Potential wound healing activity of Quercus infectoria formulation in diabetic rats

Preparation of extract and topical agent

Nutgalls from Quercus infectoria were purchased from Thai Herbal shop, Hat Yai, Songkhla, Thailand. Botanical identification of the material was obtained by a special botanist. A voucher specimen of them is collected at Natural Research Center of Excellence, Prince of Songkla University. The powder of nutgalls was extracted as previously described (Chusri & Voravuthikunchai, 2009). For pharmaceutical formulation, the extract was dissolved in 95% ethanol or sterile distilled water at concentrations of 15–40% (w/v) and tested for antibacterial activity. Based on antibacterial activity and solubility, the extract at concentration 30% (w/v) was used for further formulation. Twenty formulations were prepared from the extract and various concentrations of propylene glycol, polyethylene glycol 400 (PEG-400), polysorbate 20, polysorbate 60, 95% ethanol, and uniphen P-23 (Table 1). The volume of each formulation was made up to 100 mL with sterile distilled-deionized water. Each formulation was stored in a sterile bottle at room temperature until use.

Antibacterial activity

Broth microdilution method was performed to determine the minimal inhibitory concentration (MIC) of 20 Qi formulations against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) NPRC R001. The bacterial pathogens were cultured in Mueller-Hinton broth (MHB, Difco, France) at 37 °C for 4 h. The suspensions were adjusted to McFarland standard No. 0.5 and diluted with MHB to reach a cell density corresponding to 10⁶ CFU/mL. Qi formulations were diluted using two-fold dilution method. The bacterial suspension was then added with 100 µL of two-fold serially diluted of each Qi formulation in a sterile 96-well microtiter plate. After 18 h of incubation, the bacterial growth was measured using a microplate reader (Sunrise, Tecan, Switzerland) at 620 nm. The MIC is defined as the lowest concentration that produces a complete inhibition of bacterial growth. An experiment was done in triplicate. Bacterial culture with MHB without the addition of each Qi formulation was used as a control. The most active formulation with low MIC value was selected for further experiment.

Phytochemical screening

Total phenolic compounds of both the selected formula and the extract were assessed using the Folin-Ciocalteu agent (Fattahi et al., 2014). The diluted solution (100 µL) at concentration 100 µg/mL was mixed with 750 µL of 10% Folin-Ciocalteu’s reagent dissolved in sterile distilled deionized water. After incubation, 750 µL of 6% (w/v) sodium bicarbonate was added and further incubated for 30 min in dark at room temperature. The absorbance was measured at 760 nm using UV-visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). Quantification was done on a standard curve of gallic acid (Sigma, Germany). The results were expressed as gallic acid equivalents (mg of GA/g of extract).

The aluminium chloride colorimetric assay was performed to determine total flavonoid content (Fattahi et al., 2014). Each tested sample (1 mL) was added into test tubes along with 4 mL of distilled deionized water and 0.3 mL of 5% sodium nitrite solution. After 5 min of incubation, 0.3 mL of 10% aluminium chloride was added and further incubated for 6 min at room temperature. Then, 2 mL of 1 M sodium hydroxide was added and the volume of the mixture was making up to 10 mL with distilled water. The mixture was carefully mixed before determination. The absorbance was measured at 510 nm using UV-visible spectrophotometer. The blank was performed using distilled water. Catechin was used as a standard control. The samples were performed in triplicates. The data of total flavonoid were expressed as mg of catechin equivalents/100 g of dry extract.

Reverse phase high performance liquid chromatography (RP-HPLC) analysis

The quality of the selected formulation was analysed by HPLC using High Performance Liquid Chromatograph 1100 (Aligent Technologies, Santa Clara, CA, USA) according to the previous report (Pin et al., 2006). Gallic acid (Sigma, Darmstadt, Germany) was used as a chemical marker. The analytical column used was Hypersil ODS 4.0 × 250 mm with packing material of 5µm particle size at a flow rate of 1 mL/min at room temperature. The mobile phase consisted of 0.1% phosphoric acid (solvent A) and acetonitrile (solvent B). The gradient elution employed was as follows: 85%A at 0–12 min, 75%A at 12–20 min, 85%A at 0–22 min, 85%A at 22–25 min. Each injection contained 10 µL of the sample. Detection was monitored at wavelength 280 nm.

Anti-oxidant activity

An anti-oxidant activity of both the selected formulation and the extract was performed by DPPH and ABTS scavenging assay (Meda et al., 2005; Thaipong et al., 2006). For DPPH scavenging assay, the tested formulation and the extract were dissolved in methanol and mixed with 1.5 mL of 80 µM DPPH solution in methanol. After 15 min of incubation in the dark, the absorbance of the reaction was determined at 517 nm UV–V is spectrophotometer.

For ABTS scavenging assay, an aliquot solution of 7 mM ABTS (Merck, Germany) was oxidized with potassium persulfate for 16 h in the dark at room temperature. The ABTS+solution was diluted with phosphate buffer saline (PBS; Merck, Darmstadt, Germany) pH 7.4 to initial an absorbance of 0.70 ± 0.02 at 734 nm. An aliquot (20 µL) of each formulation was mixed with 2 mL ABTS+solution for 6 min in the dark. The absorbance was read at 734 nm using UV-visible spectrophotometer. Trolox was used as a positive control for both experiments. The radical scavenging activity was calculated according to the following formula; ${\displaystyle \text{\%}Inhibition=\left[\left(blankabsorbance-sampleabsorbance\right)∕blankabsorbance\right]\times 100}$

Stability test

Stability of the most active Qi formulation (QiF10) at different temperatures was determined using stability test. The sample was kept under two different temperatures at 4 °C for 24 h and 60 °C for 24 h, three cycles. After incubation, the sample was observed for changes in appearance, such as colour, precipitation, separation, and pH (Reddy & Murthy, 2005).

Animals

Male wistar rats (7–8 weeks, 200–250 g) were acclimated to standard housing and fed under a protocol approved by the Animal Ethic Committee, Prince of Songkla University, Thailand (Ref.12/2014). All animals were housed in individual cages under constant temperature (22 °C) and humidity with a 12-hour light-dark cycle.

Diabetic rat model

Rats were divided into two groups: diabetes (n = 16) and non-diabetes (n = 16). All animals were fasted overnight. Diabetes was induced by intraperitoneal injection of freshly prepared solution of streptozotocin (50 mg/kg) in 0.1 M citrate buffer (pH 4.5). Non-diabetic rats were injected with citrate buffer alone. After seven days of injection, blood was taken from the tail to measure blood sugar levels by glucometer. The animals were considered as diabetes, if their blood sugar level was 250 mg/dL or higher (Ravi, Ramachandran & Subramanian, 2004).

Wound protocol

For wounding, the animals were anaesthetized with intraperitoneal injection of thiopental sodium (55 mg/kg). The dorsal area was completely shaved and sterilized with 70% ethanol. In addition, 2% lidocaine was applied as a topical anaesthesia. Treatment and control were performed in the same animal in order to minimise bias. Two 1-cm² excision wounds were created on the upper back of each animal with a scalpel. Wounds were left to chronic state for three days post-wounding. On day 4, 15 µL of the selected Qi formulation (30% w/v) was topically applied to one of duplicate wounds on each animal and physiological saline (control) was applied to the other. Each treatment was given daily until wound closure. On day 0, 3, 7 and 14 post-treatment, wounds (n = 8) in each animal group were photographed and the wound size was measured by computing software ImageJ. Data were reported as percentage wound closure calculated by the following formula; ${\displaystyle \text{\%}woundclosure=\left(A_0-A_1∕A_0\right)\times 100}$ A₀ = Wound area on day 0

A₁ = Wound area on day 3, 7, or 14 post-treatment

Histological analysis

On day 0, 7, and 14 post-treatment, animals were sacrificed and wound samples (n = 4) from each group were collected for histological analysis. Tissues were fixed in formalin and embedded in paraffin. Six-micrometer paraffin sections were stained with hematoxylin and eosin (H&E) for measurement of re-epithelialization and granulation tissue production under a light microscope. The images were photographed.

Statistical analysis

All tested groups were compared with the control groups and the results were analysed statistically using t-test. Continuous variables were described in terms of mean and Standard Error of Measurement (SEM). All calculations were performed by SPSS 14.0 (SPSS Inc., Chicago, IL, USA). The values of p < 0.05 were considered to be statistical significance.

Selection of formulation and antibacterial activity

An antibacterial activity of 20 Qi formulations was tested to determine MIC values against MRSA. Table 1 displays composition and antibacterial activity of 10 representative Qi formulations. The others were not presented because the extract was not completely soluble for antibacterial assay. MIC range was found between 0.29 to 37.5 mg/mL. It was observed that QiF10 exhibited antibacterial activity against MRSA with MIC values at 0.29 mg/mL. Thus, the formulation of QiF10 was selected for further experiment. The base solution of all formulations did not show an antibacterial activity (Table 1). The stability of QiF10 was determined by observing the formula appearance, including colour, odour, precipitation, and pH value. There was no change in all observed characteristics (Table S1).

Phytochemical screening and HPLC

Total phenolic and total flavonoid compounds were determined. The results demonstrated that the ethanolic extract consisted of high concentration of total phenolic compounds (954.07 mg GAE/100 g) which was close to standard gallic acid (960.85 mg GAE/100 g), while QiF10 had total phenolic compound at 580.34 mg GAE/100 g (Table 2). Both the extract and the formulation showed less total flavonoid contents than the standard control. There were 247.22 mg CE/100 g and 101.39 mg CE/100 g for the extract and the formulation, respectively (Table 2). Furthermore, HPLC analysis of gallic acid in the extract and the formulation were 32.09 mg/g and 12,583.66 mg/L, respectively. The chromatograms of gallic acid quantified in of the extract and QiF10 were presented in Fig. 1.

Anti-oxidant activity

DPPH and ABTS radical scavenging ability of QiF10 and the extract are shown in Table 2. High percentage of DPPH and ABTS inhibition was noticedin the extract testing group with percentage inhibition at 97.40% and 98.70%, respectively. The formulation also exhibited a high percentage of DPPH and ABTS inhibition with 94.48% and 96.43%, respectively. Interestingly, these inhibition values were very close to standard control trolox which were 97.62% and 99.14% against DPPH and ABTS, respectively (Table 2). An anti-oxidant activity did not observe in the base formulation of QiF10 (Table 2).

Streptozotocin-induced diabetic condition in rats

Figure 2A shows blood sugar levels in diabetic and non-diabetic rats during the experiment. After seven days of injection, streptozotocin injection (50 mg/kg) was found to induce marked hyperglycaemia, compared with citrate-injected controls. Diabetic rats exhibited high blood sugar level at 398 mg/dL, while non-diabetic rats had their blood sugar levels less than the endpoint. Throughout the experimental period, high blood sugar levels in diabetic rats were observed, while those in non-diabetic groups remained stable approximately 106 mg/dL. Moreover, the clinical signs were presented in diabetic rats, including polyuria, polydipsia, and weight loss during the experiment (Fig. 2B).

Rate of wound closure

Figure 3 demonstrates wound healing activity in the treatment and the control groups of both diabetic and non-diabetic rats. After the wounds were left for three days, crust was observed in all groups. QiF10 and normal saline solution were initially applied on day 4 post-wounding in the treatment and the control groups, respectively. The rate of wound closure in diabetic rats was profoundly delayed throughout the experiment, compared with non-diabetic rats (Fig. 3A). Diabetic wounds treated with QiF10 appeared to heal faster than the saline-treated wounds (Fig. 3B). On day 3 after treatment, the wound area in both the treatment and the control groups was not significantly different (p < 0.05). In contrast, on day 7, the significant difference (p < 0.05) in percentage of wound closure in response to QiF10 treatment was observed with 94%, when compared with normal saline-treated wounds (50.18%).

In the non-diabetic group, percentage wound closure and wound characteristics between the treatment and the control groups were not different (Fig. 3C). On day 7, an increasing rate of wound closure was found in both the treatment and the control groups with 82.39% and 76.66%, respectively (Fig. 3C). By the end of the study, percentage wound closure in the treatment and the control group was noticed at 87% and 80%, respectively.

QiF10 enhances wound healing process

To observe tissue changes, histological analysis was performed by staining with H&E. Fig. 4 illustrates the histological images of diabetic wounds and non-diabetic wounds treated with or without QiF10. Before initial treatment, incomplete epithelialization was observed with less number of cells and collagen deposition in all groups. Moreover, a long gap in wound area was presented in the dermis layers. After treatment for seven days, QiF10-treated diabetic wounds showed better re-epithelialization and collagen deposition than the control wounds (Fig. 4A). In addition, it has been developed abundant granulation tissue with a large number of cells containing fibroblast, collagen, capillaries, as well as inflammatory cells (Fig. 4A). In contrast, minimal cellular infiltration and collagen deposition were indicated in wounds treated with normal saline solution (Fig. 4A). Loosely packed collagen fibre and partial re-epithelialization were noted in normal saline treated wounds (Fig. 4A). This lack of cellular proliferation and granulation tissue formation led to delayed wound healing process.

Figures 4B–4C demonstrates histological images of non-diabetic wounds treated with or without QiF10. After seven days of the intervention, both QiF10 and normal saline-treated wounds were partially re-epithelialized (Fig. 4B). A complete re-epithelialization that contained granulation tissue rich in fibroblasts, collagen, and inflammatory cells in the dermis layers was shown in both groups by day 14 (Fig. 4C). Compared with diabetic wounds, non-diabetic ulcers had normal wound healing process with large numbers of cellular infiltration and collagen production (Figs. 4A, 4B). Moreover, regular differentiated keratinocyte cells were clearly observed in both the treatment and the control wounds from the non-diabetic group, resulting in rapid and complete re-epithelialization (Fig. 4C).