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Phytochemical screening and in vitro antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing attributes of Senna auriculata (L.) Roxb. leaves
⁎Corresponding authors at: Bioprocess and Biomaterials Laboratory, Department of Microbial Biotechnology, Bharathiar University, Coimbatore, India (S. Sadhasivam); Research Department of Plant Biology and Biotehnology, Loyola College, Chennai, Tamil Nadu, India (A. Khusro). armankhan0301@gmail.com (Ameer Khusro), sadhaofficial@buc.edu.in (Subramaniam Sadhasivam)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
This study was aimed to determine total phytochemical components as well as in vitro antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing attributes of Senna auriculata (L.) Roxb. leaves extract. Among different solvent extracts tested, methanolic extract revealed maximum alkaloid, flavonoids, phenolic, and tannin contents of 90.41 ± 0.15 mg atropine equivalent/g, 156.64 ± 0.97 mg quercetin equivalent/g, 155.44 ± 1.65 mg gallic acid equivalent/g, and 121.31 ± 2.34 mg gallic acid equivalent/g of extract, respectively. Antibacterial activity of methanolic extract was determined against indicator pathogens using agar well diffusion assay which exhibited potential activity against Bacillus subtilis. Antioxidant, anti-inflammatory, anti-diabetic, and wound healing attributes of methanolic extract were analyzed using standard methodologies. The extract exhibited potential antioxidant traits by scavenging ABTS (IC50 value − 224.2 µg/mL) and DPPH (IC50 value − 76.24 µg/mL) in a concentration dependent manner. Further, the extract demonstrated significant anti-inflammatory activities at varied doses with IC50 value of 55.7 µg/mL. The methanolic extract exhibited promising rates of α-amylase and α-glucosidase inhibition with IC50 values of 49.45 and 38.72 µg/mL, respectively. Further, the extract showed no toxicity on L929 mouse fibroblast cell lines (99.66 ± 0.14% cell viability). Most importantly, the treatment of cell monolayer of L929 mouse fibroblast cell lines with methanolic extract showed high rate (100%) of wound healing trait. GC–MS analysis of methanolic extract revealed the presence of Mome inositol, 13-Docosenamide, (Z)-, Cycloheptasiloxane, tetradecamethyl-, and Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester as predominant compounds. In conclusion, the biological properties of S. auriculata leaves suggested its vast role as ideal antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing agents in future.
Keywords
Biological properties
GC–MS
Phytochemicals
Senna auriculata (L.) Roxb
Wound healing
1 Introduction
Senna auriculata (L.) Roxb. (Cassia auriculata, Family: Fabaceae or Leguminosae) is a common traditional and Asian beverage nutritional plant which is widely used in Indian traditional medicine for the treatment of diabetes mellitus and rheumatism (Juan-Badaturuge et al., 2011; Begum et al., 2013; Vijayakumar and Nachiappan, 2017). In India, different parts of S. auriculata such as bark, flowers, leaves, roots, and unripe fruits mixtures are used as “Avarai Panchaga Choornam” which is employed for the management of blood glucose level, urinary infections, conjunctivitis, and ophthalmia (Deshpande et al., 2013; Khader et al., 2017). Another preparation of beverage is called “kalpa herbal tea” containing S. auriculata (dried flowers) as its major component which is widely consumed by people who are suffering from diabetes, urinary tract diseases, and constipation (Thabrew et al., 2004; Khader et al., 2017). Likewise, S. auriculata leaves have been used for treating ulcers, skin disease, leprosy, and herpetic eruption. The roots and flowers of this plant have shown alexeteric and anti-diabetic properties too (Begum et al., 2013; Esakkirajan et al., 2014).
Phytochemicals are classic secondary metabolites of the plants which have numerous pharmacological benefits (Dhiman et al., 2016; Wang et al., 2018). Phytoconstituents such as alkaloids, glycosides, flavanoids, and triterpenes have been reported in methanolic extract of S. auriculata (Devi et al., 2014). Previously, the ethanolic extract of S. auriculata flowers and buds exhibited in vitro antioxidant and anti-diabetic properties (Nambirajan et al., 2018). However, investigation on some other important biological properties of S. auriculata leaves is limited (Kolar et al., 2018; Khyade et al., 2020). Considering this, the present context was aimed not only to analyze phytochemical constituents but also investigate in vitro antibacterial, antioxidant, anti-inflammatory, anti-diabetic, and wound healing potentials of S. auriculata leaves extract.
2 Materials and methods
2.1 Plant collection and identification
Fresh leaves of Senna auriculata were collected from Bharathiar University, Coimbatore, India and stored at room temperature. The authenticity of the plant specimen (BSI/SRC/5/23/2017/Tech./1781) was confirmed at the Botanical Survey of India (BSI), Southern Regional Centre, Coimbatore, by referring the deposited plant specimen.
2.2 Extract preparation
The fresh plant leaves were cleaned, washed using tap water, shade dried (37 °C) for five days, and ground into powder form. The powdered leaves (30 g) were subjected to successive extraction using a) petroleum ether, b) chloroform, c) ethyl acetate, d) methanol, and e) distilled water by soxhlet apparatus at 40 °C for 6–8 h. Distilled water was used for the maceration to extract the polar and non-polar compounds and then subjected to different solvent extraction (Senguttuvan et al., 2014). After extraction, crude liquids were kept in the water bath (60 °C) for the evaporation of any excess solvents. The extracts obtained were collected and stored in the sealed container for further use.
2.3 Phytochemical screening (qualitative analysis)
Presence of varied phytochemicals viz. alkaloids, carbohydrates, flavonoids, cardiac glycosides, phenols, proteins, saponins, tannins, and terpenoids in S. auriculata leaves extracts was determined according to the methodology of Prabhavathi et al. (2016).
2.4 Quantitative estimation of metabolites
2.4.1 Total alkaloid content (TAC)
The TAC in each extract was determined using the methodology of Tambe and Bhambar (2014) with some modifications. The plant extract (50 µg/mL) was dissolved in dimethyl sulphoxide and mixed with 1 mL of 2 N HCl. Further, 5 mL of bromocresol green solution and 5 mL of phosphate buffer were added into the mixture. The mixture was added into 4 mL of chloroform and diluted with chloroform. An atropine solution (50 µg/mL) was prepared and used as a standard. The absorbance of the solution was recorded at 470 nm using UV/Vis spectrophotometer. The TAC was expressed as milligrams of atropine equivalent (mg of AE)/g of extract.
2.4.2 Total flavonoid content (TFC)
The TFC in each extract was determined using the methodology of Dirar et al. (2019) with some modifications. The extract (50 µg/mL) was added into a 96-well plate containing 50 µL of ethanol. Further, 5 µL of 10% (w/v) AlCl3 (prepared in methanol) and 140 µL of distilled water were added into the wells. The mixture was shaken vigorously and the absorbance was read at 420 nm using UV/Vis spectrophotometer after 30 min of incubation. Quercetin was used as a standard. The TFC was calculated as milligrams of quercetin (mg of quercetin)/g of extract.
2.4.3 Total phenolic content (TPC)
The TPC in each extract was estimated using the methodology of Dirar et al. (2019) with some modifications. The extract (50 µg/mL) was added into a 96-well plate containing 50 µL of ultra pure water. Further, 10 µL of Folin–Ciocalteu phenol reagent was added into each well and incubated for 5 min. After incubation, 100 µL of 0.7% (w/v) NaHCO3 was added into each well and incubated further at 40 °C for 45 min under dark condition. Gallic acid was used as standard and the absorbance was measured at 550 nm using UV/Vis spectrophotometer. The TPC was calculated as milligrams of gallic acid equivalent (mg of GAE)/g of extract.
2.4.4 Total tannin content (TTC)
The TTC in each extract was determined using the method of Tambe and Bhambar (2014) with some modifications. The extract (50 µg/mL) was added into 7.5 mL of distilled water and 0.5 mL of Folin-Ciocalteu phenol reagent. The mixture was added further into 1 mL of 35% (w/v) Na2CO3 solution and diluted to 10 mL using distilled water. The mixture was shaken vigorously and incubated at room temperature for 30 min. Gallic acid was used as standard and the absorbance was measured at 725 nm using UV/Vis spectrophotometer. The TTC was calculated as milligrams of gallic acid equivalent (mg of GAE)/g of extract.
2.5 Biological activities
2.5.1 Antibacterial activity
In view of the qualitative and quantitative phytochemical analysis, methanolic extract showed better results compared with other solvents extracts, hence, all the further experiments were performed using methanolic extract only.
The antibacterial efficacy of the methanolic extract was assessed using agar well diffusion method (Kanmani and Lim, 2013). Gram-positive bacteria such as Bacillus subtilis and Methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa were procured from Coimbatore Medical College, Coimbatore, Tamil Nadu, and were used as indicator pathogens. Indicator bacteria were sub-cultured in Mueller-Hilton broth (HiMedia) medium overnight at 37 °C. Mueller-Hilton agar medium (HiMedia) was prepared and poured aseptically into the sterile Petri plates. Further, the bacterial pathogens were swabbed into the agar plates and wells were made using cork borer. Different concentrations (20, 50, and 80 µg/mL) of the extract as well as standard antibiotic (vancomycin − 30 µg) were added into the respective wells and plates were incubated at 37 °C for 48 h. After required incubation period, the zone of inhibition was measured in millimetres (mm).
2.5.2 Antioxidant activities
2.5.2.1 ABTS radical scavenging assay
ABTS radical scavenging assay of the methanolic extract was carried out according to the methodology of Ismail et al. (2017). In brief, 2 mM of ABTS diammonium salt and 3.5 mM of potassium persulphate were mixed with distilled water and kept in dark for 16 h. Subsequently, 290 µL of ABTS solution were mixed with the extract (100–500 µg/mL) and incubated for 10 min in a 96-well plate. The absorbance was recorded at 750 nm. Ascorbate was used as positive control. The ABTS radical scavenging percentage (%) was calculated as: ABTS scavenging (%) = [Ac-As/Ac] × 100 where Ac and As are the absorbance of control and absorbance of extract, respectively.
The IC50 value was calculated as the concentration of extract causing 50% scavenging of ABTS radical.
2.5.2.2 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay
DPPH scavenging assay of the methanolic extract was carried out according to the method of Tshibangu et al. (2017) with some changes. The stock concentration of 0.1 mM DPPH was prepared in methanol and 5 mL of the stock solution were mixed with 1 mL of the extract (100–500 µg/mL). Ascorbic acid (100–500 µg/mL) was used as standard. Samples were incubated in dark for 30 min and then absorbance was recorded at 517 nm. The DPPH radical-scavenging was calculated using following formula: DPPH scavenging (%) = [Ac-As/Ac] × 100 where Ac and As are the absorbance of control and absorbance of extract, respectively.
The IC50 value was calculated as the concentration of extract causing 50% scavenging of DPPH radical.
2.5.3 Anti-inflammatory activity
The inhibitory effect of protein denaturation was determined according to the methodology of Gunathilake et al. (2018) with minor modifications. The reaction mixture containing 0.2 mL of bovine albumin (1%), 4.78 mL of phosphate-buffered saline (PBS, pH 6.4), and 20–100 µg/mL of extract were incubated in a water bath at 37 °C for 15 min. After the incubation, the reaction mixture was heated at 70 °C for 4 min. The reaction mixture was cooled and the turbidity was measured at 660 nm using PBS as control and aspirin as a standard. The inhibition of protein denaturation was calculated as: Inhibition (%) = 100 × [1–A2/A1] where A1 and A2 are the absorbance of control and absorbance of extract, respectively.
The IC50 value was calculated as the concentration of extract causing 50% inhibition of protein denaturation.
2.5.4 Anti-diabetic activities
2.5.4.1 α-amylase inhibition assay
The inhibitory activity of α-amylase was investigated as per the method of Wan et al. (2013) with some modifications. Briefly, the methanolic extract (20–100 µg/mL) and acarbose were mixed with 10 µL of α-amylase (pre-incubated in 20 mM of sodium phosphate buffer, pH 6.7) and incubated at 37 °C for 5 min. The starch solution (0.2% w/v) was added into the reaction mixture to make the total volume up to 2 mL and incubated at 37 °C for 5 min. After incubation, 1 mL of dinitrosalicylic acid reagent was added and kept in a boiling water bath. After 5 min, the solution was allowed to cool, followed by the addition of deionized water. The absorbance was read at 540 nm and α-amylase inhibition was calculated as: α-amylase inhibition (%) = [Ac-As/Ac] × 100 where Ac and As are the absorbance of control and absorbance of extract, respectively.
The IC50 value was calculated as the concentration of extract causing 50% inhibition of α-amylase.
2.5.4.2 α-glucosidase inhibition assay
α-glucosidase inhibitory activity was determined according to the methodology of Wan et al. (2013) with some changes. The extract (20–100 µg/mL) and acarbose were mixed with 75 µL of α-glucosidase (prepared in 0.1 M potassium phosphate buffer; pH 6.8) and incubated at 37 °C for 15 min. Further, 20 μL of 5 mM para-nitrophenyl-α-D-glucopyranoside (PNPG) prepared in 0.1 M potassium phosphate buffer were added to initiate the reaction and incubated further for 15 min. After incubation, the reaction was terminated by adding 80 μL of 0.2 M Na2CO3 and the absorbance was read at 405 nm. α-glucosidase inhibition was calculated as: α-glucosidase inhibition (%) = [Ac-As/Ac] × 100 where Ac and As are the absorbance of control and absorbance of extract, respectively.
The IC50 value was calculated as the concentration of extract causing 50% inhibition of α-glucosidase.
2.6 Cytotoxicity assay
2.6.1 Cell line used
Mouse Fibroblast cell line (L929 cells) was procured from NCCS, Pune and maintained in Minimum Essential Medium (MEM) containing high glucose with 10% fetal bovine serum (supplemented with 1% antibiotic) and kept in CO2 incubator.
2.6.2 MTT assay
L929 cells with a cell density of 2 × 104 cells were seeded in a 96-well plate containing MEM medium supplemented with 10% fetal bovine serum and cultured overnight for cells attachment. After cells attachment, MEM medium was replaced with 200 µL of fresh MEM medium containing different concentrations of methanolic extract (5–100 µg/mL). After 24 h of incubation, 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL prepared in PBS) was added to each well and kept it for 4 h in CO2 incubator. Finally, the medium containing MTT was discarded, 200 μL of DMSO was added into each well, and allowed to incubate for 20 min. After incubation, the absorbance was read at 570 nm using ELISA reader (Koohpar et al., 2015). The viability of cells was calculated using following formula: Cells viability (%) = [As/Ac] × 100 where As and Ac are the absorbance of treated cells and absorbance of control cells, respectively.
2.6.3 Trypan blue assay
L929 cells (5 × 105 cells) were seeded in 6-well plates and allowed to grow overnight in MEM medium supplemented with 10% FBS for the cell attachment. The medium was further replaced with MEM medium containing various concentrations of methanolic extract (5–100 µg/mL) and incubated for 24 h. After required incubation period, the medium was removed, trypan blue stain (0.4% w/v) was added in each well, and then viewed under inverted microscopy (Zhang et al., 2015).
2.7 In vitro wound scratch assay
L929 cells were cultured in 24-well plate at a cell density of 2 × 105 cells and cultured up to 80% confluency. At this stage, a small linear wound was formed on the surface of the monolayer by gentle scraping as per the methodology of Liang et al. (2007). Cells were washed with PBS to remove the debris and cells were further treated with methanolic extract (25 and 50 µg/mL). The cell migration was monitored up to 24 h and the images were captured using inverted phase-contrast microscopy (Raghavan et al., 2015).
2.8 Gas chromatography–mass spectrometry (GC–MS) analysis
The Elite-MS (5% biphenyl 95% dimethylpolysiloxane, 30 m × 0.25 mm ID × 250 µm df) fused silica column (Clarus 680 GC) was used for GC–MS analysis. For the compound separation, helium was used as transporter gas at a constant flow of 1 mL/min. During the chromatographic run, the injector temperature was set at 260 °C, and 1 µL of the extract was injected into the instrument. The oven temperature was maintained at 60 °C for 2 min, followed by 300 °C at the rate of 10 °C/min; and 300 °C for 6 min. The mass detector conditions were as follows; transfer line temperature of 240 °C, ion source temperature of 240 °C and ionization mode electron impact at 70 eV, 0.2 sec scan time, and 0.1/sec scan time interval. The volatile low-molecular-weight fragments were analyzed between 40 and 600 Da. The spectrums of the test components were compared with the known components stored in the GC–MS NIST (2008) library.
2.9 Statistical analysis
All the experiments were carried out in triplicates, and the data were represented as mean ± standard deviation. The statistical analysis was performed by one-way ANOVA using SPSS version 12.0 (SPSS, Inc., Chicago, IL). The differences between experimental groups were considered significant when the P value was <0.05 (*P < 0.05).
3 Results and discussion
3.1 Extract yield
The yields of S. auriculata leaves extract from different solvents (petroleum ether, chloroform, ethyl acetate, methanol, and distilled water) are presented in Fig. 1. Among different solvents used, maximum yield was obtained for methanol extract (39.9%), followed by ethyl acetate (19%), petroleum ether (6.6%), distilled water (5.4%), and chloroform (3.9%) extracts.
3.2 Phytochemical analysis
Phytochemical analysis showed the presence of alkaloids, carbohydrates, flavonoids, cardiac glycosides, phenols, proteins, saponins, tannins, and terpenoids in S. auriculata leaves extract. However, methanolic extract showed the presence of comparatively more phytoconstituents than other solvents. Petroleum ether and aqueous extracts depicted the presence of fewer phytochemicals (Table 1). The quantitative estimation of major phytocomponents is shown in Table 2. The methanolic extract revealed maximum TAC, TFC, TPC, and TTC of 90.41 ± 0.15 mg AE/g, 156.64 ± 0.97 mg QE/g, 155.44 ± 1.65 mg GAE/g, and 121.31 ± 2.34 mg GAE/g of extract, respectively.
| Tests | Solvent extracts | ||||
|---|---|---|---|---|---|
| Petroleum ether | Chloroform | Ethyl acetate | Methanol | Distilled water | |
| Alkaloids | + | + | + | ++ | + |
| Carbohydrates | − | − | ++ | + | − |
| Flavonoids | + | + | ++ | ++ | + |
| Glycosides | + | − | − | + | − |
| Phenols | + | + | + | ++ | + |
| Proteins | − | ++ | + | − | − |
| Saponins | − | ++ | − | + | − |
| Tannins | + | + | ++ | ++ | + |
| Terpenoids | − | − | + | + | − |
− = Absent; + = Present; ++ = Highly present.
| Tests | Extracts | ||||
|---|---|---|---|---|---|
| Petroleum ether | Chloroform | Ethyl acetate | Methanol | Distilled water | |
| TAC (mg AE/g) | 34.22 ± 0.09d | 32.76 ± 0.03e | 85.71 ± 0.49b | 90.41 ± 0.15a | 67.86 ± 4.67c |
| TFC (mg QE/g) | 38.26 ± 0.16d | 60.49 ± 0.13c | 109.69 ± 0.64b | 156.64 ± 0.97a | 31.52 ± 0.16e |
| TPC (mg GAE/g) | 60.24 ± 0.06b | 33.77 ± 0.20e | 42.83 ± 0.21c | 155.44 ± 1.65 s | 39.79 ± 1.41d |
| TTC (mg GAE/g) | 53.92 ± 0.51c | 38.11 ± 0.34d | 79.35 ± 0.23b | 121.31 ± 2.34a | 30.32 ± 0.11e |
Note: Each value is expressed as mean ± SD. abcdeValues in each row varied significantly (P < 0.05).
Plant-based compounds have several biological applications. An alkaloid compound has been reported to exhibit lethal effects against colon and breast cancer cells and has been used for antimicrobial, antiviral, antiprotozoal, and antitumor applications (Rinaldi et al., 2017). Flavonoids have been used for anti-diabetic, antimicrobial activities, anti-inflammatory, and anti-aging preparations (Wang et al., 2018). Previous researches have shown that the plant phenolic compounds offer the role of potential natural antioxidants (Dhiman et al., 2016; Takaidza et al., 2018). Flavonoids and phenols have raised particular interest because of their potential biological characteristics as antioxidant, antiestrogenic, anti-inflammatory, immunomodulatory, cardioprotective, and anticarciogenic compounds (Kumar and Baskar, 2015). Besides, tannins play an essential role in many biological applications because of their anti-inflammatory, cardio-protective, and antimicrobial properties (Zarin et al., 2016; Huang et al., 2018).
3.3 Antibacterial activity
The methanolic extract exhibited potential antibacterial activities against the tested pathogens in a concentration-dependent manner with maximum zone of inhibition of 13.33 ± 0.61 mm against B. subtilis, followed by MRSA (11.66 ± 0.14 mm), P. aeruginosa (11 ± 0 mm), and E. coli (9.66 ± 0.38 mm) (Table 3). Further, scanning electron microscopy (SEM) analysis revealed the accumulation of extract on the bacterial cell wall, thereby inhibiting its growth (Fig. 2). Previously, the phenolic extracts of berry plants demonstrated significant antibacterial activities against E. coli, S. aureus, B. cereus, and Salmonella enteric (Tian et al., 2018). Al-Matani et al. (2015) reported the antibacterial activity of Ficus sycomorus leaves extract against Haemophilus influenza, E. coli, and S. aureus. Likewise, the antibacterial activity of Artemisia nilagirica (Clarke) Pamp leaves extract was demonstrated against E. coli, S. aureus, P. aeruginosa, and B. subtilis at different concentrations (Parameswari et al., 2019).
| Pathogens | Extract and antibiotic | |||
|---|---|---|---|---|
| 80 μg/mL | 50 μg/mL | 20 μg/mL | Vancomycin (30 μg) | |
| B. subtilis | 13.33 ± 0.61a | 9.33 ± 0.28a | 6.33 ± 0.17a | 14.66 ± 0.71a |
| MRSA | 11.66 ± 0.14b | 9 ± 0a | 4.33 ± 1.1b | 8.33 ± 0.62d |
| E. coli | 9.66 ± 0.38c | 7.33 ± 0.63c | 6.66 ± 0.16a | 12 ± 0b |
| P. aeruginosa | 11 ± 0b | 8.66 ± 0.17b | 3 ± 0c | 10.66 ± 0.33c |
Note: Values are expressed as the mean ± SD. abcdValues in each column varied significantly (P < 0.05).
![SEM analysis of untreated and treated [methanolic extract (50 µg/mL) and vancomycin (30 μg)] bacterial pathogens. Arrows indicate the destruction of bacterial cell wall due to the inhibitory actions of methanolic extract and vancomycin.](/content/184/2021/14/9/img/10.1016_j.arabjc.2021.103345-fig2.png)
3.4 Antioxidant activities
Fig. 3 shows the antioxidant abilities of the methanolic extract of S. auriculata leaves against the free radicals. The extract exhibited free radical scavenging activities of 26.27 ± 0.17, 41.49 ± 1.15, 66.83 ± 2.21, 73.03 ± 1.42, and 81.12 ± 0.46% against ABTS (Fig. 3a). The extract showed potential antioxidant activities of 62.36 ± 2.72, 69.28 ± 0.31, 73.16 ± 0.61, 88.78 ± 1.13, and 96.33 ± 0.64% by degrading DPPH (Fig. 3b). Results further implied that the antioxidant potential of the extract was comparable to ascorbic acid. The IC50 values of extract against ABTS and DPPH were estimated as 224.2 and 76.24 µg/mL, respectively.
The phenols and flavonoids are reported as the significant counterparts in phytochemical mediated free-radical scavenging. Cinnamon zeylanicum and Eugenia polyantha have shown significant free radical scavenging abilities due to the presence of phenols and flavonoids (Ismail et al., 2017). The methanolic extract of Digera muricata and Mangifera indica exhibited antioxidant properties by reducing Fe3+ to Fe2+ upon electron donation. In the present study, the antioxidant activities of methanolic extract might be mediated through the presence of notably higher phenolic content, thereby favouring the findings of Mehwish et al. (2019). Likewise, the methanolic leaves extracts of Pistacia lentiscus and Ruscus aculeatus have been reported as better antioxidant agents against DPPH and ABTS radicals (Chaves et al., 2020).
3.5 Anti-inflammatory activity
The methanolic extract of S. auriculata leaves demonstrated significant anti-inflammatory activities (23.38 ± 0.28–84.2 ± 1.15%) in a dose dependent manner (Fig. 4). The obtained results were comparable to the aspirin mediated anti-inflammatory activity which exhibited 39.6 ± 0.62–94.08 ± 1.62% inhibition at different concentrations. The IC50 value of the extract towards the inhibition of protein denaturation was estimated as 55.7 µg/mL. Previous studies suggested potential anti-inflammatory activities of coffee beans and Pedalium murex by protein denaturation method (Chandra et al., 2012; Sangeetha and Vidhya, 2016). In a recent study, the stem bark water–acetone extract of Guibourtia tessmanii showed significant inhibition of ovalbumin denaturation activity with an IC50 value of 102.25 ± 10.21 µg/mL, while water–ethanol extract showed IC50 value of 180.27 ± 5.27 µg/mL (Obiang et al., 2021).
3.6 Anti-diabetic activities
The methanolic extract exhibited α-amylase inhibition of 37.2 ± 0.26–93.2 ± 0.37% in a dose-dependent manner (20–100 µg) (Fig. 5a). Further, the extract depicted α-glucosidase inhibition of 42.2 ± 0.28–80.72 ± 0.64% at distinct concentrations (Fig. 5b). The IC50 values of extract against α-amylase and α-glucosidase inhibition were estimated 49.45 and 38.72 µg/mL, respectively. Nair et al. (2013) investigated α-amylase and α-glucosidase inhibition activities of methanolic extract of Artocarpus heterophyllus and Piper betel leaves, Cinnamomum zeylanicum bark, and Artocarpus altilis fruit. Wan et al. (2013) studied in vitro hypoglycemic activity of Swertia kouitchensis ethanolic extract in terms of α-amylase and α-glucosidase inhibition. The ethanolic extracts of stems and leaves of Myrica gale possessed significant anti-diabetic effects by inhibiting α-amylase (IC50 = 62.65 mg/L) and α-glucosidase (IC50 = 27.20 mg/L) (Sekhon-Loodu and Rupasinghe, 2019).
3.7 Cytotoxicity assay
The cell anti-proliferative effect of methanolic extract was assessed in L929 mouse fibroblast cell line by MTT assay. The cell morphology and cell viability at various concentrations are shown in Fig. 6 which revealed no morphological changes in treated cells. Results showed 99.66 ± 0.14% cell viability at all concentrations of the extract (5–100 µg/mL). Further, the trypan blue assay confirmed that the extract showed no toxicity on L929 cell lines (Fig. 7). These results strongly confirmed that methanolic extract had no cytotoxic effect on healthy L929 mouse fibroblast cells.
The cytotoxic assessment of different plant extract was investigated against chick embryo fibroblast cell line in the past. High cell viability was reported for Tinospora cordifolia extract, followed by Plumbago zeylanica and Withania somnifera (Kakad and Dhembare, 2014). Similarly, the cytotoxicities of Ricinus communis leaves extracts were evaluated against Human Caucasian skin fibroblast (BUD-8) cell line which showed <90% of cell viability by acetone and methanol extracts, whereas hexane and dichloromethane extracts showed >90% cell viability after 24 and 48 h of exposure (Nemudzivhadi and Masoko, 2014). In another study, the methanolic leaves extracts of Aristolochia saccata exhibited cytotoxic activity against L929 mouse fibroblast cell line for 48 h. Results confirmed that the lower concentrations of methanolic extract had no cytotoxic effect but the higher concentrations of extract exhibited slight toxic effects on L929 cells. Results concluded that methanolic leaves extracts of A. saccata were highly biocompatible (Bolla et al., 2019).
3.8 In vitro wound healing assay
In vitro wound healing scratch assay was performed to find out the potential effect of S. auriculata leaves extract on the promotion of wound healing process in L929 mouse fibroblast cell lines. Cell monolayer treated with the methanolic extract showed a significantly higher rate of wound healing as compared to control after 8, 16, and 24 h. Cells treated with 25 µg/mL of extract showed 51.33 ± 0.13, 89.6 ± 0.81, and 97.66 ± 0.36% of healing at 8, 16, and 24 h, respectively. The cells treated with 50 µg/mL of extract showed 100% wound healing at 24 h (Fig. 8). Results revealed rapid wound healing trait of methanolic extract of S. auriculata leaves than the control cells without any cytotoxic effect.
Wound healing assays are widely used as in vitro technique to investigate the cell migration and to examine the influence of effective drugs on the rates of cell motility and proliferation (Johnston et al., 2016). Haddadi et al. (2019) demonstrated potential effect of Scrophularia striata hydro-alcoholic extract on the wound healing process in human dermal fibroblasts (HDF) and human umbilical vascular endothelial cells (HUVECs) cell lines. Juneja et al. (2019) evaluated in vitro wound healing efficacy of methanolic and chloroform extracts of Boerhavia diffusa leaves against the keratinocytes HaCaT cells. The methanolic extract closed 59% wound area within 12 h and 97% wound closure in 60 h in HaCaT cells; whereas the chloroform extract showed comparatively lesser wound closing (68%) at 60 h. Result concluded that both methanol and chloroform extracts stimulated the cell migration of keratinocytes into the denuded area.
3.9 GC–MS analysis
GC–MS analysis showed the presence of distinct compounds in the methanolic extract (Fig. 9). However, Mome inositol, 13-Docosenamide, (Z)-, Cycloheptasiloxane, tetradecamethyl-, and Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester were dominantly present (Table 4). These compounds might be responsible for varied medicinal properties of methanolic extract of S. auriculata leaves. Previous studies demonstrated diversified applications, particularly anti-neuropathic, lipotropic, anti-cirrhotic, cholesterolytic, anti-alopecic, and sweetener properties of Mome inositol (Das et al., 2014). Amongst these compounds, 13-Docosenamide, (Z)- has been known for antiviral properties (Hasan et al., 2019). Cyclodecasiloxane, eicosamethyl is a bioactive compound that has several biological properties such as antirheumatic, hepatoprotective, and anti-spasmodic (Bobade, 2019).
| S. No. | Compounds | Area (%) | Retention time | Chemical formula | Molecular weight |
|---|---|---|---|---|---|
| 1 | 7,10-Epoxytricyclo[4.2.1.1(2,5)]decane, 1-trimethylsilyl- | 1.25 | 6.01 | C13H22OSi | 222 |
| 2 | Dimethoxyglycerol Docosyl Ether | 1.61 | 7.40 | C27H56O5 | 460 |
| 3 | Cycloheptasiloxane, tetradecamethyl- | 4.94 | 8.37 | C14H42O7Si7 | 518 |
| 4 | Cyclooctasiloxane, hexadecamethyl- | 1.18 | 9.73 | C16H48O8Si8 | 592 |
| 5 | Mome inositol | 56.14 | 10.76 | C7H14O6 | 194 |
| 6 | Mome inositol | 2.56 | 11.09 | C7H14O6 | 194 |
| 7 | 6-(4-Chlorophenyl)-2,5,5-triphenyl-5,8-dihydro-6H-azeto[1, 2-a][1,3]thiazolo[4,5-d]pyrim Idine | 2.58 | 12.96 | C31H22ClN3S | 503 |
| 8 | Cyclodecasiloxane, eicosamethyl- | 1.48 | 15.19 | C20H60O10Si10 | 740 |
| 9 | 1H-Purin-6-amine,[(2-fluorophenyl)methyl]- | 0.86 | 17.64 | C12H10FN5 | 243 |
| 10 | Silicone oil | 1.13 | 18.96 | NA | 0 |
| 11 | Cyclodecasiloxane, eicosamethyl- | 0.84 | 20.17 | C20H60O10Si10 | 740 |
| 12 | Silicone oil | 2.70 | 21.62 | NA | 0 |
| 13 | Silicone oil | 1.07 | 22.77 | NA | 0 |
| 14 | (Z)-1-(3,4,5-Trifluorophenyl)-2-(naphthyl)ethane | 0.30 | 24.13 | C18H11F3 | 284 |
| 15 | Silicone oil | 1.43 | 24.55 | NA | 0 |
| 16 | Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester | 4.13 | 25.34 | C21H42O4 | 358 |
| 17 | 13-Docosenamide, (Z)- | 5.84 | 26.07 | C22H43NO | 337 |
| 18 | Silicone oil | 1.21 | 26.36 | NA | 0 |
| 19 | Octasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- | 1.37 | 27.78 | C16H50O7Si8 | 578 |
| 20 | Octasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- | 1.17 | 29.49 | C16H50O7Si8 | 578 |
| 21 | Octasiloxane,1,1,3,3,5,5,7,7,9,9,11,11,13,13,15,15-hexadecamethyl- | 0.57 | 30.11 | C16H50O7Si8 | 578 |
NA – Not available.
4 Conclusions
Herbal plants have been found to possess significant biological properties such as antimicrobial, antioxidant, antidiabetic, cell migration/proliferation, and wound healing effects. Results of this study depicted that S. auriculata leaves extracts were potential sources of essential phytochemicals such as alkaloids, flavonoids, phenols, and tannins. The methanolic extract of S. auriculata leaves possessed significant antibacterial activities against certain human bacterial pathogens. It also displayed potential in vitro antioxidant, anti-inflammatory, and anti-diabetic activities due to the rich source of phytochemical contents. The methanolic extract enhanced the wound healing efficacy without inducing any cytotoxic effect on L929 mouse fibroblast cell lines. In addition, GC–MS analysis showed the presence of Mome inositol, 13-Docosenamide, (Z)-, Cycloheptasiloxane, tetradecamethyl-, and Octadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester as predominant compounds.
Acknowledgement
The author, Dr. Subramaniam Sadhasivam acknowledges DBT, India for the financial support provided by the Ramalingaswami Re-entry fellowship (Order No.BT/RLF/Re-entry/55/2013). Authors extend their appreciation to the Researchers supporting project number (RSP-2021/190), King Saud University, Riyadh, Saudi Arabia.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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