Phenolic constituents, anti-radicals, and enzymes inhibitory potentials of Brachystegia eurycoma seeds: Effects of processing methods

ABSTRACT

Impact of roasting and boiling on the phenolic constituents, anti-radicals, xanthine oxidase (XO), and angiotensin 1-converting enzyme (ACE) inhibitory potentials of Brachystegia eurycoma seeds (BES) was evaluated. Raw BES contained caffeic acid, ellagic acid, rutin, and quercetin as the predominant phenolics, and strongly scavenged DPPH* and ABTS*⁺, and inhibited XO and ACE. Both roasting and boiling resulted in decrease in the levels of phenolics and bioactivities of the seeds, but roasted seeds retained higher levels of the phenolics and the bioactivities than the boiled seeds. Roasting could be recommended as a valuable processing method for the retention of the phenolics and bioactivities of BES.

KEYWORDS:

• Brachystegia eurycoma seeds
• Phenolic compounds
• Anti-radicals
• Xanthine oxidase
• Angiotensin 1-converting enzyme

Introduction

Brachystegia eurycoma Harms, belonging to the Caesalpinoideae family, is an underutilized legume that grows mostly in the tropical rain forest of West Africa. It is regarded as an economic tree crop due to its wide range of uses, including in food and ethnomedicine^[1] and as a source of timber.^[2] In West Africa, especially in southeastern part of Nigeria, many rural dwellers use the edible seeds flour as a natural thickening agent in traditional soups.^[1,3] The seeds flour improves the consistency and flavour of traditional soups. Previous studies on the nutrients composition of the seed indicated that it is a rich source of nutrients supplement.^[3,4]

In the rural areas of West Africa where B. eurycoma seeds and other underutilized legume seeds are used to thicken traditional soups, it has been observed that there are fewer incidences of nutritionally related diseases such as obesity, cardiovascular diseases, diabetes, and protein malnutrition.^[5] Ndukwu^[6] further noted that consumption of the B. eurycoma seeds helps in softening bulky stools and could protect against colorectal cancer. A recent in vitro study reported that the extract of B. eurycoma seeds flour inhibited some enzymes (α-amylase, α-glucosidase, and aldose reductase) that are associated with type 2 diabetes.^[7] In a related study, the seeds flour was reported to be rich in flavonoids and phenolic acids and to display antidiabetic activity in type 2 diabetic rats.^[1]

The various health benefits of legume seeds and other plant foods have been attributed to their bioactive compounds such as flavonoids and phenolic acids.^[1,8] Hence, phenolic-rich extracts of some plant foods have been reported to be useful for the management of gout and hypertension through the inhibition of xanthine oxidase (XO) and angiotensin 1-converting enzyme (ACE), respectively, and scavenging of free radicals.^[9,10] XO (EC 1.1.3.22) catalyzes the last two reactions in the catabolism of purine nucleotides, in which hypoxanthine is first converted to xanthine, and finally to uric acid; with a concomitant reduction of molecular oxygen to superoxide, a reactive oxygen species (ROS).^[11] Excessive activity of XO leads to over-production of uric acid, and consequently, hyperuricaemia. Subsequently, the deposition of monosodium urate monohydrate crystals in tissues of the body, particularly the joints, eventually results in gout.^[12] When in excess, the ROS and other uric acid-derived radicals generated from XO-catalyzed reactions can activate the renin/angiotensin pathway,^[13] thereby leading to the development of cardiovascular diseases such as hypertension.^[14]

In the renin/angiotensin pathway, ACE catalyzes the cleavage of angiotensin I to angiotensin II. The Angiotensin II produced is a vasoconstrictor that stimulates the secretion of aldosterone, and inactivates bradykinin, a vasodilator and hypotensive peptide, thereby raising the blood pressure.^[15] Hence, overactivity of ACE can cause the development of hypertension.^[16] Studies in different human populations have revealed a positive correlation between hypertension and the incidence and prevalence of gout.^[17]

One of the important clinical approaches for the treatment of gout and hypertension is the inhibition of XO and ACE, respectively.^[9,10] Chemically synthesized inhibitors of XO (e.g. allopurinol) and ACE (e.g. captopril) available for the treatment of each of these two diseases,^[18,19] however, have certain adverse effects. For example, XO inhibitors are associated with the risk of developing hypersensitivity syndrome, which is characterized by side effects such as hepatic dysfunction, renal impairment, rashes, and fever.^[20] Similarly, ACE inhibitors have been associated with such adverse effects as cough, skin rashes, proteinuria, and hypotension.^[21] To address the adverse effects of the synthetic inhibitors and the high cost of buying them, research efforts have been intensified recently in finding natural inhibitors of these enzymes.

Traditionally, B. eurycoma seeds are processed in various ways, including boiling in water and roasting. After any of these thermal treatments, they are shelled and ground into flour, which is then used to thicken and flavour traditional soups.^[22] However, processing methods are known to affect the levels of phenolic compounds in various dietary sources and their health-promoting bioactivities.^[23,24] Hence, this study was designed to evaluate the effect of two processing methods (roasting and boiling) on the phenolic constituents, anti-radicals and enzymes (XO and ACE) inhibitory potentials of B. eurycoma seeds.

Materials and methods

Chemicals and reagents

All chemicals were of analytical grade. Methanol, acetonitrile, acetic acid, gallic acid, caffeic acid, and chlorogenic acid were purchased from Merck (Darmstadt, Germany). Quercetin, rutin, luteolin; DPPH, l-ascorbic acid, ABTS, Trolox, xanthine, allopurinol, rabbit lung ACE, captopril and hippuryl-histidyl-leucine were bought from Sigma Chemical Co. (St. Louis, MO, USA).

Samples collection and preparation

About 1.5 kg of dry B. eurycoma seeds sample was purchased at Bodija market in Ibadan, Oyo State, Nigeria. The seeds were authenticated at the Herbarium in Department of Botany, University of Ibadan, Nigeria. Subsequently, the seeds were sorted and later divided into three portions of about 500 g each. The first portion served as raw (untreated) B. eurycoma seeds. The second portion was roasted in a thermostatically regulated hot-air oven at 150 ± 2°C for 20 min; while the third portion was boiled in tap water at 1:10 (seeds:water) ratio in a thermostatically regulated water bath (100 ± 0.5°C) for 20 min. After the boiling time, the seeds were placed on filter paper at room temperature for 15 min to drain out the water retained on the seeds. Thereafter, the boiled seeds were oven-dried at 40°C for 48 h to attain a constant moisture content of about 10%. Each sample portion was manually shelled, and later milled into flour of 0.5 mm particle size. The flour samples were packed in an airtight container and stored at 4°C for further analysis.

Preparation of polyphenolics-rich extracts

Polyphenolics-rich extracts of the raw, roasted, and boiled B. eurycoma seeds flours were prepared according to the method described by Kuo et al.^[25] In brief, 100 g portion of each sample was extracted with 300 mL absolute methanol at 50°C for 3 h in three successive times. Each sample was filtered with Whatman (No. 2) filter paper, and the extract was collected, following each successive extraction. Thereafter, the extract was partitioned with 200 mL of hexane in a separatory funnel, and the aqueous phase was further extracted with 180 mL of ethyl acetate for three successive times. The resulting extract of each sample was dried in a rotary evaporator at 45°C under reduced pressure. The residue obtained after drying of each sample was used for the various analyses.

Quantification of phenolic acids and flavonoids by HPLC-DAD

Phenolic acids and flavonoids constituents of the extracts were quantified using a reverse phase high-performance liquid chromatography (HPLC-DAD) system (Shimadzu, Kyoto, Japan), equipped with a Shimadzu Prominence Auto-sampler (SIL-20A) and Shimadzu LC-20AT reciprocating pumps connected to a DGU 20A5 degasser with a CBM 20A integrator, SPD-M20A diode array detector, and LC solution 1.22 SP1 software. Each extract was injected by means of the auto-sampler at a concentration of 10 mg/mL. Chromatographic separations were carried out using Phenomenex C₁₈ column (4.6 mm × 250 mm × 5 μm particle size). The mobile phase was HPLC grade water with 1% acetic acid (v/v) (solvent A) and HPLC grade acetonitrile (solvent B) at a flow rate of 0.6 mL/min and injection volume 40 μL. The gradient programme composition was 5% solvent B reaching 15% at 10 min; 30% solvent B at 25 min, 65% solvent B at 40 min, and 98% solvent B at 45 min, followed by 50 min at isocratic elution until 55 min. At 60 min the gradient reached the initial conditions again, following the method described by Adefegha et al.^[26] with slight modifications. The extracts and mobile phase were filtered through 0.45 μm membrane filter (Millipore) and then degassed by ultrasonic bath before use. Stock solutions of standards references were prepared in the acetonitrile: water (1:1, v/v) at a concentration range of 0.025–0.400 mg/mL. Quantifications were carried out by integration of the peaks using the external standard method at 254 nm for gallic acid and ellagic acid; 327 nm for caffeic acid; and 366 nm for quercetin, rutin, and luteolin. The chromatography peaks were confirmed by comparing their retention time with those of reference standards and by DAD spectra (200–600 nm). Calibration curve for gallic acid: Y = 16745x + 1329.8 (r = 0.9998); caffeic acid: Y = 12546x + 1165.3 (r = 0.9999); ellagic acid: Y = 11784x + 1094.5 (r = 0.9995); rutin: Y = 15083x + 1193.4 (r = 0.9997); luteolin: Y = 13749x + 1285.1 (r = 0.9999); and quercetin: Y = 11645x + 1425.9 (r = 0.9998). All chromatographic operations were carried out at ambient temperature and in triplicate.

Limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation (SD) of the responses and the slope using three independent analytical curves, as defined by Fernandes et al.^[27] LOD and LOQ were calculated as 3.3 and 10 σ/S, respectively, where σ is the SD of the response and S is the slope of the calibration curve.

DPPH* scavenging assay

The ability of each extract to scavenge DPPH* was determined according to the method reported by Cervato et al.^[28] A portion of 1.0 mL of various concentrations (15, 30, 45, and 60 μg/mL) of the extracts was mixed with 3.0 mL of DPPH* solution (60 µM) in test tube. The test mixture was kept in the dark for 30 min, and thereafter the absorbance was read at 517 nm in a UV/visible spectrophotometer. A reference test (with methanol in the place of the extract) and a positive control test (with ascorbic acid solution in the place of the extract) were included in the assay. The ability of each extract to scavenge DPPH* was subsequently calculated as % scavenging ability and expressed as half-maximal scavenging concentration (SC₅₀). The SC₅₀ was obtained from the non-linear regression equation of the % scavenging ability versus extract concentration curve.

where A517_reference is the absorbance of the reference test, and A517_sample is the absorbance of the test containing the extract.

ABTS*⁺ scavenging assay

The ability of each extract to scavenge ABTS*⁺ was according to the method described by Re et al.^[29] ABTS*⁺ reagent was prepared by mixing equal volume of 7 mM ABTS*⁺ and 2.45 mM K₂S₂O₈ aqueous solutions in amber reagent bottle, and incubating same in the dark at room temperature for 16 h. Thereafter, the absorbance of the reagent was adjusted to 0.7 ± 0.02 with 95% ethanol at 734 nm. A portion of 2.0 mL of the ABTS*⁺ reagent was mixed with 0.2 mL of the extract, and the mixture was kept in the dark at room temperature for 15 min. Subsequently, the absorbance was read at 734 nm in a UV/visible spectrophotometer. The ABTS*⁺ scavenging ability of each extract was later calculated from a standard curve prepared using Trolox, and expressed as Trolox equivalent antioxidant capacity (TEAC).

Preparation of rats’ liver homogenate for XO inhibition assay

Four adult male Wistar strain albino rats weighing of 200 ± 10 g were used to prepare liver homogenate that served as the source of XO for the XO inhibition assay. The rats were acquired from the experimental animal breeding unit of the Department of Veterinary Medicine, University of Ibadan, Nigeria. The rats were given humane treatment by following the guidelines specified in the public health service policy on humane care and use of laboratory animals of the National Academy of Science, USA.^[30] The rats were initially allowed to acclimatize under ambient laboratory conditions for 7 days, during which they fed on commercial rodent feed and table water ad libitum. Subsequently, liver homogenate was prepared following the method previously described by Nakamura et al.^[31] Briefly, the rats were anaesthetized mildly with ether and then sacrificed. The liver tissue was quickly harvested, washed in ice-cold 0.15 M KCl, and then blotted dry with filter paper. One gram portion of it was homogenized in 9 mL ice-cold 50 mM Tris–HCl buffer (pH 7.4) containing 1 mM ethylene diamine tetraacetic acid. Thereafter, the homogenate was sonicated twice under ice-cold condition for 30 s and centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was decanted and used immediately as the source of the XO for the XO inhibition assay.

XO inhibition assay

The method reported by Umamaheswari et al.^[32] was followed to determine the ability of each extract to inhibit XO, with slight modification. Briefly, 300 μL of 50 mM sodium phosphate buffer (pH 7.5), 100 μL of various concentrations (20, 40, 60, and 80 μg/mL) of each extract in dimethyl sulphoxide (DMSO), 100 μL of the freshly prepared liver tissue homogenate, and 100 μL of distilled water were mixed in test tube. The mixture was pre-incubated at 37°C for 15 min, and thereafter 200 μL of freshly prepared 0.15 mM of xanthine solution (substrate) was added. The mixture was incubated at 37°C for 30 min, following the reaction was terminated with the addition of 200 μL of 0.5 M HCl. Subsequently, the absorbance was read at 295 nm in a UV spectrophotometer against a reagent blank containing the phosphate buffer in the place of the liver tissue homogenate. Allopurinol was used as a positive control, while a reference test containing 100 μL of DMSO in the place of the extract was included in the assay in order to obtain the maximum uric acid formed under the experimental conditions. The percentage inhibition of XO by each extract was calculated, and the IC₅₀ was obtained from the non-linear regression equation of the % inhibition versus extract concentration curve.

where A295_reference is the absorbance of the reference test without the extract, and A295_sample is the absorbance of test extract.

ACE inhibition assay

The method reported by Cushman and Cheung^[33] was followed to determine the ability of each extract to inhibit ACE. Briefly, 50 μL of various concentrations (15, 30, 45, and 60 μg/mL) of the extract was mixed with 50 μL of ACE solution (4 mU/mL), and the resulting mixture was incubated at 37°C for 15 min. Next, 150 μL of 8.33 mM of the substrate (hippuryl-histidyl-leucine) in 125mM Tris–HCl buffer (pH 8.3) was added to the reaction mixture, and this was incubated at 37°C for 30 min. Thereafter, the reaction was terminated with the addition of 250 μL of 1 M HCl to the mixture, and the hippuric acid (HA) produced was extracted with 1.5 mL of ethyl acetate. The ethyl acetate layer of the mixture was separated by centrifugation, and 1.0 mL portion of it was dispensed into a clean test tube in which it was evaporated to dryness in a hot-air oven. The HA residue in the test tube was dissolved in 1.0 mL of deionized water, and its absorbance was read in a UV spectrophotometer at 228 nm. A positive control test containing 64 nmol/L captopril and a reference test (both without the extract) were included in the assay. The percentage inhibition of ACE by each extract was calculated, and the IC₅₀ was obtained from the non-linear regression equation of the % inhibition versus extract concentration curve.

where A228_reference is the absorbance of the reference without the extract, and A228_sample is the absorbance of test containing the extract.

Statistical analysis

Results of replicate experiments were presented as mean ± SD. Analysis of variance was carried out on the result data at 95% confidence level using SPSS statistical software package, version 17.

Results and discussion

The HPLC profiles of the phenolic acids and flavonoids constituents of raw, roasted, and boiled B. eurycoma seeds extracts are shown in Figs. 1a, 1b, and 1c, respectively. Raw seeds (Fig. 1a) contained gallic acid (retention time – t_R = 9.85 min, peak 1), caffeic acid (t_R = 22.53 min, peak 2), ellagic acid (t_R = 31.45 min, peak 3), rutin (t_R = 40.13 min, peak 4), quercetin (t_R = 49.06 min, peak 5), and luteolin (t_R = 60.11 min, peak 6). Roasted seeds contained gallic acid (peak 1), ellagic acid (peak 3), rutin (peak 4), and quercetin (peak 5); while boiled seeds contained gallic acid (peak 1), rutin (peak 4), and quercetin (peak 5).

Figure 1. Representative high-performance liquid chromatography profiles of raw (A), roasted (B), and boiled (C) B. eurycoma seeds. Gallic acid (peak 1), caffeic acid (peak 2), ellagic acid (peak 3), rutin (peak 4), quercetin (peak 5), and luteolin (peak 6).

The levels of the various phenolic acids and flavonoids are presented in Table 1. In the raw seeds, phenolic acids were present in the order of caffeic > ellagic > gallic acids; while flavonoids were present in the order of quercetin > rutin > luteolin. In a previous study, the presence of these phenolic acids and flavonoids except luteolin in raw B. eurycoma seeds flour had been reported.^[1] However, the levels of the various phenolic acids (gallic acid: 1.04 ± 0.05; caffeic acid: 2.95 ± 0.01; ellagic acid: 2.18 ± 0.01 mg/g) and flavonoids (rutin: 4.57 ± 0.03; quercetin: 4.61 ± 0.01 mg/g) detected in the raw seeds in this study were generally lower than levels (gallic acid: 1.17 ± 0.03; caffeic acid: 18.13 ± 0.02; ellagic acid: 9.26 ± 0.01; rutin: 5.92 ± 0.01; quercetin: 6.04 ± 0.01 mg/g) earlier reported.^[1] These differences may be attributed to variations in biotic and abiotic factors, time of sample collection, as well as variation in sample extraction method used, which may all affect the levels of phytochemicals in the seeds.^[34,35]

Table 1. Phenolic acids and flavonoids constituents of raw, roasted, and boiled B. eurycoma seeds.

Compounds	Raw (mg/g)	Roasted (mg/g)	Boiled (mg/g)	LOD μg/mL	LOQ μg/mL
Gallic acid	1.04 ± 0.05^a	0.35 ± 0.02^b	0.28 ± 0.01^c	0.009	0.030
Caffeic acid	2.95 ± 0.01	ND	ND	0.021	0.068
Ellagic acid	2.18 ± 0.01^a	0.41 ± 0.03^b	ND	0.028	0.092
Rutin	4.57 ± 0.03^a	2.73 ± 0.01^b	2.15 ± 0.02^c	0.015	0.051
Quercetin	4.61 ± 0.01^a	2.09 ± 0.01^b	1.03 ± 0.04^c	0.013	0.043
Luteolin	0.32 ± 0.02	ND	ND	0.024	0.079

Results are expressed as mean ± SD of three determinations. Averages followed by different superscript letters along the same row differ at p < 0.05. ND: not detected; LOD: limit of detection; LOQ: limit of quantification.

Relative to the raw seeds, the two processing methods used (roasting and boiling) resulted in significant (p < 0.05) decrease in the levels of gallic acid, rutin, and quercetin. Ellagic acid reduced significantly (p < 0.05) in the roasted seeds, but it was not detected in the boiled seeds. The result further shows that both roasting and boiling methods led to total loss of caffeic acid and luteolin as these were not detected in both the roasted and boiled seeds. Although there is no previous literature on loss of phenolic compounds in B. eurycoma seeds due to heat treatments, our findings are consistent with those of similar studies on tree nuts and vegetables. Lemos et al.^[36] reported reductions in the levels of some phenolic acids, including gallic, caffeic, and ellagic acids in roasted baru nut, relative to the raw nuts. Similarly, Gębczyński and Lisiewska^[37] reported that boiling resulted in the decrease of the levels of phenolic acids and flavonoids in broccoli. The reduction in the levels or the absolute loss of some the phenolic acids and flavonoids observed in this study may be due to their thermal decomposition as polyphenols are known to get degraded easily when subjected to heat treatment.^[38] Phenolic acids such as caffeic acids are known to volatilize easily during heating for a long period.^[36] Flavonoids and ellagic acid are susceptible to heat, possibly due to their highly hydroxylated structure, which is vulnerable to redox reactions, thereby promoting their losses during heat treatment.^[36]

For the roasted seeds, Maillard reactions that possibly took place at the roasting temperature (150°C) might have contributed to the decrease of the levels of the phenolic acids and flavonoids.^[39] More of the phenolic acids and flavonoids were lost in boiled seeds than in the roasted seeds. This may be due to heat-induced oxidation and leaching of the water-soluble phenolics into water during boiling.^[39,40] It is also possible that chemical rearrangements involving binding of the phenolic compounds with other organic substances during boiling resulted in the decrease of the levels of the phenolic acids and flavonoids.^[41] The report of Francisco et al.^[42] had earlier indicated that different types of phenolic compounds even within the same group are affected differently by the same cooking method. Thus, the differential reductions in the quantities of the individual phenolic acids and flavonoids after boiling may be a reflection of variations in their individual thermostability and solubility in hot water.^[43]

The free radical scavenging abilities of raw, roasted, and boiled B. eurycoma seeds, as tested using DPPH* and ABTS*⁺ scavenging assays, are presented in Table 2. The half-maximal scavenging concentration (SC₅₀) of raw seed (25.10 ± 1.39 μg/mL) against DPPH* was significantly (p < 0.05) lower than those of roasted and boiled seeds (27.38 ± 1.48 and 48.06 ± 2.03 μg/mL, respectively). These results clearly indicate that both roasting and boiling methods decreased the free radical scavenging ability of the seeds since lower SC₅₀ values represent stronger DPPH* scavenging ability. However, ascorbic acid (SC₅₀: 7.36 ± 0.74 μg/mL), used as a reference antioxidant in this study, exhibited stronger DPPH* scavenging activity than the raw seeds. Similarly, the ABTS*⁺ scavenging ability of the seeds decreased significantly from 1.77 ± 0.20 mmol TEAC/g in the raw seeds to 1.54 ± 0.07 and 0.83 ± 0.02 mmol TEAC/g in the roasted and boiled seeds, respectively.

Table 2. DPPH* SC₅₀ and ABTS*⁺ scavenging abilities of raw, roasted, and boiled B. eurycoma seeds extract.

Antioxidant activity	Raw	Roasted	Boiled	Ascorbic acid
DPPH*SC50 (μg/mL)	25.10 ± 1.39^c	27.38 ± 1.48^b	48.06 ± 2.03a	7.36 ± 0.74^d
ABTS*^+ scavenging ability (mmol TEAC/g)	1.77 ± 0.20^a	1.54 ± 0.07^b	0.83 ± 0.02^c	NA

Results are expressed as mean ± SD of triplicate analysis. Values followed by different superscript letters along the same row differ significantly at p < 0.05. NA: not applicable.

The DPPH* and ABTS*⁺ scavenging abilities (SC₅₀: 25.10 ± 1.39 μg/mL and 1.77 ± 0.20 mmol TEAC/g, respectively) of the raw seeds observed in this study are much stronger when compared with the DPPH* and ABTS*⁺ scavenging abilities (IC₅₀: 5.05 ± 0.64 mg/mL and 29.78 ± 0.07 µmol TE/g, respectively) previously reported for methanol extract of B. eurycoma seed flour.^[7] As stated earlier, variations in biotic and abiotic factors, time of sample collection, and method of sample extraction may have contributed to the difference in the free radical scavenging potential we observed, relative to the previous report.^[34,35] The decrease in the DPPH* and ABTS*⁺ scavenging potentials of the seeds due to roasting and boiling observed in this study is consistent with the findings of other researchers. Rawson et al.^[39] reported a decrease in the DPPH* scavenging ability of fennel bulb due to boiling and roasting. Similarly, Girgin and El^[44] reported a decrease in DPPH* and ABTS*⁺ scavenging ability of cauliflower due to boiling, while Borges et al.^[45] also reported a decrease in the DPPH* scavenging potential of cashew nut due to roasting. Since the free radical scavenging abilities of the roasted and boiled seeds decreased in a similar trend as the levels of the individual phenolic acids and flavonoids, these phenolics might be the major contributor to the antioxidant activity of the seeds. Phenolic acids and flavonoids have unique structures that confer them with redox properties, enabling them to act as hydrogen donors, singlet oxygen quenchers, and reducing agents.^[46] Rawson et al.^[39] noted that loss in antioxidant capacity is due to the chemical oxidation of polyphenolic compounds during thermal processing treatments.

The abilities of the raw, roasted, and boiled seeds extracts to inhibit XO and ACE, expressed as their half-maximal inhibitory concentrations (IC₅₀), are presented in Table 3. The IC₅₀ of the seeds against XO increased from 45.17 ± 2.14 μg/mL in the raw seeds to 46.58 ± 2.37 and 60.74 ± 2.96 μg/mL in the roasted and boiled seeds, respectively. This trend indicates a decreasing order in the XO inhibitory potential of the seeds due to roasting and boiling. Allopurinol, which was used as a reference XO inhibitor in this study, inhibited XO more than the raw and thermally processed seeds. However, the IC₅₀ of the raw seeds against XO is comparable with those of Olea europaea leaf (IC₅₀: 42 μg/mL)^[47] and Tetrapleura tetraptera fruit (IC₅₀: 45.71 ± 1.44 μg/mL),^[48] also against XO. By inhibiting XO, raw B. eurycoma seeds may be useful for retarding the production of uric acid, and consequently, preventing hyperuricaemia and gout since the overactivity of XO contributes to the development of these two conditions.^[12]

Table 3. IC₅₀ of raw, roasted, and boiled B. eurycoma seeds extracts against xanthine oxidase (XO) and angiotensin 1-converting enzyme activities (ACE).

Sample	XO IC50 (μg/mL)	ACE IC50 (μg/mL)
Raw	45.17 ± 2.14^c	29.08 ± 1.74^c
Roasted	46.58 ± 2.37^b	32.87 ± 1.81^b
Boiled	60.74 ± 2.96^a	48.93 ± 2.15^a
Allopurinol	5.48 ± 0.27^d	NA
Captopril	NA	4.75 ± 0.15^d

Results are expressed as mean ± SD of replicate analysis. Values followed by different superscript letters along the same column differ significantly at p < 0.05. NA: not applicable.

Similarly, the IC₅₀ of the seeds against ACE increased from 29.08 ± 1.74 μg/mL in the raw seeds to 32.87 ± 1.81 and 48.93 ± 2.15 μg/mL in the roasted and boiled seeds, respectively, indicating a decreasing order in the ACE inhibitory potential of the seeds following roasting and boiling treatments. Captopril, the reference ACE inhibitor in this study, had the strongest ACE effect (IC₅₀: 4.75 ± 0.15 μg/mL). Whereas the raw B. eurycoma seeds had a stronger ACE inhibitory potency than Ocimum gratissimum (IC₅₀: 56.63 ± 3.12 μg/mL),^[49] it had a weaker ACE inhibitory effect than guava leaves (IC₅₀: 21.06 ± 2.04 μg/mL).^[10] Raw B. eurycoma seeds extract may therefore be useful for preventing excessive production of angiotensin II (a vasoconstrictor) through the inhibition of ACE, which is an important clinical approach for treating and managing hypertension.^[9]

The XO and ACE inhibitory effects of the seeds extracts may be attributed to their constituent phenolic acids and flavonoids. Previous studies had shown that plants extracts rich in these two classes of polyphenolic compounds, including cocoa and guava leaves, inhibited XO and ACE.^[10,50] Thus, the decrease in the XO and ACE inhibitory capacities of the roasted and boiled seeds is a reflection of the decrease in the levels or the outright loss of some the phenolic acids and flavonoids constituents following these two thermal processing methods.

Conclusion

Raw B. eurycoma seeds contained major phenolic acids and flavonoids, including caffeic acid, ellagic acid, rutin, and quercetin. The seeds extract strongly scavenged free radicals (DPPH* and ABTS*⁺) and inhibited XO and ACE. Roasting and boiling led to decrease in the levels or outright loss of some the phenolic acids and flavonoids; and in the free radical scavenging, and XO and ACE inhibitory effects of the seeds. However, roasted seeds retained higher levels of the phenolic acids and flavonoids, and displayed stronger free radical scavenging, and XO and ACE inhibitory effects than the boiled seeds. Hence, raw B. eurycoma seeds may be useful in retarding the production of uric acid and angiotensin II, through XO and ACE inhibition, respectively, which are important strategies for managing gout and hypertension, respectively. Roasting of the seeds is recommended as a more valuable processing method than boiling, for the retention of the phenolic constituents and bioactivities, for optimum health benefits.