Bigel formulations of St. John’s wort extract in wound healing: toxicological aspects

Abstract

This study aimed to investigate the toxicological profile of hyperforin (HP) in silico and to assess it in vivo after topical application of an HP-rich St. John’s wort (SJW) extract. The former analysis predicted low toxicity because of HP’s inability to bind DNA or proteins, but structural alerts for skin irritation/corrosion, carcinogenicity, and mutagenicity were found. Animal studies involved the treatment of excision wounds in Wistar rats with poloxamer 407/borage oil formulations (bigels; Bs) containing HP-rich SJW extract previously developed by us. The effects of semisolids comprising ‘free’ extract (B/SJW) or extract loaded in nanostructured lipid carriers (B/NLC-SJW) were compared to positive (commercial herbal product) and negative (untreated) controls after 2-, 7-, 14-, and 21-day applications. Malondialdehyde (MDA) and ABTS assays evaluated the degree of oxidative stress—treatment with bigels did not affect MDA favorably but led to an increased radical-cation scavenging capacity (compared to controls). Gamma-glutamyl transferase (GGT), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and lactate dehydrogenase (LDH) enzyme levels were measured as indicators for liver/tissue damage. Treatment with both B/SJW and B/NLC-SJW for 21 days resulted in lower GGT and ASAT levels than those in controls. Two-day application of the biphasic semisolids contributed to normalized ALAT levels (lower than in both negative and positive controls), and the same trends were observed in LDH levels after a 7-day treatment. The promising results obtained after the B/NLC-SJW application suggest that this drug delivery system may not only preserve HP in SJW extract effectively but also ‘expose’ its cyto-/hepatoprotective potential.

Keywords:

• Bigels
• hyperforin
• QSAR
• Hypericum perforatum
• in vivo toxicity
• wounds

Introduction

Transdermal delivery has many key advantages, such as escaping hepatic first-pass metabolism, avoiding gastrointestinal side effects, and minimizing fluctuations in plasma drug concentrations. Application of skin-treatment pharmaceuticals can target skin disease sites without a significant risk of systemic absorption or metabolic side effects [1]. However, toxicological monitoring is required, and various animal and nonanimal studies can be employed for this purpose.

Computer-aided drug discovery methods are gaining more recognition because of their ability to address the challenges that conventional experimental approaches encounter, e.g. scale, time, and cost. One of the traditional in silico methods used to assess the theoretical molecular toxicity of an active pharmaceutical ingredient (API) is the quantitative structure–activity relationship (QSAR). Its principal applicability is associated with reproducing and predicting xenobiotics’ metabolic activation reactions and pathways, which may induce various in vivo genotoxic effects [2–6].

Despite the advantages of QSAR, in vivo models provide the opportunity to observe complex biological responses, such as immune reactions, inflammation, and organ-specific toxicity, which cannot be accurately predicted solely by in silico approaches [7]. Moreover, the assessment of blood liver enzymes may provide data about the safety of new formulations. In addition, oxidative status parameters, such as plasma antioxidant capacity and malondialdehyde (MDA) levels, appear informative as indicators of redox balance and possible reactive oxygen species (ROS) injuries [8, 9].

Recently, our team obtained a high-hyperforin (HP)-yield St. John’s wort (SJW) extract to investigate its wound-healing potential. As the chemical instability of the principal acylphloroglucinol is well known, a protective environment had to be built prior to dermal application. Nanostructured lipid carriers (NLCs) were selected for this purpose—on the grounds of their numerous skin-beneficial properties and HP’s hydrophobicity. After elaborating on different NLC models and choosing the one with favorable properties, the next step was the development of a semisolid vehicle, viz., bigel (B). Formulations with different hydrogel-to-oleogel ratios were characterized, and the preferable one was tested in an incision wound model; thus, the therapeutic effect of SJW on wounds, known from traditional medicine, was demonstrated [10, 11].

This study aims to expand our research with an investigation of HP’s toxicological profile—both in silico and in vivo after the topical application of bigels comprising 0.5% (w/w) HP-rich SJW extract—either ‘free’ or loaded in NLCs (NLC-SJW). Since there is no other data about the topical administration of such formulations, no information about the toxicological aspects concerning it is available. Therefore, we believe this study will supplement our contribution to modern wound management.

Materials and methods

Materials

The following reagents were used in the study: 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (∼98%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), uric acid (99+%; ∼98%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), potassium persulfate (99.99%; ∼98%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), PBS (pH 7.4, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), trichloroacetic acid (≥99.5%, Merck KGaA, Darmstadt, Germany), thiobarbituric acid (≥98%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), malondialdehyde (≥96%, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), ketamine 5% (Bremer Pharma GmbH, Warburg, Germany), xylazine 2% (Alfasan Int., Woerden, Netherlands), jodseptadon 10% (Chemax Pharma Ltd., Sofia, Bulgaria), and sodium chloride 0.9% solution (В. Braun Melsungen AG, Melsungen, Germany). A bigel containing SJW extract (B/SJW) and another bigel comprising NLC-SJW (B/NLC-SJW) were prepared as previously described by us [10].

Methods

Quantitative structure–activity relationship

To evaluate HP’s metabolic activation and toxicological profile in silico, the freely available Organization for Economic Co-operation and Development (OECD) QSAR Toolbox version 4.5 was used [3].

The skin metabolism simulator was applied to determine the bioactivation of HP. It mimics the metabolism of chemicals in the skin compartment. The transformation reactions can be divided into two main types: rate-determining (Phase I and Phase II reactions) and non-rate-determining (molecular transformations of highly reactive intermediates) [3].

The following profilers were also used to elicit the toxicological profile of HP and its metabolites:

• DNA and protein binding by OASIS—scrutinizes the presence of alerts within target molecules that may interact with DNA and/or proteins;

• Protein binding for skin—investigates the presence of alerts within the target molecules responsible for interaction with proteins, especially skin proteins;

• Skin irritation/corrosion inclusion rules—contains structural alerts that can be used for positive classification of chemicals causing skin irritation and/or corrosion;

• Carcinogenicity and in vitro and in vivo mutagenicity—both profilers work as a decision tree for estimating carcinogenicity/mutagenicity based on a list of structural alerts [2–5].

Experimental animals

Ethics statement

The protocol of the study was approved by the Commission for Ethical Treatment of Animals at the Bulgarian Food Safety Agency (permit number: 265/02.06.2020). All experiments were conducted under the EU Directive 2010/63/EU for animal experiments, the Basel Declaration, and the International Council for Laboratory Animal Science ethical guidelines for researchers [12–14].

Housing conditions

One hundred nineteen male Wistar rats (Rattus norvegicus albinus) weighing 200–250 g were provided by the Vivarium of the Medical University of Varna. The experimental animals were housed individually in standard plastic cages (size: 23 cm width × 42 cm length × 14 cm height) under standard environmental conditions: 22 ± 1 °C with a relative humidity of about 55% and 12 h light/dark cycles. They were provided food (a standard pellet diet; Vasil Kostov Fodder Factory, Aksakovo, Varna, Bulgaria) and drinking water ad libitum during the whole research period. Prior to the experiments, the rats were allowed to acclimatize for 10 days.

Excision wound model

An excision wound model was used to study the toxic effects of the skin wound application of newly formulated semisolid dosage forms (bigels) in rats.

Prior to the injury, rats were anesthetized by intramuscular injection of 35.0 mg/kg ketamine (5%) and 5.0 mg/kg xylazine (2%). The dorsal hair of each rat was shaved and disinfected with povidone-iodine (10% cutaneous solution). Two full-thickness excisional wounds were created using a 6-mm biopsy punch, diametrically opposite to the dorsal line of the animals [15, 16].

Grouping, topical application of semisolids, and wound monitoring

Prior to the experimental procedure, the animals were randomly assigned into the following five groups:

• Group C (C): a control group without an excision wound (N = 7);

• Group 0 (G0): an untreated, negative group (N = 28);

• Group 1 (G1): a positive control group treated with a commercial herbal semisolid formulation (CP) containing extracts from Aloe vera, Prunus amygdalus, Vitex negundo, and Rubia cordifolia (N = 28);

• Group 2 (G2): a group treated with B/SJW (N = 28);

• Group 3 (G3): a group treated with B/NLC-SJW (N = 28).

In G1–G3, the test formulations were applied topically once daily. During the experiments, no deaths occurred in any of the groups.

Seven animals from each group (G0, G1, G2, and G3) were euthanized on the 2nd, 7th, 14th, and 21st post-operative days by injecting ketamine and xylazine in doses of 70.0 mg/kg and 15 mg/kg, respectively [16]. The death of the animals was confirmed by cardiopulmonary arrest, algor mortis, and areflexia in the hind legs, as described elsewhere [17]. The rats from group C were euthanized at the end of the study period.

Blood samples were taken from the jugular vein and heparinized, and the analyses were performed immediately after the blood collection.

Oxidative status parameters

Plasma antioxidant capacity

The plasma antioxidant capacity was measured using ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical-cation scavenging activity (according to Ref. [18]). The sample (10 µL) was added to 1 mL of ABTS cation radical solution in PBS (pH 7.4). Absorption was measured at 734 nm using a spectrophotometer (M501 single beam UV/Vis, Spectronic CamSpec Ltd., Leeds, UK) just before and 6 min after sample addition, and PBS served as a blank. The net sample absorption (A), used for the calculation of radical scavenging activity, was calculated by the following equation: $$A=\left[\left(A_{sample}{}_{0min}-A{}_{sample}{}_{6min}\right)-\left(A_{blank}{}_{0min}-A{}_{blank}{}_{6min}\right)\right]$$

Uric acid was used as a standard, and plasma antioxidant capacity was presented as mmol/L uric acid equivalents (UAE). Each sample was measured in triplicate, and the results are presented as mean values with standard deviation (±SD).

Malondialdehyde

Malondialdehyde (MDA) concentration was determined spectrophotometrically in blood plasma by the method of Porter et al. [19]. Trichloroacetic acid (250 µL) was added to the sample (100 µL), and protein precipitate was removed. Then, thiobarbituric acid (150 µL) was added to the supernatant. Samples were incubated at 96 °C for 20 min and cooled to room temperature. Absorption was measured at 532 nm against thiobarbituric acid as a blank. MDA was used as a standard, and plasma MDA concentration was presented in nmol/L. Each sample was measured in triplicate, and the results are presented as mean values ± SD.

Biochemical analyses

Activities of gamma-glutamyl transferase (GGT) (cat. No. BAOSR6x19.01), aspartate aminotransferase (ASAT) (cat. No. BAOSR6x09.01), alanine aminotransferase (ALAT) (cat. No. BAOSR6x07.01), and lactate dehydrogenase (LDH) (cat. No. BAOSR6x27.01) enzymes were measured in plasma using commercially available kits (Beckman Coulter, Miami, FL, USA) on an automatic biochemical analyzer (Beckman Coulter AU640, Beckman Coulter, Miami, FL, USA).

Statistical analysis

The values of oxidative stress and biochemical parameters were represented as mean ± SD. Comparison between groups was performed using Student’s t-test, and differences were considered statistically significant at the level of p < 0.05. Data processing was performed using the statistical software product Graph Pad Prism (version 6.0, GraphPad Software, Inc., San Diego, CA, USA).

Results

Quantitative structure–activity relationship

Quantitative structure–activity relationship of HP

The QSAR Toolbox analysis showed that HP (the structure presented in Figure 1) could not bind to DNA or (skin) proteins. However, this phytochemical can cause skin irritation and/or corrosion because of a ketone structural alert. In addition, it was predicted to have carcinogenic and mutagenic effects (in vitro and in vivo) owing to the presence of alpha, beta-unsaturated carbonyl structural alert.

Figure 1. Chemical structure of HP (parent molecule).

The applied mathematical metabolism prediction model determined the biotransformation of HP in the skin. The ‘skin metabolism’ simulator gave five possible HP metabolites obtained through Phase I metabolism reactions (Table 1).

Table 1. Numbers and structure of predicted metabolites of HP obtained via ‘skin metabolism’ simulator.

1	2	3	4	5

Quantitative structure–activity relationship of HP metabolites

To evaluate the toxicological profile of HP metabolites, their ability to bind DNA and proteins (including protein binding for skin sensitization) was determined (Table 2).

Table 2. In silico-predicted binding of HP metabolites to DNA, proteins, and protein binding for skin sensitization.

	DNA binding	Protein binding	Protein binding for skin sensitization
Structural alert	No alert found	No alert found for metabolites No. 2 and 4. 1, 2-dicarbonyls and 1, 3-dicarbonyls for metabolites No. 1, 3, and 5.	No alert found
Mechanistic alert	–	Direct acting Schiff base formers	–
Mechanistic domain	–	Schiff base formation	–

According to the results in Table 2, no structural alerts for DNA or skin sensitization proteins binding were found among possible HP metabolites. However, some can bind to proteins via Schiff base formation because of the presence of 1,2-dicarbonyl and 1,3-dicarbonyl structures.

As expected, all HP metabolites, similar to the parent structure, gave a structural alert for ketones—a functional group responsible for skin irritation and/or corrosion. No structural alerts were found for the carcinogenicity or in vitro mutagenicity of HP metabolites. However, there is one for in vivo mutagenicity—the H-acceptor-path3-H-acceptor (Table 3).

Table 3. Carcinogenicity (genotoxic and nongenotoxic), in vitro mutagenicity (Ames test), and in vivo mutagenicity (Ames test) of HP metabolites predicted via in silico studies.

	Carcinogenicity (genotoxic and nongenotoxic)	In vitro mutagenicity (Ames test)	In vivo mutagenicity (Ames test)
Structural alert	No alert found	No alert found	H-acceptor-path3-H-acceptor for metabolite No. 1–5

Oxidative status parameters

Data about plasma antioxidant capacity and MDA levels are presented in Figure 2. The results demonstrate significantly reduced plasma antioxidant capacity in the tissue damage model (G0) during all periods, compared to the nontreated control without wound (Figure 2(A)). Treatment with B/SJW and B/NLC-SJW increased the plasma antioxidant status of animals on day 7, day 14, and day 21 compared to G0. The application of semisolids containing HP-rich SJW extract led to a more pronounced effect than treatment with marketed herbal formulation in all study periods. At the same time, the antioxidant capacity on day 21 was significantly lower in G2 (p = 0.00000224) and G3 (p = 0.00000004) as compared to control C but still significantly higher than in the wound group (G0) (p = 0.0000223 for G2; p = 0.0000127 for G3).

Figure 2. Plasma oxidative status parameters of healthy unwounded animals (C), nontreated wounded animals (negative control; G0), animals treated with a commercially available product (positive control; G1), animals treated with bigel containing St. John’s wort extract rich in hyperforin (G2), and animals treated with bigel containing nanoencapsulated hyperforin-rich St. John’s wort extract (loaded in nanostructured lipid carriers) (G3). Results are obtained at the 2nd, 7th, 14th, and 21st post-operative days. Subfigure a represents the antioxidant capacity measured by the ABTS method, and Subfigure B represents the MDA concentration. Asterisks and letters indicate statistically significant differences: * vs. C; a vs. G0, b vs. G1, and c vs. G2. Values are means ± SD (n = 3).

In the tissue damage model, elevated MDA levels were found on the second and seventh days, and values decreased on the fourteenth day to levels lower than the control group (0.34 vs. 0.61 nmol/L, p = 0.000062) (Figure 2(B)). Treatment with CP and HP-rich SJW extract-containing formulations did not have a favorable effect on the changes in this marker. The values remained higher than the negative control in all other groups for all treatment periods. In animals treated with bigels (G2 and G3), the MDA values were higher than in those from G1 on day 2 and day 7. After 21 days of treatment with the CP, the MDA values were significantly lower than in all other groups.

Biochemical analyses

Plasma levels of enzymes GGT, ASAT, ALAT, and LDH as markers of tissue/liver damage were measured in animals from all experimental groups (C, G0, G1, G2, and G3) after 2, 7, 14, and 21 days (Supplemental Table S1). The GGT levels increased by 185% (p = 0.0006) and 120% (p = 0.0121) in animals treated with B/SJW (G2) and by 142% (p = 0.0038) and 175% (p = 0.0091) in those treated with B/NLC-SJW (G3) compared to the untreated control group on day 2 and day 7, respectively. The GGT levels were higher by 138% (p = 0.0001) and 86% (p = 0.0368) in G2 and by 102% (p = 0.0064) and 132% (p = 0.0174) in G3 compared to the tissue damage model (G0) at the same research periods. However, the levels of GGT normalized on day 14 and day 21, and after the application of B/NLC-SJW for 21 days, the values were 64% (p = 0.0271) lower than those measured in the animals with nontreated wounds.

The ASAT levels on day 21 were lower in the animals treated with both bigel formulations compared to those in control (C) and wounded (G0) rats. Nevertheless, a significant difference was found only in the B/SJW group (G2) (by 24%, p = 0.0362 vs. C and 19%, p = 0.0500 vs. G0).

On day 2, the application of semisolids containing HP-rich SJW extract and CP contributed to normalized/lower ALAT levels—by 27% (p = 0.0004), 25% (p = 0.0183), and 23% vs. wounded rats, respectively. Evidently, the effectiveness of the developed formulations was similar to that of CP in the research period discussed. On day 14, both bigels were significantly more effective in lowering the ALAT plasma levels (53.74 ± 6.94 U/L p = 0.0031 vs. G1; 51.58 ± 9.77 U/L p = 0.0079 vs. G1) compared to CP (65.63 ± 4.16 U/L). On the same day, the ALAT plasma levels significantly decreased in the B/NLC-SJW treatment group (51.58 ± 9.77 U/L p = 0.0179 vs. G0) compared to the wound group (65.08 ± 8.59 U/L). In the latter, the ALAT levels were higher than those in the control group by 47% (p = 0.0001 vs. C).

Seven-day application of HP-rich SJW extract-comprising semisolids contributed to a decrease in LDH levels by 58% (p = 0.0334) and 67% (p = 0.0205) decrease vs. compared to no wound treatment. Also, in the tissue damage model (G0), the values on day 7 were significantly increased—by 203% (up to 389.42 ± 164.14, p = 0.0200 vs. C); however, on day 14 and day 21, the LDH levels in the nontreated wounded animals normalized.

Discussion

Quantitative structure–activity relationship

There is a lack of literature data on the cutaneous metabolism of HP; therefore, in silico predictions were performed initially in this study. In addition, the possibility of skin irritation and/or corrosion was assessed since this could cause rashes, dermatitis, or even irreversible skin damage [20]. According to the obtained results, HP was predicted to possess potential carcinogenic and mutagenic effects. However, this has not been confirmed by other researchers, indicating that not all reactions in the body are possible [21–23]. Thus, it is necessary to conduct in vitro tests like the Ames test. Also, Hokkanen et al. [24] have described 57 metabolites of HP, none of which are associated with skin metabolism. Because of the potential of some HP metabolites to bind to proteins, the resulting conjugates can directly affect cells by disrupting their primary functions or indirectly lead to damage.

The skin irritation/corrosion profiler results contradict the indications for HP in topical application: according to the Patent No. EP1131063B1, it is suitable for treating cancer and/or precancerous diseases, skin aging, and inflammatory and bacterial skin diseases, including in veterinary medicine [25]. In turn, the resulting metabolites may cause adverse drug reactions, such as skin irritations, in some patients.

The structural alert ‘H-acceptor-path3-H-acceptor’ (Figure 3) for in vivo mutagenicity indicates the possibility of chemical interaction of HP metabolites with DNA and/or proteins via noncovalent binding, such as DNA intercalation or groove binding [26]. Among the descriptors potentially accounting for noncovalent interactions, the presented molecular framework results in increased sensitivity/specificity for what concerns the micronucleus training set [3, 27].

Figure 3. Structural alert for H-acceptor-path3-H-acceptor. Any atom except hydrogen is marked as ‘A,’ and ‘H-bond-Acc’ represents any atom that is a potential hydrogen bond acceptor.

Oxidative status parameters

A number of natural products possess antioxidant properties, and they can be expressed by different pathways, such as direct free radical scavenging, transition metal chelation, or maintenance of endogenous antioxidant systems [28, 29]. As one of the most widely applied analytical methods for evaluating the antioxidant capacity of natural products [30], the ABTS assay was employed in our experiment.

The results of this study demonstrate reduced antioxidant capacity in the tissue damage model (G0) during all periods compared to the control (C). That result is in accordance with the observation that skin wounds could be a source of free radicals [31]. Application of HP-rich SJW extract-containing formulations increased the plasma antioxidant status of animals on day 7, day 14, and day 21 compared to the nontreated wounded rats (G0). Application of B/SJW and B/NLC-SJW led to a more pronounced effect than treatment with CP in all research periods. SJW extracts are rich in polyphenols, including HP, and exert pronounced antioxidant properties [32, 33]. In this regard, the higher antioxidant potential established in animals treated with HP-rich SJW extract-containing formulations is explainable. Furthermore, in silico analysis showed that HP’s molecule has no structural alerts to potentiate ROS formation. Other studies also demonstrated good skin tolerance of the oil-containing SJW extract applied for the treatment of burn wounds in rats as well as different skin irritations in humans [33, 34]. The latter may be due to the presence of a ketone structural alert, as mentioned earlier. However, the antioxidant capacities of SJW extract may switch to pro-oxidative at very high concentrations [35], which could explain the measured lower values on day 21.

MDA, a widely used marker of oxidative lipid injury and oxidative stress [36], results from the lipid peroxidation of polyunsaturated fatty acids. Elevated MDA levels were established in G0 on day 2 and day 7. Treatment with CP (G1) and bigels with HP-rich SJW extract (G2 and G3) did not affect the changes in this marker favorably—the values remained higher than the control for all treatment periods. On day 21, the MDA levels were still significantly higher in G2 and G3 than in C and G0. Recent studies provided evidence that different nanoparticles could trigger oxidative stress by generating intracellular ROS. Their production could result from interactions between nanoparticles and cells, thus leading to cytokine secretion, which triggers secondary oxidative stress due to free radicals [37].

Biochemical analyses

In this study, the GGT, ASAT, ALAT, and LDH plasma levels were measured as markers for liver/kidney/tissue damage. GGT is a well-known serum biomarker for liver damage, atherosclerosis, heart failure, arterial stiffness and plaque, gestational diabetes, and impaired or inadequate antioxidant defense [38]. LDH is another primary biomarker for tissue damage, including erythrocytes and liver-specific isoforms [39, 40]. ALAT and ASAT are the most specific markers for hepatic injury [41, 42]. Thus, when analyzing GGT, ASAT, ALAT, and LDH levels, the cyto/hepatotoxic or cyto/hepatoprotective potential of the formulations containing HP-rich SJW extract could be assessed in a wound model in rats. Because the in silico analysis predicted low metabolic activation of HP and the small potential of its metabolites to bind to DNA and proteins, HP was not expected to exert cytotoxic effects.

We have detected that both bigel formulations increased GGT levels compared to the untreated wound group (G0), commercial product treatment (G1), and control animals (C) on day 2 and day 7 (Supplemental Table S1). HP, one of the primary phytochemicals in SJW preparations, has not been widely analyzed for its toxicity. A study reports that high doses of hyperforin-ethylene diammonium salt (2000–5000 mg/kg p.o./once per day) for 14 days caused an increase in ALAT and ASAT levels in mice [43]. These mice were diagnosed with an initial stage of liver injury, which was reversed by the end of the treatment [43]. On the other hand, after adding 1% dried SJW to the diet, broiler turkeys fed for 126 days did not show any significant change in GGT, ASAT, or ALAT [44].

A study from 2018 showed that Hypericum perforatum L. quercetin-biapigenin poly(Ɛ-caprolactone)-loaded nanoparticles or free compounds were effective in protecting HepG2 cells against tert-butyl hydroperoxide-induced toxicity [45]. An earlier paper demonstrated that an intraduodenal injection of H. perforatum alcoholic extract possesses a hepatoprotective effect against CCl₄-induced liver injury in mice by increasing bile secretion and reducing barbiturate sleeping time [46]. In this study, the authors found that treating wounded animals with both HP-rich SJW extract-containing formulations significantly reduced ALAT levels on day 2 and LDH levels on day 7. However, only B/NLC-SJW treatment reduced ALAT activity compared to the negative control (G0) on day 14, while the application of B/SJW led to a significant reduction in ASAT levels on day 21 (Supplemental Table S1). We speculate that the B/NLC-SJW formulation may possess a slightly stronger hepatoprotective effect, considering that this requires additional future experiments.

Another important observation is that on day 14, we may distinguish a significantly stronger effect of bigels (G2 and G3) compared to that of CP (G1) (Supplemental Table S1). According to a previous report, intraperitoneal injection of Hypericum perforatum L. extract at a dose of 50 mg/kg body weight significantly reduces hepatic ischemia-reperfusion injury-induced levels of ALAT, ASAT, and LDH activities in the liver and increases the activities of antioxidant enzymes catalase and glutathione peroxidase [47]. Herein, we report that both HP-rich SJW extract-comprising semisolids effectively reduced wound-induced tissue/liver damage biomarkers ALAT and LDH.

Clinical [48] and experimental [49] research showed that hypoxia and high lactate are characteristics of wound healing [50]. Injuries from lacerations of the skin are associated with disruption of the vascular network. Through the destruction of the local network of vessels, a hypoxic gradient between the vascularized wound margins and the avascular center of the wound is created. Furthermore, this is linked to increased lactate production, transfer, and local availability, inducing angiogenesis, extracellular matrix generation, fibroblast-induced collagen deposition, and TGF-β synthesis even without acidosis [50]. Also, under wound-healing conditions, some cells, particularly neutrophils, macrophages, and fibroblasts, depend to a large extent on glycolysis for cellular energy and produce large amounts of lactate [51, 52]. Other studies have shown that high lactate stimulates collagen deposition and angiogenesis [51, 53, 54]. These processes are mediated by LDH, the decline of the NAD + pool, and the subsequent downregulation of NAD-mediated polyadenosine diphosphoribose (pADPR) and adenosine diphosphoribose (ADPR) [51].

It was recently reported that a steady increase in functional LDH activity in sub-acute burn wounds is linked to cellular infiltration [55]. Researchers found increased LDH activity related to cellular infiltration, including CD3+, CD4+, CD8+ lymphocytes, CD20+ B-cells, and CD68+ macrophages, which are connected to changes in the distribution of lactate and pyruvate [55]. An ex vivo model of equine corneal epithelial wound healing showed altered LDH expression within 24 and 48 h in both wounded and unwounded corneas [56]. Elevated serum levels of LDH were also found after tissue damage and surgical procedures: higher values were found in the large-stitch closure group compared with the small-stitch closure group [57].

It is known that the most significant morphological changes occur during the first 7 days of wound healing [58]. During this period, the inflammatory phase occurs, characterized by infiltration of the wound with granulocytes or polymorphonuclear leucocytes, followed by macrophages and lymphocytes. Pro-inflammatory mediators and growth factors are secreted [59]. Proliferative processes are also in progress as the tissue begins to heal with the effective closure of the wound [60]. This phase is characterized by fibroblast migration, collagen synthesis, angiogenesis, granulation tissue formation, and epithelialization [59]. The measured high LDH levels on day 2 and day 7 suggest hepatotoxic events. On the other hand, we may assume that the dynamic processes that take place in the injured tissue also contribute to an increase in the total pool of lactate dehydrogenases in the plasma. Therefore, both analysis of total LDH activity and further identification of LDH isoenzymes would be useful to clarify the results we present.

The tissue/hepatoprotective potential of SJW may be due to the known antioxidant hyperoside, one of the main constituents isolated from the herb [61, 62]. It is known that hyperoside may possess in vitro and in vivo protection against oxidative stress-induced liver injury by inducing superoxide dismutase levels and decreasing those of MDA [63]. On the basis of our findings that GGT serum levels decreased, respectively normalized, since day 14 in G2 and G3, together with the literature data [47], we hypothesize that potential recovery of liver GGT activity may promote the activation of glutathione peroxidase possibly by recovering intracellular glutathione levels. These findings point to a possible molecular mechanism concerning the hepatoprotective potential of SJW formulations. However, the suggested mechanism of hepatoprotection by induced antioxidant activity should be analyzed in more detail in future studies.

Conclusions

There is limited literature data about the hepatoprotective/toxic effects of HP in SJW. Our study represents the first attempt to predict its toxicological profile and assess its effects on plasma levels of tissue/liver damage biomarkers in rat skin wound models. After the application of in silico methods (QSAR Toolbox software for metabolic activation in the skin to the OECD), the parent structure (HP) was found to possess a good toxicological profile because of lack of ability to bind to DNA and proteins and low metabolic activation. The generated HP metabolites, however, exhibit different reactivity; therefore, they may be among the causes of the previously reported adverse reactions after topical application of HP. The absence of structural alerts in the HP molecule that can potentiate ROS formation correlates with the established antioxidant potential through MDA and ABTS assays. The GGT, ASAT, ALAT, and LDH levels suggested that NLC could be considered a suitable colloidal carrier—in addition to the successful preservation of HP in SJW extract, its cyto- and hepatoprotective potential was demonstrated. Further studies regarding the wound-healing process on a tissue and cellular level after B/NLC-SJW application would complement the present findings.

Authors’ contributions

Conceptualization: Y.K.-K. and V.A.; collection of biological material: O.T., S.S., M.H., Y.K.-K., and V.A.; experimental work and data acquisition: Y.S., D.V., and S.S.; formal analysis: D.V. and I.I.; data analysis: Y.S., D.V., O.T., S.G., M.H., Y.K.-K., and V.A.; literature research and original draft preparation: Y.S., D.V., O.T., and I.I.; visualization: O.T., I.I. and Y.K.-K.; writing—review and editing: Y.S., D.V., O.T., S.G., Y.K.-K., and V.A.; funding acquisition: V.A.; supervision: V.A. All authors have read and approved the final version of the manuscript and agree to be responsible and accountable for all aspects of the reported work.

Disclosure statement

The authors declare no conflict of interest.

Data availability statement

All data are available from the corresponding author upon reasonable request.

Additional information

Funding

This work was funded by Fund ‘Nauka’ at the Medical University of Varna, Bulgaria, through Project No. 18027, ‘Lipid nanoparticles—a modern technological approach for the inclusion of hyperforin with improved chemical stability in topical formulations for accelerated wound healing,’ Competition-Based Session for Scientific Research Projects, 2018.