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. 2023 Jan 12;13(2):46. doi: 10.1007/s13205-023-03464-8

Isolation, purification and characterization of quercetin from Cucumis sativus peels; its antimicrobial, antioxidant and cytotoxicity evaluations

Anjani 1, Nidhi Srivastava 2, Jyoti Mathur 1,✉
PMCID: PMC9837362  PMID: 36643397

Abstract

The peels of C.sativus are produced in large quantities in food processing industries and as kitchen-waste, resulting into tremendous loss of valuable bioactive components. Considering this, the flavanoids from C.sativus peels (CSP) were isolated and characterized by the column chromatography, high-performance thin-layer chromatography (HPTLC), Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (NMR) methods. The antioxidant and antibacterial activity of CSP extract against Escherichia coli, Streptococcus mutans and Pseudomonas aeruginosa was assessed. Scanning electron microscopy (SEM) was also used to investigate the killing efficacy of CSP extract against selected bacterial strains. Cytotoxic activity of extract was analyzed on L929 (connective tissue, Mouse, Mus Muscular) cell lines to check their viability. According to the results, total flavanoid content in CSP extract was found as 55.3 ± 1.154 µg/g QE and the chromatographic and spectral data of the isolated compound was elucidated as quercetin—a flavanoid. The CSP extract has also found to give significant antioxidant activity for both DPPH (2, 2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant power) assay. The CSP extract was found to inhibit growth of E. coli, P. aeruginosa and S. mutans by forming inhibitory zones of 11.36 ± 0.47, 6.7 ± 0.36 mm and 10.16 ± 0.11 mm, respectively. The SEM results also confirm the rupturing of cells or biofilms of bacterial cells as compare to control strains. Additionally, CSP extract was found to not inhibit the proliferation of L929 cells and more than 90% viability of cells was achieved. The present finding highlights the phytochemical profile and therapeutic applications of C.sativus peels.

Keywords: Antimicrobial, Antioxidant, Cucumis sativus peels, Cytotoxicity, Flavanoids, HPTLC, SEM

Introduction

Generally, plants have a wide range of secondary metabolites with pharmacological activity. Therefore, the discovery of lead components from plants, leads to drug development and performed application in therapeutic use. Plant source provides a number of bioactive components such as phenolic compounds, flavanoids, alkaloids, steroids, saponin tannins, terpenoids, glycosides, proteins, etc. having wide range of therapeutic applications in combating a number of diseases. Flavanoids are considered as polyphenolic components which are classified into, flavanols, anthocyanides, flavones, flavanones, isoflavonens. The functional hydroxyl group of flavanoids are mediating the antioxidant activity (Nair et al. 2009; Dehshahri et al. 2012), anticancer (Adlercreutz 2002; Cornwell et al. 2004), antimicrobial and other aging diseases (Rice-Evans et al. 1995), hepatoprotective activity (Zhu et al. 2012) and anti-inflammatory activity (Pan et al. 2010).

Cucumis sativus or Cucumber belongs to Cucurbitaceae family such as, watermelon, melon, zuacchini etc. It is widely used as fresh vegetable salads, pickles or as a cooked vegetable (Sotiroudis et al. 2010). In Indian system of medicine, C.sativus seeds and fruits have important therapeutic values, particularly in Ayurveda where more than two-thousand herbs and formulations are available for management of aging. The medical properties of C.sativus have been described since ancient times. Additionally, it is used for a number of skin problems, including sunburn and swelling under the eye (Franco et al. 2002). C.sativus exhibits broad range of in vivo and in vitro biological activities (Miller et al. 2000; Stratil et al. 2006; Mukherjee et al. 2013). Their fruit and seed extracts are reported to have antifungal and antibiotic activity (Sood et al. 2012; Mallik et al. 2013). The peels and fruits of C.sativus are also known to have hypochloesterolemic and antidiabetic activity (Saidu et al. 2014).

The C.sativus seed and fruit have been studied for pharmacological properties but comparatively less number of studies has been reported on the therapeutic potential of C.sativus peels. The plant and their parts such as leaves, fruits, peels, flowers and stems provide a majority of natural medicines. In general, vegetable and fruit peels are considered as agro waste ad throw away instead of their use as an antimicrobial agent (Singh et al. 2019). The use of these peels as an antimicrobial agent provides opportunity to solve the problems in the area of antibiotic application in the cost-effective way and as environmental friendly in reducing the pollution from the disposal of such agro waste (Szabo et al. 2019). Hence in the present finding, an attempt was made to isolate and characterize the flavanoid (quercetin) by chromatographic and spectral methods from peels of C.sativus with their antioxidant, antimicrobial and cytotoxicity activities.

Materials and methods

Materials, collection and processing

Extraction of CSP was carried out using 80% ethanol (SRL, India). Peel of C. sativus were collected from the hostel mess hall, Banasthali Vidyapith, Rajasthan, India. All peels were washed with distilled water and dried in shade at room temperature (25 ± 1 °C) for two weeks. The dried peels were then ground into a fine powder with a mechanical blender and stored at room temperature.

Microorganisms

The standard strains of Escherichia coli (MTCC 568), Streptococcus mutans (MTCC 890), and Pseudomonas aeruginosa (MTCC 647) were obtained from IMTECH (Institute of Microbial Technology), Chandigarh, used for antimicrobial activity of CSP extract.

Extraction of C. sativus peels and phytochemical analysis

Up to 50 g of fine powder of CSP were subjected to maceration for 72 h using 80% ethanol (Jang et al. 2012 and Zhu et al. 2019). The evaporation of was carried out using rotary evaporator and dried with the help of desiccators. The ethanolic extract of CSP was then utilized for further evaluations.

Preliminary phytochemical tests for qualitative analysis in CSP extracts were performed using the methods of Petchi et al. (2013), to identify the secondary metabolites in the extract.

Estimation of total flavanoid content

The estimation of TFC in the extract of CSP was carried out by aluminum chloride colorimetric method of Zhinsen et al. (1999), with slight modifications. In 0.5 ml of 5% NaNO2 solution, 500 µg/ml extract was mixed and allowed to stand for 5 min. Then 0.3 ml of 10% AlCl3 solution and 1 ml of 1 M NaOH solution were added into the mixture. The final volume of mixture was made up to 5 ml using distilled water. Mixture was well mixed and kept for 15 min. Reading was taken at 510 nm using UV Visible double beam spectrophotometer (Systronics 2201) and calibration curve was drawn using quercetin as standard (50–300 µg/ml).

Isolation, identification and characterization of quercetin

Extract of CSP are typically a mixture of several type of bioactive compounds. The major phytochemical markers such as flavanoids (quercetin) were aimed to identify and characterized using some chromatographic and spectral methods. The isolation of quercetin from CSP was carried out according to the methods of Patel et al. (2017). This extract was the used for further evaluations.

Column chromatographic fractionation and TLC of ethanolic extract

The isolation and separation from CSP was carried out using column chromatography and TLC, respectively (Gini and Jothi 2018). 10 gm of dried sample was packed on a silica gel column (Marck, India). Elution was begins with 100% hexane and polarity was then increased using ethyl acetate, ethanol and methanol. Each gradient of mobile phase was passed through the column and fractions (5 ml) were collected at the rate of 10 ml/min. The collected fractions were then subjected to TLC plate and Rf values were then compared with standard flavanoids. TLC of eluted fractions was performed on pre-coated 20 × 20 cm aluminum sheets. The column fractions were spotted on TLC plates of 0.2 mm above from bottom line with capillary tubes (5 µl) three times to apply greater concentration of sample. The solvent system was chloroform: methanol (1:1) as running mobile phase. Suitable amount of mobile phase was loaded in the tank and kept for half an hr was expected to achieve balance of solvent vaporization. Colored and colorless bands emerged on the plate were detected under UV light λ = 365 nm and the fractions showing Rf values equal or close to the standards were selected for the structural identification.

Rf value was calculated as:

Rf=Distance travelled by compounds/Distance travelled by solvent

The eluted fractions showing identical Rf on TLC plates were pooled together and detected through HPTLC chromatography.

HPTLC analysis

The HPTLC of CSP column fractions was performed according to the methods of Patel et al. (2017) with slight modifications. 2 mg of standard quercetin was dissolved in methanol to prepare the stock solutions. The different concentrations from 500, 1000, 2000, 3000, 4000 and 5000 ng were prepared from the stock solution. For HPTLC analysis, pre-coated aluminum TLC plates (20 × 10 cm) with 0.2 mm silica gel layer (60 F254 by MERCK, India) were used. The standard and selected column fractions (Rf equal to standard quercetin on TLC plates) were applied to the silica-coated plate with the help of automatic TLC sampler (CAMAG, LINOMAT 5) for HPTLC analysis. The plate was then developed using toluene-ethyl acetate-formic acid in the ratio of 5:4:0.2 (v/v/v) as a mobile phase inside the twin chamber and 15 min pre-saturation was done using mobile phase. The plate was dried after development and scanned (CAMAG TLC Scanner 3) at 254 nm. For the photo-documentation of plate, a TLC plate visualize was used. ICH (International Conference on Harmonization) guidelines (ICH 1996, 2005) were used for method validation.

For the calibration curve, peak area was plotted against the amount applied. The LOD (limits of detection) and LOQ (limits of quantification) were determined by the equation as per ICH guidelines. The content of quercetin in the samples was quantified using the regression equation obtained from the standard curve.

FTIR analysis

The FTIR spectrum helps in compound identification on the basis of existence of different functional group. The spectrum was obtained by FTIR spectrometer (Bruker) connected to software of OPUS operating system. The sample was prepared in hydraulic pellet press (SR. NO, 1436) by using KBr pellet. The scanning of sample disc was then carried out between 4000 and 400 cm−1 by 25 scans. The spectrum was generated by instrument and peak picking was finished and outcomes were analyzed (Sasidharan et al. 2011).

1H NMR spectroscopy analysis

The NMR experiment was carried out in 400 MHz Bruker Avance III HD Nanobay Spectrometer. 5 mg of sample was dissolved in deuterated solvent DMSO. TMS was used as an internal standard. Sample was loaded into to a glass tube and inserted in a plastic spinner. Spinner contains the sample inserted into the magnet and chemical shifts were expressed in ppm. The hydrogen atoms present in the compounds are detected by 1H-NMR (Blunder et al. 2017).

In vitro antioxidant activity of CSP extract

To determine the antioxidant activity of CSP crude extract, two different assays were used, including 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and FRAP (ferric reducing antioxidant power) assay on the basis of methods of Brand-Williams et al. (1995) and Benzie and Strain (1996) with some experimental modifications, respectively. All the experiments were performed in triplicates.

DPPH assay

The determination of radical scavenging activity of CSP extract was carried out by DPPH assay. In this assay, different concentrations (50–300 µg/ml) of CSP extract were prepared using methanol and into this 1 ml of 0.1 mM DPPH radical was then added. The absorbance of reaction mixture was taken at 515 nm after 30 min of incubation, using UV Visible double beam spectrophotometer (Systronics 2201). The ascorbic acid was used as a standard and results were expressed in terms of ascorbic acid equivalents. The percentage of radical scavenging activity (RSA) was calculated using the formula:

\% RSA=Absorbance of control-Absorbance of sample/Absorbance of control×100

FRAP assay

CSP extract was also used to determine antioxidant power by FRAP assay and the assay was based on the reducing power of extract. TPTZ (tripyridyltriazine) was used as FRAP reagent. The different concentrations of CSP extract (50–300 µg/ml) were added into freshly prepared FRAP reagent and mixed well. The incubation was carried out in dark for 30 min and absorbance was taken at 593 nm. Ascorbic acid was used as a standard antioxidant and results were expressed in terms of ascorbic acid equivalents. Relative percentage of reducing power was calculated using the following equation:

Relative%of reducing power=Absorbance of sample-Absorbance of control/Highest absorbance of standard-Absorbance of control×100

Antibacterial activity and minimum inhibitory concentration (MIC)

Antibacterial activity of CSP extract at different concentrations was checked against E. coli, S. mutans and P. aeruginosa using agar well diffusion method (Langfield et al. 2004). Autoclave process (at 121 °C for 15 min) was followed for sterilization of all equipments. MHA (Muller Hinton Agar) was used as growth medium for all the bacterial strains. 20 ml of MHA was poured aseptically in all petri plates and solidified for 10 min. The strains of bacteria were spreaded on agar plates (106 CFU/ml) with the help of sterile swab. Wells were created on MHA agar plates with the help of sterile cork borer and different concentrations of CSP extract were pipetted into them. Test drug ampicillin was used as positive control (1 mg/ml) and 80% ethanol was used as negative control. All the procedure was performed in laminar air flow hood to avoid any kind of microbial contamination and all the plates were then incubated at 37 °C for 24 h. Zone of inhibition were measured in mm by using measuring scale.

The MIC (minimum inhibitory concentration) was determined by using serial dilution method with slight modifications (Usman et al. 2014; Chhetry et al. 2022). 100 µl of MHB was transferred into 96 well plate followed by the addition of 100 µl extract of CSP of different concentrations (30 mg/ml up to 0.93 mg/ml) in plate. Twofold serial dilution method was performed for dilutions.100 µl of test bacterial strain was then inoculated into plate and incubated for 24 h at 37 °C. Same procedure was followed for all the tested bacterial strains. All the experiments were carried out in triplicates.

Scanning electron microscopy

Morphological changes in the bacterial cell walls can be observed by using Scanning electron microscopy (Benli et al. 2008). The morphology of E. coli, S. mutans and P. aeruginosa was observed before and after the addition of CSP extract. For this, the overnight cultures of bacterial strains were pelleting down (at 9300 rpm) for 15 min. The pellets obtained were then washed by using PBS (phosphate buffer saline) of pH 7.4 and resuspended. The MIC concentration of CSP extract was added to the respective bacterial pallets and incubation was carried out at 37 °C for 24 h. Accordingly, the control samples were also prepared in the same way in the exception of CSP extract addition. The pellets were then washed with PBS (pH 7.4) and fixed by using glutaraldehyde (2.5%). The dehydration of fixed bacterial pellets was carried out by the graded alcohol series (90–10%). After the dryness, pellets were protected horizontally by suing double-sided adhesive tape. Palladium thin conductive coat was applied to samples by using sputter coater (Quorum) for 1 min to maintain low vacuum (10–2 mbar/P3).The observations of samples were carried out under SEM (Oxford Instrument, XManN, Tescan) to obtain the comprehensive ultra-structure of the exterior surface.

Cytotoxic study of Cucumis sativus peels extract

The in vitro cytocompatibility of CSP extract was studied on L929 fibroblast cell lines of mouse connective tissue by using MTT assay (Sugeena and Tharalakshmi 2020). The L929 cells were purchased from NCTC National Centre for Cell Science, Pune, India. The L929 fibroblasts were incubated in 90% DMEM (Dulbecco’s Modified Eagle Medium, Himedia) containing 10% FBS (Fetal Bovine Serum, Himedia) and 1% antibiotic solution (penicillin–streptomycin) and incubated at 37 °C for 24 h with 5% CO2 atmosphere. The cells were maintained by changing the growth media after every second day. When the cells reached the confluence they were detached with the help of 0.05% trypsin solution (Himedia) and transferred into new culture flask. After adding fresh medium, the cells at the concentration of 1 × 104 cells/ well were transferred into 96 well plates and incubation was carried out for 24 h at 37 °C with 5% CO2. To this, media extract of different concentration was added after removing the old medium and incubated at 37 °C for 24 h with 5% CO2. The test media containing CSP extract was removed from each well by adding 50 µl of MTT reagent (5 mg/ml). The plates were incubated at 37 °C for 24 h with 5% CO2 for 4 h. MTT solution was then discarded after the incubation period and 100 µl DMSO (dimethyl sulfoxide) was added in each well to dissolve the formazan crystals. The viability of cells was checked after 24 and 48 h in separate 96 well plates. After 24 and 48 h of incubation, absorbance was recorded at 570 nm by using microplate reader (Thermo Scientific, Multiskan FC). The experiments were repeated three times and the cell viability was assessed.

Statistical study

The present work experiments were repeated three times and the result values were expressed in mean ± SD, (n = 3).

Results and discussion

Phytochemical study and total flavanoid content

The results of phytochemical study showed the CSP contains flavanoid, terpenoids, phenols, tannins, alkaloids, carbohydrates, proteins and glycosides. While the steroids and saponins were found to be absent in CSP extract. According to the results, the total flavanoid content of CSP ethanolic extract was found to be 55.3 ± 1.154 µg QE/g dw of sample. Local knowledge, accessible availability, and literature on the curative effects of CSP helped in the selection under research. In the present study, variations in phytochemicals of CSP observed between the present and prior investigations as John et al. (2018). Also, the flavanoids are beneficial because of their capacity to inhibit enzymes, as well as for their anti-inflammatory and antibacterial activities. The differences of present work with previous studies might be due to geographic location, weather and genetic makeup variations of plants and the processes used for extraction of phytochemicals.

Isolation, identification and characterization of flavanoids

Column and thin layer chromatography

Silica gel chromatography was used for the separation of flavanoids from CSP extract (Fig. 1a). The elations were collected and subjected to TLC plate. The TLC provides excellent qualitative data for estimating CSP extract flavanoids. A number of sports of CSP extract after column separation were found to be present on TLC plates, but the fraction number 32–36 (named as F32–F36) with Rf of 0.63 coincided with that of standard quercetin (Rf 0.64). The color of spots was found to be dark brown matched with standard flavanoid (Fig. 1b).

Fig. 1.

Fig. 1

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Separation and identification of quercetin. a Column chromatography of Cucumis sativus peels extract. b TLC plate of column fractions F32-36 under UV λ = 365 nm

HPTLC analysis

Quercetin was quantified by HPTLC from CSP extracted fractions (F32–36). The presence of quercetin was confirmed by HPTLC scanned at 254 nm (Fig. 2a) and at 366 nm (Fig. 2b). The peaks at Rf 0.63 in test sample were found to be superimposable (Fig. 3b–f) with the respective standard of quercetin (Fig. 3a). The 3D graph of HPTLC of column fractions of CSP is representing peak purity of the samples (Fig. 3g). The quantification of selected fractions was also determined by HPTLC chromatogram (Table 1). The content of quercetin in fractions F32, F33, F34, F35, F35, and F36 was found to be 2.58, 3.08, 2.40, 1.28 and 2.10 µg, respectively. The calibration curve indicates that the peak-area response was a polynomial function of the amount of standard quercetin in the range of 0.5, 1, 2, 3, 4 and 5 µg. The calibration coefficient was found to be 0.910 and the limit of detection (LOD) and limit of quantification were determined to be 3.846 and 11.654 ng/spot, respectively. HPTLC chromatographic fingerprinting is a precise, linear, accurate method for identification of compounds in herbal extract identification and can be used in characterization and authentication of medicinally important plants (Gomathi et al. 2012). The confirmed fractions of quercetin were then pooled together for the characterization by FTIR and NMR analysis.

Fig. 2.

Fig. 2

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HPTLC plates of standard and fractions, track 1–6 represent standard quercetin and track 7–11 represent F32-36. a At 254 nm. b At 366 nm

Fig. 3.

Fig. 3

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Densitometric HPTLC chromatogram of Cucumis sativus peels column fractions and standard quercetin at 254 nm. a Standard quercetin. b–f Chromatograms of column fractions F32-36. g 3D HPTLC chromatogram of standard and sample fractions (peak purity of the samples)

Table 1.

HPTLC results of Cucumis sativus peels column fractions F32–36

Tracks Fraction number Area X (cal) Rf Substance
7 F32 15,354 3.109 µg 0.63 Quercetin
8 F33 18,193 4.225 µg 0.63 Quercetin
9 F34 15,053 2.991 µg 0.63 Quercetin
10 F35 10,297 1.123 µg 0.63 Quercetin
11 F36 14,137 2.631 µg 0.63 Quercetin
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FTIR analysis

FTIR peaks showed at 3242.22 cm−1 (OH stretching) of phenols, 1661.86 cm−1 (C=O stretching) of ketone. Whereas, 1605.54 cm−1 (C–C), 1559.22 cm−1 (C=O) and 1451.85 cm−1 (C=C) assigned to aromatic stretching vibration. Vibrations of phenols were observed at 1378.35 cm−1. The C–H bond was observed at 1314.9 cm−1. Vibrations of aryl ether and phenols were observed at 1257.29 cm−1 (C–O bond), and 1199.61 cm−1 (C–O bond), respectively. Banding vibrations and C–CO–C starching were observed at 1163.62 cm−1. The peaks at lower frequency between 1092.12 cm−1 and 638.42 cm−1 were assigned to C–H banding vibrations of aromatic hydrocarbons. The frequencies of the peaks in the FTIR spectrum are found to be in the same range as the frequencies of functional groups observed in standard quercetin and also with good references of Sambandam et al. (2016). FTIR spectrum of an isolated compound depicts in Fig. 4, lists the associated typical peak locations.

Fig. 4.

Fig. 4

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FT-IR spectra of isolated compound

NMR analysis

Nuclear magnetic resonance spectroscopy was used to confirm the position of protons by 1H-NMR in isolated compound. The NMR experiments were carried out by using 400 MHz (Bruker Avance III nanobay spectrometer). 5 mg of purified compound dissolved in DMSOd6 and TMS (tetramethyl silane) used as an internal standard. The chemical shifts of spectra were expressed in ppm. The spectrum of isolated compound shows signals corresponding to a tri-substituted β-ring of the flavanoid nucleus at δ ppm: 12.51 (s, 1H), 11.22 (s, 1H), 8.63 (s, 1H), 7.38 (s, 1H), 7.16 (s, 1H), 6.19 (s, 1H), 6.41–6.42 (d, 1H), 6.88–6.90 (d, 1H), 7.53–7.55 (d, 1H), 7.56–7.67 (d, 1H), represented in Fig. 5. The 1H NMR spectra of isolated compound revealed aromatic hydrogen groups, which are close to those reported for quercetin moiety (Sambandam et al. 2016; Thangadurai and Velavan 2018).

Fig. 5.

Fig. 5

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NMR Spectra of isolated compounds

Antioxidant activity using DPPH and FRAP assay

The DPPH activity was used for radical scavenging activity of CSP extract in which the electron paired off in the presence of hydrogen donor and the DPPH color becomes purple to yellow (Fig. 6b). In the results, the final concentration of CSP extract has shown significant activity (54.15%) when compared with standard (81.31%) ascorbic acid (Fig. 6a). A comparison of DPPH antioxidant activity of Cucumis sativus peels with some common waste peels are also represented in Table 3.

Fig. 6.

Fig. 6

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Antioxidant activity of Cucumis sativus peels extract. a DPPH assay. b Reaction mechanism of DPPH (2, 2-diphenyl-1-picrylhydrazyl). c FRAP assay. d TPTZ reduction reaction

Table 3.

Comparison of antioxidant and antimicrobial activities of Cucumis sativus peels with some common waste peels

Name of common waste peels Antioxidant activity Antimicrobial activity References
Assay used Percentage activity Micro-organism used Zone of inhibition (in mm)
Cucumis sativus DPPH 54.15 Escherichia coli 11.36 Present study
Beta vulgaris DPPH 50.46 Escherichia coli 15 John et al. (2018)
Allium cepa DPPH 53.43 Streptococcus aureus 8 Fredotovic et al. (2021); Santhosh et al. (2021)
Allium sativum DPPH 39 Streptococcus aureus 10 Lasalvia et al. (2022); Santos et al. (2022)
Raphanus sativus DPPH 36 Pseudomonas aeruginosa 11 Janjua et al. (2013); Yucetepe et al. (2019)
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The FRAP assay was performed for CSP extract antioxidant activity based on the reducing power of extract in which TPTZ (a potential antioxidant) reduced from Fe3+ into Fe2+ ion with the production of blue color (Fig. 6d). The results of FRAP assay of CSP extract at their final concentration has shown 71% of reducing power. The reducing power was expressed in relation to the ascorbic acid as reference standard.

Antimicrobial study of Cucumis sativus peels extract

The antimicrobial efficacy of CSP extract was assessed by agar well diffusion method against P. aeruginosa, S. mutans and E. coli on MHA plates. All bacterial strains were found to inhibit by CSP extract and no inhibitory zone was observed in negative control wells for all bacterial strains. At 30 mg/ml concentration of CSP extracts, the maximum inhibition was reported in E. coli (11.36 ± 0.47 mm) followed by S. mutans (10.16 ± 0.11 mm) and P. aeruginosa (6.7 ± 0.36 mm), as represented in Table 2. The antimicrobial activity of CSP extract in comparison with some common waste peels are also represented in Table 3. The minimum inhibitory concentration for E. coli, S. mutans and P. aeruginosa were found at 1.87, 3.75 and 7.5 mg/ml, respectively. Similarly, higher antibacterial effectiveness of Cucumis sativus peels methanol and chloroform extracts has been found against E.coli, Staphylococcus aureus, Shigella flexneri and Klebsiella pneumonia (John et al. 2018). Additionally, the antibacterial effectiveness of Cucumis sativus and their parts were found against some microbial strains (Cho et al. 2004; Sotiroudis et al. 2010) but limited literatures are observed on the antimicrobial activity of Cucumis sativus peels. The present research work demonstrated the highest inhibitory effectiveness of CSP extract was found against E. coli and S. mutans as compare with P. aeruginosa.

Table 2.

Inhibition zone (in mm) of Cucumis sativus peels extract against E. coli, S. mutans and P. aeruginosa

Bacteria Cucumis sativus peels extract
10 mg/ml 20 mg/ml 30 mg/ml Standard (Ampicillin) Control (80% ethanol)
Escherichia coli 5.83 ± 0.05 7.76 ± 0.15 11.36 ± 0.47 16.26 ± 0.20 0.46 ± 0.05
Pseudomonas aeruginosa 3.83 ± 0.11 4.63 ± 0.05 6.7 ± 0.36 14.1 ± 0.06 0.13 ± 0.05
Streptococcus mutans 5.3 ± 0.26 6.43 ± 0.30 10.16 ± 0.11 15.13 ± 0.11 0.43 ± 0.05
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Each value represented in table are mean ± SD (N = 3)

Scanning electron microscopy

CSP extract was used as a natural antimicrobial agent. According to the results, disruption or irregular morphology of cells were observed for CSP extract treated cells. CSP extract causes the cell membrane damage and facilitates the release of cell constituents, resulting in inactivation of microbes. The structure of treated and control (untreated) microbial cells viewed under SEM revelled the features, represented in Fig. 7. The control cells were smooth and originality of native bacteria was observed, while treated cells were appeared as damaged in cell structure. The treated cells were found fragmented, different in shape as compared to control cells, many pits and gaps were observed. The cell barrier is a barrier between the cytoplasm and the extracellular medium. The extracts of plants and their parts studied for their bactericidal activities and they were able to disrupt the cell membrane, by change in its pH, and increase cell membrane permeability (Eduardo et al. 2010).

Fig. 7.

Fig. 7

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Scanning electron microscopy of bacterial cells as control (without treatment) and after treatment with Cucumis sativus peels extract. a E. coli cells without treatment. b E. coli cells after treatment. c S. mutans cells without treatment. d S. mutans cells after treatment. e P. aeruginosa cells without treatment. f P. aeruginosa cells after treatment

Cytotoxicity activity of Cucumis sativus peels extract

According to the results, cell viability of L929 cells after 24 and 48 h of incubation after treatment with CSP extract are shown in Fig. 8. The different concentrations of CSP extract were tested for cytotoxic evaluations. The control cells absorption values were adjust at 100% viability. The cytotoxicity of samples was rated on the % viability basis relative to control group. The non-toxicity of samples are considered if the viability is more than 90%, slight toxic between 60 and 90% viability, moderate toxicity between 30 and 59% and severely toxic to the cells if < 30% viability (Sjogren et al. 2000). In the present study, all tested concentrations (2–20 mg/ml) of CSP extract showed > 90% viability of L929 cells after 24 and 48 h treatment. The results of present study demonstrate that the CSP ethanolic extract is safe and non-cytotoxic at the tested concentrations.

Fig. 8.

Fig. 8

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Percentage viability of L929 mouse connective tissue cells after 24 and 48 h incubation with 80% ethanolic extract of Cucumis sativus peels using MTT assay

Conclusion

The ethanolic extract prepared by Cucumis sativus peels was having significant antibacterial activity against E. coli, P. aeruginosa and S. mutans. The presence of various phytochemicals in CSP extract considered as responsible for the antimicrobial activities. The extract shows effective inhibitory effect in the growth of gram negative (P. aeruginosa and E. coli) and gram positive bacteria (S. mutans) by rupturing or destroying their cell wall which was confirmed by scanning electron microscopy (SEM). The present finding shows that the antimicrobial property can be helpful in preventing and controlling the bacterial infections. The CSP extract has also found to give significant antioxidant activity for both DPPH and FRAP assay. From the present study, our finding also provide evidence that the ethanolic extract of Cucumis sativus peels are a potential source of flavanoid-quercetin, which has been identified and characterized by chromatographic and spectral studies. Since, the CSP extract may be use for the humans, so it was necessary to check the toxicity. The cytotoxicity of herbal formulation was determined on L929 cell lines by using microculture tetrazolium (MTT) assay. The results of present study demonstrate that the CSP extract is safe and non-cytotoxic with the cell viability rate of > 90%. However, further investigations need to be performed to determine the in vivo biological activities of the extract. This information would be helpful for applying CSP extract as a traditional-based antimicrobial agent in therapeutic drug for the implication of human health.

Acknowledgements

The authors are thankful to Prof. Ina Aditya Shastri, Banasthali Vidyapith and Prof. Dipjyoti Chakraborty, Department of Bioscience and Biotechnology, Banasthali Vidyapith for their encouragement and providing scientific platform to carry out present work. We are also thankful to DBT supported Bioinformatics Center, for providing computational assistance and Dr. Swati Paliwal, Department of Bioscience and Biotechnology, Banasthali Vidyapith, for providing guidance during cell culture work.

Author contributions

JM and NS conceived the present idea of writing the research and designed the content. JM and NS encouraged to investigate and supervised the findings of this work. AS developed the theory and performed the experiments. AS contributed to the analysis of the results and writing of the manuscript.

Funding

Authors do not have any funding support.

Data availability

The datasets generated during the current study is included in this article.

Declarations

Conflict of interest

The authors confirm that they have no conflicts of interest with respect to the work described in this manuscript.

Ethical approval

Ethical approval is not applicable. This article does not contain any studies with human participants or animals performed by any of the authors.

Contributor Information

Anjani, Email: sihaganjani264@gmail.com.

Nidhi Srivastava, Email: nidhi1.srivastava@niperraebareli.edu.in.

Jyoti Mathur, Email: contact.srivastava@gmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated during the current study is included in this article.

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