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Asian Pacific Journal of Cancer Prevention : APJCP logoLink to Asian Pacific Journal of Cancer Prevention : APJCP
. 2022 Aug;23(8):2671–2686. doi: 10.31557/APJCP.2022.23.8.2671

In Vitro Study on Effect of Zinc Oxide Nanoparticles on the Biological Activities of Croton tiglium L. Seeds Extracts

Wael Mahmoud Aboulthana 1,*, Nagwa Ibrahim Omar 1, Amal Mohamed El-Feky 2, Enas Ahmed Hasan 3, Noha El-Sayed Ibrahim 4, Ahmed Mahmoud Youssef 5
PMCID: PMC9741903  PMID: 36037121

Abstract

Objective:

Croton tiglium L. seeds were studied against colon cancer induced chemically in rats after incorporating silver nanoparticles (Ag-NPs) but the body has no the ability to discrete silver or silver ions. Therefore, the present study was designed to reveal the biological activities of different C. tiglium L. seeds extracts incorporated with zinc oxide nanoparticles (ZnO-NPs).

Results:

It was found that C. tiglium L. seeds provided with high contents of total protein (27.43 g/100g), carbohydrate (18.29 g/100 g) and lipid (46.31 g/100 g). The chromatographic techniques revealed that concentrations of the predominant compounds increased in all studied extracts (ethanolic, aqueous and petroleum ether) after incorporating ZnO-NPs. The in vitro biological activities showed that the aqueous extract possessed the highest antioxidant and scavenging activities. It exhibited the highest inhibitory effect on α-amylase (41.89%) and acetyl cholinesterase (AChE) (23.00%) in addition to its higher anti-arthritic activity. All the biological activities increased after incorporating ZnO-NPs. It showed the highest cytotoxic activity that increased after incorporating ZnO-NPs against human colon carcinoma (CACO-2) cells. Therefore, this extract was selected for undergoing further studies on CACO-2 cells. The aqueous extract incorporated with ZnO-NPs arrested growth of CACO-2 cells at G2/M and increased percentage of total apoptotic cells and necrosis. The median lethal dose (LD50) showed that the extracts incorporated with ZnO-NPs were safer than the native extracts.

Conclusion:

The study showed that the aqueous extract was the most active extract that consequently exhibited promising biological activities after incorporating ZnO-NPs.

Key Words: Croton tiglium Seeds, Zinc oxide nanoparticles, Green Nanotechnology, Biological Activity, Toxicity

Introduction

Croton tiglium L. is categorized as the second largest genus under the Family “Euphorbiaceae”. Approximately 1300 species of largely trees, shrubs and herbs that grow under varied environmental conditions in tropical and sub-tropical regions were belonged to this large genus (Torres et al., 2008). Due to the presence of essential oils, the leaves, seeds, stem, roots, and the juice from the bark were widely utilized for medicinal and nutritional purposes among different global communities (Harkat-Madouri et al., 2015; Sai Prassana and Karpaga, 2015). Hu et al., (2010) emphasized that C. tiglium L. seeds are rich in phorbol esters, crotonic acid and fatty acids in addition to active phytoconstituents that are responsible for severe purgative effect of the C. tiglium L. seeds extract. Due to the strong toxic effect of the chemical synthetic products, components of natural essential oil are gaining frequent presence and increasing interest in the recent studies investigating their potential activity and functional utility (Babahmad et al., 2018; Rakmai et al., 2018). These essential oils possessed purgative, analgesic, antibacterial, anti-oxidation, anti-inflammatory and anti-tumor activities. Therefore, they are abundantly utilized in cosmetic industries and indigenous medicines (El Gendy et al., 2015). It was reported that linoleic acid, oleic acid and eicosenoic acid are the most abundant fatty acids exist in a methyl-esterified sample obtained by reflux method (Mei et al., 2012). It increases gastrointestinal motility, has a smoothening effect on skin, reduces inflammation, expectorant and rubefacient. Traditionally, C. tiglium L. is used for kidney stones, bronchial irritation, convulsions and skin disorders. It has been utilized widely among the Asian communities for treating gastrointestinal problems (Ojo et al., 2017).

Development of nanotechnology led to astonishing distribution in progression of medicine and health care system. The mechanism by which the metal nanoparticles (M-NPs) were synthesized using plant extracts by mean of green nanotechnology may be associated with concept of the phytoremediation (Selvarajan and Mohanasrinivasan, 2013). It is well known that silver nanoparticles (Ag-NPs) are utilized widely as an effective antimicrobial agent coating on catheter and wound dressing (Konop et al., 2016). There is no clear regulation enough for managing risk of the Ag-NPs in implant medical devices. On the other side, the human body has no the ability to discrete silver or silver ions, leading to an accumulation of silver as well as destruction of DNA and / or red blood cells (Chen et al., 2015 ; Ferdous and Nemmar, 2020).

As reported by Aboulthana et al., (2019), aqueous C. tiglium L. seeds (the most effective) extract used for green synthesis of Ag-NPs. Although the silver C. tiglium L. nano-extract showed ameliorative effect against colon cancer induced chemically in rats as demonstrated by Aboulthana et al., (2020), Aboulthana et al., (2021) postulated that incorporating Ag-NPs into aqueous C. tiglium L. seeds extract caused deleterious effect on brain tissue when administrated orally due to its high affinity to accumulate in the brain affecting the enzymatic and non-enzymatic antioxidant system. Zinc oxide (ZnO) or zinc oxide nanoparticles (ZnO-NPs) differ from the other metal oxides. They are characterized by their biodegradability, low toxicity and economy. Therefore, they are approved by Food and Drug Administration (FDA) in the past two decades to be utilized in biomedicine applications, especially in discipline of antibacterial or anticancer fields in addition to its utilization in the industrial products such as coating, paint and cosmetics (Parihar et al., 2018). Many recent studies demonstrated that ZnO-NPs are characterized by good biocompatibility and antibacterial properties (Ozkan et al., 2018 ; Jiang et al., 2018 ; Hameed, 2018). The ZnO-NPs that were prepared by the chemical and physical methods are not suitable to be utilized in the medical field due to limitation of chemical reagents as well as their residue after reaction. Development of green chemistry has attracted more attention because it is believed to be biocompatible, nontoxic and eco-friendly. Many recent studies proved that green technology-based synthesis of nanoparticles is considered as promising methods for large-scale production of M-NPs to be utilized for biomedical applications. Results of the recent studies proved that the ZnO-NPs that were biosynthesized using a wide variety of plant extracts are safe for human use alternative to Ag-NPs or other M-NPs (Song and Yang, 2016 ; Ali et al., 2016 ; Ji et al., 2017). The current experiment was designed to reveal the biological activities of different C. tiglium L. seeds extracts after incorporating ZnO-NPs.

Materials and Methods

Determination of nutritional value of C. tiglium L. seeds

Total protein content was estimated in C. tiglium L. seeds by micro-kjeldahl method as stated by El-Feky et al., (2020). Total carbohydrate content was quantified as glucose by phenol sulfuric acid method suggested by El-Feky et al., (2018). Total lipid content was gravimetrically determined based on the method suggested by AboulNaser et al., (2020). Consequently, the obtained values (proteins, carbohydrates and lipids) were used for calculating the caloric value of C. tiglium L. seeds using the equation suggested by Coelho et al., (2014).

Preparation of different plant extracts

C. tiglium L. seeds were obtained from Agricultural Research Center, Giza, Egypt. The crushed dried seeds were successively extracted in soxhlet apparatus with petroleum ether (60 - 80°C), ethyl alcohol, and then with distilled water for 20 hrs for each solvent, the obtained three extracts were separately filtered then concentrated in a rotary evaporator at 45°C under reduced pressure to dryness.

Isolation and identification of the major compounds from different C. tiglium seeds L. extracts

The petroleum ether extract was chromatographed on silica gel column. Elution was successively carried out by methylene dichloride (CH2Cl2) and increasing the polarity with ethyl acetate. The fractions were successively collected and individually concentrated to 5 ml then screened by thin layer chromatography (TLC) using toulene : ethyl acetate (7:3 v/v) as solvent system. The separated fractions were visualized by spraying with 10% H2SO4 and heating at 110οC for 5 min. The resulting similar fractions were collected from the column together according to Rf values. Structures of the isolated compounds were interpreted based on the spectral analyses (FT-IR, 1H-NMR, 13C-NMR and Mass spectroscopy).

The ethanolic extract was applied on preparative TLC using chloroform: methanol (80:20 and 70:30 v/v) as developing system. The plates were examined under the UV light at 254 and 366 nm, respectively. The selected bands were scratched and collected. The Rf values of the isolated compounds were recorded and the structural elucidation of the isolated compounds were interpreted based on their spectral analyses (UV, MS, IR, 1H-NMR and 13C-NMR spectrometry).

Preparation of zinc oxide C. tiglium L. seeds nano-extract

ZnO-NPs were synthesized using plant extracts as reducing agents in order to prepare ZnO-NPs with optimum yield and particle size. For preparation of ZnO-C. tiglium seeds nano-extract, the ZnO-NPs were synthesized by sol-gel method suggested by Bao et al., (2012) with some modifications. The plant extract was added into zinc acetate solution till obtaining the white precipitate that dried then converted into powder to be ready for characterization.

Characterization of the biosynthesized zinc oxide nanoparticles

The ZnO-NPs spectra were assayed by Shimadzu UV-VIS recording spectrophotometer UV-240 at λ 200 - 800 nm after diluting the samples (10-fold) with deionized water. The crystalline nature and grain size of the synthesized ZnO-NPs were analyzed by a Philips X-Ray Diffractometer (XRD) (PW 1930 generator, PW 1820 goniometer) equipped with Cu Kα radiation as an X-ray source (45 kV, 40 mA, with λ = 0.15418 nm). Shape and size of the synthesized ZnO-NPs were determined at high resolution level (200 KV) using Transmission Electron Microscope (TEM) (model JEM-1230, Japan) operated at accelerating voltage of 120 kV, with maximum magnification of 600X103 and a resolution until 0.2 nm. The average hydrodynamic size of the synthesized ZnO-NPs was determined by Dynamic Light Scattering (DLS) (Malvern Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, United Kingdom) according to method suggested by Murdock et al., (2008).

Determination of the phyto-constituents in C. tiglium L. seeds extracts before and after incorporating zinc oxide nanoparticles

1. Quantitative determination of major phyto-constituents

Concentration of total polyphenols was determined using Folin Ciocalteu reagent according to method suggested by Singleton and Rossi (1965). Total tannins contents were determined using tannic acid as a reference compound based on the method described by Broadhurst and Jones (1978).

2. Investigation of the lipoidal constituents

The P. ether extracts of C. tiglium L. seeds before and after incorporating ZnO-NPs were subjected to gas chromatograph coupled with a mass spectrometer (GC/MS analysis) (model Shimadzu GC/MS–QP5050A) on an Agilent 6890, 70 eV. MS spectrometer; Finnigan Model 3200, Mass spectrometer at 70 eV. The constituents have been identified by comparison of their spectral fragmentation patterns with those of the available database libraries Wiley (Wiley Int.) USA and NIST (Nat. Inst. St. Technol., USA) and/or published data. Quantitative determination was carried out based on integration of the area under peak according to the method suggested by Elsawi et al., (2020).

3. Investigation of the phenolics and flavonoidal compounds

The different phenolic and flavonoidal compounds were identified in the aqueous and ethanolic extracts of C. tiglium L. seeds before and after incorporating ZnO-NPs using high pressure liquid chromatography (HPLC) (Shimadzu-UFLC Prominence) equipped with an auto sampler (Model-SIL 20AC HT) and UV-Vis detector (Model-SPD 20A) (Japan). The separation process was carried out through analytical column of an Eclipse XDBC18 (150 X 4.6 µm; 5 µm) with a C18 guard column (Phenomenex, Torrance, CA). The mobile phase is consisting of solvent system of acetonitrile (solvent A) and 2% acetic acid in water (v/v) (solvent B). Before the chromatographic run, all samples were filtered through a 0.45 µm Acrodisc syringe filter (Gelman Laboratory, MI). Each sample (50 µl) was injected automatically by the injector piece. The flow rate was kept at 0.8 m min−1 for a total run time of 70 min and the gradient program was as follows: 100% B to 85% B in 30 min, 85% B to 50% B in 20 min, 50% B to 0% B in 5 min and 0% B to 100% B in 5 min. The peaks were monitored simultaneously at 280 and 320 nm for the benzoic acid and cinnamic acid derivatives, respectively. When the chromatographic run was finished, the peaks were identified by congruent retention times and UV spectra and compared with those of the standards (El-Sayed et al., 2018).

In vitro study on different C. tiglium L. seeds extracts before and after incorporating zinc oxide nanoparticles

1. Antioxidant activity

Total antioxidant capacity (TAC) and total iron reducing power were assessed according the methods suggested by Prieto et al., (1999) and Oyaizu (1986), respectively. The scavenging activities were assessed against 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals according to the methods suggested by Rahman et al., (2015) and Arnao et al., (2001), respectively.

2. α-amylase inhibitory assay

It was carried out using acarbose as standard drug for calculating percentage of α-amylase inhibition (%) using 3.5-dinitrosalicylic acid (DNSA) method (Wickramaratne et al., 2016).

3. Acetyl cholinesterase (AChE) enzyme activity

It was measured for calculating percentage of AChE inhibition (%) using Ellman’s method (Ellman et al., 1961).

4. Antiarthritic activity

4.1. Protein denaturation

Percentage of protein denaturation inhibition can be calculated according to the method described by Lavanya et al., (2010) and Das and Sureshkumar (2016). All results were compared with standard (diclofenac sodium) and the control represents 100% protein denaturation.

4.2. Proteinase inhibitory activity

This assay was carried out by calculating percentage of the proteinase inhibitory activity according to the method suggested by Oyedapo and Famurewa (1995).

5. Cytotoxic activity

Cytotoxic activities were determined against human hepatocellular (HEPG-2) and colon carcinoma (CACO-2) cells using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as suggested by Vichai and Kirtikara (2006). Percent of cell-growth inhibition (%) and median inhibitory concentration (IC50) were calculated using IC50 calculation software.

6. DNA content analysis

The CACO-2 cells (3×105/well) were seeded into 6-well plates, cultured overnight and treated with different C. tiglium L. extracts and their ZnO nano-extracts individually for 24 hrs. The cells were fixed in 75% ethanol at -4oC overnight then incubated with 50 ng/mL PI staining solution and 0.1 mg/mL RNase A in dark place at room temperature for 15 min. The DNA content was quantified in the cells by flow cytometry (BD FASCCalibur-USA).

7. Cell apoptosis

The cell apoptosis was assessed by Annexin V-FITC apoptosis detection kit (Annexin V-FITC-BD Bioscience PharmingenTM, USA). The CACO-2 cells (3 ×106/well) were seeded into 6-well plates then cultured overnight before the exposure to different concentrations of C. tiglium L. extracts and their ZnO nano-extracts (50, 100, 150, 200 µg/ml). Furthermore, both of each extract and its ZnO nano-extract (100µg /ml) added to CACO-2 cells for different times (24, 36, 48 and 48 hrs). Cells were collected by cold centrifugation at 300 x g for 10 min and consequently washed twice and re-suspended in 500 µl of cold (+4oC) 1X PBS buffer then precipitated again by centrifugation. This step was followed by adding 100 μl 1X Binding Buffer per sample. Annexin V (100 μl) was added to treated cell samples (106 cell/ml) then incubated. Annexin V-FITC (5μl) and propidium iodide (PI) (5μl) were incubated in the dark for 15 min. at room temperature, then 400 μl of 1X binding buffer was added. The cells were immediately analyzed by flow cytometry (BD FASCCalibur-USA) within 1hr for a maximal signal (Koopman et al., 1994).

8. Extraction of RNA and quantitative RT-PCR

The CACO-2 cells were cultured in six-well plates and exposed individually to C. tiglium L. extracts and their ZnO nano-extracts (100 μg/ml). Total RNAs were extracted from treated cells using the RNeasy Mini Kit (Qiagen RNA extraction/BioRad syber green PCR MMX) based on the method suggested by Pfaffl (2001). For quantifying expressions of EGFR, Bcl-2 and Casp3 genes, the total RNAs (10 ng) from each sample was processed by reverse transcription for cDNA synthesis using the High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, USA). Consequently, the cDNA was amplified with the Syber Green I PCR Master Kit (Fermentas) in a 48-well plate using the Step One instrument (Applied Biosystems, USA), as a following: 10 min at 95ºC for enzyme activation followed by 40 cycles of 15 sec at a temperature of 95ºC, 20 sec at 55ºC and 30 sec at 72ºC for the amplification step. The changes in expression of each target gene were normalized relative to the mean critical threshold (CT) values of GAPDH as a housekeeping gene by the ΔCt method. One μl of both primers specific for each target gene (EGFR (F: 5’-GACTCCGTCCAGTATTGATCG-3’; B: 5’-GCCCTTCGCACTTCTTACACTT-3’), Bcl2 (F: 5’-TCCCTCGCTGCACAAATACTC-3’; B: 5’-ACGACCCGATGGCCAAGA-3’), Casp3 (F: 5’-TGTTTGTGTGCTTCTGAGCC-3’; B: 5’-CACGCCATGTCATCATCAAC-3’) and GAPDH (F: 5’-GAAGGTGAAGGTCGGAGTCA-3’; B: 5’-TTGAGGTCAATGAAGGGGTC-3’)) was added. The mRNA levels were quantified using the 2 ΔΔCq method that was suggested by Wang et al., (2014). GAPDH was used as the internal control. Experiments for each gene were conducted in triplicate.

Median lethal dose of different extracts (LD 50 )

The different native C. tiglium L. extracts and their ZnO nano-extracts (after incorporating ZnO-NPs) were studied separately for evaluating the LD50. Three hundred and twenty four adult albino mice (weight 20-25 g) were divided into 9 groups (6 mice in each group) for calculating the LD50 of the native ethanolic C. tiglium L. extract, 9 groups for ZnO ethanolic nano-extract, 9 groups for native aqueous extract, 9 groups for ZnO aqueous nano-extract, 9 groups for native P. ether extract and 9 groups for ZnO P. ether nano-extract. For each extract, the groups were treated orally by stomach tube with rising doses of 1,000, 2,000, 4,000, 6,000, 8,000, 10,000, 12,000, 14,000 and 16,000 mg/Kg. Number of dead mice was recorded after 24 hrs of extract administration. The LD50 was calculated according to the methods suggested by Paget and Barnes (1964).

Results

The nutritional composition of C. tiglium L. seeds

It was found that C. tiglium L. seeds provided great values of total protein (27.43 g/100g), carbohydrate (18.29 g/100 g) and lipid contents (46.31 g/100 g). Consequently, the caloric value of C. tiglium L. seeds was calculated as 599.67 kcal/100g.

The major compounds isolation from different C. tiglium L. seeds extracts

The steroidal and terpenoidal compounds were isolated from petroleum ether extract and identified by column chromatography technique. The collected fractions were tested using Liebermann–Burchard reagent, then they were spotted on TLC plate and sprayed with 10% H2SO4 reagent. The positively tested fractions were applied on TLC silica gel plates against the available authentics. By comparing Rf values and the chromatographic appearance under UV, two steroidal compounds (β-sitosterol and stigmasterol) beside to one pentacyclic triterpenoid (α-amyrin) were identified and isolated in pure form. Stigmasterol was isolated for the first time from C. tiglium L. seeds. It was identified at Rf value 0.20 and obtained as white crystalline powder with melting point 170°C. Furthermore, isopimara-7,15-dien-3β-ol was also isolated by silica gel column with CH2Cl2: EtOAc (90:10 v/v) as amorphous powder and identified at Rf value 0.46 in toluene : ethyl acetate solvent system (7:3 v/v). UV λmax was 226 nm; FT-IR bands at 3320 cm−1 for OH stretching, 2945, 3850 cm−1 for C-H stretching, and 1645cm−1 for –C=C– Stretch. EI-MS 70 eV m/z (rel. int.): 288 [M]+ (10) calculated for molecular formula C20H32O, 273 [M-CH3]+ (15), 270 [M-H2O]+ (8), 255 (30), 245 (13), 213 (16), 200 (22), 185 (25), 171 (29), 145 (51), 132 (69), 129 (100), 119 (81), 105 (70), 91 (25). As well as, stigmast-4-en-3-one was isolated for the first time from C. tiglium L. seeds by silica gel column with CH2Cl2–EtOAc (80:20 v/v) as white needles, melting point 155°C. It was identified at Rf value 0.63 in toluene :ethyl acetate solvent system (7:3 v/v). UV λmax was 255 nm; FT-IR bands at 3315 cm−1 for OH stretching, 2927, 3810 cm−1 for C-H stretching, 1715 cm−1 for C=O stretching, and 1,620cm−1 for –C=C– Stretch. EI-MS 70 eV m/z (rel. int.): 412 (35) calculated for molecular formula C29H48O, base peak at m/z 124 (100). The characteristic fragmentations were m/z 43 (55), 55 (40), 71 (25), 95 (28), 134 (10), 150 (15), 178 (12), 213 (20), 229 (35), 255 (20), 273 (11), 329 (16), 381 (9), 396 (17).

Quercetin-7-O-β-D-glucopyranoside was isolated from ethanolic extract with CHCl3: MeOH (80:20 v/v) as yellow crystals with melting point 220°C. It was identified at Rf value 0.67 and gave positive test for flavonoids. UV λmax in MeOH (256,269sh, 372), MeOH+NaOMe (244sh, 291, 366,456), MeOH+AlCl3 (258sh, 273, 340, 458), MeOH+AlCl3/HCl (268, 303sh, 365, 425) stable UV absorbance was observed in band I and II indicating presence of hydroxyl group at C-3 and C-5, MeOH+NaOAc (286, 377, 428sh), MeOH+NaOAc/H3BO3 (261, 290sh, 385) bathochromic shift of band I indicates the presence of 3`, 4`dihydroxy group. 1H-NMR (400 MHz, CD3OD, δ ppm): 6.45 (1H, d, J=2.8Hz, H-6), 6.79 (1H, d, J=2.8Hz, H-8), 7.90 (1H, d, J=2.7Hz, H-2’), 6.93 (1H, d, J=8.5Hz, H-5’), 7.65 (1H, dd, J=2.7, 8.5Hz, H-6’), 5.00 (H-1``). 13C-NMR (125 MHz, DMSO, δ): 157.53 (C-2), 127.48 (C-3), 180.73 (C-4), 162.14 (C-5), 93.67 (C-6), 161.43 (C-7), 93.76 (C-8), 160.45 (C-9), 99.50 (C-10), 121.47 (C-1`), 103.94 (C-2`), 144.25 (C-3`), 154.33 (C-4`), 101.43 (C-5`), 114.93 (C-6`), 100.00 (C-1``), 72.85 (C-2``), 75.64 (C-3``), 63.80 (C-4``), 75.21 (C-5``), 60.25 (C-6``).

13-O-Acetylphorbol-20-(9Z,12Z-octadecadienoate) was identified at Rf value 0.41 in CHCl3 : MeOH (70:30 v/v) and isolated as amorphous powder. UV λmax was 245 nm; FT-IR bands at 3390 cm−1 for stretching, 1,720 cm−1 for C=O stretching, 1695 cm−1 for ester linkage, 1,630 cm−1 for –C=C– stretch. EI-MS 70 eV m/z (rel. int.): 669 (25) calculated for molecular formula C40H61O8, 632 (31) [M-2H2O]+, 590 (37) [M-H2O-CH3COOH]+, and 388 (28) [M-linoleic acid]+. 13-O-Tigloylphorbol-20-(9Z,12Z-octadecadienoate) was identified at Rf value 0.46 in CHCl3:MeOH (70:30) and isolated as amorphous powder. UV λmax was 241 nm; FT-IR bands at 3,420 cm−1 for stretching, 1,715 cm−1 for C=O stretching, 1,700 cm−1 for ester linkage, 1,625 cm−1 for –C=C– stretch. EI-MS 70 eV m/z (rel. int.): 709 (17) calculated for molecular formula C43H65O8, 590 (29) [M-tiglic acid-H2O]+, 410 (34) [M-linoleic acid]+ and 328 (21) [M-tiglic acid-linoleicacid-H2O]+.

The structural properties of prepared ZnO-NPs

The UV-Visible spectroscopy was used for studying the optical properties of the synthesized ZnO-NPs. Data presented in Figure 1a showed that preparation of ZnO-NPs from the biosynthesis route was confirmed from the sharp peak identified in the UV-visible spectrum at 382 nm. Data of the XRD pattern illustrated in Figure 1b and showed that peaks identified at 2 θ = 31.7, 34.4, 36.2, 47.5, 56.5, 62.7, 67.8 and 68.1 and assigned to (100), (002), (101), (102), (110), (103), (112) and (201) confirmed formation of ZnO-NPs with high quality. The TEM data were in compatible to the UV and XRD data. The TEM analysis was utilized for revealing size and the crystalline characteristics of the biosynthesized ZnO-NPs. As revealed in Figure 1c, the TEM image confirmed synthesis of ZnO-NPs with morphological shape and hexagonal polycrystalline structure with approximately size ranged from 20 to 70 nm. The catalytic activity of the synthesized ZnO-NPs is strongly related to their hexagonal structure that implies more ionicity. The ZnO-NPs are well dispersed and some of them are irregular in their shapes. Furthermore, it was emphasized that the particles are almost spherical with a slight variation in thickness. The particle size that was determined by TEM technique is almost close to that obtained by XRD analysis. DLS is an accurate procedure to use a monodisperse dilute solution for measuring particle size ranged from 5 nm to 5 mm. Data of the DLS illustrated in Figure 1d, showed that the particle size distribution and the hydrodynamic size of the fabricated ZnO-NPs has main diameter around 164 nm.

Figure 1.

Figure 1

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Characterization of the Synthesized Zinc Oxide Nanoparticles (ZnO-NPs) Showing. a) Ultraviolet-visible (UV-VIS) spectrum, b) X-Ray Diffraction (XRD) spectrum, c) Transmission Electron Microscope (TEM) image and d) Dynamic Light Scattering (DLS)

The phyto-constituents in C. tiglium L. seeds extracts before and after incorporating ZnO-NPs

Data of GC/MS analysis of petroleum ether C. tiglium L. extract depicted in Table 1 showed that 38 compounds representing 88.53% were identified; where the identified lipoidal components consist of 17 hydrocarbons (41.21%), 9 fatty alcohols (23.0%), 7 aldehydes (18.21%), 3 ketones (5.27%) and 2 sterols (0.84%). It was found that n-octadecane, hentriacontane and tetracosane were the major identified hydrocarbons in petroleum ether extract representing 5.61, 7.04 and 6.54%, respectively. While 2-nonen-1-ol (12.25%) was considered as the main identified fatty alcohol. In addition, 9-octadecenal and 1-methyl-bicycloheptan-6-one are the most common aldehydic and ketonic compounds with the highest concentration representing 11.78 and 5.08%, respectively. Beside to identification of 2 steroidal compounds ergosterol and β-sitostero representing 0.45 and l0.39%, respectively.

Table 1.

GC/MS Analysis of the Lipoidal Constituents in Petroleum ether C. tiglium L. seeds Extract before and after Incorporating ZnO-NPs

Class Compound Mol. Weight BP Relative area %
Before After
Hydrocarbons 4-Decene 140 55 0.06 0.07
4-Methylnonane 142 41 1.04 1.05
9-methyl-1-decene 154 56 0.97 1.11
2-Methyldecane 156 43 1.65 2.06
5-Methyl-4-undecene 168 55 0.11 0.13
2-Methylundecane 170 43 2.03 3.15
2,5,6-Trimethyldecane 1 184 57 1.54 2.01
2-Methyltridecane 198 43 0.15 0.18
2,4,6,8-Tetramethyl-1-undecene 210 43 0.24 0.26
2-Methyltetradecane 212 57 1.03 1.05
n-Hexadecane 226 43 2.02 2.11
n-Heptadecane 240 43 3.34 3.37
n-Octadecane 254 57 5.61 6.24
6-Methyl octadecane 268 57 2.08 2.22
n-Docosane 310 43 3.76 4.09
Tetracosane 338 57 6.54 6.57
Hentriacontane 436 57 7.04 7.12
Fatty alcohols Hept-4-en-1-ol 114 41 1.98 2.01
2-Octen-1-ol 128 57 0.93 0.96
2-Nonen-1-ol 142 57 12.25 13.02
2-Decen-1-ol 156 57 2.08 2.1
10-Dodecyn-1-ol 182 68 0.98 0.99
Dodecanol 186 41 1.54 1.57
Tetradecanol 214 41 1.96 2.02
6,9-Pentadecadien-1-ol 224 67 1.14 1.14
Octadecanol 270 41 1.04 1.06
Aldehydes 2,2-Dimethyl-4-pentenal 112 55 0.87 1.03
Heptanal 114 57 1.15 1.17
2-Isononenal 140 43 1.01 1.04
Nonanal 142 41 0.19 0.21
2,4-Decadienal 152 81 1.06 1.07
Decanal 156 43 2.15 2.15
9-Octadecenal 266 41 11.78 13.11
Ketones 1-Methyl-bicycloheptan-6-one 124 67 5.08 5.13
2-Nonanone 142 43 0.14 0.14
Campherenone 220 41 0.05 0.06
Sterols Ergosterol 396 69 0.45 0.47
β-Sitosterol 414 41 0.39 0.41
Total hydrocarbons 41.21 42.79
Total fatty alcohols 23 24.87
Total aldehydes 18.21 19.78
Total ketones 5.27 5.33
Total sterols 0.84 0.88
Total identified compounds 88.53 93.65
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BP, Base Peak

It was worthy to mention that the phyto-constituents identified in petroleum ether extract were not changed or missed after incorporating ZnO-NPs, but their values increased. It was noticed that concentration of the total hydrocarbons in that extract elevated from 41.21 to 42.79%, total fatty alcohols increased from 23.0 to 24.87%. Additionally, the total aldehydes and ketones concentration raised from 18.21 to 19.78%, and from 5.27 to 5.33%, respectively. Beside to increasing concentration of sterols content from 0.84 to 0.88%. Increasing concentration of the lipoidal components was responsible for improving the biological activities by synergetic mechanism between all compounds of the petroleum ether extract.

Data of HPLC analysis compiled in Table 2 showed that the phenolic compounds that were identified in the ethanolic and aqueous C. tiglium L. seeds extracts were about 14 and 11, respectively. Moreover, 4 flavonoidal compounds were noticed in both ethanolic and aqueous extracts. It was noticed that catechin and vanillic acid were the most predominant phenolic compounds identified in the ethanolic C. tiglium L. extract and their concentration increased from 315.404 and 151.947 µg/g to 401.273 and 156.284 µg/g, respectively after incorporating ZnO-NPs. As regard to the aqueous C. tiglium L. extract, gentisic acid and chlorogenic acid were considered as the most common phenolic compounds and their concentrations increased from 99.581 and 112.869 µg/g to 99.984 and 117.352 µg/g, respectively after incorporating ZnO-NPs. On the other hand, rutin was identified as the major flavonoidal compound in both ethanolic and aqueous C. tiglium L. seeds extracts. Its concentration increased from 41.625 and 39.233 µg/g to 43.289 and 47.814µg/g, respectively after incorporating ZnO-NPs.

Table 2.

HPLC Analysis of the Phenolics and Flavonoids in Ethanolic and Aqueous C. tiglium L. seeds Extracts before and after Incorporating ZnO-NPs

Compound Concentration (µg/g)
Ethanolic extract Aqueous extract
Before After Before After
Phenolics Gallic acid 32.715 35.278 27.214 29.716
Protocatechuic acid 59.866 61.425 32.086 32.981
p-Hydroxybenzoic acid 52.587 54.842 27.264 28.094
Gentisic acid 77.349 81.028 99.581 99.984
Catechin 315.404 401.273 69.793 71.428
Chlorogenic acid 94.764 101.254 112.869 117.352
Caffeic acid 50.341 51.753 17.104 20.587
Syringic acid 8.979 9.015 15.889 16.102
Vanillic acid 151.947 156.284 8.171 8.987
Ferulic acid 22.473 24.897 36.934 37.891
Sinapic acid 11.429 18.349 0.000 0.000
p-coumaric acid 13.241 19.257 0.000 0.000
Rosmarinic acid 9.427 11.025 0.000 0.000
Cinnamic acid 8.869 10.266 15.29 18.301
Flavonoids Quercetin 27.482 32.487 26.908 33.873
Kaempferol 34.172 36.981 29.941 33.427
Rutin 41.625 43.289 39.233 47.814
Chrysin 27.362 29.476 12.406 19.452
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In vitro study on different C. tiglium L. seeds extracts before and after incorporating zinc oxide nanoparticles

As presented in Table 3, total polyphenolic compounds and condensed tannins are the major active phyto-constituents in the different C. tiglium L. seeds extracts. The aqueous C. tiglium L. seeds extract contains the highest concentration of total polyphenolic compounds (124.44 ± 2.19 mg gallic acid/100 gm) and condensed tannins (26.95 ± 0.12 μg/mL) more than the other C. tiglium L. extracts. Incorporation of ZnO-NPs into the aqueous C. tiglium L. seeds extract increased concentrations of total polyphenolic compounds and condensed tannins to 236.44 ± 4.16 mg gallic acid/100 gm and 36.93 ± 0.10 μg/mL, respectively.

Table 3.

Antioxidant Activity of the Different Extracts of C. tiglium L. seeds before and after Incorporating ZnO-NPs

Extract Total Polyphenols
(mg gallic acid/100 gm)		 Total Condensed Tannins (μg/mL) Total Antioxidant Capacity (mg gallic acid/gm) Iron Reducing Power
(µg/mL)
Before Ethanolic 60.02 ± 1.37 22.10 ± 0.18 103.79 ± 1.37 98.94 ± 1.02
Aqueous 124.44 ± 2.19* 26.95 ± 0.12* 140.53 ± 2.00* 138.82 ± 4.51*
P. Ether 22.71 ± 1.03 - 75.76 ± 1.65 64.55 ± 1.42
After ZnO-Ethanolic 114.04 ± 2.61 31.13 ± 0.09 185.78 ± 2.44 178.09 ± 1.84
ZnO-Aqueous 236.44 ± 4.16* 36.93 ± 0.10* 251.55 ± 3.59* 249.88 ± 8.12*
ZnO-P. Ether 43.15 ± 1.96 - 135.61 ± 2.96 116.18 ± 2.55
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* denotes the most effective extract, Values expressed as mean of three replicates ± SE.

The biological activities of the different C. tiglium L. seeds extracts were assayed by measuring the total antioxidant capacity and iron reducing power in addition to the scavenging activity against DPPH and ABTS radicals using ascorbic acid as standard. The total antioxidant capacity and iron reducing power are closely related to concentrations of the total polyphenols and total condensed tannins. Therefore, it was noticed that the aqueous C. tiglium L. seeds extract exhibited the highest the antioxidant activity (140.53 ± 2.00 mg gallic acid/gm) and iron reducing power (138.82 ± 4.51 µg/mL) as illustrated in Table 3. Incorporation of ZnO-NPs into the aqueous C. tiglium L. seeds extract increased the antioxidant activity and iron reducing power to 251.55 ± 3.59 mg gallic acid/gm and 249.88 ± 8.12 µg/mL, respectively.

As regard to the scavenging activity against DPPH and ABTS radicals, it was found that the aqueous C. tiglium L. seeds extract possessed the highest scavenging activity. Strong scavenging activity against DPPH radical was indicated by low IC50 value. As presented in Table 4, the lowest IC50 value (12.91 ± 0.11 μg/mL) was noticed with the aqueous C. tiglium L. seeds extract as compared to the other extracts. Moreover, it showed the highest scavenging activity against ABTS radical (41.11 ± 0.07%) at equal concentrations of all studied extracts, whereas at the same concentration, the standard acorbic acid was 36.78 ± 0.03%. Incorporation of ZnO-NPs into the aqueous C. tiglium L. seeds extract increased the scavenging activity by lowering the IC50 value required to inhibit DPPH radical to 3.78 ± 0.02 μg/mL and increasing inhibition of ABTS radical to 62.91 ± 0.10%.

Table 4.

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radicals Scavenging Activity of the Different Extracts of C. tiglium L. seeds before and after Incorporating ZnO-NPs

Extract IC50 (µg/mL) Inhibition of ABTS Radicals (%)
Before Ethanolic 18.92 ± 0.05 35.23 ± 0.04
Aqueous 12.91 ± 0.11* 41.11 ± 0.07*
P. Ether 28.89 ± 0.06 22.15 ± 0.12
After ZnO-Ethanolic 7.17 ± 0.03 51.78 ± 0.04
ZnO-Aqueous 3.78 ± 0.02* 62.91 ± 0.10*
ZnO-P. Ether 11.23 ± 0.04 38.99 ± 0.09
Ascorbic acid (Standard) 3.83 ± 0.02 36.78 ± 0.03
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* denotes the most effective extract, Values expressed as mean of three replicates ± SE.

As revealed in Figure 2a, it was found that the aqueous C. tiglium L. seeds extract exhibited the highest inhibitory effect at equal concentrations on α-amylase and AChE activities (41.89 and 23.00%, respectively). Incorporating ZnO-NPs into the aqueous extract increased their inhibitory effect against α-amylase and AChE activities to 67.97 and 49.91%, respectively (Figure 2b).

Figure 2.

Figure 2

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Percent of the Inhibitory Effect on Activities of α-amylase and Acetyl Cholinesterase (AChE) for a) native C. tiglium seeds extracts and b) ZnO-C. tiglium L. seeds nano-extracts. Values expressed as mean of three replicates ± SE. * denotes the most effective extract

Data depicted in Table 5 showed percentage of protein denaturation and proteinase inhibitory activity. It was noticed that the aqueous C. tiglium L. seeds extract showed higher protein denaturation (52.44 ± 0.67%) and proteinase inhibitory activity (52.22 ± 0.42%) compared to diclofenac sodium that was used as a powerful non-steroidal anti-inflammatory standard drug. Incorporation of ZnO-NPs into the aqueous extract showed better protein denaturation (78.99 ± 0.44%) and proteinase inhibitory activity (75.17 ± 0.99%) than the reference drug (diclofenac sodium) and the native aqueous extract itself.

Table 5.

Anti-Arthritic Activity of the Different C. tiglium L. seeds Extracts before and after Incorporating ZnO-NPs

Extract Proteinase Denaturation (%) Inhibition of Proteinase Activity (%)
Before Ethanolic 36.99 ± 0.48 31.91 ± 0.66
Aqueous 52.44 ± 0.67* 52.22 ± 0.42*
P. Ether 26.19 ± 0.66 32.28 ± 0.60
After ZnO-Ethanolic 67.77 ± 0.39 52.46 ± 0.66
ZnO-Aqueous 78.99 ± 0.44* 75.17 ± 0.99*
ZnO-P. Ether 45.65 ± 1.11 46.75 ± 0.65
Diclofenac sodium (Standard) 48.68 ± 0.24 40.46 ± 0.73
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* denotes the most effective extract, Values expressed as mean of three replicates ± SE.

Data compiled in Table 6 and illustrated in Figure 3a showed that the aqueous C. tiglium L. seeds extract exhibited the highest cytotoxic activity (IC50 546.00 μg/mL) against HEPG-2 cells compared to the other native extracts. The Supplementary 1 showed the maximum (Conc. 1,000 μg/ml) and IC50 of different native C. tiglium seeds extracts compared to control HEPG-2 cells. It was found that incorporation of ZnO-NPs into aqueous extract increased the cytotoxic activity against HEPG-2 cells compared to native aqueous extract itself (IC50 314.40 μg/mL). The Supplementary 2 showed the maximum (Conc. 1000 μg/ml) and IC50 of different C. tiglium L. seeds extracts after incorporating ZnO-NPs compared to control HEPG-2 cells. As regard to the in vitro cytotoxic activity against CACO-2, data depicted in Table 6 and illustrated in Figure 3b showed that the aqueous C. tiglium L. seeds extract exhibited the highest cytotoxic activity (IC50 176.90 μg/mL) against CACO-2 cells compared to the other native extracts. The Supplementary 3 showed the maximum (Conc. 1,000 μg/ml) and IC50 of different native C. tiglium L. seeds extracts compared to control CACO-2 cells. Incorporation of ZnO-NPs into aqueous extract elevated the cytotoxic activity against CACO-2 cells compared to native aqueous extract itself (IC50 100.20 μg/mL). The Supplementary 4 showed the maximum (Conc. 1000 μg/ml) and IC50 of different C. tiglium L. seeds extracts after incorporating ZnO-NPs compared to control CACO-2 cells. In the present study, it was found that the cytotoxic activity of the aqueous C. tiglium L. seeds extract increased after incorporating ZnO-NPs

Table 6.

Cytotoxic Activity of the Different C. tiglium L. Seeds Extracts against Human Liver (HEPG-2) and Colon Cancer (CACO-2) Cells before and after Incorporating ZnO-NPs

Extract IC50%
HEPG-2 CACO-2
Before Ethanolic 671.3 309.70
Aqueous 546.00* 176.90*
P. Ether 954.3 692.10
After ZnO-Ethanolic 451.5 252.50
ZnO-Aqueous 314.40* 100.20*
ZnO-P. Ether 902.8 353.30
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* denotes the most effective extract, Values expressed as mean of three replicates ± SE.

Figure 3.

Figure 3

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Cytotoxic Activity of the Different C. tiglium L. seeds Extracts before and after Incorporating ZnO-NPs against a) human liver cancer (HEPG-2) and b) human colon cancer (CACO-2)

As revealed in Supplementary 5, it was found that treatment of CACO-2 cells with the aqueous C. tiglium L. seeds extract incorporated with ZnO-NPs decreased the fold changes of EGFR and Bcl2 genes (0.43 and 0.41, respectively) compared to the native aqueous extract itself (0.77 and 0.62, respectively). As regard to Casp3 gene, the aqueous C. tiglium L. seeds extract incorporated with ZnO-NPs increased its fold changes (4.24) more than the native aqueous extract itself (2.48). Treatment of CACO-2 cells with the native aqueous C. tiglium L. seeds extract or the aqueous extract incorporated with ZnO-NPs arrested the cell growth at G2/M compared to doxorubicin that arrested the cell growth at G1/S (Supplementary 6). As illustrated in Supplementary 7, treatment of CACO-2 cells with the aqueous C. tiglium L. seeds extract incorporated with ZnO-NPs increased percentage of the total apoptotic cells (29.12%) and increased percentage of the early (4.52%) and late apoptotic cells (13.27%) as compared to control CACO-2 or those cells treated with the native aqueous extract itself. Moreover, percentage of the necrosis increased in the CACO-2 cells treated with ZnO-aqueous nano-extract (11.33%) more than those cells treated with the native aqueous extract (7.26%). Data presented in Table 7 showed that incorporation of ZnO-NPs into the aqueous C. tiglium L. seeds extract caused on significant changes in expression of the EGFR, Bcl2 and Casp3 genes in the treated CACO-2 cells as compared to control HEPG-2 or those cells treated with the native extract itself.

Table 7.

Data of the EGFR, Bcl2 and Casp3 Genes Expression in Human Colon Cancer (CACO-2) Cells Treated with Aqueous C. tiglium L. extract before and after Incorporating ZnO-NPs

Extract EGFR
Control cells Test cells FLD
Conc. (µM) GAPDH EGFR ΔCTC GAPDH EGFR ΔCTE ΔΔ CT 2 -ΔΔCT
HC TC TC-HC HE TE TE-HE ΔCTE-ΔCTC Eamp=1.849
Aqueous C. tiglium extract 23.46 26.82 3.36 24.08 27.86 3.78 0.42 0.77
ZnO-Aqueous nano-extract 23.46 26.82 3.36 23.82 28.55 4.73 1.37 0.43
Doxorubicin 23.46 26.82 3.36 23.91 29.21 5.3 1.94 0.30
Control 23.46 26.82 3.36 23.46 26.82 3.36 0.00 1.00
Extract Bcl2
Control cells Test cells FLD
Conc. (µM) GAPDH Bcl2 ΔCTC GAPDH Bcl2 ΔCTE ΔΔ CT 2 -ΔΔCT
HC TC TC-HC HE TE TE-HE ΔCTE-ΔCTC Eamp=1.849
Aqueous C. tiglium extract 23.46 28.76 5.30 24.08 30.15 6.07 0.77 0.62
ZnO-Aqueous nano-extract 23.46 28.76 5.30 23.82 30.58 6.76 1.46 0.41
Doxorubicin 23.46 28.76 5.30 23.91 30.83 6.92 1.62 0.37
Control 23.46 28.76 5.30 23.46 28.76 5.30 0.00 1.00
Extract Casp3
Control cells Test cells FLD
Conc. (µM) GAPDH Casp3 ΔCTC GAPDH Casp3 ΔCTE ΔΔ CT 2 -ΔΔCT
HC TC TC-HC HE TE TE-HE ΔCTE-ΔCTC Eamp=1.849
Aqueous C. tiglium extract 23.46 33.61 10.20 24.08 32.75 8.67 -1.48 2.48
ZnO-Aqueous nano-extract 23.46 33.61 10.20 23.82 31.62 7.80 -2.35 4.24
Doxorubicin 23.46 33.61 10.20 23.91 30.79 6.88 -3.27 7.46
Control 23.46 33.61 10.20 23.46 33.61 10.2 0.00 1.00
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Data of the flow cytometric analysis presented in Figure 4 and using Annexin V-FITC as shown in Figure 5 showed that treatment of CACO-2 cells with the native aqueous C. tiglium seeds extract or the aqueous extract incorporated with ZnO-NPs enhanced apoptosis as compared to control CACO-2 cells.

Figure 4.

Figure 4

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Data of DNA Content in a) Control CACO-2, b) CACO-2 treated with doxorubicin, c) CACO-2 treated with aqueous C. tiglium L. extract and d) CACO-2 treated with ZnO-Aqueous nano-extract

Figure 5.

Figure 5

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Data of Apoptosis Assay with Annexin V-FITC showing a) Control CACO-2, b) CACO-2 treated with doxorubicin, c) CACO-2 treated with aqueous C. tiglium L. extract and d) CACO-2 treated with ZnO-Aqueous nano-extract

Toxicity of different C. tiglium L. seeds extracts before and after incorporating ZnO-NPs

It was noticed that the LD50 values of ethanolic, aqueous and P. ether extracts were about 7667, 8083 and 5500 mg/Kg, respectively. After incorporating ZnO-NPs, the LD50 values increased to 11000, 11333 and 9500 mg/Kg, respectively (Figure 6).

Figure 6.

Figure 6

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The Median Lethal Doses (LD50) of the Different C. tiglium L. seeds Extracts before and after Incorporating ZnO-NPs.”

Discussion

The nutritional composition of C. tiglium L. seeds

During the current study, it was noticed that C. tiglium L. seeds provided great values and could be consumed for either nutritional or medicinal purposes which in agreement with Owade et al. (2019) and supported by Aboulthana et al. (2019) who stated that C. tiglium L. seeds have been utilized mainly for its oil, protein and polysaccharide contents.

The major compounds isolation from different C. tiglium L. seeds extracts

During the present study, the steroidal (β-sitosterol and stigmasterol) and the pentacyclic triterpenoid (α-amyrin) compounds were identified and isolated in pure form and this was in agreement with the study carried out by Smina et al. (2011). Their spectroscopical data were in agreement with that reported by El-Feky et al. (2018). Furthermore, isopimara-7,15-dien-3β-ol is diterpene compound isolated from leaves of Croton zambesicus by Block et al. (2004) and not identified before from C. tiglium L. seeds. Although numerous quercetin derivatives were identified by Guerrero et al. (2002) from Croton schiedeanus L, quercetin-7-O-β-D-glucopyranoside was isolated and identified for the first time from C. tiglium L. seeds during the present study. In the current experiment, 13-O-Acetylphorbol-20-(9Z,12Z-octadecadienoate) and 13-O-Tigloylphorbol-20-(9Z,12Z-octadecadienoate) were isolated and identified. These two phorbol esters were previously isolated from C. tiglium L. seeds by El-Mekkawy et al., (2000).

The structural properties of prepared ZnO-NPs

The biosynthesized ZnO-NPs was identified as sharp peak identified in the UV-visible spectrum at 382 nm and this agreed with the experiment carried out by Kavitha et al. (2013) who reported that the peak at 280 nm corresponds to the plant extract. Moreover, the strong absorption band that was assigned to the intrinsic band-gap absorption of ZnO might be attributable to transition of the electron from the valence to conduction band (Khorsand Zak et al., 2013). The DLS showed that the zeta potential denotes repulsion degree between adjacent particles (with similar charges) in dispersions (Hassan et al., 2019). Moreover, the particle size distribution and the hydrodynamic size of the fabricated ZnO-NPs has main diameter around 164 nm. This agreed with Kim et al. (2012) who reported that the synthesized ZnO-NPs can form aggregates (lumps of primary particles held together by strong chemical bonds) or agglomerates (groups of primary particles gathered by weak van der Waals forces) in a liquid dispersion of dry powder.

In vitro study on different C. tiglium L. seeds extracts before and after incorporating ZnO-NPs

Due to the presence of various active bio-components like alkaloids, terpenoids, phenolics, tannins, saponins, polysaccharides, proteins, enzymes and vitamins, plant extract can be used as a potential substitute for reducing agents (Ahmed et al., 2016). It was found that the aqueous C. tiglium L. seeds extract is in the highest concentrations of total polyphenolic compounds and condensed tannins more than the other C. tiglium L. extracts. The phenolic compounds and flavonoids are secondary metabolites exist in almost all medicinal plants. Antioxidative and anti-carcinogenic activities are considered as the most common biological activities of these phyto-constituents in addition to their role as bio-reductants of metallic ions in aqueous medium (Yuvakkumar et al., 2014). This might be attributed to the presence of the functional groups responsible for the bio-reduction and stabilization process during biosynthesis of metal and metal oxide NPs (XiuLan et al., 2017). During the ZnO-NPs biosynthesis, phenol and flavonoids in plant extract react with zinc nitrate by binding zinc surface to control the particle size after activating the ZnO-NPs biosynthesis. These phyto-constistuents are responsible for reducing and capping activity due to existence of a large number of OH groups (Bala et al., 2015).

It was noticed that aqueous C. tiglium L. seeds extract exhibited the highest the antioxidant activity and this might be attributed to the complex nature of the phenolic compounds and the other phytochemicals that have redox properties enable them to act as hydrogen donors and hence to be used as reducing agents. The plant extracts possessed higher scavenging activities against the oxidative stress induced by Reactive Oxygen (ROS) and Nitrogen Species (RNS) due to the presence of these active constituents with high concentrations as suggested by Kalim and Nikalje (2017).

DPPH and ABTS free radical scavenging assays provide an easy and rapid methods for estimating ability of the antioxidants to scavenge free radical based on quenching stable-colored radicals (Zia-Ul-Haq et al., 2012). During the current study, it was noticed that the scavenging activity of the extract incorporated with ZnO-NPs increased and this was confirmed by decreasing the absorbance at 517 nm as a result of changing color of the reaction product from purple to yellow. This agreed with Nagajyothi et al. (2015) who emphasized that the antioxidant activity of the extract increased after incorporating ZnO-NPs due to presence of the active phyto-constituents that were responsible for imparting antioxidant capabilities and utilized throughout manufacturing of ZnO-NPs for their reduction and stabilization.

α-amylase is the most essential digestive tract enzyme required for hydrolyzing carbohydrates. Therefore, inhibition of this enzyme is one of the most suitable ways for lowering postprandial hyperglycemia (Nair et al., 2013). Moreover, AChE enzyme exhibits its role in tissue synapses or neuromuscular junctions through catalyzing the hydrolysis process during which acetyl choline (a neurotransmitter) is converted into choline and acetic acid. Therefore, activation of this enzyme is one of leading cause of Alzheimer’s disease. Inhibition of this enzyme is one of the suitable ways necessary for Alzheimer treatment (Suganthy et al., 2018). It was found that incorporation of ZnO-NPs into the aqueous extract increased their inhibitory effect against α-amylase and AChE activities and this agreed with Jan et al., (2021) who emphasized that existence of ZnO-NPs in aqueous C. tiglium L. seeds extract showed higher anti-diabetic properties through suppressing α-amylase activity. This might be attributed . The biosynthesized ZnO-NPs exhibited efficient anti-Alzheimer’s activity by inhibiting AChE activity and this agreed with Khalil et al. (2019) who reported that ZnO-NPs belongs to the M-NPs that suppressed AChE followed by cobalt oxide and iron oxide nanoparticles.

Inflammation is a response process by which living tissues react towards injury. It is considered as the most common phenomenon that occurs during arthritis and induced by denaturation of protein. Inflammation and activity of immune response decreased by steroids (Nagajyothi et al., 2014). It was found that the aqueous C. tiglium L. seeds extract showed higher protein denaturation and proteinase inhibitory activity. Incorporation of ZnO-NPs into the aqueous extract showed better protein denaturation and proteinase inhibitory activity than the native aqueous extract itself. This agreed with Senthilkumar et al. (2017) who revealed that the presence of ZnO-NPs enhanced the anti-arthritic activity of the extract due to their capability for inhibiting protein denaturation and proteinase inhibitory activity. This might be related to increasing the scavenging activity against the free radicals that are critically involved in arthritis and inflammation.

Cell viability assays are considered as basic criteria to reveal the cellular response to toxic substances and to elucidate efficiency of the extract against incidence and progression of cancer cells by the MTT assay. A calcein (green fluorescent compound) that produced from calcein AM as a result of role of active esterase exists with intact membranes in living cells serves as a marker for viable cells. Moreover, there is red fluorescent nucleic acid stain called propidium iodide (PI) used for detecting the damaged cells that take up the dye and stain positive and it has no the ability to penetrate normal cells (Sanlioglu et al., 2007). Studying efficiency of the anti-cancer potential of ZnO-NPs against growth and progression of cancer cells belongs to scientific hotspots for cancer therapy (Vimala et al., 2014). During the current study, the in vitro cytotoxicity of different C. tiglium L. seeds extracts was studied after incorporating ZnO-NPs against HEPG-2 and CACO-2 in comparison with native extracts.

Data of the current study showed that the cytotoxic activity of the aqueous C. tiglium L. seeds extract increased after incorporating ZnO-NPs and this agreed with the study carried out by Modena et al. (2019) who emphasized that the cytotoxic activity might be attributed to presence of alcohols and phenolic groups in addition to C-N stretching vibrations of aromatic amines of biomolecules on the surface of the biosynthesized ZnO-NPs that physiologically act on inhibiting growth of cancer cells. Furthermore, the plant-derived ZnO-NPs showed an exciting potential as promising anti-cancer agents by enhancing the cellular death through production of the ROS that disrupt signal transduction pathways (Ismail et al., 2018). When ZnO-NPs get entry into the cancerous cell, they produced ROS species, disturbed depolarization of the membrane and damaged DNA; all these events eventually leads to apoptosis or death of cancer cell (Jan et al., 2020). During the current study, it was noticed that the aqueous C. tiglium L. seeds extract showed better cytotoxic activity against CACO-2 than HEPG-2 cells. Therefore, it was selected for undergoing further studies on CACO-2 cells before and after incorporating ZnO-NPs.

Data of the flow cytometric analysis showed that treatment of CACO-2 cells with the native aqueous C. tiglium seeds extract or the extract incorporated with ZnO-NPs enhanced apoptosis as compared to control CACO-2 cells. This agreed with Jan et al., (2021) who postulated that ZnO-NPs showed prominent apoptosis after for 24 h treatment. The cytotoxic activity of ZnO-NPs against growth and progression of human cancer cells occurred through three primary mechanisms including breakdown of the ZnO-NPs into Zn+2, ROS production and DNA damage. In addition, the physical properties (size, surface chemistry and dose) of the ZnO-NPs dictate their overall uptake, elimination and antitumor properties (Chen et al., 2019). Also, ZnO-NPs have the ability to induce apoptosis in CACO-2 cells by oxidative stress leading to cytotoxicity, inflammatory responses, mitochondrial membrane alterations and release of interleukin-8 in cancerous cells (Jain et al., 2018). It was demonstrated that ZnO-NPs showed their anticancer and antiproliferative effect via up-regulating the apoptotic and tumor suppressor genes, down-regulating the antiapoptotic genes, inducing ROS production, DNA fragmentation and caspase-3 enzyme in cancer cells (Ismail et al., 2014 ; Iswarya et al., 2017).

Toxicity of different C. tiglium L. seeds extracts before and after incorporating ZnO-NPs

It was found that administration of the different C. tiglium L. seeds extracts incorporated with ZnO-NPs orally was safer than the native extracts. This was agreement with Shaban et al., (2021) and supported recently by Aboulthana et al., (2022) who reported that no significant alterations detected in the biochemical markers after oral administration of M-NPs. Biodistribution of the M-NPs in organs and tissues depends highly on size and dose of the M-NPs. Size of the M-NPs is considered as the most important factor affecting their biodistribution. Ratio of the large surface area to volume of the NPs stimulates their interactions with the macromolecules in biological systems (Wang et al., 2020). Biosynthesis of M-NPs by mean of green nanotechnology found with less toxicity and it increased safety of plant extract in which the M-NPs incorporated (Abdel-Halim et al., 2020). This was attributed to efficiency the renal tissue to eliminate the M-NPs with low degradation rate in the body to avoid the undesirable side effects (Aboulthana et al., 2019).

Author Contribution Statement

Wael M. Aboulthana: Writing – original draft, Writing – review & editing, Visualization, Supervision and Project administration. Nagwa I. Omar: Resources, Data curation, Writing – review & editing. Amal M. El-Feky: Methodology, Validation, Formal analysis, Writing – review & editing. Enas A. Hasan: Formal analysis, Writing – review & editing. Noha E. Ibrahim: Literature collection, Writing – review & editing. Ahmed M. Youssef: Conceptualization, Data curation, Writing – review & editing. All authors have read and agreed to the published version of the manuscript.

Acknowledgments

This research was accomplished in laboratories of the National Research Centre, Dokki, Giza, Egypt and provided by researchers of different scientific fields.

Ethical statement

Handling of the animals and the experimental design were carried out based on the guidelines reported in “Guide for the care and use of laboratory animal” and according to the protocol that approved by Institutional Animal Ethical Committee of National Research Centre, Dokki, Giza, Egypt.

Conflict of 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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