Many plant species represent a large source of structurally new compounds that might serve as leads for the development of new drugs, nutraceuticals and functional foods. Much of the therapeutic activity of plants is due to their biologically active polyphenolic substances, mostly flavonoids and phenolic acids, which possess antioxidant, anti-lipoxygenase and anticancer activities [1
]. In developing countries and particularly the Gulf Countries, large segments of the population still rely on folk medicine to treat serious diseases including cancers and various types of inflammations.
Reactive oxygen species (ROS) induce oxidative damage to biomolecules like lipids, nucleic acids, proteins and carbohydrates. This damage causes the onset of many diseases such as rheumatoid arthritis, cirrhosis, arteriosclerosis, diabetes and cancer [3
]. ROS also affect food quality. Interest in finding naturally occurring antioxidants for use in food preservation, flavoring, cosmetics, and in health promotion to replace synthetic antioxidants, that are being restricted due to their carcinogenicity [2
] has increased noticeably. In addition, ROS propagate inflammation by stimulating release of cytokines and activation of enzymes such as lipoxygenases (LOXs) from inflammatory cells. LOXs are the key enzymes in the biosynthesis of leukotrienes from fatty acids producing active lipid metabolites. LOX is involved in provoking several inflammation-related diseases such as arthritis, asthma, cardiovascular, cancer and allergic diseases [4
]. For this reason, targeting inhibitors of LOX is a promising therapeutic target for treating wide spectrum of human diseases.
Histone deacetylases (HDACs) are becoming a prominent therapeutic target for treatment of cancer and other diseases [6
]. HDAC inhibitors (HDACI) represent a novel class of targeted drugs which alter the acetylation statues of several proteins. These agents, modulating both chromatin structure through histone acetylation, and the activity of several non-histone substrates, are able to determine changes in gene transcription and to induce a plethora of biological effects ranging from cell death induction, to angiogenesis inhibition or modulation of immune responses [7
]. The shortcomings of HDACs are instability and toxicity [8
]. For this reason, targeting natural inhibitors of HDAC is a promising therapeutic target for treating a wide spectrum of human diseases
(AO), belonging to the Malpighiaceae family, is widespread in tropical Africa, Asia and the Mediterranean region and in the sandy plains in the Western Gulf countries. Currently, it is being cultivated in the greenhouse and under laboratory conditions [10
]. Acridocarpus socotranus
is commonly traditionally used in Yemen for the treatment of headaches and muscle pain [11
]. The leaves and bark of Acridocarpus chloropterus
in Tanzania have been reported to have antiplasmodial, anti-trypanosomal and anti-leishmanial activities [13
]. Several species of Acridocarpus
are still used traditionally all over the World as folk medicines, and more specific research to justify this is essential.
Based on the above rationale, the objective of this research focused on the quantitative determination of the phenolic content, antioxidant, anti-lipoxygenase and anti-HDAC activities of the aqueous ethanol extract of AO obtained from Al Ain and Oman. The antioxidant potential of AO was assessed in comparison with the scavenging power of the two stable nitrogen–centered radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical (ABTS•+). The reducing power of antioxidants was assessed by the ferric reducing antioxidant power (FRAP) assay as well as anti-bleaching of β-carotene activity. LOX inhibitory activity of Acridocarpus extracts was also measured. Finally, HDAC inhibition activity of Acridocarpus extracts was measured with a HDAC Colorimetric Assay Kit (Millipore Corporation).
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.
3.2. Plant Materials
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.
3.4. Total Phenolic Content Determination
Total phenolic content was assessed using the method reported by Singleton et al
] 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.
3.5. Estimation of Total Antioxidant Activity
3.5.1. FRAP assay
The reducing power of antioxidants by the decrease of the ferric ions to the ferrous ions constitutes the basis of the FRAP assay. A blue colored ferrous-tripyridyltriazine complex is formed, as per the method reported by Nenadis et al
]. The FRAP reagent was freshly prepared by mixing 2,4,6-tripyridyltriazine (10 mM, 1.0 mL) and ferric chloride (20 mM, 1.0 mL) in acetate buffer (0.25 M, 10 mL, pH 3.6). Samples of plant species extract (50 µL) were added to the FRAP reagent (3.0 mL). The tests were carried out in triplicates. After a period of 8 min incubation at room temperature, the absorbance was measured at 593 nm. A calibration curve of ascorbic acid was developed to quantitatively determine the antioxidant capacity of the plant extracts expressed as mmol ascorbic acid equivalent per gram of dry extract.
3.5.2. ABTS Assay
The reduction of the blue-green 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS•+
) by antioxidants to its original colorless ABTS form is the basis of the ABTS assay. The ABTS•+
is decolorized by antioxidants according to their antioxidant content [25
]. A mixture of ABTS (10 mmol) and hydrogen peroxide (28.3 µmol) in acetic acid-sodium acetate buffer (30 mmol, pH 3.6, total volume of 2.0 mL) was quickly mixed with the plant species extract or a standard compound (100 µL) in a test tube. For positive referencing, Dibutyl hydroxytoluene (BHT) was used. The content of the tube was mixed and allowed to stand for 6 min and the absorbance was measured at 660 nm. Inhibition of free radical scavenging activity was calculated using the equation:
% Inhibition = 100 × (absorbance of the control − absorbance of the sample)/absorbance of the control
EC50 value (µg/mL) is the effective concentration at which ABTS•+ is scavenged by 50%. A calibration curve of ascorbic acid was established, the antioxidant content of the plant species extracts were then expressed as mmol ascorbic acid equivalent per gram of dry plant extract.
3.5.3. DPPH• Radical Assay
The DPPH stable radical scavenging capabilities of plant extract was determined according to a standard procedure reported by Nenandis et al.
]. Methanolic solution of DPPH radical (3.8 mL, 60 µg/mL) was quickly mixed with the plant extract (200 µL) in a test tube. BHT was used as a positive standard. The contents of the tube were mixed and rested for 30 min. The absorbance was then measured at 517 nm. Inhibition of free radical scavenging activity was calculated using the equation:
% Inhibition = 100 × (absorbance of the control − absorbance of the sample)/absorbance of the control.
EC50 value (µg/mL) is the effective concentration at which DPPH• radicals are scavenged by 50%. A calibration curve of ascorbic acid was developed. The antioxidant content of the plant extracts were expressed as mmol ascorbic acid equivalent per gram of dry extract.
3.5.4. β-Carotene Bleaching Assay
The prevention of β-carotene bleaching by plant extracts was assessed according to the procedure of Lim et al
]. A sample of β-carotene solution (1.0 mL, 200 µg/mL in chloroform) was mixed with of linoleic acid (200 µL) and Tween 20 as emulsifier (200 µL). The mixture was evaporated to remove chloroform in a rotary evaporator at 40 °C. Deionized water (100 mL) was added slowly to form an emulsion. Portions of β-carotene/linoleic acid emulsion (3 mL each) were mixed in test tubes with 200 µL of various plant extract concentrations. The control was a 200 µL of 50% of methanol in 3.0 mL of the above emulsion. As a positive standard, BHT was used. After an incubation period of 120 minutes at 45 °C, the absorbance of the samples, standards and control were measured at 470 nm. Inhibition of free radical scavenging activity was calculated using the equation:
% Inhibition = 1 − (absorbance of the control at time zero − absorbance of the control after 120 min)/absorbance of the sample at time zero − absorbance of the sample 120 min) × 100
EC50 value (the effective concentration at which bleaching of β-carotene is prevented by 50% µg/mL) was determined graphically.
3.6. LOX Inhibitory Assay
Lipoxygenase (EC 184.108.40.206 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.
3.7. HDAC Inhibition Activity Screening
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.
3.8. Statistical Analysis
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.