w ointment. All the groups were topically applied their respective ointments, once daily, till the complete healing achieved. Wound healing was assessed by analyzing % wound closure, hydroxyproline content, epithelialization period, tensile strength, enzymatic antioxidative status and inflammatory markers. Total phenolic and flavonoid content of the extract was estimated to be 112.32 ± 1.12 and 84.42 ± 0.47 mg/g, respectively. HPLC-DAD of nBuTP confirmed the presence of chlorogenic acid (20.74 ± 0.03), protocatechuic acid (34.45 ± 0.02 mg/g), caffeic acid (4.31 ± 0.03 mg/g) and ferulic acid (1.43 ± 0.01 mg/g). 5% and 10% w/ w nBuTP ointment significantly accelerated the wound healing process dose-dependently in both wound models, evidenced by the faster rate of wound contraction, epithelialization, increased hydroxyproline content, high tensile strength, increased antioxidant enzyme activity, decreased the level of inflammatory markers compared to the control group. Histopathological studies also revealed the dose-dependant amelioration of wound healing by re-epithelialization, collagenation and vascularization of wounded skin sample in nBuTP treated groups. These results implicate potential medicinal value of nBuTP to heal dermal wounds.

1. Introduction The wound is a common health issue all over the world [1]. It is formed as a loss of continuum of anatomical and cellular function in the normal living tissue, caused by the various cell abuses (e.g., chemical, physical, thermal injury and microbial infection) [2]. Wound healing is

a complex biological convalesced and multifaceted mechanism in response to tissue impairment. Optimal wound healing can be achieved by the fundamental principle of diminishing the further growth of tissue injury along with furnishing ample tissue perfusion, nutrition, adequate oxygenation and favorable protective environment, which ameliorates the normal anatomy and physiology of the affected part [3]. The

Abbreviations: CAT, catalase; CHFTP, chloroform fraction of Trianthema portulacastrum Linn. hydroethanolic extract; CRP, C-reactive protein; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid; EATP, ethyl acetate fraction of Trianthema portulacastrum Linn. hydroethanolic extract; EDTA, ethylene diamine tetra acetic acid; GAE, gallic acid equivalents; GSH, glutathione; GPx, glutathione peroxidase; HPLC-DAD, high performance liquid chromatography coupled with diode array detector; NBT, Nitro blue tetrazolium; nBuTP, n-butanol fraction of Trianthema portulacastrum Linn. hydroethanolic extract; RE, rutin equivalents; SOD, superoxide dismutase; TNB, 2-nitro-5-thiobenzoate; TP, Trianthema portulacastrum Linn ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Verma). http://dx.doi.org/10.1016/j.biopha.2017.09.125 Received 6 August 2017; Received in revised form 24 September 2017; Accepted 24 September 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.

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were procured from Hi Media Laboratories Pvt. Ltd., Mumbai, India. Rutin, gallic acid, chlorogenic acid, ferulic acid, caffeic acid, protocatechuic acid, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). All chemicals were of analytical and HPLC grade.

ultimate aim of wound healing is tissue reconstruction, accomplished by a cascade of stages that involves hemostasis (release of clotting factors to check bleeding) with concurrent inflammatory response (neutrophils, monocytes and macrophages move towards the wound area), proliferation (granulation of tissue by regeneration of blood, lymphatic vessels and collagen fibers with the help of fibroblast and myofibroblast), re-epithelialization (keratinocytes approach over the granulation tissue), differentiation (formation of new outer layer), maturation (injured tissue healed, development of scar and further strengthening of granulation tissue by building structural protein) and restitution of tensile strength of wounded cell [4,5]. Due to unavoidable side effects of presently used synthetic drugs for wound management, it is necessary to detect an alternate remedy with minimum side effects and more effectiveness [6,7]. Since past few decades, traditional folklore medicine and their extracts are becoming a promising choice for the management of wound by the researchers, due to the presence of secondary metabolites of diverse nature [8,9]. However, owing to government's strict forest policies, extinction of plant species and other factors, the collection of plant drugs from the jungle is not appropriate. Therefore, the use of weeds, as medicine, provides a better option in this regard [10]. Trianthema portulacastrum Linn. (TP) is an annual and perennial weed of tropical America, Africa, Southeast Asia and other parts of the world [11]. TP is a potentially utilized herb in Indian traditional medicinal system, belonging to Aizoaceae family. It is commonly known as horse purslane, sabuni, santhi, vishakhapara, bishkapra [10]. The weed is rapidly growing, branched, succulent and prostrate. It grows during rainy season along with various agricultural crops [12,13]. Leaves are opposite, petiolated, ovate obovate in shape, entire margin, purple or green in color and 0.5–3 cm in size. Flowers are solitary, bisexual, and pale pink and white in color [13,14]. In India, it is used as a green leaf vegetable and is considered to have high nutrition value due to the presence of fiber, proteins, riboflavin, potassium, sodium and iron [15,16]. Traditionally it is used for the treatment of anemia, ulcer, jaundice, inflammation, liver disease, migraine and night blindness [13,17,18]. The different plant parts of TP have analgesic, antipyretic, anti-inflammatory and antibacterial properties [19,20]. Literature study reveals the presence of following biologically important phytoconstituents in TP: flavonoids, C-methyl flavones, leptorumol, β-sitosterol, stigmasterol, quercetin, ferulic acid, oxalic acid, trianthenol, 5-hydroxy-2-methoxy benzaldehyde, 3-acetyl aleuritolic acid, p-methoxy benzoic acid, p-propoxy benzoic acid beta-cyanin and 3,4-dimethoxy cinnamic acid [21–24]. On the basis of experimental research, TP is known to possess various therapeutic activities, namely, hepatoprotective [25] hepatic and mammary anti-tumor [26,27], antifungal [24], antioxidant [28,29], analgesic [30], hypoglycemic, hypolipidemic [31], anthelmintic [32], mosquito larvicidal [33], diuretic [34], anti-inflammatory [35], etc. In African countries, TP is traditionally used for the treatment of wound dressing [36]. TP leaves are reported to contain bioactive compounds, such as, phenolic and flavonoid compounds, which are responsible for antioxidant, antibacterial and anti-inflammatory effect [37–39]. With this consideration, the present study was designed to scientifically explore the folklore use of TP leaves as wound healing agent by incision and excision wound models in wistar albino rats along with biochemical estimation. There is no scientific study about wound healing activity of TP leaves so far.

2.2. Collection and authentication of plant Fresh leaves of TP were collected in the rainy season from Bhotwas village of Rewari district, Haryana, India. Plant material was scientifically identified and authenticated by Prof. R.M. Kadam, Head of Department, Department of Botany, Mahatma Gandhi Mahavidyalaya, Latur, Maharastra, India. 2.3. Extraction and fractionation of plant extract The collected plant leaves were thoroughly washed with water, dried under shade and then coarsely powdered. The dried plant material (2.5 kg) defatted with petroleum ether (60–80° C) and air dried. The resultant powder was macerated with 70% ethanol for three times at room temperature and filtered. Consequently, the filtrate was evaporated under vacuum to obtain a concentrated hydroethanolic extract (112 g) followed by suspending it in water in a separating funnel and partitioned with chloroform, ethyl acetate and n-butanol, respectively. Different fractions obtained by partitioning with all three solvents [ethyl acetate (EATP) (15.1 g, 13.48% w/w), chloroform (CHFTP) (30.6 g, 27.32% w/w), and n-butanol (nBuTP) (45.4 g, 40.53% w/w)] were filtered using Whatman filter paper (no. 1) and the respective filtrate was stored at −20 °C till further study [40]. 2.4. Colorimetric determination of total phenolic compounds Total phenolic content of TP was estimated by Folin-Ciocalteu colorimetric method described by Singleton V et al. [41]. The result was determined from the calibration curve of a standard solution of gallic acid and expressed as mg gallic acid equivalents (GAE)/g of extract. The experiment was repeated thrice. 2.5. Colorimetric determination of total flavonoid compound Total flavonoid content was determined by aluminium chloride colorimetric technique [42]. The flavonoid content was expressed in mg rutin equivalents (RE)/g of extract from standard rutin solution calibration curve. The experiment was repeated thrice. 2.6. In vitro antioxidant activity All three fractions (ethyl acetate, chloroform, n-butanol) were evaluated for their ability to scavenge free radicals by 2,2-diphenyl-1picrylhydrazyl (DPPH) method, as described by Kedare and Singh [43]. The reaction was started with the addition of 3.3 mM DPPH (150 μl) solution, prepared in methanol, in different concentrations of each fraction of TP. The resultant solution was placed in the dark at 25° C for 30 min and then scanned spectrophotometrically at 517 nm. Ascorbic acid was used as a standard to compare the antioxidant activity. Following formula was used to calculate the antioxidant capability of the sample.

2. Material and methods 2.1. Chemicals and reagents

Activity of radical scavenging (%) Absorbance of control − Absorbance of sample x100 = absorbance of control

Sodium chloride, sodium hydroxide, sodium azide, aluminium chloride, hydrogen peroxide, hydrochloric acid, acetic acid were purchased from Qualigens (Mumbai, India). Folin and Ciocalteau’s phenol reagent was obtained from Merck KGaA (Darmstadt, Germany). Nitro blue tetrazolium (NBT) and ethylene diamine tetra acetic acid (EDTA)

The IC50 value of each fraction was analyzed by using log (dose) vs % antioxidant activity graph. 87

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24 h of shaving back of rats, test ointments were applied and regularly observed for adverse skin symptoms (redness, irritation, itching and inflammation) for 15 days.

2.7. Quantitative analysis of phenolic compounds in nBuTP High performance liquid chromatography (HPLC) (Shimadzu, Japan) fabricated with a diode array detector (DAD-MZOA) and a binary pump was used for analysis of phenolic compounds in nBuTP. The sample was filtered by using 0.2 μm nylon filter (Millipore, Germany) and the filtrate was injected in 250 mm × 4.6 mm (i.d.), 5 μm C18 reverse phase column (Merck, Darmstadt, Germany). The chromatographic separation was performed by using the mobile phase [0.1% (v/v) orthophosphoric acid (solvent A) in HPLC-grade methanol (solvent B) in 60:40 ratio]. The solvent gradients for the analysis were 0–15% B in 15 min, 15–40% B in 40 min, and 45–100% B in 25 min with a flow rate of 0.8 ml/min. The standard compounds, i.e., ferulic acid, protocatechuic acid, chlorogenic acid and caffeic acid, were used for identification and quantification of phenolic compounds in nBuTP by comparison of their respective retention time.

2.12. Animal grouping for wound healing assay Two wound models (incision and excision) were used to assess the wound healing activity of test samples. Rats were randomly divided into four groups, containing six animals in each group. Similar animal groups were used for both wound models. Group I– Treated with vehicle (ointment base) Group II– Treated with standard ointment, i.e., povidone iodine ointment USP (5% w/w) Group III– Treated with 5% w/w nBuTP ointment Group IV–Treated with 10% w/w nBuTP ointment The experimental study of wound healing assessment was performed with once in a day topical application of their respective treatment, till the wound was completely healed.

2.8. Animals 48 healthy adult male wistar albino rats (180–200 g) were selected for the present study. Rats were housed in polypropylene cages, allowed free access to the standard pellet diet and water ad libitum throughout the experiment. Seven days prior to the start of the study, animals were acclimatized to the standard laboratory conditions, i. e., temperature (25 ± 2° C), relative humidity (44–56%), light and dark cycles (12:12 h), of the well ventilated animal house. The experiment protocol was permitted by the Institutional Animal Ethics Committee, SHUATS, Allahabad, India (Approval Number IAEC/SHIATS/PA16III/SEYAV09).

2.13. Excision wound model All the animals from four groups were shaved on their dorsum region with the help of depilatory cream (Reckitt Benckiser Inc., UK) 24 h prior to the creation of wound. Rats were anesthetized with mild ether. Excision wound outline of approximately 500 mm2 circular area was drawn on the shaved dorsal portion of all the rats followed by the creation of wound of 2 mm thickness at the marked area by using a sterilized sharp surgical blade and scissor according to the described method [47]. The wound was cleaned with sterile cotton wipe dipped in normal saline and treated with their above mentioned respective group treatment, considered as day 0, till the wound was observed as completely healed. Wounds were kept without dressing throughout the experimental duration. Various parameters pertaining to wound healing (wound area, epithelialization period, wound closure and hydroxyproline content) were evaluated.

2.9. Preparation of nBuTP formulation Simple Ointment (British Pharmacopoeia) was used as a base for formulation [44]. Separately nBuTP was added into prepared ointment base to obtain test formulation at two dose levels, 5% and 10% w/w. 2.10. Acute oral toxicity

2.13.1. Collection of blood and tissue At the end of the experiment (18th day), animals from the excision wound model were euthanized by cervical dislocation, blood was collected from each group in EDTA containing collection tubes and used for analysis of inflammatory markers. The skin sample was taken out from the wound region of all groups of excision wound model. A major part of dissected wound tissue was used for analysis of enzymatic antioxidant defense system and the remaining part was preserved for histology. For antioxidant enzyme profile studies, the collected wound tissue was homogenized in TBS buffer [Tris-HCl (50 mM): NaCl (150 mM), 1:2] at pH 7.4 in Ultra-Turax homogenizer. Resultant homogenates were centrifuged for 15 min at 4 °C, 5500g and the supernatants were stored at −80 °C till further use.

Adult male wistar albino rats were selected for acute oral toxicity study. Suspension formulation of nBuTP was prepared by using 30% DMSO and administered orally to overnight fasted rats at four different dose levels, i.e., 2000, 3000, 4000 and 5000 mg/kg, for 14 days according to the OECD guidelines. Control group rats were treated with 2 ml/kg DMSO [45]. After administration of the respective dose, rats were examined for general behavior, activity, food and water intake, mortality or toxic symptoms, such as anorexia, muscle cramping, salivation, diarrhea, convulsions, if any, for 48 h and then daily for 14 consecutive days. Next day (15th day), rats were euthanized and blood was collected from orbital sinus for examination of various hematological alterations [46]. EDTA was used as an anticoagulant. Vital organs (kidney, brain and liver) of various groups were carefully dissected, cleaned, blotted and immediately weighed accurately and preserved in 10% formalin solution. The ratio of each organ to the body weight of control and nBuTP (2000, 3000, 4000 and 5000 mg/kg) administered rats was calculated. Histological alteration of vital organs, such as liver and kidney, was also examined. Before blocking the liver and kidney tissues in paraffin wax, they were dehydrated with alcohol and then processed in xylene. Tissue sections (5 μm) were stained with hematoxylin and eosin dye. The microphotographs of histological structure of prepared sections were obtained by using a light microscope.

2.13.2. Evaluation of wound healing parameters 2.13.2.1. Percentage wound closure rate and epithelialization. Wound area was calculated by tracing the wound contour on a transparent sheet followed by placing the sheet of a traced wound on 1 mm2 graph paper. For visual assessment, photographs of the wound were clicked on every third day till the wound was completely healed. Wound closure rate was determined by following formula [48].

Wound closure (%) Area of wound on day 0 − Area of wound on nth day x100 = area of wound on day 0

2.11. Acute dermal irritation and toxicity study

Where n represents the number of days, i. e., 3rd, 6th, 9th, 12th, 15th and 18th. Epithelialization period (number of days taken to drop off the dead tissue without any sign of raw wound) was also recorded [49].

Acute dermal irritation and toxicity study was performed at 2000 mg/kg dose of nBuTP according to OECD guideline 402. After 88

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2.13.2.2. Hydroxyproline estimation. The content of hydroxyproline was estimated by using granulation tissues resulted on wounds of excision wound model [50]. Granulation tissues were cleaned with 0.9% w/v cold saline solution followed by drying in the oven at 60 °C. Dried tissues were hydrolyzed by HCl (6N) at 130 °C for 4 h. Sodium hydroxide (10 N) was poured to neutralize the tissue hydrolysate to pH 7.0 and placed aside for 20 min to attain chloramine-T oxidation. The reaction was completed by the addition of perchloric acid (0.4 M) and then color was formed by using Ehrlich reagent. Color intensity was recorded spectrophotometrically (Shimadzu UV-1800, Japan) at 557 nm. Hydroxyproline content was calculated with the help of hydroxyproline calibration curve and results were expressed as mg/g of dry granulation tissue.

Hemostasis was attained with the normal saline cotton swab and then the parted skin was stitched by intermittent sutures using a curved needle (no. 11) and black silk surgical thread (no. 000) at a distance of 1 cm. To obtain good closure, suture thread was tightly knotted on both the edges of the wound. After 24 h of stitching, wounds were topically treated with their respective treatment, considered as day 1, once a day, till complete healing. On the 13th post-wounding day, the sutures were removed, while ointment application was continued till 15th day and tensile strength of healed wound was measured by continuous constant water flow technique [57].

2.13.2.3. Determination of enzymatic antioxidant profile of granulation tissue. Enzymatic antioxidant activity, such as Superoxide dismutase (SOD), Catalase (CAT) and Glutathione peroxidase (GPx), was evaluated by using supernatants from homogenates of wound tissues of excision wound model. SOD activity was measured by using the method described by Winterbourne et al. [51], which is based on the principle of inhibition of photoreduction of NBT dye by SOD enzyme. Absorbance was measured at 560 nm and enzyme activity was expressed as unit/mg of protein in tissue. A unit of enzyme defined as the amount of enzyme that inhibits 50% of the oxidation reaction. CAT assay was initiated with the addition supernatant into phosphate buffer (0.01 M) followed by addition of H2O2 (0.16 M). After incubating the reaction mixture for 1 min at 37° C, the assay was completed by mixing of dichromate: acetic acid reagent. Absorbance was examined spectrophotometrically at 570 nm according to the method of Sinha et al. [52]. CAT activity was expressed as μM of H2O2 consumed/mg protein in tissue. GPx activity was examined using the procedure given by Flohe and Gunzler [53]. The assay reaction is based on the rate of formation of 2nitro-5-thiobenzoate (TNB) from DTNB coupled with the oxidation of glutathione (GSH) by GPx enzyme. Absorbance was measured at 412 nm and the result was expressed as μM/mg protein in tissue.

All data were expressed as mean ± standard error mean (SEM) of six animals in each group and the results were compared statistically using one-way analysis of variance (ANOVA) followed by Dunnett’s test using GraphPad Prism software. Mean values were considered statistically significant at p < 0.05, p < 0.01 and p < 0.001.

2.15. Statistical evaluation

3. Results 3.1. Evaluation of total phenolic and total flavonoid content Colorimetric method was used to estimate the total phenolic and flavonoid concentration in TP hydroethanolic extract. Total phenolic content was found to be 112.32 ± 1.12 mg of gallic acid equivalents (GAE)/g of dry extract. Flavonoid content was observed to be 84.42 ± 0.47 mg of rutin equivalents (RE)/g of dry extract.

3.2. Antioxidant activity All three TP fractions were used to detect their hydrogen or electron donation ability by the stable free radical DPPH colorimetric method. The results were evident of antioxidant activity in order of nBuTP > CHFTP > EATP with IC50 values of 35.95 ± 1.11, 56.08 ± 1.05, 93.07 ± 1.21 μg/ml, respectively (Table 1) (Fig. 1A). The strongest free radical scavenging activity was exhibited by ascorbic acid with the IC50 value of 0.74 ± 0.08 μg/ml (Fig. 1B). All samples showed the antioxidant activity in concentration dependant manner.

2.13.2.4. Determination of inflammatory markers. At the end of wound healing study, blood was collected from a retro orbital vein of every rat and analyzed for inflammatory markers by estimation of C-reactive protein (CRP) turbidimetrically in serum with the help of commercial kit (Beacon Diagnostics Ltd, India). Plasma fibrinogen was detected according to the colorimetric method described by Deepa et al. [54]. Briefly, the assay It the with mixing of plasma sample in CaCl2 (2.5%), which results in the formation of a clot. Then, the clot was rinsed repeatedly with distilled water and dissolved in NaOH (0.25 N) in a water bath. H2SO4 was added to neutralize the clot followed by mixing of Folin-Ciocalteau reagent and Na2CO3 (20%). The resultant reaction mixture was incubated for 30 min at 37° C and absorbance was measured at 620 nm.

3.3. Quantitative determination of phenolic compounds in nBuTP by HPLCDAD method Since nBuTP showed the highest antioxidant activity, it was selected for further study. The phenolic concentration of nBuTP was detected by HPLC-DAD, which confirmed the presence of protocatechuic acid, chlorogenic acid, caffeic acid and ferulic acid as shown in Fig. 2. Concentrations of phenolic compounds are shown in Table 2. It was observed that nBuTP contains the higher amount of protocatechuic acid (34.45 ± 0.02 mg/g of extract) among other detected phenolic compounds.

2.13.2.5. Histological study. For observation of histological alterations, the collected healed wound skin specimens were separately preserved in 10% formalin, dehydrated with alcohol, processed in xylene and then blocked in paraffin wax. Skin sections of 5 μm were cut and stained with hematoxylin and eosin dye [55]. The stained section was observed under a light microscope and microphotographs were taken for each group.

Table 1 Antioxidant activity of different fractions of TP and ascorbic acid.

2.14. Incision wound model Pre-shaved rats were anaesthetized and 6 cm longitudinal line was marked on either side of the vertebral column parallel to the paravertebral region. An incision was made with the full thickness of 2 mm through the marked skin, with the help of sterile surgical scissor [56].

Sample

Log IC50 (μg/ml)

IC50 (μg/ml)

EATP CHFTP nBuTP Ascorbic acid

1.9688 1.7488 1.5557 −0.1258

93.07 ± 1.21a 56.08 ± 1.05b 35.95 ± 1.11c 0.74 ± 0.08

Each value is reported as mean ± SD (n = 3). Statistically significant at p < 0.05, where a >

89

b > c

in each column.

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Fig. 1. (a) Antioxidant activity of different TP fractions on DPPH assay, (b) Antioxidant activity of ascorbic acid on DPPH assay.

observed in the standard group than control group. Since day 9, values of wound contraction for nBuTP10 group (p = 0.0072, 40.03%) was found comparable to the standard group (p = 0.0047, 40.61%). Till 18th day, the rate of wound closure was significantly improved in nBuTP10 group (p < 0.0001), more or less equal to standard group (p < 0.0001). Since day 9, there was significant dose dependant reduction in wound area was observed in nBuTP5 group as compared to control group (day 9, p = 0.0287; day 12, p = 0.0193; day 15, p = 0.0102; day 18, p = 0.0054). In addition to this, rapid epithelialization was also detected in standard and nBuTP10 groups (18.14 ± 0.7 and 18.62 ± 0.5 days, respectively) and moderate epithelialization in the nBuTP5 group (20.04 ± 0.2 days) as compared to control group (22.25 ± 0.8 days).

Fig. 2. HPLC-DAD chromatogram of nBuTP fraction. Peaks marked with 1-Protocatechuic acid, 2-Chlorogenic acid, 3-Caffeic acid, 4-Ferulic acid.

Table 2 Individual phenolic content in nBuTP fraction (mg/g extract). Phenolic compound

nBuTP (mg/g extract)

Chlorogenic acid Protocatechuic acid Caffeic acid Ferulic acid

20.74 ± 0.03 34.45 ± 0.02 4.31 ± 0.03 1.43 ± 0.01

3.6. Hydroxyproline content Rats treated with the standard drug (p = 0.0011) showed the highest hydroxyproline content, which was equivalent to the nBuTP10 group (p = 0.0048) when compared with control group. While nBuTP5 group exhibited slightly less hydroxyproline content compared to standard group and significantly higher (p = 0.0478) than the control group (Table 4).

Each value is reported as mean ± SD (n = 3).

3.7. Enzymatic antioxidant profile of granulation tissue

3.4. Acute oral and dermal toxicity studies

Tissue antioxidant enzymatic activity of control group was found less as compared to other groups, which is evident of the oxidative stress condition in the control group (Table 5). However, after application of nBuTP ointment (5 and 10% w/w), the SOD activity (p = 0.0320 and p = 0.0012, respectively), CAT activity (p = 0.0299 and p = 0.0038, respectively) and GPx activity (p = 0.0445 and p = 0.0061, respectively) significantly increased in dose-dependent manner as compared to control group.

There was no mortality or sign of toxic symptoms, such as anorexia, salivation, convulsions, muscle cramping, breathing trouble and hypo activity, were observed as compared to control group throughout the duration of toxicity study. Hematological parameters, i.e., RBC, WBC and hemoglobin count, were found unaltered as compared to control group. LD50 of nBuTP was estimated to be higher than 5000 mg/kg. In case of dermal toxicity test, the limit test dose did not show any adverse reaction on rat skin, i. e., irritation, redness, swelling and itching, as compared to control group. There was no alteration in body weight and weight of vital organ as well as the histological structure of liver and kidney tissues during the whole experimental period. (Table 3, Figs. 3 and 4)

3.8. Inflammatory markers Inflammatory status of different groups was evaluated by examining the level of CRP and fibrinogen, which are well known proteins in the inflammatory condition. Increased concentration of these proteins indicates the state of inflammation. CRP content was found to be significantly less in standard group (p = 0.0003) and nBuTP10 group (p = 0.0005), followed by nBuTP5 group (p = 0.0164) as compared to control group (Table 6). Results of fibrinogen level were observed to be significantly alleviated in standard (p = 0.0004), nBuTP10 group (p = 0.0011) and nBuTP5 group (p = 0.0208) when compared to control group.

3.5. Effect of nBuTP on percentage wound closure and epithelialization period Variation in wound area of excision model at every third day was observed (Fig. 5). From the results (Table 4), it was noticed that on the 3rd day of post wounding, there was no significant change in wound closure in all treatment groups as compared to control. Whereas on the 6th day, significant (p = 0.0072) reduction in wound area was 90

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Table 3 Effect of nBuTP on hematological parameters, body weight and organ weights relative to body weight in acute oral toxicity. Parameters

Control

nBuTP (2000 mg/kg)

nBuTP (3000 mg/kg)

nBuTP (4000 mg/kg)

nBuTP (5000 mg/kg)

WBC (×103 cells/μl) RBC (×103 cells/μl) Hemoglobin (g/dl) Body weight (g) Liver (g%) Brain (g%) Kidney (g%)

13.2 ± 0.5 9.2 ± 0.7 19.4 ± 0.9 189 ± 6.12 2.78 ± 0.17 0.49 ± 0.05 0.53 ± 0.04

12.2 ± 0.4a 9.1 ± 0.3b 20.2 ± 1.1c 200 ± 5.23d 2.67 ± 0.12e 0.50 ± 0.02f 0.54 ± 0.01g

12.8 ± 0.4a 10.2 ± 0.5b 19.4 ± 1.0c 189 ± 4.67d 2.69 ± 0.11e 0.47 ± 0.03f 0.54 ± 0.03g

13.7 ± 0.7a 10.4 ± 0.3b 19.8 ± 0.8c 188 ± 4.51d 2.7 ± 0.1e 0.49 ± 0.04f 0.52 ± 0.02g

14.3 ± 0.8a 9.1 ± 0.2b 20.4 ± 1.2c 200 ± 5.61d 2.75 ± 0.13e 0.48 ± 0.03f 0.53 ± 0.02g

All the values are expressed as mean ± SEM, n = 6. Statistically significant at p < 0.05, where a,b,c,d,e,f,g in each row indicates statistically non significant (p > 0.05), when compared to control.

(Fig. 6c). However, the slide of nBuTP5 (Fig. 6d) showed a potential increase in collagen fibers, fibroblast cells along with well re-epithelialization, keratinization and lack of inflammatory cells in comparison to control group (Table 7) [58,59].

3.9. Histopathological examination Histopathological parameters of excision wound tissue collected from every group on day 18 were evaluated, as shown in Fig. 6(a–d). Section of the control group showed huge inflammatory cells, decreased the level of collagen fibers, pus cells, necrosis and fibroblast (Fig. 6a). Standard group section showed complete tissue healing, which was observed by the appearance of re-epithelialization, decreased the level of inflammatory cells, whereas collagen fibers, blood vessels and fibroblast cell were increased (Fig. 6b). Section of nBuTP5 group rat showed re-epithelialization, fibroblast, platelets, less inflammatory cells, elevated concentration of collagen fibers and blood vessels

3.10. Tensile strength On the 15th day, tensile strength for each group from incision wound model was measured and reported in Table 8. Breaking strength was found to be highest in the standard group (p < 0.0001) followed by nBuTP10 group (p < 0.0001) and nBuTP5 group (p = 0.0014) as compared to control group. Fig. 3. Photomicrograph (20X) of haematoxylineosin stained rat kidney in acute toxicity study of nBuTP at dose level of (a) 2000 mg/kg, (b) 3000 mg/ kg (c) 4000 mg/kg and (d) 5000 mg/kg.

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Fig. 4. Photomicrograph (20X) of haematoxylineosin stained rat liver in acute toxicity study of nBuTP at dose level of (a) 2000 mg/kg, (b) 3000 mg/ kg (c) 4000 mg/kg and (d) 5000 mg/kg.

4. Discussion

nBuTP ointment (5% and 10%) showed the prohealing effect in both wound models (excision and incision) when topically applied for 18 days, validated by significant improvement and increase in rate of wound closure, hydroxyproline content, reduction in epithelialization period of granulation tissue and inflammatory marker levels. The significant effect of wound repairing was further supported by escalated enzymatic antioxidant level as well as the histopathological examination of healed tissue. A number of experimental studies have reported that secondary metabolites of a plant, such as phenolic compounds, flavonoids and terpenoids accelerates wound healing process, mainly due to their antioxidant, anti-inflammatory and antibacterial properties, which is principally known to be responsible for rapid contraction of the wound and faster rate of epithelialization [65]. Higher content of total phenolic and flavonoid compounds confirmed the presence of these biomolecules in TP. The highest antioxidant fraction, nBuTP, was quantitatively analyzed by HPLC-DAD and revealed abundant protocatechuic acid along with other phenolic compounds, i. e., caffeic acid, chlorogenic acid and ferulic acid. Protocatechuic acid is well known for its antioxidant and anti-inflammatory activity, which is estimated to exhibit a synergistic effect in the prompt healing of wound [66]. Caffeic acid contributes the wound

Wound healing is an intricate process comprising a chain of biochemical events, commencing with coagulation followed by inflammation, collagenation, wound contraction and ends by epithelialization [60]. Such precise injury mending movement can be thwarted by many elements that include a weak immune system, lacking oxygenation and microbial contaminations, which frequently leads to serious health intricacies. For that reason, the production and advancement of powerful wound recuperating agent, who is primarily capable of efficient wound healing in short span and lessen undesired health inconveniences, is a critical research zone of current biomedical sciences [61]. The wound is a common health issue among the people worldwide, its clinical treatment with a therapeutic agent having the potency to recover one or more phases of wound repairing with minimal side effects is always the biggest challenge in surgical practice [62,63]. The potency of herbal extracts in the form of ointment could ameliorate wound healing and tissue repair with minimum side effects [64]. In the present study, results provide the scientific justification to the traditional information of wound healing potency of Trianthema, since 92

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Fig. 5. Photographic representation of wound healing process in excision wound model. Control (Group 1), standard drug (Group 2), nBuTP5 treated (Group 3) and nBuTP10 treated (Group 4) at 0, 3, 6, 9, 15 and 18 days of post wounding.

collagenation, wound contraction and epithelialization, as it owes antibacterial, anti-inflammatory and antioxidant properties which is concordant with the previous reports [70,71]. As far as safety profile of nBuTP ointment is concerned, it was found safe at a dose level of 5000 mg/kg orally. Moreover, no adverse skin reaction was noticed in case of dermal toxicity study, therefore, it was

healing potential of nBuTP by accelerating the collagen-like polymer production along with decreasing in oxidative stress level and it also possesses anti-inflammatory ability [67]. Additionally, chlorogenic acid and ferulic acid are reported to have anti-inflammatory activity [68,69]. It is estimated that these phenolic compounds impart a major role in the promotion of various phases of wound healing, i. e.,

Table 4 Effect of nBuTP on various parameters of excision wound model. Group

Control Standard nBuTP5 nBuTP10

Wound closure (%) Day 3

Day 6

4.15 ± 2.31 12.78 ± 1.98 9.08 ± 3.01 10.58 ± 2.44

14.37 25.89 19.58 22.67

Day 9 ± ± ± ±

3.21 2.32y 2.21 1.39

29.71 40.61 38.15 40.03

Day 12 ± ± ± ±

1.65 1.65y 3.45x 0.87y

48.73 68.32 55.95 67.38

± ± ± ±

Day 15 1.87 1.79z 1.64x 1.51z

65.67 86.23 76.33 85.71

± ± ± ±

Each value is reported as mean ± SEM (n = 6). Significantly different at xp < 0.05, yp < 0.01, zp < 0.001, when compared to control.

93

Hydroxyproline content (mg/g of tissue)

Epithelialization period (Days)

25.85 32.65 29 ± 31.62

22.25 18.14 20.04 18.62

Day 18 2.87 2.11z 1.76x 2.32z

72.55 ± 2.10 100.00 ± 1.43z 82.32 ± 2.26y 98.78 ± 1.88z

± 1.35 ± 1.22y 95 ± 1.11x ± 0.76y

± ± ± ±

0.8 0.7y 0.2x 0.5y

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function of myofibroblasts. Collagen is an essential and vital component of granular tissue, composed of hydroxyproline amino acid, which strengthens and contributes to the integrity of extracellular tissue as well as confers epithelialization in the final phase of healing [74]. Hydroxyproline content is considered as an indicator of collagen synthesis, maturation and strength in wound healing process [75]. In our study, we found that hydroxyproline content was significantly elevated in wounded granulation tissue on the application of nBuTP (5% and 10%) ointment dosedependently, signifies that collagen synthesis and maturation collectively aids in the healing of the wound. Increased concentration of hydroxyproline provides strength to the tissue for repair. Histopathological studies also support the effectiveness of nBuTP as a wound healing agent, since rats treated with test ointment showed complete reepithelialization, matured collagen fibers, neovascularization, fibroblast and less inflammatory cells. Tensile strength is a major index of wound repairing and strength since it indicates the subdermal formation and deposition of the newly formed collagen fibers [59,76]. A best wound curative agent must have the potential of accelerating the viability of collagen fibers in the wound region, which increases the tensile strength of wound [56]. Results of the present study revealed a significant increase in tensile strength in rats treated with nBuTP (5 and 10%) due to strengthening effect and maturation of collagen fibers synthesized in granulation tissue. Niwa et al. suggested that formation of free radicals and lack of ability to scavenge reactive oxygen species are causative factors of various skin lesions and it interferes in the proliferation of fibroblast [77]. Tissue wound responds to increase in oxidative stress followed by the damaging effect on cellular membrane, DNA, proteins and lipid molecules results in the delay in healing process [78]. According to Dissemond et al., acceleration of healing process could be achieved by removal of these reactive oxygen species. Hence, granulation tissue estimation for antioxidant enzyme activity is pertinent to pace up the repairing of wound tissue [79]. The results of present study showed that

Table 5 Effect of nBuTP on enzymatic antioxidant profile. Group

SOD (U/mg Protein)

CAT (μMH2O2/mg Protein)

GPx (μM/mg Protein)

Control nBuTP5% nBuTP10%

15.67 ± 1.12 19.79 ± 1.25x 22.35 ± 0.87y

34.55 ± 2.51 42.15 ± 1.79x 45.05 ± 1.53y

0.07 ± 0.02 0.12 ± 0.01x 0.14 ± 0.01y

Each value is reported as mean ± SEM (n = 6). Significantly different at xp < 0.05, yp < 0.01, when compared to control. Table 6 Effect of nBuTP on inflammatory markers level. Group

C-reactive protein (mg/l)

Fibrinogen (g/l)

Control Standard nBuTP5 nBuTP10

3.08 2.01 2.45 2.11

5.05 2.53 3.48 2.79

± ± ± ±

0.05 0.11z 0.24x 0.06z

± ± ± ±

0.54 0.23z 0.42x 0.21y

Each value is reported as mean ± SEM (n = 6). Significantly different at xp < 0.05, yp < 0.01, zp < 0.001, when compared to control.

considered safe at 2000 mg/kg dose. The results of the current study revealed the enhanced rate of wound closure, decreased healing time and epithelialization period in rats treated with nBuTP ointment in dose dependant manner. It has been reported that rapid wound contraction results in decreased healing time as it decreases the wound size and reduces the amount of extracellular matrix, which is responsible for the delay in wound repairing. Wound closure further assists re-epithelialization by diminishing the remoteness of wanderer keratinocytes [72]. Moreover, the faster rate of wound closure is also supported by the efficiency of medication [73]. Thus, the potential of nBuTP on wound closure and reepithelialization advocates enhanced migration as well as the proliferation of epithelial cells along with the production, migration and

Fig. 6. Photomicrograph (20×) of haematoxylineosin stained histological dermal section of wound area for (a) Control group, (b) Standard group, (c) nBuTP5 group and (d) nBuTP10 group. DE-Disrupted epidermis, SE-Separation of epidermis from dermis, IC-Inflammatory cells, PC-Pus cells, F-Fibroblast, NVNeovascularization, C-Collagen, P-platelets, CRComplete re-epithelialization.

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Table 7 Histological data of the granulation tissue obtained from different groups. Groups

Re-epithelialization

Fibroblast

Collagenation

Neovascularisation

Inflammatory cells

Control Standard nBuTP5 nBuTP10

− +++ ++ +

++ ++ +++ ++

− +++ ++ +++

++ ++ ++ +++

+++ − ++ +

Hematoxylin and eosin stained wound skin sections were scored as −: Absence, +: Slight, ++: Moderate, +++: Extensive.

healing of the wound.

Table 8 Effect of nBuTP on tensile strength of healed wound. Group

Tensile Strength (g)

Control Standard nBuTP5 nBuTP10

450.18 556.65 520.53 548.81

± ± ± ±

Conflict of interests Authors declare that they have no conflict of interests.

10.30 11.20z 11.50y 13.70z

Acknowledgements Authors are thankful to Shalom Institute of Health and Allied Sciences, SHUATS, Allahabad (U.P), India for providing research facilities. Authors are also grateful to Dr Vikas Kumar, Department of Pharmaceutical Sciences, SHUATS for his generous help in histological studies.

Each value is reported as mean ± SEM (n = 6). Significantly different at yp < 0.01, zp < 0.001, when compared to control.

nBuTP treatment significantly elevated the level of antioxidant enzymes, i. e., SOD, CAT and GPx. Increased concentration of SOD leads to a reduction of oxidative stress in tissue matrix by inhibiting the further formation of free radicals by dismutation of considerably toxic superoxide radicals into hydrogen peroxide and dioxygen radicals [80]. It may be considered that resultant radicals of hydrogen peroxide were catalyzed by increased CAT enzymes. An increase in GSH level, a thiol based endogenous antioxidant, acts as a co-substrate to accelerate the GPx assisted reaction leading to the reduction of peroxides, such as lipid and hydrogen peroxides [46]. It may be implied that nBuTP showed the effective in vivo antioxidant activity by reducing the oxidative stress in wound area due to the presence of biologically active molecules, such as phenolic and flavonoid compounds, which ultimately caused a synergistic effect in wound repairing. Tissue injury, especially at barrier organs, such as the skin, is a possible doorway for invading pathogenic microorganisms. As a result, inflammation process acts as a crucial part of wound healing to avert the growth of pathogens in the wound area, which leads to shun an infection and ultimately increase the number of fibroblast cells along with the formation of collagen [81,82]. Since nBuTP showed less inflammatory content in blood plasma compared to control group, the rate of wound contraction might be hastened due to activation of αchemokine and interleukin-8, which further influence the function of inflammatory cells, such as fibroblasts and keratinocytes, and ultimately speed up the collagenation process and granulation tissue maturation [48]. It might be proposed that protocatechuic acid and other phenolic components have reduced the inflammatory response, probably due to correlation with certain pathways of inflammation. However, further research is required to corroborate this claim. It has been reported that the plants containing abundant phenolic and flavonoid compounds have a strong potential to act as antioxidant, antibacterial and anti-inflammatory, which encourage the repairing of damaged tissue [55,83]. Therefore, it might be estimated that various components of nBuTP synergistically acted upon and showed the prohealing outcome in the dermal wound model.

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