One single method cannot precisely assess the antioxidant capacities of plant extracts due to the complex nature of the different phytochemical classes present in plants. In the present work, the FRAP, ABTS, DPPH• and β-carotene assays were used to test the antioxidant activities of Acridocarpus orientalis extracts. The results of the four assays are summarized in Table 1.

The total phenolic content of the medical plants extracts was measured with the Folin-Ciocalteu reagent assay and the results is shown in Table 2. The values varied from 184.24 to 149.23 mg gallic acid/g of dry extract. Acridocarpus-Al Ain exhibited the highest amount of total phenolics (184.24 mg gallic acid equivalent/g). Phytochemicals have been of great interest as a source of natural antioxidants used for health promotion, food preservation, food flavoring and cosmetics since they are safer for consumption and more environmentally friendly than their synthetic counterparts.

The linear correlation coefficients between the antioxidant capacity (measured by FRAP, DPPH, ABTS and β-carotene assays and total phenolic content of the four fractions are shown in Table 3. R² values varied between 0.99 for β-carotene assay and 0.87 DPPH assays, which leads to believe that the presence of phenolic compounds contributes substantially to the antioxidant activity of the tested extracts, especially in the β-carotene assay. The above results are in agreement with previous reports that showed a liner correlation between the total phenolic content and the reducing antioxidant capacity of some plant extracts [16,20].

LOX catalyzes dioxygenation of polyunsaturated fatty acids to yield cis,trans-conjugated diene hydroperoxides. Results for LOX inhibitory activity (IC₅₀) are shown in Figure 2. The Acridocarpus-Al Ain extract showed a strongest ability (p < 0.05) to inhibit LOX activity (IC₅₀ = 50.58 µg/mL) in relation to the Acridocarpus-Oman extract (IC₅₀ = 58.61 µg/mL). It is important to note that that both crude ethanol extracts possessed significantly lower (p < 0.05) LOX inhibitory activity than that of an NDGA positive standard (IC₅₀ = 4.87 µg/mL). Finally, LOX inhibition of Acridocarpus orientalis extracts were higher than those for other common plants such as Thespesia lampas (600 µg/mL) [21]. The results reported here suggest that Acridocarpus orientalis has potentially high anti-LOX effect, which might be related to the polyphenolic content and antioxidant property of the extract.

HDAC inhibitors (HDACi) are becoming a prominent therapeutic target for treating a wide spectrum of human diseases, including cancer. HDAC inhibitors arrest cell growth and lead to apoptosis in tumor cells [6]. In previous studies, Cardiotoxicity, poor pharmacokinetic and non-selectivity were repeatedly reported for different HDACi and considered as the major drawback for the future development of synthetic HDACi [8,22]. For this reason, targeting natural inhibitors of HDAC is a promising therapeutic target for treating wide spectrum of human diseases. Curcumin, a phenolic compound isolated from turmeric, and sulforaphane, an isothiocyanate isolated from broccoli, have recently been identified to possess anticancer properties via HDACi activity [22,23]. In the present work, the ethanolic extract of Acridocarpus orientalis showed a high ability to inhibit HDAC activity (IC₅₀ = 93.28 µg/mL) in Al Ain species in relation to the Oman plant extract (IC₅₀ = 102.5 µg/mL) and this inhibitory effect was concentration dependent (Figure 3). This inhibitory effect of plant extract could be attributed to a direct ionic interaction of the active ingredients of plant with the active zinc site of HDAC enzyme where the classical HDACs are zinc-dependent enzymes bearing a highly conserved catalytic domain with a zinc ion [6].

Ascorbic acid, ferric chloride, Folin-Ciocalteu reagent, dibutylhydroxytoluene (BHT), 2,4,6-tripyridyltriazine, gallic acid, sodium carbonate, 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,4,6-tripyridyl triazine, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), β-carotene (type I ≥ 93%) Trolox, soybean LOX and NDGA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). HDAC colorimetric assay kit was purchased from Millipore Corporation (Temecula, CA, USA). All other chemicals were purchased from local commercial suppliers.

Fresh Acridocarpus orientalis samples were collected from a dry river, on Jebel Hafeet mountain near Al Ain, UAE (N 24.19, E 55.62) and from Nizwa mountain in Oman (N22.9, E57.5). The collected samples were identified and representative specimens were deposited at the Biology Department Herbarium, Faculty of Science, UAE University.

An air-dried and ground aerial part sample of AO (10 g) was extracted with 70% (v/v) aqueous ethanol (200 mL). The mixture was macerated for 72 h at 4 °C. The resulting compound was then filter dried under reduced pressure in a rotary evaporator at 40 °C. An aqueous ethanol crude extract was generated. This crude extract was weighed, dissolved in 50% ethanol (typically 30 mg/mL) and kept at −20 °C for further analysis.

Total phenolic content was assessed using the method reported by Singleton et al. [24] using the Folin-Ciocalteu reagent. For a typical plant extract, a sample of the residue obtained from the crude extract described in the section above (100 µL) was thoroughly mixed with the Folin-Ciocalteu reagent (200 µL) and de-ionized water (2 mL). An incubation period at room temperature for 3 min followed. After incubation, a sample of 20% aqueous sodium carbonate (w/w, 1 mL) was added to the mixture. After one hour of incubation at room temperature, the total polyphenols were determined by measuring the absorbance of the resulting substance at 765 nm with a PerkinElmer, Lambda 25 UV/VIS spectrophotometer. Values were expressed in milligrams of gallic acid equivalent per gram dry weight of plant species extract. Data presented here are averages of three replicates.

Lipoxygenase (EC 1.13.11.12 type 1-B) (LOX) was assayed according to the method reported by Wu [26]. A mixture of a solution of sodium borate buffer (1 mL, 0.1 M, pH 8.8) and soybean LOX (10 µL, final conc. 8,000 U/mL) was incubated with plant species extract sample (10 µL) in a 1 mL cuvette at room temperature for 5 min. The reaction was initiated by the addition of linoleic acid substrate (10 µL, 10 mmol). The absorbance of the resulting mixture was measured at 234 nm over time at a rate of one measurement/min (3 readings). Inhibition of LOX was assessed using the following equation: % Inhibition = 100 × (absorbance of the control − absorbance of the sample)/absorbance of the control)

The effective concentration (µg/mL) at which LOX activity is inhibited by 50% (IC₅₀) was represented in a graph. Nordihydroguaiaretic acid (NDGA) was used as a positive standard.

HDAC inhibitory activity of Acridocarpus extracts was measured with HDAC Colorimetric Assay Kit (Millipore Corporation, Catalog number: 17-374). Plant extracts and trichostatin A, an inhibitor of HDAC, were mixed with Hela nuclear extract that contains a variety of HADC enzymes and has HDAC activity. HDAC colorimetric substrate was added to inhibitor and Hela nuclear extract mixture. A color is developed after a one hour treatment with the lysine developer. Absorbance at 405 was measured by micro plate reader model.

% Inhibition = 100 × (absorbance of the Hela nuclear extract − absorbance of the sample)/absorbance of the Hela nuclear extract)

The effective concentration (µg/mL) at which HDAC activity is inhibited by 50% (IC₅₀) was determined graphically.

Reported data are expressed as means ± SEM. Correlation analysis of antioxidants and the total phenolic content was conducted using SPSS (SPSS Inc., Chicago, IL, USA). When significant differences were detected, an analysis of differences between the means was performed by using Tukey’s HSD multiple comparison tests. Significance levels were set at p < 0.05.