FTIR spectroscopic analysis and effect of Diplotaxis Acri’s lower extract on pro-in lammatory cytokines in THP-1 human macrophages

Plants and natural compounds are extensively reported for diverse biological activities, including their effects on the in lammation pathway. The annual winter herb Diplotaxis Acris (D.Acris) is found only slopes of sandy and stony valleys in the desert. This research is intended to make a contribution to the literature regarding the employment of the species of plant ethnomedicinally by undertaking FTIR spectroscopic analysis and examining several concentrations of the plant’s extract for anti-in lammatory activity in vitro with activated THP-1 human macrophages to examine its mediating effect on in lammation. Cell viability was also evaluated, and there was no severe cytotoxic effect from D.Acris extract with any of the concentrations being assessed on cells. ELISA was used to assess the pro-in lammatory cytokines and chemo kines that were produced. It has been noted that the plant extract led to a signi icant decrease in levels of the pro-in lammatory cytokines, including interleukin (IL)-1β, tumour necrosis factor (TNF)α and IL-6. Inhibition of pro-in lammatory cytokines indicates the anti-in lammatory trends ofD.Acris. This plant can be investigated further by isolation of natural compounds from the extract and effects of these compounds can be evaluated on the same in lammatory markers to show the main active constituent responsible for anti-in lammatory activities.


INTRODUCTION
Diplotaxis DC is a genus of Brassiceae, the most valuable (economically) branch of the Brassicaceae family (Hussein et al., 2017). There are between 27 and 36 species, and their habitat ranges from the Mediterranean areas of Europe to Northwest India Larsen and Boulos (1999). The winter herb Diplotaxis Acris (D. acris), Figure 1 commonly called salad rocket (Grillo et al., 2012), grows on the slopes of sandy and stony valleys in the desert; (Oueslati et al., 2015) it is similar in taste to Eruca Sativa Mill. (Osman et al., 2019) and favoured by grazing animals (Shaye et al., 2020). As well as being economically vital, the plant has antioxidant properties (Atta et al., 2004). Locally (in Saudi Arabia) named as gahag, it is an annual glabrescent plant. Identi ication of the plant is possible through its leshy leaves and substantial purple lowers (Al-Jaber et al., 2011).
Chemically analyzing the plant demonstrated that it holds several chemical compounds including quercetin 7-rhamnoside-3 ′ -methyl ether, kaempferol 3-O-glucoside, luteolin 7rhamnoside, isorhamnetin 3-O-glucoside, luteolin 7-diglucoside, apigenin, apigenin 7-diglucoside and quercetin (Hussein et al., 2017). Clinical studies also showed that fatty acids are present alongside Figure 1: Diplotaxisacris sterols such as β-sitosterol, α-linolenic acid, and benzyl benzoate; alkaloid choline chloride is also present (Hussein et al., 2017). Additionally, with doses between 200 and 400 mg/kg D.Acris was shown to have peripheral and central antinociceptive activity, thus giving it antioxidant properties  demonstrated that the high segments of D.Acris have anti-diarrheal properties, shown from an evaluation of diarrhoea induced by castor oil, rat gastrointestinal movement, and newly slaughtered rabbit duodenal motility. Doses between 200 and 400 mg/kg have been shown to have a notable anti-diarrheal in luence on diarrhoea induced by castor oil in rats (Atta et al., 2005).
As far as can be ascertained, no previous detailed mechanistic research has been undertaken into the anti-in lammatory properties of the D.Acris lower; in the light of new global initiatives to discover new sources of medicines and food, this research intends to review and analyze the pharmacological properties of this plant in terms of its ability to act as an anti-in lammatory.
Cytokines are elemental to chronic in lammatory disease pathogenesis (Calamia, 2003), with several factors, including oxidative stress, modulating their secretion (Taylor et al., 2004). Tumour necrosis factor-alpha (TNF-α) is a cytokine that is multifunctional (Aggarwal, 2000), regulating the way activated leukocytes grow, reproduce, are differentiated, and become viable (Dempsey et al., 2003). This cytokine is also a trigger for cells to release other cytokines, chemokines, and in lammatory mediators (Hsu et al., 1995); it has demonstrable antiviral and antimicrobial properties (Wajant et al., 2001). This research is intended to examine the direct in luence of the extract of the D.Acris lower concerning pro-in lammatory cytokine secretion.

Analysis of plant extract on Fourier Transform Infrared Spectrophotometer (FTIR)
FTIR may be the most potent form of identi ication for chemical bonds (functional groups) within compounds. The chemical bond has a character- Through an interpretation of the spectrum of infrared absorption, a molecule's chemical bonds can be identi ied. For this FTIR analysis, for plant material, a dry powder of a variety of solvent extracts was employed. A 100 mg KBr pellet was used to encapsulate 10 mg of the dried extract powder for the preparation of translucent sample discs. The FTIR spectroscope was loaded with the powder sample; the spectroscope has a resolution of 4 cm −1 and a scan range of 400 and 4000 cm −1 .

Cell culture
Human monocytic cell (THP-1) lines were cultured using the RPMI 1640 medium in an atmosphere holding 5% CO 2 and controlled humidity at 37 • ; 10% FBS, 100 µg/mL streptomycin and 100 IU/mL penicillin were supplemented in the medium along with 2mM pyruvate and 2mM glutamine. A 24-well plate was seated with monocytic cells; these underwent differentiation into macrophagelike cells through being stimulated for 72 hours with phorbol myristate acetate (40 ng/mL) Parrado et al. (2017) . Cells created in this way are named THP-1 macrophages.

Cytotoxicity/cytokine production assay
Incubation of the (5.105 cells/mL) macrophages was undertaken using a variety of concentrations (0.01, 0.1, 1 and 10 µg of D. acris extract), some of which were activated with LPS (1 µg/mL) for 5 hours (for determination of TNF-α), some for 24 hours (for determination of IL-1β) or 48 hours (for determination of IL-6). Collection and storage (at -70 • C) of supernatants for future analysis was performed. An assessment of cell cytotoxicity was made by determining the release levels of lactate dehydrogenase (LDH) employing a CytoTox 96 LDHcytotoxicity kit following the directions of the manufacturer. The cytotoxicity percentage was assessed using the following equation: Determination of the cytokine levels was made using ELISA commercial kits (Mabtech, Inc. Cincinnati, OH, United States) following the directions of the manufacturer. An ELISA reader was used to measure ODs. The concentrations of cytokines were determined using interpolation with a standard curve. The results were expressed as fold changes (mean ± SEM) against a control which had no LPS (given a value of 1); these results came from between three and four independent experimental processes. Figure 2 shows the FTIR spectrum for lower extracts that underwent preparation in ethanol of D.Acris. Table 1 shows the data regarding peak values and likely functional groups (found through FTIR analysis) existing in the ethanol-prepared lower extracts of D.Acris.

CELL VIABILITY
For the evaluation of the cytotoxic effects of D.Acris extract, the macrophages underwent stimulation using a variety of extract concentrations, with or without LPS. There was no severe cytotoxic effect from D.Acris extract with any of the evaluated concentrations, whether in basal conditions (A) or in lammatory conditions (B).

Effect on LPS activated THP-1 macrophages
For the investigation of the in luence of D.Acris extract in terms of modulating in lammatory responses, ELISA was used to analyze proin lammatory cytokine levels.
When LPS was not present, cytokine levels were around those of the controls Figure 3. As predicted, adding the proin lammatory stimulus of LPS led to a signi icant increase in the levels of all cytokines. Nevertheless, if the cells were cultured with D.Acris extract present (10 µg/mL ethanolic extract), the rise in cytokine levels was signi icantly reduced, with the values obtained being on a par with those found in basal conditionsFigure 4.

CONCLUSION
The results of this research lead to the conclusion that D.Acris extract and its phytoconstituents can operate as a provider of immunomodulators. The different functional groups found within the extract appear to show that cellulose, glycogen, lipids, phosphates, amides, amino acids, glycogen, carotenoid, and carbohydrates can be present in this plant. One of the functional groups that were found in the extract was the OH group, which has the capability of forming hydrogen bonding capacity; this group's presence in the D.Acris ethanol extract is likely to indicate that the extract has excellent potential for the inhibition of in lammatory activities.

ACKNOWLEDGMENT
The NFA would like to thank XYZ University for its provision of research facilities and iscal support for this research.