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Phytobiological-facilitated Production of Silver Nanoparticles From Selected Non-cultivated Vegetables in Nigeria and Their Biological Potential.

Objectives
Plant-mediated synthesis [silver (Ag) to form Ag nanoparticles (AgNPs)] is becoming progressively well accepted in many scientific and pharmaceutical fields. The aim of this study was to synthesize AgNPs using air-dried leaves of four neglected vegetables, i.e. Ceratotheca sesamoides, Ceiba pentandra, Crassocephalum crepidioides, and Launaea taraxacifolia.


Materials and Methods
Ultraviolet-visible (UV-Vis) spectroscopy, fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM) were used for characterization. Cell stabilization membrane and lipoxidase assays were used to determine used to assess the antiinflammatory activities while 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) assays were used to assess the antioxidant activities of AgNPs [L. taraxacifolia-AgNPs, C. sesamoides Ag nanoparticles (CS-AgNPs), C. pentandra Ag nanoparticles (CP-AgNPs), and C. crepidioides AgNPs (CC-AgNPs)].


Results
The UV-Vis spectra of the synthesized NPs displayed absorption bands at around 360-440 nm, which is a characteristic band for AgNPs. The SEM image showed that the AgNPs formed were spherical in morphology. CC-AgNPs exhibited the most significant inhibitory activity against human red blood cell membrane stabilizasyonu [median inhibitory concentration (IC50): 32.2 μg/mL] while CS-AgNPs displayed the most significant inhibitory activity against lipoxygenases (IC50: 32.8 μg/mL). CP-AgNPs exhibited the most significant antioxidant effect against both ABTS and DPPH (IC50: 5.5 and 6.4 μg/mL) when compared to ascorbic acid (IC50: 4.7 μg/mL).


Conclusion
The synthesized AgNPs were found to be stable and the FTIR evidence suggested that the phytochemicals in the vegetables might have played an important role in the reduction and stabilization of AgNPs. This work showed that the synthesized AgNPs from non-cultivated vegetables can find relevance and application in health, drugs, food and environmental science. The evidences herein further confirmed their ethnopharmacological applications.


INTRODUCTION
For centuries, cultures around the world have continuously employed and taken advantage of edible but non-cultivated plants for sufficient nutrition, food security, and wealth creation. [1][2][3] These non-cultivated plants supply necessary and essential components of the human diet, supplying the body with various minerals, protein, and certain precursors of human hormones besides helping in the build-up of energy. [4][5][6] Some of the plants studied here are non-cultivated due to their being tagged as "poor man's" vegetables but are eaten by the locals. Ceratotheca sesamoides 7 belongs to the family Pedaliaceae. It is mostly found in Africa and it grows as a wild and non-cultivated plant. However, in some parts of Africa, it is being cultivated, and because of its similarities with common sesame (Sesamum indicum), some call it false sesame. 8,9 Although widely regarded as a delicacy in most West African countries, literature on this plant and its consumption is scanty and not sufficient. 10 C. sesamoides is traditionally employed in the management of diarrhea in Nigeria. The plant is used as an aphrodisiac and in the treatment of jaundice, snake bites, and skin ailments. C. sesamoides leaf infusions are used to facilitate delivery in both humans and animals. [10][11][12][13] In northern Nigeria, C. sesamoides seeds are used to relieve circumcision pains.
Ceiba pentandra belongs to the family Malvaceae. 7 It is native to the Caribbean, Central America, northern South America, Mexico, and tropical West Africa. Besides its young leaves' nutritional benefits, in Nigeria many locals use its leaves for treating many ailments. This plant has many ethnobotanical uses (Table 1), i.e. to treat headache and diabetes and as a diuretic and aphrodisiac. Its use as one of the main ingredients in a hallucinogenic drink has also been reported. 14,15 Crassocephalum crepidioides 7 is also called thickhead, fireweed, Okinawa spinach, and red flower ragleaf in English, Ebolo, or Ebire (Yoruba) in Nigeria. Its use is widespread in many tropical and subtropical regions, but is especially prominent in tropical Africa. It has also been widely cultivated in Asia due to its medicinal and nutritional properties. 16,17 In southern Nigeria, C. crepidioides' leaves have been reported to be valuable in the management of indigestion, stomach ache, and fresh wounds (in Uganda) and its leaves' decoction is employed in Nigeria against headache (Table 1). In Tanzania, a mixture of the leaf sap of C. crepidioides and Cymbopogon giganteus is taken by mouth against epilepsy. Its dried leaves are used to stop nose bleeds and aid in sleeping. 18 Launaea taraxacifolia (synonymous to Lactuca taraxacifolia) 7 is a greenish leafy vegetable that is mainly eaten in the western part of Nigeria. This vegetable is eaten in most countries in Africa either cooked or as salad, i.e. Dahomey, Ghana, Senegal, and Sierra Leone. 19 Most people in West Africa call L. taraxacifolia by the name African lettuce or wild lettuce. 20 There are many ethno-medicinal applications of L. taraxacifolia. This leafy vegetable has been employed in managing many ailments for centuries, ailments such as diabetes, eye diseases (conjunctivitis), measles, skin diseases, and yaws (Table 1). Some cultures in Nigeria rubbed a concoction of its leaves on the limbs of toddlers to facilitate walking. 21,22 Many studies have reported the green synthesis of leafy vegetable extracts employing various metals, i.e. the green synthesis of copper nanoparticles (NPs) using Ocimum sanctum 23 green synthesis of palladium NPs employing Origanum vulgare leaf extract 24 lemon fruits and turmeric powder to steady the green synthesis employing manganese NPs 25 and the synthesis of silver NPs (AgNPs) from Curcuma longa 26 and Calotropis. Beside their nutritional benefits, leafy and non-cultivated vegetables ( Figure 1) are known to possess therapeutic uses. 13,27,28 However, many of these cheap but disease-preventing plant species are yet to be sufficiently studied and exploited. Hence, the aim of the present study was to investigate the phytochemical screening of these noncultivated vegetables' leaves extract and experimentally carry out characterization and application of these medicinal plants species' AgNPs as antiinflammatory and antioxidant agents and acetylcholinesterase inhibitors.

MATERIALS AND METHODS
Fresh green plants of Crassocephalum crepidioides (I.U. 0345), Ceratotheca sesamoides (I.U. 011), Ceiba pentandra (UILH/001/957), and Launaea taraxacifolia (UILH/002/1020) were obtained in December 2016 from "Oja-Oba" market in Ilorin, in Kwara State of Nigeria located in the rain forest zone at latitude 10°00' North of the equator and longitude 8°00′ East of the Greenwich meridian. The plants were identified and authenticated at the Plant Biology Department, University of Ilorin, and voucher numbers collected. The authenticated plant materials were air-dried at ambient temperature for 2 weeks to completely remove the moisture content and to effectively prepare the plants for the next stage of preparation. After drying, the dried leaves were crushed into fine powder using a ceramic pestle and mortar and the samples were kept in an air-tight plastic container.

Equipment and reagents
The equipment used comprised a pestle and mortal, extraction jar, rotary evaporator, centrifuging machine, ultravioletvisible (UV-Vis) spectrophotometer, and fourier transform infrared spectrophotometer (FTIR). The reagents included n-hexane, methanol, silver nitrate, ferric chloride, potassium ferricyanide, chloroform, sulfuric acid, lead acetate, acetic anhydride, potassium hydroxide, and Fehling solution. They were purchased from Labtrade and Sunaf Nig. Ltd. All solvents used were of analytical grade.  crepidioides were macerated in 3 L of n-hexane in an extraction jar such that the level of the solvent was above that of the plant materials. The macerated mixtures were then left for 72 h at ambient temperature. The extracts were filtered out from the macerated mixture using Whatman 185 µm filter paper. The n-hexane extracts were concentrated in a vacuum rotary evaporator under reduced pressure and suitable temperature, transferred to appropriately labeled 250 mL beakers, and allowed to stand at ambient temperature to permit evaporation of residual solvents. The procedure was repeated using methanol after the residue of the n-hexane extract has been air-dried.

Phytochemical screening
Preparation for the test was done by pouring 3 mL of the leaf extracts into separate test tubes and diluting with 2-4 mL of deionized water. The various tests were carried out following the procedures described below. Standard techniques of screening and detecting secondary metabolites in plants were used. 29,30 The metabolites tested for were alkaloids, anthraquinones, cardiac glycosides, carbohydrates, flavonoids, saponins, steroids, phenolics, tannins, and triterpenes.

Synthesis of silver nanoparticles
The synthesis of AgNPs was carried out according to the method described in our previous study. 31 Ten milliliters of the leaf extract was measured and poured into a clean 250 mL beaker and reacted with 100 mL of 0.01 M AgNO 3 (prepared from stock AgNO 3 -0.1 M of AgNO 3 ) from a burette (titration method) using AgNO 3 as the titrant and the extracts as the titrant at ambient temperature. A color change to yellow was observed. The synthesized mixture was left for 24 h and then separated by centrifugation using a centrifuging machine. Clear liquid was decanted and the settled layer (NPs) was stored in a 5 mL plastic sample vial and labeled accordingly. The following nomenclature was given to the synthesized NPs: L. taraxacifolia (LT)-AgNPs, C. sesamoides (CS)-AgNPs, C. pentandra (CP)-AgNPs, and C. crepidioides (CC)-AgNPs.

Characterization of silver nanoparticles
The characterization of LT-AgNPs, CS-AgNPs, CP-AgNPs, and CC-AgNPs was done using a combination of analytical and spectroscopic techniques, namely UV-Vis, FTIR, and scanning electron microscopy (SEM).

Ultraviolet-visible spectroscopy
The optical properties of the AgNPs of both plants were determined by UV-Vis spectroscopy on a Biochrom Libra PCB 1500 UV-VIS spectrophotometer. The wavelength with the highest absorbance was determined. The absorbance of AgNPs dispersed in a quartz cuvette with a 1 cm optical path was measured by withdrawing a small aliquot from the reaction mixture and a wavelength scan was taken every 60 min, then 90 min, and after 24 h. The wavelength was varied from 320 nm to 620 nm for L. taraxacifolia, from 320 nm to 670 nm for C. crepidioides, from 320 nm to 620 nm for C. sesamoides, and from 320 nm to 620 nm for C. pentandra.

Fourier transform infrared spectroscopy
The functional groups present in the methanolic extract of L. taraxacifolia, C. crepidioides, C. sesamoides, and C. pentandra, which were responsible for capping and efficient stabilization of the synthesized AgNPs, were determined using a Shimadzu FTIR model IR8400s spectrophotometer. The solutions were dried at 75°C and the dried powders were characterized in the range 4000-400 cm -1 by KBr pellet method.

Scanning electron microscopy
NPs of these plants' extracts were viewed using an Ultra Plus FEGSEM (Carl Zeiss, Germany) and the size and shape of the NPs were determined using the Smart SEM Ver. 5 software (Carl Zeiss, Germany).

Biological activities
Antiinflammatory activity

Cell stabilization membrane
The antiinflammatory activity of these extracts was tested by in vitro human red blood cell (HRBC) membrane stabilization method. The reaction mixtures (4.5 mL) consisted of 2 mL hypotonic saline solution, phosphate buffer (pH 7.4), and 1 mL of test solution in normal saline; 0.5 mL of 10% rabbit RBC in normal saline was added. For control tests, 1 mL of isotonic solution was used. The mixtures were incubated at 560°C for 30 min, cooled under running water, and centrifuged, while the absorbance of the supernatants was read at 560 nm. Percentage membrane stabilizing activity was calculated as follows: The control represents 100% lysis. The result was compared with STD (100 µg/mL) treated samples. 32,33 Lipoxidase assay The inhibitory activity against lipoxygenases (LOXs) was studied using linoleic acid as substrate and lipoxidase as enzyme. Test samples were dissolved in 0. 25   added followed by incubation for 5 min at 250°C. After that, 1.0 mL of lenoleic acid solution (0.6 mM) was added followed by thorough mixing and absorbance was measured at 234 nm. Indomethacin was used as reference standard. The percent inhibition was calculated from the following equation: % inhibition=[(Abs control-Abs sample) /Abs control] x100 All tests and analyses were run in triplicate and averaged. 34,35 Antioxidant activity

2,2-Diphenyl-1-picrylhydrazyl (DPPH) activity
The method employed was the one reported by Oguntoye et al. 28 but with slight modifications. 36 Mean ± standard error of the mean of two independent experiments run in duplicate was used to present the results.

2,2'-Azino-bis-(3-ethyl) benzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging (ABTS) activity
The ABTS radical cation decolorization assay based on the scavenging of ABTS + radicals by antioxidant components of the extracts was used. The assay follows the procedure of Oguntoye et al. 28 with slight modifications. 36 All analyses were performed in duplicate.

Statistical analysis
Mean ± standard error of the mean of two independent experiments run in duplicate was used to present the results. The results are reported as mean ± standard deviation.

Phytochemical screening
The phytochemical constituents of the extracts of C. crepidioides, C. sesamoides, C. pentandra, and L. taraxacifolia are shown in Table 2. On the whole, polyphenol, flavonoids, triterpenes, and steroids were identified in all the plants' extracts. Alkaloids and saponins are absent in most of these plants except for methanol extract of C. crepidioides and hexane extract of C. pentandra. The hexane extracts of C. sesamoides gave poor results for most groups of secondary metabolites investigated as shown in Table 2. The phytochemical screening reveals that flavonoids are present in the various extracts.

UV-Vis spectroscopy study
Visual inspection showed color changes. The color changes that were witnessed indicate the formation of C. crepidioides, C. sesamoides, C. pentandra, and L. taraxacifolia AgNPs as shown in Table 3. Many studies have shown that AgNPs displayed these color changes in aqueous solution due to the excitation of surface plasmon resonance (SPR) of AgNPs, and this was the first confirmation test to show that AgNPs were formed. [37][38][39][40][41] The AgNPs formed were examined further by the use of UV-Vis spectroscopy, which is an important and popular tool used for characterization.
It was discovered that the aqueous extracts of C. sesamoides and C. pentandra were able to reduce silver nitrate to AgNPs at 450 nm, being the surface plasmon absorbance peak among others. Figure 2 shows the curve in each spectrum of synthesized AgNPs absorbed in the wavelength range 380-440 nm of AgNPs of C. sesamoides and C. pentandra. The absorption spectra showed SPR and peaks at 380 nm in the case of L. taraxacifolia (Figure 2), whereas the bands for C. crepidioides were observed at 410 nm as shown in Figure 2. This peak falls within the range of specification for NPs reported by previous authors. 42,43 Fourier transform infrared spectroscopy study FTIR spectroscopy measurements were employed to recognize and identify the biological reducing functional group, which will give a hint about the likely group of organic compounds present in these wild and non-cultivated vegetables responsible for the reduction of the Ag + ions to elemental Ag 0 and the ensuing capping resulting in efficient stabilization of the AgNPs formed. 44 The FTIR spectra of the synthesized AgNPs of the   This indicated the presence of alkaloids, flavonoids, and others in this plant extract. The peaks at 1587 cm -1 correspond to C-H stretching of the aromatic compounds. As shown in Figure 3, most of these spectra proved distinctive functional groups of compounds, i.e. alkaloids, coumarins, flavonoids, and phenolic acids, which may all have had an active role in the reduction and capping of the synthesized AgNPs.

Scanning electron microscope
The scanning electron microscope identifies the surface characteristics, morphology, and the distribution of the CC-AgNPs, CS-AgNPs, CP-AgNPs, and LT-AgNPs depicted in the SEM micrograph (Figure 4), to determine the silver concentration of the NPs. AgNPs generally show a typical absorption characteristic peak at approximately 3 keV due to the surface plasma resonance phenomenon. 45 The cracked lines in the SEM micrographs ( Figure 1A-1D) would enhance laminar flow, indicating the potential of the AgNPs for toxicant removal. 46,47 The NPs synthesized by these non-cultivated vegetables were highly agglomerated except for CC-AgNPs, which displayed a scattered morphology (Figure 1). MubarakAli et al. 48 ascribes this cluster to a dehydration-induced combination of Ag NPs. However, CS-AgNPs, CP-AgNPs, and LT-AgNPs showed a trend in terms of differences in the dimensions and magnitude of the synthesized NPs. This can be accredited to the fact that the bigger and bulkier NPs can hold more Ag.

Antioxidant activity
The methanolic extracts of the four non-cultivated vegetables with their corresponding synthesized NPs were evaluated and compared employing two different assays for their antioxidant activity as shown in Table 4. The AgNPs and the methanol extract for each of these plants were evaluated for in vitro activity employing DPPH and ABTS assays. The results are expressed in terms of IC 50 (the concentration that caused 50% inhibition) and are presented in Table 4. These were obtained by in vitro method at various concentrations (100, 200, 300…500 µg/mL) of the extracts and AgNPs formed. The synthesized AgNPs of the non-cultivated vegetables and the extracts tend to display significant antioxidant activity at the dose 100 µg/mL concentration; this was noted with the positive control as well. The higher the concentration the lower the antioxidant effect that was observed, although there was a climax at 400 µg/mL as shown in Table 4. Table 4 shows that there is an obvious trend: the synthesized AgNPs displayed better activity when compared to the extracts of these plants, i.e. AgNPs from C. crepidioides, C. sesamoides, L. taraxacifolia, and C. pentandra displayed better in vitro antioxidant activity (IC 50 : 11.4, 12.4, 11.3, and 5.5 µg/mL) with the ABTS assay and (IC 50 : 15.4, 9.4, 13.3, and 6.4 µg/mL) using the DPPH assay but the methanol extracts of these plants displayed values lower than those of the former. CP-AgNPs, CC-AgNPs, and LT-AgNPs exhibited the most significant antioxidant effect against ABTS (IC 50 : 5.5, 11.3, and 11.4 µg/mL), while CP-AgNPs and CS-AgNPs displayed the most significant antioxidant activity against DPPH (IC 50 : 6.4 and 9.4 µg/mL) when compared to the positive control used, ascorbic acid (IC 50 : 4.7 µg/mL). Most of the AgNPs formed showed the most significant result at 100 µg/mL, although the positive control gave the best result at this dose as well (Table  4). Higher plants always contain constituents and substances with antioxidant effects. Flavonoids are among these naturally occurring substances that are widely renowned to exert scavenging ability against superoxide, free, and hydroxyl radicals. 49 In the present study, we assessed the antioxidant effects of the AgNPs of the non-cultivated vegetables and their methanolic extracts because of the multifaceted and complex nature of compounds in plants; the antioxidant nature of these AgNPs and their extracts cannot be studied by only a single method. As a result of this, the generally accepted assays, i.e. DPPH and ABTS methods, were used in the present study. CP-AgNPs displayed significant antioxidant activity in both assays employed, but CS-AgNPs only showed good antioxidant activity in the DPPH assay only. The DPPH and ABTS antioxidant assays proved that these neglected vegetables with their synthesized AgNPs show antioxidant activity. Bello et al. 15 examined the antioxidant effects of the leaves of L. taraxacifolia and C. pentandra (methanol extracts). These plant species displayed significant antioxidant activity when the ABTS assay was employed as compared with ascorbic acid.

Antiinflammatory activity
The methanolic extracts of the four non-cultivated vegetables with their corresponding synthesized NPs were evaluated and compared using cell-based assays for their antiinflammatory activity as shown in Table 5. The AgNPs and the methanol extract for each of these plants were evaluated for in vitro activity employing the HRBC membrane stabilization method and lipoxidase assay. The results are expressed in terms of IC 50 (the concentration that caused 50% inhibition) and are presented in Table 5. These were carried out with an in vitro method at various concentrations (100, 200, 300….500 µg/ mL) of the extract. The extract tends to display a significant antiinflammatory activity at 100 µg/mL concentration; this was noted with the positive control as well. The higher the concentration the lower the antiinflammatory effect that was seen, although there was a climax at 400 µg/mL as shown in Table 5. Table 5 shows that there is an obvious trend: the synthesized AgNPs displayed better activity when compared to the extracts of these plants, i.e. AgNPs from C. crepidioides, C. sesamoides, C. pentandra, and L. taraxacifolia displayed better in vitro antiinflammatory activity (IC 50 48.5 µg/mL) against LOXs. CC-AgNPs and CP-AgNPs exhibited the most significant inhibitory activity against HRBC (IC 50 : 32.2 and 34.7 µg/mL), while CS-AgNPs and LT-AgNPs displayed the most significant inhibitory activity against LOXs (IC 50 : 32.8 and 48.5 µg/mL) when compared to the positive control using indomethacin (IC 50 : 28.1 µg/mL). Most of the AgNPs formed showed the most significant result at 100 µg/mL, although the positive control gave the best result at this dose as well. CS-AgNPs and LT-AgNPs displayed good activity against the LOX assay employed; they could serve as good LOX inhibitors. It was very surprising that they displayed moderate activity in the other assay used. Some authors have reported the antiinflammatory activity of C. pentandra through the LOX assay. It was reported that the methanol extract of its leaves displayed inhibitory activity against LOX with an IC 50 of 102.4 µg/mL when compared with that of the positive control, 90.4 µg/mL (indomethacin). 22 This neglected vegetable's (C. pentandra) extracts exhibited inhibitory activity against LOX with an IC 50 of 53.6 µg/mL. LOXs are present in the airway and stomach epithelium, leukocytes, and gut cells, and they aid in the introduction of an oxygen molecule to the 5-position of arachidonic acid to give the intermediate (5S)hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid or 5-HETE. This is an important aspect of antiinflammatory activity in the LOX assay, hence inhibiting the biological genesis of leukotriene and 5-HETE. Hence, the search for specific inhibitors of LOX activity from medicinal plants is ongoing and imperative. LOX inhibitors, i.e. CS-AgNPs and LT-AgNPs, could possess some great advantages for the treatment of allergic rhinitis, arthritis, asthma, atherosclerosis, cancer, osteoporosis, and psoriasis. 50,51 CONCLUSION Future studies will be carried out using various chromatographic techniques, spectroscopic techniques, and mass spectrometry to isolate and elucidate the bioactive compounds in the active fractions of wild and non-cultivated vegetables. The specific receptors these active plants' extracts and their corresponding synthesized AgNPs might be acting on to elicit antiinflammatory effects will be determined. There should be in vivo testing on small mammals to verify the antiinflammatory effects of these compounds in living organisms. Because AgNPs of both C. crepidioides and C. sesamoides significantly inhibited inflammatory response, it would be interesting to assay other plants from these families for antiinflammatory activity. The phytobiological facilitated production of AgNPs from selected non-cultivated vegetables proves to be ecofriendly and successful. In the current research, it has been shown that the synthesis of AgNPs by a simple, costeffective, nontoxic, and reproducible green chemistry method allows for better antioxidant and antiinflammation worth. This study reports for the first time the synthesis, characterization, and antiinflammatory and antioxidant activities of CS-AgNPs, CP-AgNPs, and LT-AgNPs. The synthesized AgNPs were found to be stable and the FTIR evidence suggested that the phytochemicals might have played an important role in the reduction and stabilization of AgNPs. This work showed that the synthesized AgNPs from non-cultivated vegetables can find relevance and application in health, drugs, food, and environmental science. The evidence herein further confirmed their ethnopharmacological applications.