ABSTRACT

Objectives:

Increased generation of free radicals exceeding the antioxidant capacity of the host is deleterious. Thus new, potent, and safe antioxidants will be a valuable addition to the limited antioxidant arsenals available. Therefore, the antioxidant activity, cytotoxicity potential, and phytochemical constituents of the methanol extract of Spondias mombin seed (MESSM) were investigated

Materials and Methods:

2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H₂O₂) were the antioxidant assays used. The cytotoxicity of MESSM was evaluated against a rhabdomyosarcoma (RD) cell line in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide based assay. The phytochemical constituents of MESSM were identified using gas chromatography-mass spectrometry.

Results:

MESSM produced better antioxidant activity in the DPPH (IC₅₀=58.64±1.49 μg/mL) and H₂O₂ (IC₅₀=44.03±5.57 μg/mL) assays than in the NO (IC₅₀=494.55±12.68 μg/mL, p<0.0001) assay. Moreover, MESSM was nontoxic (CC50=139.6±0.54 μg/mL) in comparison to cyclophosphamide (CC50=0.97±0.03 μg/mL) against the RD cell line. The major compounds in MESSM were dodecanoic acid (22.48%), tetradecanoic acid (17.95%), n-hexadecanoic acid (15.35%), capsaicin (12.11%), and dihydrocapsaicin (5.23%).

Conclusion:

The seed extract of Spondias mombin contains nontoxic antioxidant compounds that could be explored in the pharmaceutical and cosmetics industries for the development of antioxidant agents.

INTRODUCTION

Free radicals such as reactive oxygen species (ROS) in the form of superoxide anion (O₂), hydroxyl radical, and hydrogen peroxide (H₂O₂), and reactive nitrogen species (NOS) in the form of nitric oxide (NO) radical are by-products of the human body’s metabolism and can also be generated from exogenous stimuli.¹ A physiological amount of ROS is considered to function in signal delivering while an excessive amount damages DNA, proteins, and lipids, which can induce cell death, implicated in the pathogenesis of cancer, ageing,² ophthalmological diseases,³ cardiovascular diseases,⁴ and many general neurodegenerative pathologies like Alzheimer’s disease,⁵ Parkinson’s disease,⁶ and prion disease.⁷

Excessive ROS are neutralized by a controlled and balanced complex web of antioxidant defenses in the body.⁸ Antioxidants help to minimize the effect of free radicals on biomolecules by scavenging them. However, if production of free radicals overwhelms the biological antioxidants, there will be a need for introduction of an external source of antioxidants. An external source of antioxidants offers a promising way to prevent the deleterious effect of excessive exposure to ROS.⁸ It has been reported by previous studies that phytochemicals such as flavonoids and polyphenols can help in preventing the development of chronic diseases.⁹ Moreover, the use of extracts from pomegranate, green tea, grape seed, and mushrooms as antioxidants in skin care products to prevent the clinical signs of photoaging is on the increase.^2,10 Thus, the interest in naturally occurring antioxidants in foods, cosmetics, and pharmaceutical products has significantly increased. Prolonged use of antioxidants necessitates the need for safe and effective antioxidants that the natural agents symbolize, unlike the synthetic antioxidants that have been associated with toxicity¹¹ and carcinogenic effects¹² after prolonged use.

There is a need to search for more antioxidants of natural origin that can be added to the limited arsenals of antioxidants available. Spondias mombin (S. mombin) is a fructiferous tree that is commonly found in Nigeria, Brazil, Peru, southern Mexico, Sierra Leone, Equatorial Guinea and Côte d’Ivoire.¹³ In Nigeria, it is commonly known as iyeye in the Yoruba ethnomedicine. Antidiarrheal, antimicrobial, diuretic, febrifuge, and emetic activities of the fruit, leaf, or bark of S. mombin leaf have been reported.^{14,15,16,17,18} The flower, leaf, or bark are used for wound healing and to treat stomachache and various inflammatory conditions.¹⁹ Antioxidant activity of the leaf and fruit has been reported.^20,21 Despite the information on the leaf, fruit, flower, and stem bark of S. mombin, little is known about the usefulness of its seed, which is considered a waste product. Therefore, we report the antioxidant, cytotoxicity, and phytochemical composition of the seed of S. mombin.

MATERIALS AND METHODS

Reagents

Sodium bicarbonate, sodium hydroxide, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), rutin, Folin-Ciocalteu reagent, rutin, gallic acid, ascorbic acid, sodium nitroprusside, sulfanilamide, N-naphthyl-ethylenediamine dihydrochloride, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma Aldrich Co., St. Louis, MO, USA.

Plant collection

The fresh fruit of S. mombin was collected behind the Pharmacology and Therapeutics building, University of Ibadan on 20 September 2017. Identification of the plant was done by Mr. D. P. Esimekhuai of the Botany Department, University of Ibadan (UIH-22764).

Plant extraction

The fleshy part of the S. mombin fruit was removed completely; thereafter the seed was oven dried at 40 °C and pulverized. The maceration method of extraction was employed by soaking 203 g of S. mombin seed in a mixture of methanol:water (70:30) for 72 h. The solvent was removed at reduced pressure and temperature in order to obtain the extract, which was kept at 4 °C.

Total phenolic content (TPC)

The determination of the TPC of methanol extract of Spondias mombin (MESSM) was carried out by following the procedures of a previously reported method.²² Briefly, 0.1 mL of MESSM (1 mg/mL) and 0.1 mL of Folin-Ciocalteu phenol were mixed and left for 5 min. Thereafter, 1 mL of 7% Na₂CO₃ and 1.3 mL of distilled water were added. The mixture was allowed to stand for 90 min at 29 °C. Optical density (OD) was obtained at 750 nm. The TPC was estimated in triplicate and expressed as milligrams of gallic acid equivalents (GAE) per gram of the dried seed of S. mombin.

Total flavonoid content (TFC)

The determination of MESSM TFC was done according to a previously reported method.²³ First, 0.3 mL of MESSM (1 mg/mL), 0.15 mL of AlCl₃.6H₂O (0.3 M),  0.15 mL of NaNO₂ (0.5 M), and 3.4 mL of methanol (30%) were mixed. After 5 min, the reaction was stopped with 1 mL of NaOH (1 M). The absorbance was obtained at 506 nm. The TFC was estimated in triplicate and expressed as milligrams of rutin equivalents (RE) per gram of the dried seed of S. mombin.

Antioxidant assays of MESSM

DPPH assay

A modified version of the method described by Silva et al.²⁴ was followed. Briefly, gradient concentrations of MESSM (6.25-400 µg/mL) or the standard drug ascorbic acid (0.25-16 µg/mL) were prepared in ethanol and incubated with 0.04 mg/mL DPPH (1:1.5 vol/vol) for 30 min at 29 °C in the dark. OD was obtained at 517 nm and expressed as percentage of the control.

Hydrogen peroxide assay

A previously reported method was used.²⁵ H₂O₂free radical was generated from H₂O₂ solution. Briefly, 2 mM of freshly prepared H₂O₂ (6:1 v/v) was mixed with gradient concentrations of MESSM (6.25 to 400 µg/mL) or ascorbic acid (2.5-160 µg/mL). The mixture was left in the dark for 10 min at 29 °C. Thereafter, absorbance was recorded at 230 nm.

Nitric oxide assay

Briefly, sodium nitroprusside in aqueous solution (40 mM) was added to graded concentrations (50-800 µg/mL) of MESSM (1:4 v/v) and kept in the dark at 29 °C for 2 h. Subsequently, the resulting solution was mixed with Griess reagent (1:1 v/v) and kept for 15 min in the dark at 29 °C.²⁶ Absorbance was obtained at 550 nm and the amount of NO in each of the wells was estimated from the sodium nitrite curve. Ascorbic acid at 2.5-160 µg/mL served as the standard drug.

Data analysis for antioxidant assays

OD values of MESSM or ascorbic acid in all the antioxidant assays were calculated and represented as the percentage of the negative control. Percentage scavenging activity in respect to the negative control was calculated. The 50% inhibitory concentration of MESSM (IC₅₀) was determined using linear regression in a commercial statistical package, Origin^®.

Toxicity assessment of MESSM against the rhabdomyosarcoma (RD) cell line

A published method was used.²⁷ Briefly, a ten-fold serial dilution of 1 mg/mL MESSM was prepared in culture media resulting in 0.01-1000 µg/mL. A confluent monolayer of RD cells cultured in a microtiter plate for 24 h was incubated with the graded concentrations of MESSM or cyclophosphamide (positive control) at 37 °C in 5% CO₂. After 72 h, 25 µL of 2 mg/mL MTT solution was added and further incubation was done for 1.5 h at 37 °C in 5% CO₂. Finally, 125 µL of DMSO was added to the cells and they were agitated for 15 min. Absorbance was recorded at 492 nm and expressed as percentage of the negative control. The 50% cytotoxic concentration (CC₅₀) was determined using nonlinear regression in GraphPad Prism 5^®.

Chemical composition of MESSM using gas chromatography-mass spectrometry (GC-MS)

In the identification of the phytochemical constituents of MESSM, GC-MS was used according to a previously described method.²⁸ The model of the instrument used was an Agilent Technologies 7890 GC system with a 5975 MC detector. The mobile phase was a carrier gas, helium (99.99% purity), and the column was an HP5 MS 30 m in length, 0.320 mm in internal diameter, and 0.25 µm in thickness. To identify the compounds, the retention time and fragmentation pattern were compared to the NIST library database.

Statistical analysis

The antioxidant and cytotoxicity assays were performed in duplicate and repeated in three independent experiments. The IC₅₀ or CC₅₀ values were expressed as mean ± standard error of mean. The mean IC₅₀ or CC₅₀ of the MESSM was compared to that of the standard drug using the Mann-Whitney U test and difference was significant at p<0.05.

RESULTS

The percentage yield, following the extraction of MESSM in 70% methanol, was 2.72%. The TPC and TFC of MESSM were 239.50±7.9 mg GAE/g sample and 105.3±3.6 mg RE/g sample, respectively. In addition, the IC₅₀ of MESSM in the DPPH, hydrogen peroxide, and NO scavenging assays was 58.64±1.49, 44.03±5.57, and 494.55±12.68 µg/mL, respectively (Table 1). Ascorbic acid, the standard drug used in the 3 antioxidants assays, gave an IC₅₀ of 4.31±0.26, 10.63±0.31, and 48.74±1.46 µg/mL, respectively. Moreover, MESSM and cyclophosphamide showed CC₅₀ of 139.6±0.54 and 0.97±0.03 µg/mL, respectively, on the RD cell line in the cytotoxicity assay. The GC-MS chromatogram of MESSM showed 21 distinct peaks (Figure 1) corresponding to 21 compounds (Table 2). The identified compounds can be grouped into alkanes (42.86%), fatty acids (28.57%), phenol amide (9.52%), phenolic lipids (9.52%), saponin (4.76%), and terpenoids (4.76%). Peaks 3, 7, 12, 18, and 19 represent dodecanoic acid, the most abundant (22.48%), followed by tetradecanoic acid (17.95%), n-hexadecanoic acid (15.35%), capsaicin (12.11%), and dihydrocapsaicin (5.23%). The m/z, fragmentation pattern, and structures of dodecanoic acid, tetradecanoic acid, n-hexadecanoic acid, capsaicin, and dihydrocapsaicin are presented in Figures 2, 3, 4, 5, 6.

Table 1

Figure 1

Table 2

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

DISCUSSION

The seed extract of S. mombin contains nontoxic antioxidant components. Ascorbic acid, a standard drug, had better activity than the MESSM, a crude extract. Purification of this extract might yield more potent compounds. In the present study, MESSM was 8-11 times less active in scavenging NO free radicals than scavenging the DPPH and H₂O₂ free radicals. In water, plants, the human body, food, microorganisms, and air, H₂O₂ occurs in low quantities.¹² The decomposition of H₂O₂ yields hydroxyl radicals, which can damage DNA and cause lipid peroxidation.²⁹ Similar reports on the antioxidant activity of other components of S. mombin have been published.^20,21,30 However, it appears this is the first report of the antioxidant activity of the seed of S. mombin. The seed, a waste product, has not been investigated. In the spirit of turning waste into wealth, this study was performed. In order to rule out the toxicity of MESSM, its cytotoxicity was evaluated. The MESSM was 143 times less toxic than cyclophosphamide, the standard drug against RD cells. This implies MESSM is nontoxic to RD cells. In addition, MESSM showed high total flavonoid and phenolic contents. Phenolic compounds are secondary metabolites that contain hydroxyl groups that confer them with scavenging ability. The MESSM had a higher phenolic content (>100) than some fruits such as S. mombin, guava, strawberry, pineapple, soursop, and passion fruit.^21,31

The following major compounds were identified in the seed of S. mombin: dodecanoic acid (22.48%), tetradecanoic acid (17.95%), n-hexadecanoic acid (15.35%), and phenol amides capsaicin (12.11%) and dihydrocapsaicin (5.23%). Dodecanoic acid, also known as lauric acid, showed in vitro antimicrobial activity against Propionibacterium acnes and beneficial effects in a mouse ear model of Propionibacterium acnes-induced inflammation.³² Lauric acid is one of the reagents for making soaps and cosmetics.³³ Tetradecanoic acid (myristic acid), another constituent of MESSM, is used as a dietary supplement and a flavoring agent in the food industry. It is also used in the cosmetic industry, for making toiletries, emulsifiers, facial creams, and lotions, and in the pharmaceutical industry.^34,35 Furthermore, hexadecanoic acid (palmitic acid) possesses antioxidant, anticancer, and anti-inflammatory activities.^36,37,38,39 Capsaicin and dihydrocapsaicin, other constituents of MESSM, are phenol amides, majorly found in pepper. Capsaicin and dihydrocapsaicin possess antioxidant activity.⁴⁰ Capsaicin is also used for the treatment of pain. It can be administered in topical ointments, nasal sprays, and dermal patches.⁴¹ A transdermal patch of capsaicin (Qutenza^®) was approved by the Food and Drug Administration for the management of pain due to postherpetic neuralgia.⁴² Other minor compounds in the seed of S. mombin with antioxidant activity that have been previously reported include (9Z, 12Z)-9,12-octadecadienoic acid (linoleic acid), 4-H-pyran-4-one, 2.3-dihydro-3.5-dihydroxy-6-methyl, and hexadecanoic acid-methyl ester.⁴³

CONCLUSION

The seed extract of S. mombin lacked cytotoxicity potential. It contains nontoxic antioxidant compounds that could be explored in the pharmaceutical and cosmetics industries for the development of antioxidant agents.