Introduction

Hypertension and oxidative stress are key risk factors for cardiovascular disease, highlighting the need for novel interventions1. While therapeutic options exist, preventive strategies including dietary approaches remain a global priority, particularly among asymptomatic populations. Oil Palm Phenolics (OPP), derived from crude palm oil (CPO) by-products, are rich in bioactive polyphenols with potential health benefits2. OPP, known for their antioxidant (AOX) properties have been studied for their blood pressure (BP) regulation effects3,4. During CPO extraction, water-soluble phenolics by-products are recovered through a patented process to produce OPP liquid. This polyphenols filtrate has demonstrated physiological potential in preclinical and clinical studies, while its safety and biomedical effects are being progressively updated3,4,5. Preclinical2,6,7,8,9,10 and clinical studies11,12,13 highlight OPP’s benefits in lipid metabolism, BP regulation, and AOX functions3,4. However, the clinical efficacy of spray-dried encapsulated OPP remains unverified, warranting further trials4.

The liquid OPP’s low tolerability at high volumes11 suggests encapsulation may improve efficacy while preserving bioactive properties. We hypothesize that encapsulated OPP promotes BP regulation and AOX function. The safety evaluation and tolerance profiles from the current trial was initially published where minimal and no serious adverse effect (AE) were observed, and haematology, liver, renal profiles were physiologically normal13. Recorded AE were classified based on their causality-relation to OPP intake. Based on current evidence2,3,4,10, this Phase One trial investigates the physiological effects and dose-response profile of encapsulated OPP on triglycerides (TG), total cholesterol (TC), LDL-C, HDL-C, systolic BP (SBP), diastolic BP (DBP), and AOX markers in healthy individuals to support the planning of subsequent trials involving individuals with increased risk of hypertension or cardiovascular disease.

Results

The trial began with 100 volunteers comprising 32 males and 68 females (Table 1), with three dropouts in the OPP group, resulting in 97 completing the study (Fig. 1). To optimize human dosing, the current study selected 250 and 1000 mg/d OPP based on prior dose-exploration research9, while the 1500 mg/d dose was included to assess its effects at the upper tolerability threshold established in safety evaluations5. Therefore, the current clinical trial is crucial for identifying the optimal OPP dosage for clinical use. Volunteers’ demographics, BP and baseline profiles are tabulated in Table 1, while the safety and tolerance reports, haematology, renal and liver function profiles, and volunteers’ feedback are systematically presented recently13. There was a significant reduction of SBP at day-60 compared to the baseline day-1 (p = 0.017) in Group B who consumed 250 mg/d OPP (Table 2). SBP measures before and after all treatment groups were within physiological range. Treatment with 250 mg/d OPP again demonstrated a unique pattern after DBP was declined significantly at day-30 and 60 (p < 0.001) (Table 3).

Table 1 Baseline demographic, anthropometric, blood pressure (BP) and lipid biochemistry profiles of volunteers (Mean ± SD).
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Fig. 1
figure 1

CONSORT diagram of the OPP and placebo treatments.

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Table 2 Changes of sistolic blood pressure (SBP) after placebo and OPP treatments (Mean ± SD).
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Table 3 Changes of diastolic blood pressure (DBP) after placebo and OPP treatments (Mean ± SD).
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These results indicate that supplementation with 250 mg/d of encapsulated OPP produced significant reduction in both SBP and DBP in healthy adults. Since all BP values remained within the normotensive physiological range, the observed effect is more appropriately interpreted as a modulatory or homeostatic influence on vascular function rather than a direct antihypertensive action. This regulatory response suggests a potential preventive role of OPP in maintaining cardiovascular stability among healthy individuals. Moreover, no further reduction observed at higher doses (1000 or 1500 mg/d). This pattern implies that OPP’s efficacy in reducing SBP and DBP may plateau or attenuate at higher intakes. Such non-linear dose-response effects are consistent with findings in both human and animal studies of polyphenols, where moderate dosing produced stronger physiological effects than higher dosages. Interestingly, the 250 mg/d dose significantly reduced DBP by day-30 and sustained the reduction until day-60. In contrast, SBP reduction at the same dose was significant only at day-60, suggesting a differential response timeline between SBP and DBP. This may reflect a more immediate vascular relaxation effect on DBP, while SBP changes might require longer exposure.

Unlike BP, none of the OPP treatments resulted in statistically significant changes in serum lipid profiles over the intervention period. However, a significant reduction in HDL-C was observed in the placebo group (p = 0.029), while HDL-C levels were preserved in all OPP groups (Table 4). There was a significant TC increment at day-60 following all OPP treatments. TC during 1000 mg/d OPP treatment was eventually increased significantly from baseline after 60-days (p = 0.015), while the significant increment during 250 mg/d (p = 0.007) and 1500 mg/d (p < 0.001) treatments was observed only at day-60 when compared to their respective level at day-30. Among all OPP treatments, only 250 mg/d dose did not demonstrate any significant changes in LDL-C, compared to the 1000 mg/d (p = 0.046) and 1500 mg/d (p = 0.003) treatments. Unlike the 1000 mg/d treatment in which the LDL-C was increased significantly from baseline after 30 days, the significant increment of LDL-C during 1500 mg/d OPP was only observed at day-60 when compared to day-30. Similarly, TG was increased significantly at day-60 compared to day-30 only during 250 mg/d OPP (p = 0.013).

Table 4 Changes in serum lipid biochemistry profiles after placebo and OPP treatments (Mean ± SD).
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Although there was no significant difference between pre-and post-supplementation with 1000 and 1500 mg/d OPP, glutathione peroxidase (GSH-Px) significantly declined at day-60 with placebo (p = 0.015) and 250 mg/d OPP (p = 0.001) (Table 5). Even though GSH-Px values declined across all groups, the reductions were less severe with higher OPP doses (1000 and 1500 mg/d), implying that encapsulated OPP may help mitigate AOX depletion over time. This aligns with previous studies where polyphenol interventions stabilized or delayed the decline in AOX enzymes. Glutathione reductase (GR) increased significantly after 30 and 60 days during placebo, 1000 and 1500 mg/d OPP treatments (p < 0.001). Despite a significant GR increment at day-30, only supplementation with 250 mg/d OPP resulted in significant GR increment at day-60 from day-30. GR increased significantly by day-30 with 1000 and 1500 mg/d OPP, and remained elevated thereafter. However, OPP’s effectiveness remains uncertain as the placebo group also showed significant increases at days-30 and -60. At day-30, GR increased more rapidly with 1000 and 1500 mg/d OPP than with 250 mg/d OPP or placebo. By day-30, GR levels reached 53.79 µmol/L with 1000 mg/d and 51.82 µmol/L with 1500 mg/d OPP, while placebo and 250 mg/d OPP resulted in lower levels of 45.59 and 38.91 µmol/L, respectively (Table 5). This observation supports the hypothesis that OPP at moderate-to-high doses may promote early activation of endogenous AOX defences.

Table 5 Plasma AOX profiles changes after placebo and OPP treatments (Mean ± SD).
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Total AOX capacity (T-AOC) increased significantly with 1500 mg/d OPP, emerging by day-30 (p = 0.038, following Bonferroni adjustment); however, this effect was not sustained by day-60, suggesting a transient enhancement of the AOX status. Total superoxide dismutase (T-SOD) declined significantly by day-60 in the placebo group (p = 0.003), whereas T-SOD levels remained relatively stable across all OPP treatment groups, indicating a possible protective AOX role of encapsulated OPP supplementation.

Discussion

Liquid OPP ability in reducing hyperlipidaemia without compromising the safety aspects was documented in animals9, followed by safety and physiological evaluations in humans11, then further substantiated by comparable investigation employing alternative methodologies using distinct preclinical models and slightly varied phytochemical extracts derived from the oil palm fruits to characterize the OPP health-related effects14. Despite the trial limitation where the appearance, colour and taste of the placebo that did not replicate the OPP liquid, it was proven safe for human’s consumption for 60 days11. Nevertheless, the practicality and cost-effectiveness of liquid OPP for future studies are concerns, as supplying large volumes to volunteers is impractical. The diverse sensory perceptions of OPP liquid drinks have posed challenges for volunteers, leading to reluctance for participating in future trials. As a result, volunteers proposed a powdered form, which the investigator acknowledged. Differences in polyphenols kinetics, bioavailability and physiological functions has been carefully measured following ingestion of a specific dose of polyphenols supplied either as plant extract, pure compound, whole food or beverages15. Our preclinical3,4,5,6,7,8 and clinical studies11,12,13 compared the benefits and limitations of liquid and spray-dried OPP3,4. While liquid phenolics retain bioactivity, they are prone to oxidation and microbial growth, reducing shelf life. Spray-drying improves stability by lowering moisture content but may alter bioactive compounds due to heat exposure4.

The current trial explores the preliminary use of spray-dried encapsulated OPP, addressing concerns about their therapeutic efficacy from liquid towards encapsulated extracts. In conjunction with the tolerability and safety evaluation of the encapsulated OPP13, the current dose-response trial that included 250 mg/d OPP (lower dose), 1000 and 1500 mg/d OPP (higher doses) treatments also investigate their modulatory effect assessment. Unlike our pilot trial11 that was initiated based on earliest animal study6, the OPP dose used in the current trial was suggested from improvised preclinical works5 that applied methodical extrapolation and conversion from animal dose to human equivalent dose9. Previous studies suggest that specific polyphenol doses may effectively lower BP in humans16,17,18,19. The BP-lowering effects of OPP are attributed to active polyphenols such as caffeoylshikimic acid, protocatechuic acid, p-hydroxybenzoic acid, and hydroxytyrosol3,4.

The lack of BP reduction at higher OPP doses aligns with previous findings on freeze-dried strawberry powder (FDSP) in pre and stage one hypertensive postmenopausal women, where a significant SBP decrease occurred only with the lower dose (25 g FDSP, containing 102 mg anthocyanins), while the higher dose (50 g FDSP, containing 204 mg anthocyanins) had no effect20. Despite differences in polyphenol dosage between studies, these results suggest that certain polyphenols may exert therapeutic benefits more effectively at lower doses. Animal study suggest that the BP-lowering effects of polyphenols depend on the delivery method3,4 with single acute dose of 250 mg/kg OPP significantly reduced BP by 24–27 mmHg, whereas long-term supplementation with a higher dose (1500 mg/L GAE for 20 weeks) showed no effect. Findings suggest BP reduction may stem from an acute vasodilatory response, likely driven by increased endogenous nitric oxide (NO), in which clearly warranting further research on optimal dosing3,4.

In humans, BP outcomes may differ based on polyphenol dosage and duration, with low doses and short-term supplementation duration showing negligible effects, whereas extended moderate-to-high doses can decrease SBP/DBP particularly in hypertensive populations19,21. BP reductions are potentially mediated by OPP-induced endothelial NO synthase activation, facilitating vasodilation, smooth muscle relaxation, and cardiovascular homeostasis2. OPP may scavenge reactive oxygen species (ROS) and reduce oxidative stress, therefore potentially preventing hypertension10. Notably, doses lower than 250 mg/d may be sufficient to regulate circulating NO in hypertension models. Prior investigations in healthy individuals have reported significant BP reductions, either in SBP/DBP alone, or both SBP and DBP16,17,22. Previous findings align with our current BP data trend where healthy individuals consuming a capsule containing 210 mg punicalagins, 328 mg pomegranate, and 0.37 mg anthocyanins showed a significant DBP reduction after eight weeks23. However, given that DBP was measured at multiple time points, a decline may have occurred earlier, though post-hoc analysis was not performed to confirm statistical significance. Similarly, in a study on 25 mg epicatechin supplementation, significant time effects were observed, but the exact time points of significance remained unclear24. A 12-week study on pre-hypertensive individuals demonstrated that 400 mg/d of grape seed proanthocyanidin extract significantly reduced SBP and DBP by 13.1 and 6.5 mmHg, respectively, while a 200 mg/day dose reported no effect22. Some polyphenols have demonstrated faster BP-lowering effects, where supplementation with a pomegranate fruit capsule for 14 days significantly reduced both SBP and DBP, with further reductions by day-2817. The antihypertensive potential of polyphenols, though clinical trials often show significant reductions only in DBP, with a non-significant trend toward SBP reduction25.

Polyphenol supplementation in liquid demonstrated a borderline statistical effect on DBP reduction in healthy individuals, indicating less significant BP reduction and variability compared to hypertensive individuals26. A placebo-controlled crossover trial on 15 normotensive and 20 hypertensive subjects consuming flavanol-rich dark chocolate for 15 days found significant BP reductions in hypertensive individuals, while normotensive subjects showed changes only in SBP21. Analysis by tertiles indicated that baseline BP affects responses to polyphenol treatment. High baseline BP subjects significantly reduced SBP after eight weeks of consuming a bilberries, lingonberries, and black currant/strawberry puree mixtures16. As expected, polyphenol supplementation benefits hypertensive individuals more than healthy ones, with significant BP improvements observed in short and long-term19. We hypothesize that OPP supplementation uniquely affects BP while sharing similar mechanisms with various polyphenol groups. Generally, polyphenols reduce BP through multiple combined mechanisms including endothelium-dependent vasodilation, endothelium-derived hyperpolarizing factor, and reduction of oxidative stress15, as well as from an acute vasodilatory response, likely driven by increased endogenous NO, in which clearly warranting further research on optimal dosing3,4. Furthermore, the distinctive chemical structure of polyphenols too is crucial in their BP-lowering efficacy15.

NO regulates BP through vasodilation, making it a key target for hypertension management by enhancing its production, bioavailability, and stability27. It is synthesized from L-arginine by NO synthase (NOS), with neuronal (nNOS) and endothelial (eNOS) producing small amounts, while inducible (iNOS) generates higher levels in response to inflammation. NO supports vascular health by activating soluble guanylyl cyclase in smooth muscle cells, increasing cyclic guanosine monophosphate, leading to muscle relaxation, reduced vascular resistance, and improved blood flow27. In NO-deficient rat models, OPP supplementation (1500 and 3000 mg/L GAE for four weeks) significantly mitigated BP increases over an eight-week period compared to the NG-nitro-L-arginine methyl ester treatment induced hypertension group10. These findings demonstrating that OPP-induced vascular relaxation is mediated via eNOs3,4. Using a mesenteric vascular bed model, OPP treatment induced dose-dependent relaxation in the aorta, suggesting its efficacy in lowering BP in both normotensive Wistar Kyoto and spontaneously hypertensive rats2. Collectively, these preclinical studies propose that OPP enhances NO production and activity, promoting vasodilation and BP reduction4. The OPP-induced BP reduction may result from an acute vasodilatory effect, likely triggered by a surge in endogenous NO and reduced peripheral resistance3,4.

The current trial demonstrated notable improvements in AOX status, as indicated by significant increases in GSH-Px, GR, T-AOC, and T-SOD from baseline. GR levels increased across all OPP treatment groups, but a similar trend was also observed in the placebo group, raising the possibility that this may reflect a compensatory physiological response to oxidative stress rather than a direct effect of OPP supplementation. The significant T-AOC increase observed at day-30 with 1500 mg/d OPP was not sustained thereafter, suggesting a short-lived enhancement of AOX capacity.

While GSH-Px levels did not rise, supplementation with 1000 and 1500 mg/d OPP appeared to prevent the enzymatic decline seen in lower-dose and placebo groups, suggesting a potential protective role against oxidative stress-induced depletion. This interpretation aligns with findings from prior studies using 50 ml/d of acai berry-based juice, which maintained GSH-Px and GR over six weeks28. However, GR remained unchanged in resting phases before and after treatment, with only total GSH showing a significant post-treatment increase relative to baseline28. Moreover, few studies have evaluated GSH-Px and GR simultaneously28,29,30, and those that did often required longer durations such as six months of roselle-based polyphenol intake29 to eventually observed the significant increases. Notably, a study involving 16 young men reported no significant changes in either enzyme after six weeks of 500 mg/d encapsulated green tea extract30, underscoring the need for longer intervention periods. It is then postulated that OPP supplementation would yield similar effects if administered over a longer duration, as seen in previous studies29. Polyphenols enhance glutathione production and detoxification by activating key enzymes, supporting increased glutathione levels and redox balance. By modulating oxidative stress pathways, polyphenols upregulate glutathione-related enzymes such as epigallocatechin gallate and γ-glutamylcysteine synthetase28, thus enhancing intracellular glutathione synthesis and GSH-Px activity. However, these effects vary with polyphenol type and dosage. Taken together, these findings suggest that encapsulated OPP may selectively support AOX homeostasis under certain conditions, though the evidence does not support a generalized or sustained enhancement of the AOX system. Previously, OPP preparations demonstrated functional synergistic AOX effects4, attributable to their hydrophilic phenolic structures that combat ROS3 and inhibit copper-mediated LDL-oxidation2.

To more accurately interpret the AOX effects of OPP, all AOX measurements should be evaluated in relation to the placebo group. While higher doses of OPP appeared to elevate GR activity, a similar increase was also observed with placebo, making it difficult to attribute the effect solely to OPP. In contrast, the lowest dose showed a delayed GR response, with initial suppression followed by a rebound, suggesting a distinct and possibly dose-specific pattern. This delayed response warrants further investigation, while the similar trends seen with higher doses and placebo suggest the GR increase may not be treatment-specific. Unlike placebo, the effectiveness of OPP supplementation in maintaining T-SOD status for 60-days is evident. This aligns with studies where 400 mg/d encapsulated catechin-rich grape seed extract and 150 mg/d of resveratrol treatments for 28 days, showed no significant SOD changes31, while 980 mg/d of encapsulated green tea polyphenols over four weeks reduced SOD in male athletes32. The decline in SOD during placebo treatment highlights the role of OPP in sustaining AOX status, likely due to its polyphenol content.

Several studies have identified effective polyphenol dosages, with higher doses often required to enhance T-AOC. Most human trials assess T-AOC using the Ferric-Reducing Antioxidant Potential (FRAP) method, which was also applied in this study. Here, only the highest OPP dose led to a significant short-term increase in FRAP levels, observed at mid-intervention but not sustained by the end. This suggests that high-dose OPP may offer transient AOX benefits. Therefore, any future studies should include an additional assessment between days-1 and -30 to confirm the acute effects of high-dose OPP on FRAP levels. A previous trial in 40 female athletes showed no significant changes in FRAP after eight weeks of 300 mg/d grape seed extract supplementation33. However, our findings align with a crossover study in 48 healthy subjects, where 250 ml/d of Sicilian red wine consumption significantly increased FRAP after four weeks34. Notably, when subjects discontinued the red wine, FRAP levels returned to baseline, while those who started supplementation at week-five showed a similar four-weeks increase. This parallel with our results where FRAP levels rose significantly 30 days after the highest OPP dose treatment. The red wine study however did not include a washout period, potentially allowing residual effects from prior treatment34. However, the normalization of FRAP levels to baseline after eight weeks without polyphenol supplementation suggests that polyphenols exert only short-term AOX effects, with no lasting impact once intake ceases. This aligns with the concept of transient AOX effects, as evidenced by temporary increases in T-AOC34 which emphasize the need to optimize polyphenol dosage and timing to maximize AOX efficacy.

The T-AOC measured reflects the combined action of major endogenous AOX circulating in the blood35. Our findings suggest that GSH-Px, GR, and SOD play crucial roles in the AOX defence system, with SOD catalyzing superoxide dismutation while GSH-Px and GR neutralizing peroxides within the glutathione system. As integral components of the body’s AOX network, variations in these enzyme activities influence T-AOC values. Our study revealed complex interactions between endogenous and exogenous AOX, demonstrating how polyphenols upregulate multiple AOX enzymes, creating a coordinated defence mechanism. While liquid OPP showed greater efficacy8, encapsulation was introduced to address limitations in previous work11. Although the liquid OPP was more effective compared to the spray-dried form8, the delivery method of OPP in the current trial using capsules was proposed following discovered limitations from previous investigation11. This approach was actually adjusted from the similar methods that were previously done by other investigator in evaluating other polyphenols treatment25. Previous investigation even used the polyphenol extract in a powder form, packed in a protective sachet before dissolving in a water as a drink26. Encapsulation enhances polyphenol stability, protecting against oxidation and degradation while preserving therapeutic efficacy. However, contrary to our expectations and previous findings, several statistically significant increases in serum lipid parameters were observed following OPP supplementation. Although these changes remained within normal clinical reference ranges for healthy individuals, they indicate a non-neutral lipid response to OPP in this population. Such results should be interpreted with caution, as modest lipid fluctuations are common in normolipidemic, healthy individuals and may reflect physiological adaptation rather than adverse metabolic effects. Nevertheless, given the well-established cardiometabolic risks associated with elevated LDL-C and TG, these observations warrant careful consideration. Future trials involving chronic hyperlipidemic or metabolically at-risk populations are needed to clarify whether these trends represent true biological effects or context-dependent responses influenced by baseline lipid status, dose, or duration of supplementation.

Growing evidence suggests that polyphenols may exert more pronounced lipid-lowering effects in individuals with metabolic disorders rather than in healthy populations. Preceding investigation have reported significant improvements including reductions in TC, LDL-C, and TG following polyphenol supplementation in hyperlipidemic individuals36. This is partially supported by our own Phase Two clinical trial employing the identical 250 mg/d encapsulated OPP treatment, in which TC and LDL-C levels significantly decreased from baseline after 60 days of supplementation12. However, these reductions were also observed in the placebo group, suggesting that factors such as dietary control, or participant behaviour may have contributed to the observed effects. Interestingly, HDL-C levels increased significantly after 30 days of OPP supplementation in that study and remained elevated throughout the intervention period. Although OPP is suggested to reduce lipids2,3,4,10,12, other polyphenols achieve this action by activating AMP-activated protein kinase in hepatic cells and inhibiting Niemann-Pick C1-like 1 protein, thereby impairing micelle formation and reducing cholesterol absorption37.

Variability in polyphenol bioavailability in humans, their metabolic responses and hyperglycaemia states may influence their effectiveness. Additionally, trial limitations such as sample size, baseline health, and metabolic variability may explain the stronger lipid-lowering effects observed in individuals with metabolic disorders compared to healthy populations. Nevertheless, a key limitation of this trial is the absence of dietary and lifestyle monitoring throughout the intervention period. Volunteers were advised to maintain their usual diet and physical activity routines, but no formal dietary assessments or physical activity logs were collected. As such, it is not possible to rule out the influence of uncontrolled dietary factors on the observed lipid profile changes. This limitation is particularly relevant given the significant reduction in HDL-C in the placebo group and the increase in TC, LDL-C, and TG observed in the OPP treatment groups.

Future perspectives

Future studies should incorporate standardized dietary monitoring or controlled feeding protocols to minimize confounding and strengthen causal interpretations. Moreover, investigator should isolate pure phenolic compounds for further evaluation to optimize formulations for future applications. While OPP delivery methods vary, further studies are needed to determine the best approach for human consumption. Key factors include population suitability and optimal supplementation duration12,13. Comparative studies11,26 suggested that OPP’s effects are influenced more by health status than formulation type. Future studies should include pharmacokinetic profiling to clarify the absorption and metabolism of OPP-derived phenolics, providing insight into their biological relevance and mechanisms of action.

The findings of this study demonstrating the BP-lowering and AOX improvement effects of encapsulated OPP at specific doses that will have significant implications for addressing cardiovascular health challenges. While hypertension remains a leading global risk factor for cardiovascular diseases, contributing to approximately 11 million deaths globally in 20191, the potential of OPP to improve BP coupled with existing AOX properties postulated its suitability as a functional dietary supplement for hypertensive or pre-hypertensive populations. As demonstrated, the 250 mg/d OPP is currently the most effective dose in reducing SBP and DBP without a dose-response relationship observed at higher concentrations. However, it is important to interpret this reduction within the physiological context of normotensive individuals. Rather than indicating a therapeutic or clinical antihypertensive effect, the observed BP changes may reflect an early regulatory response, possibly through improved endothelial function or vasodilatory homeostasis. This finding supports the hypothesis that OPP may serve as a preventive nutritional agent that helps maintain vascular health in otherwise healthy individuals. Future studies in hypertensive or pre-hypertensive populations with larger number of volunteers with stratified analyses by gender are needed to evaluate whether these modulatory effects translate into clinically meaningful outcomes given that a recent meta-analysis indicates that polyphenol-rich dietary interventions can reduce SBP by 2–5 mmHg18.

OPP antioxidative potential was highlighted by improvements in GR and T-AOC, with high doses preserving GSH-Px and maintaining T-SOD levels. These findings position encapsulated OPP as a promising candidate for preventive cardiovascular support through dietary means. Given the normotensive status of our study population, the observed improvements in BP and AOX biomarkers are more appropriately interpreted as early modulatory effects within physiological limits. Moreover, the superior tolerability of encapsulated OPP compared to its liquid counterpart ensures better compliance, which is critical for long-term intervention strategies. Future phase two trial should focus on evaluating these effects over longer durations to confirm therapeutic potential and broader public health relevance. Inclusion of relevant biomarkers measurements such as plasma nitrate/nitrite, cGMP, oxidized LDL, and endothelial-related cytokines is therefore suggested for subsequent studies to better elucidate the mode of OPP action.

Conclusion

In summary, this phase one clinical trial demonstrates that spray-dried encapsulated OPP is well tolerated and may support BP regulation and AOX defense in healthy individuals. These findings suggest that OPP has potential as a preventive nutraceutical, warranting further investigation in clinical populations and over longer intervention periods.

Materials and methods

Encapsulation

OPP is a water-soluble extract derived from the aqueous vegetation liquor produced during oil palm fruit milling. It contains no oil or lipid residues and is distinct from palm oil. The primary phenolic compounds in OPP include caffeoylshikimic acid, p-hydroxybenzoic acid and protocatechuic acid2. The liquid OPP was produced at the Malaysian Palm Oil Board (MPOB) Phenolics Pilot Plant (Labu, Malaysia), spray-dried by Biotropics Malaysia Berhad (Shah Alam, Malaysia) and encapsulated by Prima Nexus Sdn Bhd (Puchong, Malaysia). A Halal, plant-based capsules were utilized for both OPP and placebo treatments. The encapsulation utilized a white opaque capsule made of hypromellose and 5% purified water. The OPP treatment consists of pure OPP extract, with each capsule containing 62.5 mg, 250 mg, or 375 mg of OPP, while the placebo comprised solely of dextrose sugar.

Protocols

This mono-centric, parallel, placebo-controlled, randomized, double-blind trial followed the Malaysian Good Clinical Practice38 and Declaration of Helsinki39 to investigate the effects of 60-day OPP supplementation in 100 healthy volunteers. This trial was approved by the Research Ethic Committee, National University of Malaysia (UKM PPI/111/8/JEP-2019-100) and registered with clinicaltrial.gov (NCT04164446; registration date: 15/11/2019) and Australian New Zealand Clinical Trial Registry (ACTRN12619001786189; registration date: 17/12/2019). Volunteers briefed and provided written information and consent form.

After the screening process as documented13, 100 of 214 recruited healthy volunteers, including 68 women and 32 men were randomized into the following groups1: Group A, placebo treatment2; Group B, OPP at 250 mg/d3; Group C, OPP at 1000 mg/d, and4 Group D, OPP at 1500 mg/d, where volunteer consumed four capsules of placebo or OPP treatments, once/day. Following 11-h fasting, a 20 mL blood was sampled at day-1, day-30 and day-60. The collected blood was divided into EDTA tubes and plain serum tubes (non-EDTA). A thorough medical examination was conducted at each intervention day. BP was measured in sitting position using Advanced® VSM-300 Vital Signs Monitor (Advanced Instruments Inc., MA, USA). Volunteers received optimal care from the trial team, utilizing Naranjo Classification to identify AE13. The trial protocol is presented in Fig. 1.

Serum lipid profile measurement

The serum tubes were centrifuged at 3000×g for 20 min at 7 °C to obtain serum samples, which were analyzed for lipid profiles measurements (TG, TC, LDL-C, HDL-C) by COBAS 6000 Integrated Chemistry and Immunoassay Electrochemiluminescence (ECLIA) platform (Roche, Indianapolis, USA).

Antioxidant (AOX) status measurement

Blood in EDTA tubes was processed similarly to obtain plasma samples, which were used to quantify four different AOX status measures. All measurements were performed using colorimetric assay kits according to the manufacturer’s protocols (Elabscience Biotechnology Co., Ltd, Hubei, China) and readings were obtained using a microplate reader (VERSAmax Tunable Microplate Reader, Molecular Devices, California, USA). Plasma sample was used directly without prior dilution. All measurement was performed at 37 °C. Glutathione peroxidase (GSH-Px) was measured using Elabscience® E-BC-K096-M kit. The assay quantifies enzyme activity by measuring the consumption of reduced glutathione (GSH) during its conversion to oxidized glutathione (GSSG) in the presence of hydrogen peroxide, catalyzed by GSH-Px. Residual GSH reacts with DTNB to form a yellow chromophore, detected at 412 nm. Non-enzymatic reactions were subtracted by parallel controls. All reagents were equilibrated to room temperature before use. Glutathione reductase (GR) activity was measured using Elabscience® E-BC-K099-M kit which detects the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The rate of NADPH oxidation, indicated by a decrease in absorbance at 340 nm, reflects GR activity. Total superoxide dismutase (T-SOD) activity was assessed using WST-1-based assay kit Elabscience® E-BC-K020-M. The assay measures the inhibition of formazan dye formation resulting from the reaction between WST-1 and superoxide anions generated by xanthine oxidase. Since SOD competes with WST-1 for superoxide anions, its activity is inversely proportional to the amount of formazan produced. Absorbance was measured at 450 nm after a 20-min incubation. Total AOX capacity (T-AOC) in plasma samples was measured using Elabscience® E-BC-K136-M kit based on the FRAP principle. In this method, AOX present in the sample reduce ferric ions (Fe3+) to ferrous ions (Fe2+), which then form a stable-coloured complex with phenanthroline. The intensity of the resulting color is proportional to the AOX capacity and was measured at 520 nm.

Statistical analysis

Stratified randomization was performed using STATA software (StataCorp LLC, Texas, USA). Identification of three stratification covariates including age, gender, and TC was performed prior to the formation of 16 stratified groups, after which volunteers were randomized accordingly.

Statistical Package for the Social Sciences (SPSS®), version 22.0 (SPSS Inc. Chicago, USA) was used to detect significant difference in parameter of interest. The analysis followed an Intention-To-Treat (ITT) approach, in which all randomized participants (n = 100) were included in the analysis of primary outcomes. For volunteers who withdrew from the study, the missing outcome data were imputed using a simple imputation method, consistent with ITT principles. The study design incorporated three time points from day-1, day-30, and day-60, which were treated as within-subject factors to assess changes over time. Differences between intervention groups, based on daily OPP dosage, were treated as between-subject factors. One-Way ANOVA was used to examine differences in lipid, BP and AOX profiles between the placebo, 250, 1000 and 1500 mg/d OPP treatments at all time points. Repeated Measures ANOVA was used to compare changes over time within each treatment group, followed by the Bonferroni adjustment. Values were considered significant at P < 0.05.