# Extracts ofEucalyptus alba Promote Diabetic Wound Healing by Inhibiting α-Glucosidase and Stimulating Cell Proliferation

Authors: Rabia Mumtaz; Muhammad Zubair; Muhammad Asaf Khan; Saima Muzammil; Muhammad Hussnain Siddique
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Treatment of diabetic wounds has always been a challenge for primary and acute health care.Eucalyptus alba has been reported to be used for the treatment of wounds and oxidative stress. Effects of using different temperatures and solvents for the extraction of Eucalyptus alba leaves were investigated in terms of diabetic wound healing activity. Leaves of E. alba were dried at 10°C, 30°C, 50°C, and 100°C, and dissolved in ethanol, methanol, and acetone to obtain a total of 12 extracts. All the extracts have remarkable antidiabetic, antioxidant, and cell proliferation activities. Among the tested extracts, highest activities were observed with leaves dried at 10°C and 30°C, whereas drying at 100°C resulted in the lowest activities. Ethanol-based extracts exhibited significantly increased cell proliferation compared with methanol- and acetone-based extract. The present study suggests that leaves of E. alba should be dried at temperature not more than 30°C and extracted in ethanol for optimum results. However, further studies should focus on the identification of specific bioactive compounds in E. alba leaves.


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## Body

## 1. Introduction

Wounds have always been a major threat to the public health and economy around the world. Patients with diabetes mellitus are at the highest risk to develop chronic wounds due to underlying conditions such as elevated blood glucose level, neuropathy, reduced blood flow, and chronic inflammation at the site of injury. According to World Health Organization, 422 million people were affected by diabetes in 2014 compared with 108 million in 1980 [1]. The another report published by the International Diabetes Federation (IDF) declared the expected rise of diabetic patients from 463 million in 2019 to 578 million by 2030 [2]. The prevalence rate of chronic diabetic wounds is one in every three to five patients with an alarming recurrence rate (40% within one year and 65% within 5 years) [3]. The overall, lifetime incidences of diabetic foot ulcers have been estimated between 19 and 34%. Thus, it is not surprising that chronic wounds in diabetics result in lower limb amputations, reduce quality of life, and require proper treatments [4]. A 2018 retrospective review of the Medicare beneficiaries analyzed that the treatment cost for acute and chronic wounds ranged from 28.1 to 96.8 billion dollars in which surgical and diabetic wounds are most expensive to treat [5]. Diabetic foot ulcer market alone is set to increase from 7.03 billion dollars in 2019 to 11.05 billion dollars in 2027, making it imperative to develop cost-effective diagnosis and treatment strategies [6].Despite the possible impairments in wound healing, various treatments ranging from traditional wound care management to surgical practices are available. Recent treatments involve the use of antibiotics, hyperbaric oxygen therapy to supply the skin tissues with adequate amount of oxygen, and tropical dressings to provide the moist environment to stimulate the healing process. Unfortunately, most of the available treatments have their own limitations such as high cost, development of microbial resistance, and allergic reactions. There is a need to find an effective and safe alternate from natural resources, which is believed to have minimum toxic effects [1].Eucalyptus is a medicinally valuable genus in the family Myrtaceae with more than 800 species grown in different regions of the world [7]. Eucalyptus alba possess various pharmacological properties including antioxidant, antimicrobial, cytotoxic, and pesticidal effects. Extracts of Eucalyptus have also been used to treat sore throat, urinary tract infections [8], inflammation [9], wounds, fungal infections, and respiratory illnesses, that is, common cold, sinus congestion, and influenza [10].Polyphenols are the most abundant secondary metabolites in plants with approximately 8000 different compounds and have attracted great attention as potential therapeutic agents in the treatment of various diseases [11]. Eucalyptus species are rich in polyphenols such as rutin, quercetin, ellagic acid, chlorogenic acid, and catechin and are well studied for their pharmacological activities [12]. Prevention and treatment of disease by polyphenols is mainly due to their antioxidant property. Moreover, ample evidence from experimental studies has confirmed their role in the prevention of cancer, diabetes, wound healing, and cardiovascular diseases [13, 14]. Therefore, plant materials and polyphenols represent the main target of research as they could be the potential agents to prevent and treat oxidative stress-related diseases [15, 16].The aim of the present study was to investigate the diabetic wound healing potential ofE. alba leaf extract using α-glucosidase inhibition assay, DPPH assay, and scratch wound healing assay. Cytotoxic effects of extracts on retinal pigment epithelial (RPE) and human hepatocellular carcinoma (Huh-7) cell lines were also determined. Moreover, polyphenolic content of the E. alba extracts was also determined using HPLC analysis.


## 2. Methods and Methods

### 2.1. Selection and Collection of Plant Samples

Fresh leaves of single genotype ofE. alba were obtained in April 2019 from Ayub Agriculture Research Institute, Faisalabad, Pakistan. A voucher specimen of the plant sample (B & BEAL-19-1a) has been deposited in the departmental herbarium. The plant name has been confirmed with https://www.theplantlist.org. Leaves of E. alba were separated, washed, weighted, and then dried in convection oven (Memmert, Schwabach, Germany) at different temperatures, that is, 10°C, 30°C, 50°C, and 100°C. Each sample was then ground to make fine powder and stored at −20°C before further analysis [17].


### 2.2. Solvent Extraction of Plant Samples

The extraction of the plant sample was performed at room temperature. Finely powdered plant material (1 g of each sample) was macerated in pure ethanol, methanol, and acetone (10 mL) for 24 hours with constant shaking. The extracts were filtered with Whatman filter paper No. 1. and then concentrated by evaporating the solvents at room temperature. Stock solutions were prepared by dissolving the dried extracts in an equal volume of phosphate-buffered saline (PBS). Extracts were further diluted from 0.1 mg/mL to 0.0125 mg/mL to calculate IC50 value for DPPH assay and α-glucosidase inhibition assay [18].


### 2.3. Antioxidant Activity: DPPH Assay

Free radical scavenging activity of theE. alba was estimated using DPPH assay with some modifications from Ref. [19]. The stock solution of DPPH (0.3 mM) was prepared in ethanol, and 10 μL of plant sample or gallic acid as standard was mixed with 190 μL of DPPH in a 96-well plate with subsequent incubation for 30 minutes in dark. The absorbance was measured using microplate reader (Biotek ELx808IU, USA) at 517 nm, and measurements were used to calculate percentage inhibition of the DPPH solution.(1)%inhibition=Absorbance of control−Absorbance of test sample/Absorbance ofcontrol∗100.


### 2.4. Antidiabetic Activity:α-Glucosidase Inhibition Assay

Theα-glucosidase inhibition activity of plant extracts was determined using 5 mM p-nitrophenyl-β-D-glucopyranoside (PNPG) as a substrate slightly modified from [20]. Briefly, 12.5 μL of plant extract, 40 μL of α-glucosidase (0.5 U/mL), and 120 μL of phosphate-buffered saline (pH = 6.8) were added to each well of 96-well plate. After 5 minutes of incubation, 40 μL of substrate 5 mM was added and plate was further incubated at 37οC for 30 minutes. Acarbose, a synthetic drug, has potential inhibitory effects against α-glucosidase, which was used as positive control at a concentration of 10 mM, and phosphate-buffered saline was used as negative control. The absorbance was measured at 405 nm using microplate reader (Biotek ELx808IU, USA).Percentage inhibition ofα-glucosidase was calculated as follows:(2)%inhibition=Absorbance of control−Absorbance of test sample/Absorbance ofcontrol∗100.


### 2.5. Wound Healing Activity: Scratch Assay

The role ofE. alba extracts on the migration of retinal pigment epithelial (RPE) cells was determined using cell scratch assay slightly modified from [21]. Cells were cultured in 12-well plates at 2x105 cells per well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Plates were incubated at optimum conditions, that is, 37°C and 5% CO2 in a CO2 incubator to produce 80–100% confluent monolayer. Linear wound was produced using a sterilized 200-μL pipette tip. Medium was removed, and cells were washed with PBS. Fresh medium with different concentrations of E. alba extracts was added, and again, plate was again incubated for 24 hours. Microscopic images were taken with an inverted microscope (Meiji Techno TC5200) using 40x magnification at 0, 4, 8, 16, and 24 hours. Images were evaluated at 1712×1368 pixels in ImageJ 1.440 software for Windows. The percentage of the scratch area covered was determined by following equation:(3)%percentage wound healing=Wound area at4h−Wound area at0h/Wound area at4h∗100.


### 2.6. Cell Viability: MTT Assay

The human hepatocellular carcinoma cell line Huh-7 and the noncancerous retinal pigment epithelial (RPE) cell line were used for the assessment of toxicity ofE. alba extracts. The cells were cultured in the same medium as described above, but in 96-well cell culture plates with an initial concentration of 2 × 104 cells per well. Each well was treated with one of the 12 different E. alba extracts at a concentration of 0.1 mg/mL. Phosphate-buffered saline (PBS) was used as negative control. After incubation of 48 hours, 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to 96-well plate and then incubated for another 4 hours. After incubation, 150 μL of 0.1% dimethyl sulfoxide (DMSO) was added that is reduced to purple-colored formazan crystals by viable cells. The optical density was determined at 560 nm. Pictures were captured with an inverted microscope using 40x magnification [22].


### 2.7. Polyphenols: HPLC Analysis

Polyphenol content inE. alba extracts was determined using a Chromera HPLC system (PerkinElmer, Shelton, CT, USA) equipped with a Flexar Binary LC pump and a UV/Vis LC Detector, both operated by software V.4.2.6410. Polyphenols were isolated at 30°C using a C-18 column (internal diameter 250 × 4.6 mm, particle size 5 m). Compounds were extracted by isocratic HPLC at a flow rate of 0.8 mL/min. The mobile phase was the mixture of solvent A (70% acetonitrile and 30% methanol) and solvent B (0.5% glacial acetic acid with double distilled water). The identification of phenolic compounds was done by comparing the retention time and spike-in of the samples with standards. Compounds were quantified based on an external standard method at 275 nm. HPLC separation efficacy was evaluated by separation factor and resolution [17, 18].


#### 2.7.1. Calibration

Standard solutions containing six different concentrations (100, 200, 400, 600, 800, and 1000μg/ml) of each identified compound were prepared in mobile phase for calibration purpose. The subsequent peak area was plotted against concentration in a calibration graph.


### 2.8. Statistical Analysis

Each experiment was performed in triplicate, and data were presented as mean ± standard deviation. Data were analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc analysis for estimating significant difference among different treated groups using GraphPad Prism software. A value ofp<0.05 was considered as significant.


## 2.1. Selection and Collection of Plant Samples

Fresh leaves of single genotype ofE. alba were obtained in April 2019 from Ayub Agriculture Research Institute, Faisalabad, Pakistan. A voucher specimen of the plant sample (B & BEAL-19-1a) has been deposited in the departmental herbarium. The plant name has been confirmed with https://www.theplantlist.org. Leaves of E. alba were separated, washed, weighted, and then dried in convection oven (Memmert, Schwabach, Germany) at different temperatures, that is, 10°C, 30°C, 50°C, and 100°C. Each sample was then ground to make fine powder and stored at −20°C before further analysis [17].


## 2.2. Solvent Extraction of Plant Samples

The extraction of the plant sample was performed at room temperature. Finely powdered plant material (1 g of each sample) was macerated in pure ethanol, methanol, and acetone (10 mL) for 24 hours with constant shaking. The extracts were filtered with Whatman filter paper No. 1. and then concentrated by evaporating the solvents at room temperature. Stock solutions were prepared by dissolving the dried extracts in an equal volume of phosphate-buffered saline (PBS). Extracts were further diluted from 0.1 mg/mL to 0.0125 mg/mL to calculate IC50 value for DPPH assay and α-glucosidase inhibition assay [18].


## 2.3. Antioxidant Activity: DPPH Assay

Free radical scavenging activity of theE. alba was estimated using DPPH assay with some modifications from Ref. [19]. The stock solution of DPPH (0.3 mM) was prepared in ethanol, and 10 μL of plant sample or gallic acid as standard was mixed with 190 μL of DPPH in a 96-well plate with subsequent incubation for 30 minutes in dark. The absorbance was measured using microplate reader (Biotek ELx808IU, USA) at 517 nm, and measurements were used to calculate percentage inhibition of the DPPH solution.(1)%inhibition=Absorbance of control−Absorbance of test sample/Absorbance ofcontrol∗100.


## 2.4. Antidiabetic Activity:α-Glucosidase Inhibition Assay

Theα-glucosidase inhibition activity of plant extracts was determined using 5 mM p-nitrophenyl-β-D-glucopyranoside (PNPG) as a substrate slightly modified from [20]. Briefly, 12.5 μL of plant extract, 40 μL of α-glucosidase (0.5 U/mL), and 120 μL of phosphate-buffered saline (pH = 6.8) were added to each well of 96-well plate. After 5 minutes of incubation, 40 μL of substrate 5 mM was added and plate was further incubated at 37οC for 30 minutes. Acarbose, a synthetic drug, has potential inhibitory effects against α-glucosidase, which was used as positive control at a concentration of 10 mM, and phosphate-buffered saline was used as negative control. The absorbance was measured at 405 nm using microplate reader (Biotek ELx808IU, USA).Percentage inhibition ofα-glucosidase was calculated as follows:(2)%inhibition=Absorbance of control−Absorbance of test sample/Absorbance ofcontrol∗100.


## 2.5. Wound Healing Activity: Scratch Assay

The role ofE. alba extracts on the migration of retinal pigment epithelial (RPE) cells was determined using cell scratch assay slightly modified from [21]. Cells were cultured in 12-well plates at 2x105 cells per well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Plates were incubated at optimum conditions, that is, 37°C and 5% CO2 in a CO2 incubator to produce 80–100% confluent monolayer. Linear wound was produced using a sterilized 200-μL pipette tip. Medium was removed, and cells were washed with PBS. Fresh medium with different concentrations of E. alba extracts was added, and again, plate was again incubated for 24 hours. Microscopic images were taken with an inverted microscope (Meiji Techno TC5200) using 40x magnification at 0, 4, 8, 16, and 24 hours. Images were evaluated at 1712×1368 pixels in ImageJ 1.440 software for Windows. The percentage of the scratch area covered was determined by following equation:(3)%percentage wound healing=Wound area at4h−Wound area at0h/Wound area at4h∗100.


## 2.6. Cell Viability: MTT Assay

The human hepatocellular carcinoma cell line Huh-7 and the noncancerous retinal pigment epithelial (RPE) cell line were used for the assessment of toxicity ofE. alba extracts. The cells were cultured in the same medium as described above, but in 96-well cell culture plates with an initial concentration of 2 × 104 cells per well. Each well was treated with one of the 12 different E. alba extracts at a concentration of 0.1 mg/mL. Phosphate-buffered saline (PBS) was used as negative control. After incubation of 48 hours, 10 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to 96-well plate and then incubated for another 4 hours. After incubation, 150 μL of 0.1% dimethyl sulfoxide (DMSO) was added that is reduced to purple-colored formazan crystals by viable cells. The optical density was determined at 560 nm. Pictures were captured with an inverted microscope using 40x magnification [22].


## 2.7. Polyphenols: HPLC Analysis

Polyphenol content inE. alba extracts was determined using a Chromera HPLC system (PerkinElmer, Shelton, CT, USA) equipped with a Flexar Binary LC pump and a UV/Vis LC Detector, both operated by software V.4.2.6410. Polyphenols were isolated at 30°C using a C-18 column (internal diameter 250 × 4.6 mm, particle size 5 m). Compounds were extracted by isocratic HPLC at a flow rate of 0.8 mL/min. The mobile phase was the mixture of solvent A (70% acetonitrile and 30% methanol) and solvent B (0.5% glacial acetic acid with double distilled water). The identification of phenolic compounds was done by comparing the retention time and spike-in of the samples with standards. Compounds were quantified based on an external standard method at 275 nm. HPLC separation efficacy was evaluated by separation factor and resolution [17, 18].


### 2.7.1. Calibration

Standard solutions containing six different concentrations (100, 200, 400, 600, 800, and 1000μg/ml) of each identified compound were prepared in mobile phase for calibration purpose. The subsequent peak area was plotted against concentration in a calibration graph.


## 2.7.1. Calibration

Standard solutions containing six different concentrations (100, 200, 400, 600, 800, and 1000μg/ml) of each identified compound were prepared in mobile phase for calibration purpose. The subsequent peak area was plotted against concentration in a calibration graph.


## 2.8. Statistical Analysis

Each experiment was performed in triplicate, and data were presented as mean ± standard deviation. Data were analyzed using analysis of variance (ANOVA) followed by Tukey’s post hoc analysis for estimating significant difference among different treated groups using GraphPad Prism software. A value ofp<0.05 was considered as significant.


## 3. Results

### 3.1. Antioxidant Activity: DPPH Assay

Results of current study showed that variation of solvent and drying temperature effect on the antioxidant activity ofE. alba leaf extracts (Figure 1). DPPH radical scavenging activity of analyzed extracts was decreased with increasing drying temperature. Among the solvents, ethanol-based extracts produced the highest results followed by methanol- and acetone-based extracts. Antioxidant activity of plant extracts was also expressed as a concentration of the extracts used to scavenge 50% of DPPH free radicals (Figure 2). E. alba extracts prepared with leaves dried at 10°C and dissolved in ethanol and methanol, which had potent inhibitory effects against DPPH with IC50 values of 0.048 mg/mL and 0.074 mg/mL, respectively. For the higher temperatures under study, leaves dried at 30oC displayed the IC50 values of 0.067 mg/mL and 0.078 mg/mL using ethanol and methanol as solvents. Results were compared to the standard drug gallic acid (IC50 0.057 mg/mL). Percentage inhibition of extracts prepared with leaves dried at 100oC was recorded <50% even at highest tested concentration (0.1 mg/mL), so the IC50 value was not observed.Figure 1
DPPH activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.Figure 2
Dose-response curves (a, c, e) and IC50 values (b, d, f) of ethanol-, methanol-, and acetone-based extracts prepared from E. alba leaves.
(a)(b)(c)(d)(e)(f)


### 3.2. Antidiabetic Activity:α-Glucosidase Inhibition Assay

The inhibitory action ofE. alba extracts against α-glucosidase was determined using PNPG as a substrate. All the extracts derived from E. alba significantly inhibited the α-glucosidase action in a dose-dependent manner with highest activity showing at 0.1 mg/mL. Among the four drying temperatures, that is, 10°C, 30°C, 50°C, and 100°C, the highest antidiabetic activity was observed at 30°C for leaves dissolved in ethanol (IC50 0.035 mg/mL) and lowest activity at 100°C for all solvents (ethanol, methanol, and acetone). Ethanol-based extracts exhibited significantly higher percentage inhibition of α-glucosidase compared with methanol and acetone (Figure 3). IC50 values of the extracts showing percent inhibition of α-glucosidase greater than 50% are represented by Figure 4.Figure 3
α-Glucosidase inhibition activity of ethanol-, methanol-, and acetone-based extracts prepared from E. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.Figure 4
Dose-response curves (a), (c) and IC50 values (b), (d) of ethanol- and methanol-based extracts prepared from E. alba leaves.
(a)(b)(c)(d)


### 3.3. Wound Healing Activity: Scratch Assay

Cell migration is important for wound healing; thus, scratch assay was performed to observe the healing activity ofE. alba extracts on retinal pigment epithelial cells. The cell migration rate in the control group (PBS) was noted to be lowest among all the tested extracts (Figure 5). Analysis of the pictures taken after wound creation at different time intervals revealed that cells treated with extracts at a concentration of 0.1 mg/mL significantly increased the migration of cells. Ethanol- and methanol-based extracts had positive effects on wound healing activity. Comparison of the variable drying temperatures and solvents proved that both the temperature and solvent had a significant effect on cell proliferation. Samples dried at 10°C and 30°C showed better cell proliferation properties than extracts derived from plant material dried at higher temperatures, regardless of whether the material had been extracted in ethanol or methanol, whereas acetone-based extracts did not stimulate the cell proliferation as compared to control group (Figure 6).Figure 5
Wound healing activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 16 hours of incubation. (a–d) are treatment with ethanol-based extracts, (e–h) represent treatment with methanol-based extracts, and (i–l) are treatments with acetone-based extracts at 0.1 mg/mL. Platelet-derived growth factor and phosphate-buffered saline were used as positive and negative control represented by (m) and (n), respectively. Scratch area immediately after it is produced is represented by (o).Figure 6
Wound healing activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration on cell proliferation of retinal pigment epithelial (RPE) cells. Images were taken and evaluated at 4, 8, 16, and 24 hours of incubation. Data are expressed as percentage scratch area covered by cell proliferation. Bars expressed the mean ± standard deviation (based on 3 repeats of each treatment and 100 measurements of scratch width for each replication).


### 3.4. Cell Cytotoxicity: MTT Assay

The investigation of cytotoxic effects ofE. alba extracts on RPE and Huh-7 cells was done using MTT assay. Retinal pigment epithelial cells exposed to E. alba extracts showed no signs of toxicity, with cell viability remaining between 85 and 99% (Figure 7), whereas the treatment of E. alba extracts on Huh-7 cells caused cell death (Figure 8). Among the 12 extracts evaluated at a concentration of 0.1 mg/mL ethanol-based extracts of leaves dried at 10°C exhibited highest inhibition of Huh-7 cells. Again, the drying temperature and solvent had a significant influence (p<0.05) on the cellular toxicity of E. alba extracts (Figure 9).Figure 7
Effects of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration on cell viability of retinal pigment epithelial (RPE) cells.Figure 8
Cytotoxicity assay of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 48 hours of incubation on Huh-7 cell line. (a–d) represent treatment with ethanol-based extracts, (e–h) represent treatment with methanol-based extracts, and (i–l) represent treatment with acetone-based extracts at 0.1 mg/mL. Phosphate-buffered saline was used as negative control represented by (m).Figure 9
Cytotoxicity assay of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 48 hours of incubation on Huh-7 cell line. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.


### 3.5. HPLC Analysis

HPLC analysis was performed to identify fourteen phenolic compounds in ethanol-basedE. alba leaf extracts. Peaks were identified by comparing their retention times to pure standards (Figure 10). Identified compounds with their respective retention times and concentrations are shown in Table 1. Significant variations were observed among analyzed extracts regarding the concentration of identified polyphenols. Chlorogenic acid, p-coumaric acid, gallic acid, sinapic acid, and quercetin were identified in all the extracts. Among the ethanol-based extracts made from leaves dried at different temperatures, highest concentrations of all the compounds were observed in the leaves dried at 30°C, and the second highest in the leaves dried at 10°C. However, with increase in temperature to 50°C, a decrease in the content of phenolic compounds was observed. Concentrations of polyphenols potentially decline as the extraction temperature reaches to 100°C.Figure 10
HPLC chromatogram of ethanol-basedE. alba leaf extracts dried at (a) 10°C, (b) 30°C, (c) 50°C, and (d) 100°C.
(a)(b)(c)(d)Table 1
Phenolic compounds identified in ethanol-basedE. alba extracts prepared from leaves dried at four different temperatures.
Phenolic compoundRetention time (min)Concentration mg/kg inE. alba leaves dried at 10°CConcentration mg/kg inE. alba leaves dried at 30°CConcentration mg/kg inE. alba leaves dried at 50°CConcentration mg/kg inE. alba leaves dried at 100°CChlorogenic acid2.8801292.671334.38619.94217.13p-Coumeric acid3.075896.991119.93413.21203.87Gallic acid3.342653.10784.38342.34112.1HB acid6.759ND47.54NDNDCaffeic acid7.49414.06621.33NDNDVanillic acid7.687ND12.86NDNDKaempferol11.0744.08NDNDNDSinapic acid12.237133.35187.1534.7423.14Ferulic acid12.46085.63104.1767.11NDSalicylic acid15.296158.63300.70ND8.13Coumarin16.085162.381077.39132.7NDQuercetin16.954196.63210.33100.1554.1Benzoic acid18.3068.6709.102.83NDRutin23.98910.1015.43ND12.16


## 3.1. Antioxidant Activity: DPPH Assay

Results of current study showed that variation of solvent and drying temperature effect on the antioxidant activity ofE. alba leaf extracts (Figure 1). DPPH radical scavenging activity of analyzed extracts was decreased with increasing drying temperature. Among the solvents, ethanol-based extracts produced the highest results followed by methanol- and acetone-based extracts. Antioxidant activity of plant extracts was also expressed as a concentration of the extracts used to scavenge 50% of DPPH free radicals (Figure 2). E. alba extracts prepared with leaves dried at 10°C and dissolved in ethanol and methanol, which had potent inhibitory effects against DPPH with IC50 values of 0.048 mg/mL and 0.074 mg/mL, respectively. For the higher temperatures under study, leaves dried at 30oC displayed the IC50 values of 0.067 mg/mL and 0.078 mg/mL using ethanol and methanol as solvents. Results were compared to the standard drug gallic acid (IC50 0.057 mg/mL). Percentage inhibition of extracts prepared with leaves dried at 100oC was recorded <50% even at highest tested concentration (0.1 mg/mL), so the IC50 value was not observed.Figure 1
DPPH activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.Figure 2
Dose-response curves (a, c, e) and IC50 values (b, d, f) of ethanol-, methanol-, and acetone-based extracts prepared from E. alba leaves.
(a)(b)(c)(d)(e)(f)


## 3.2. Antidiabetic Activity:α-Glucosidase Inhibition Assay

The inhibitory action ofE. alba extracts against α-glucosidase was determined using PNPG as a substrate. All the extracts derived from E. alba significantly inhibited the α-glucosidase action in a dose-dependent manner with highest activity showing at 0.1 mg/mL. Among the four drying temperatures, that is, 10°C, 30°C, 50°C, and 100°C, the highest antidiabetic activity was observed at 30°C for leaves dissolved in ethanol (IC50 0.035 mg/mL) and lowest activity at 100°C for all solvents (ethanol, methanol, and acetone). Ethanol-based extracts exhibited significantly higher percentage inhibition of α-glucosidase compared with methanol and acetone (Figure 3). IC50 values of the extracts showing percent inhibition of α-glucosidase greater than 50% are represented by Figure 4.Figure 3
α-Glucosidase inhibition activity of ethanol-, methanol-, and acetone-based extracts prepared from E. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.Figure 4
Dose-response curves (a), (c) and IC50 values (b), (d) of ethanol- and methanol-based extracts prepared from E. alba leaves.
(a)(b)(c)(d)


## 3.3. Wound Healing Activity: Scratch Assay

Cell migration is important for wound healing; thus, scratch assay was performed to observe the healing activity ofE. alba extracts on retinal pigment epithelial cells. The cell migration rate in the control group (PBS) was noted to be lowest among all the tested extracts (Figure 5). Analysis of the pictures taken after wound creation at different time intervals revealed that cells treated with extracts at a concentration of 0.1 mg/mL significantly increased the migration of cells. Ethanol- and methanol-based extracts had positive effects on wound healing activity. Comparison of the variable drying temperatures and solvents proved that both the temperature and solvent had a significant effect on cell proliferation. Samples dried at 10°C and 30°C showed better cell proliferation properties than extracts derived from plant material dried at higher temperatures, regardless of whether the material had been extracted in ethanol or methanol, whereas acetone-based extracts did not stimulate the cell proliferation as compared to control group (Figure 6).Figure 5
Wound healing activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 16 hours of incubation. (a–d) are treatment with ethanol-based extracts, (e–h) represent treatment with methanol-based extracts, and (i–l) are treatments with acetone-based extracts at 0.1 mg/mL. Platelet-derived growth factor and phosphate-buffered saline were used as positive and negative control represented by (m) and (n), respectively. Scratch area immediately after it is produced is represented by (o).Figure 6
Wound healing activity of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration on cell proliferation of retinal pigment epithelial (RPE) cells. Images were taken and evaluated at 4, 8, 16, and 24 hours of incubation. Data are expressed as percentage scratch area covered by cell proliferation. Bars expressed the mean ± standard deviation (based on 3 repeats of each treatment and 100 measurements of scratch width for each replication).


## 3.4. Cell Cytotoxicity: MTT Assay

The investigation of cytotoxic effects ofE. alba extracts on RPE and Huh-7 cells was done using MTT assay. Retinal pigment epithelial cells exposed to E. alba extracts showed no signs of toxicity, with cell viability remaining between 85 and 99% (Figure 7), whereas the treatment of E. alba extracts on Huh-7 cells caused cell death (Figure 8). Among the 12 extracts evaluated at a concentration of 0.1 mg/mL ethanol-based extracts of leaves dried at 10°C exhibited highest inhibition of Huh-7 cells. Again, the drying temperature and solvent had a significant influence (p<0.05) on the cellular toxicity of E. alba extracts (Figure 9).Figure 7
Effects of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration on cell viability of retinal pigment epithelial (RPE) cells.Figure 8
Cytotoxicity assay of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 48 hours of incubation on Huh-7 cell line. (a–d) represent treatment with ethanol-based extracts, (e–h) represent treatment with methanol-based extracts, and (i–l) represent treatment with acetone-based extracts at 0.1 mg/mL. Phosphate-buffered saline was used as negative control represented by (m).Figure 9
Cytotoxicity assay of ethanol-, methanol-, and acetone-based extracts prepared fromE. alba leaves dried at 10°C, 30°C, 50°C, and 100°C at 0.1 mg/mL concentration after 48 hours of incubation on Huh-7 cell line. Bars with different alphabets indicate significant differences between treatment means at p<0.05 based on Tukey’s post hoc test.


## 3.5. HPLC Analysis

HPLC analysis was performed to identify fourteen phenolic compounds in ethanol-basedE. alba leaf extracts. Peaks were identified by comparing their retention times to pure standards (Figure 10). Identified compounds with their respective retention times and concentrations are shown in Table 1. Significant variations were observed among analyzed extracts regarding the concentration of identified polyphenols. Chlorogenic acid, p-coumaric acid, gallic acid, sinapic acid, and quercetin were identified in all the extracts. Among the ethanol-based extracts made from leaves dried at different temperatures, highest concentrations of all the compounds were observed in the leaves dried at 30°C, and the second highest in the leaves dried at 10°C. However, with increase in temperature to 50°C, a decrease in the content of phenolic compounds was observed. Concentrations of polyphenols potentially decline as the extraction temperature reaches to 100°C.Figure 10
HPLC chromatogram of ethanol-basedE. alba leaf extracts dried at (a) 10°C, (b) 30°C, (c) 50°C, and (d) 100°C.
(a)(b)(c)(d)Table 1
Phenolic compounds identified in ethanol-basedE. alba extracts prepared from leaves dried at four different temperatures.
Phenolic compoundRetention time (min)Concentration mg/kg inE. alba leaves dried at 10°CConcentration mg/kg inE. alba leaves dried at 30°CConcentration mg/kg inE. alba leaves dried at 50°CConcentration mg/kg inE. alba leaves dried at 100°CChlorogenic acid2.8801292.671334.38619.94217.13p-Coumeric acid3.075896.991119.93413.21203.87Gallic acid3.342653.10784.38342.34112.1HB acid6.759ND47.54NDNDCaffeic acid7.49414.06621.33NDNDVanillic acid7.687ND12.86NDNDKaempferol11.0744.08NDNDNDSinapic acid12.237133.35187.1534.7423.14Ferulic acid12.46085.63104.1767.11NDSalicylic acid15.296158.63300.70ND8.13Coumarin16.085162.381077.39132.7NDQuercetin16.954196.63210.33100.1554.1Benzoic acid18.3068.6709.102.83NDRutin23.98910.1015.43ND12.16


## 4. Discussion

The process of wound healing is severely affected in patients suffering from diabetes due to the increased blood glucose level, colonization of infectious agents, reactive oxygen species, and prolonged inflammation stage. Therefore, the identification of bioactive compounds and development wound dressing that can stimulate the healing process in diabetic patients is a challenge [23]. Leaves from different species of Eucalyptus have previously been reported to possess phytochemicals with potential antimicrobial and antioxidant activities [24].Therapeutic uses of the plant extracts can be attributed to the presence of various phytochemicals such as polyphenols and flavonoids in different parts of plants. Recently, a number of reports on the potential effectiveness of polyphenols in the prevention and treatment of skin disorders especially wound healing have been published [25–27]. The extraction of bioactive compounds from raw plant material is significantly affected by temperature and type of the solvents used. Therefore, optimization of extraction protocol, drying temperature, and type of solvent might help to improve the concentration of bioactive compounds [28]. Drying at high temperature for longer periods leads to the evaporation of volatile compounds. Moreover, using high drying temperature >50 could promote the degradation of heat-sensitive phytochemicals and significant decrease in the total phenol and flavonoid content of plants [29]. However, a contradictory study has reported that total phenolic content in turmeric rhizome was highest when dried at 100°C [30]. Comparisons of different organic solvents for the extraction of polyphenols have usually showed ethanol, methanol, and acetone to be the most efficient alternatives among other polar solvents, with acetone producing the highest yields for the extraction of brewer’s spent grains [31]. In other cases, ethanol and methanol have proven more efficient, for example, for the extraction of polyphenols in Hibiscus sabdariffa and Severinia buxifolia, respectively [32, 33]. The variation in contents of polyphenols using different solvents might be attributed to the difference in polarity of the solvent [34]. In line with that, leaves of E. alba were dried at variable temperatures ranging from 10°C to 100°C and extracted in different polar solvents (ethanol, methanol, and acetone) to identify best possible conditions for wound healing activity of this plants. In present research, ethanol was identified as the most efficient solvent and hence chosen for HPLC analysis for the identification of phenolic compounds. The HPLC analysis revealed that concentration of phenolic compounds in ethanol-based extracts was influenced by the variable drying temperature. Overall, the concentration of identified polyphenols was highest in the leaves dried at 30°C and lowest at 100°C. The present study is in line with the previous research where the extraction of phenolic compounds from Clinacanthus nutans leaves at temperature greater than 60°C for 120 minutes caused the oxidation and degradation of the desired bioactive compounds [35].Biological systems undergoing aerobic metabolism produce reactive oxygen species that are important for normal physiological processes such as cell signaling. These reactive oxygen species are also important to get rid of pathogens present on the site of injury. However, stress can dramatically enhance the level of these reactive oxygen species resulting in damage to the cells surrounding the wound [36]. In this regard, the evaluation of antioxidant potential is important to elucidate wound healing potential of the plants. Previous research has confirmed the antioxidant potential of Eucalyptus globulus leaf extract [37]. The leaves of E. alba dried at different temperatures and extracted in different solvents have shown antioxidant activities. However, highest DPPH radical scavenging activity was observed with leaved dried at 10°C extracted in ethanol with an IC50 value of 0.048 mg/mL.Inhibition of carbohydrate digesting enzymeα-glucosidase to lower the blood glucose level is an effective approach to treat hyperglycemia. Plant extracts are gaining importance due to the negative impacts of commercially available inhibitors of α-glucosidase [38]. Interestingly, most of the ethanol- and methanol-based extracts tested in the present study displayed α-glucosidase inhibition, which is an indication of their antihyperglycemic potential. The α-glucosidase inhibition activities of acetone-based extracts were lowest among the tested extracts.Cell proliferation is a vital stage in wound healing, and a number of different plant species have been investigated for their wound healing activity using cell scratch assay [39, 40]. Ethanol-based extracts of E. alba significantly stimulated the proliferation of RPE cells followed by methanol-based extract, whereas minimal proliferation activity was observed in extracts using acetone as solvent. There is an increased interest in using plants for their pharmacological properties, but the assessment of cytotoxic effects on cells is necessary before the use of plant extract. Furthermore, the evaluation of cytotoxic effects is an important factor for the quality control of therapeutic preparations [41]. For this purpose, cytotoxicity of E. alba extracts on RPE and Huh-7 cell line was also assessed. Results revealed that none of the extract was toxic for normal human RPE cell line, as the percentage of cell viability remained 85–99% after treating the RPE cells with E. alba leaf extracts for 48 hours, whereas the treatment of tested plant extracts on Huh-7 cells causes a decrease in the cell viability of cancer cells. These results confirms that E. alba leaf extract was not cytotoxic for normal cells and can be used as a wound healing agent as well as potential candidate for anticancer activities in liver cancer cell line.


## 5. Conclusion

Eucalyptus alba leaves dried at 10°C, 30°C, 50°C, and 100°C extracted in different solvents (ethanol, methanol, and acetone) were investigated for diabetic wound healing activity. Findings of the current study revealed that extracts of E. alba effectively stimulated the in vitro cell proliferation. The antioxidant and antidiabetic activities of the extracts have also supported the diabetic wound healing potential of E. alba leaf extracts. Furthermore, the plant extracts inhibited the growth of hepatocellular carcinoma (Huh-7) cell line but showed no signs of cytotoxicity on retinal pigment epithelial (RPE) cells. Overall, best results were obtained with leaves dried at 10°C and extracted in ethanol. The study suggests that E. alba leaves might be a potential natural source for diabetic wound healing.



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