Antioxidant and In Vitro Anti-Inflammatory Screening of Ten Nutraceutical Ingredients: Black Ginger (Kaempferia parviflora) Strongly Inhibits Heat-Induced Protein Denaturation

1. Introduction

Chronic low-grade inflammation and oxidative stress are the two most common positioning claims for nutraceutical ingredients. Ingredients used to address them range from vitamins (vitamin C, tocotrienols) and dairy-derived bioactives (colostrum) to botanical extracts with traditional anti-inflammatory use, such as turmeric, ginger, garlic, ginseng, astragalus, elderberry and black ginger (Kaempferia parviflora), whose rhizome is rich in polymethoxyflavones [1]. Formulators typically compare these ingredients using literature values generated under heterogeneous conditions [2].

Simple in vitro assays offer a practical first screen. DPPH and ABTS measure direct radical scavenging [2,3,4]. Inhibition of lipoxygenase, the enzyme that initiates leukotriene biosynthesis, indicates potential to modulate an important inflammatory pathway [5], and inhibition of heat-induced albumin denaturation is a widely used surrogate for the protein-stabilising action of anti-inflammatory agents [6,7]. This study applied all four assays, in one laboratory, to ten nutraceutical ingredients, and profiled black ginger extract by untargeted mass spectrometry.

2. Materials and Methods

2.1. Samples

Ten ingredient powders were submitted on 14 September 2022 to the Faculty of Applied Sciences, TAR UMT, where all bioassays were performed as an independent paid analytical service (Table 1). Seven of the ten — the elderberry, ginseng, astragalus, black ginger, ginger, garlic and turmeric extracts — were manufactured by Bionutricia Manufacturing Sdn. Bhd., Selangor, Malaysia, under a Hazard Analysis and Critical Control Points and FSSC 22000 certified quality system; the bovine colostrum, tocotrienol and liposomal vitamin C powders were sourced by the same company from third-party suppliers. Species, plant part, extraction ratio, extraction solvent and declared marker content are those of each product’s released specification (Table 1); no specification sheet is held for the colostrum, and batch numbers are recorded in the manufacturing records and are available on request.

2.2. Sample Preparation

Elderberry extract dissolved completely in water; ginseng extract dissolved completely in DMSO; astragalus, black ginger, ginger and garlic extracts and liposomal vitamin C dissolved with minor residues; turmeric extract and colostrum dissolved with residues (colostrum gave a white precipitate consistent with protein); tocotrienol formed a cloudy solution. Stocks were centrifuged at 5000 rpm for 3 min where residues were present, and supernatants were diluted to working concentrations. Solvents and diluents per assay were: radical-scavenging assays, 50% (v/v) DMSO diluted with distilled water; lipoxygenase assay, DMSO diluted with 0.2 M borate buffer, pH 9 (elderberry prepared directly in buffer); albumin denaturation assay, DMSO diluted with 0.02 M phosphate buffer, pH 6.4 (elderberry prepared directly in buffer).

2.3. Assays

DPPH and ABTS scavenging were determined as described in the companion study [8] (analyses 12–13 and 14–17 December 2022, respectively). Lipoxygenase inhibition was determined following Chuah et al. [5] (3–7 November 2022). Inhibition of heat-induced albumin denaturation was determined following Tang et al. [7], based on Mizushima and Kobayashi [6] (9–10 November 2022). Both anti-inflammatory assays were run under the conditions used in the cited studies [5,7], which derive from the protocol of Rashid and Shafi (IOSR Journal of Pharmacy and Biological Sciences 2018, 13, 58–67): soybean lipoxygenase with linoleic acid as substrate, read at 234 nm, and heat-induced denaturation of bovine serum albumin read at 660 nm. No reference inhibitor was included in either assay. Maximum testable concentrations were limited by solubility or colour interference and are reported as assay limits.

2.4. Untargeted LC-QTOF-MS/MS

Black ginger extract was profiled with the instrument, column, gradient and annotation rules described in the companion study [8] (negative electrospray ionisation; Metabolomics Standards Initiative level 3 [9]).

2.5. Data Analysis

Results are mean ± standard deviation of three independent replicates (n = 3). IC₅₀ values were estimated from concentration–response data by linear interpolation between the two concentrations bracketing 50% inhibition. Differences between samples were tested by one-way analysis of variance with the least significant difference post hoc test, with significance set at p < 0.05 (SPSS version 20; IBM, New York, NY, USA).

3. Results

3.1. Radical Scavenging

Liposomal vitamin C was the most potent scavenger (IC₅₀ 0.12 mg/mL DPPH; 0.77 mg/mL ABTS), followed by tocotrienol (1.1; 7.3 mg/mL) and turmeric extract (3.1; 13.7 mg/mL) (Table 2). Ginger extract reached approximately 50% inhibition only at the 50 mg/mL limit. Black ginger plateaued at about 35% (DPPH) and 32% (ABTS), and ginseng, garlic and colostrum remained below 30% in both assays. Elderberry showed near-maximal DPPH response already at 12.5 mg/mL (49.5% rising only to 57.1%), so its IC₅₀ lies at or below the lowest concentration tested.

3.2. Lipoxygenase Inhibition

At the maximum soluble concentrations, all ingredients except elderberry inhibited lipoxygenase by about 50% or more (Table 3). Tocotrienol produced 59.8% inhibition at 3.125 mg/mL and liposomal vitamin C 49.7% at 6.25 mg/mL; ginseng, astragalus, black ginger, ginger, garlic, turmeric and colostrum each produced 50.5–56.4% at 12.5 mg/mL, and 69.4–76.2% at 25 mg/mL where solubility allowed. Elderberry inhibition remained constant at 23–25% across 1–20 mg/mL.

3.3. Inhibition of Albumin Denaturation

Black ginger extract was the only sample to inhibit albumin denaturation almost completely (93.8 ± 2.1% at 25 mg/mL; 91.5 ± 4.2% at 50 mg/mL), with a steep transition from no detectable activity at 12.5 mg/mL to 52.8% at 18.75 mg/mL (Table 3). Liposomal vitamin C (38.0% at 0.5 mg/mL) and tocotrienol (35.0% at 5 mg/mL) were active at low concentrations but could not be tested higher because of signal interference; on a per-milligram basis they were therefore the most potent, whereas black ginger achieved the greatest maximal protection. Ginger gave 26.1% and colostrum 12.3% at 50 mg/mL; elderberry, ginseng, astragalus, garlic and turmeric showed no detectable activity within their testable ranges.

3.4. Putative Constituents by LC-QTOF-MS/MS

In black ginger extract, accurate-mass annotation supported citric acid (Table 4); the dominant late-eluting feature (m/z 361.134, 14.5–15.1 min) could not be assigned with confidence. The background cluster at m/z 713–837 described in the companion study [8] was also present and was excluded.

4. Discussion

The ranking of radical-scavenging potency—liposomal vitamin C > tocotrienol > turmeric > the remaining botanicals—matches the known chemistry of these ingredients, and agreement between the DPPH and ABTS orders supports its reliability [2]. Most botanical extracts were weak direct scavengers, which is common for standardised extracts whose actives are present at a few percent of the powder. Two specification details bear on the weaker results. The ginseng material is a stem-and-leaf extract rather than a root extract, which is unusual for a nutraceutical ginseng and plausibly contributes to its weak showing in both assays. The declared actives of the samples also span more than three orders of magnitude, from 0.01% acetogenins in the companion study to 10–15% ginsenosides, up to 10% curcumin and at least 15% total tocotrienols and tocopherol here. Potency expressed per milligram of powder therefore tracks active loading as much as intrinsic activity.

The key finding is the behaviour of black ginger. It was a poor radical scavenger (≤35% at the solubility limit) yet gave the highest maximal inhibition of albumin denaturation of all samples, with a sharp concentration threshold. This dissociation indicates that its protein-stabilising effect does not depend on radical scavenging. Black ginger rhizome is characterised by polymethoxyflavones such as 5,7-dimethoxyflavone and 3,5,7,3′,4′-pentamethoxyflavone [1], which are relatively lipophilic and have few free hydroxyl groups—features that limit hydrogen-donating radical scavenging but are compatible with hydrophobic binding to proteins such as albumin. We propose this as a working hypothesis. The steep transition between 12.5 and 18.75 mg/mL is typical of the albumin assay, in which protection appears once ligand binding is sufficient to stabilise the protein against thermal unfolding [6]. These methoxyflavones ionise poorly in negative electrospray mode, which explains why they were not detected; a positive-mode analysis with authentic standards is the necessary next step. One qualification follows from the material itself: the extract tested is a 20:1 water extract. Polymethoxyflavones are markedly lipophilic and are carried only sparingly into an aqueous extract, so the protein-stabilising activity observed here is more safely attributed to the water-soluble fraction of the rhizome than to the methoxyflavones themselves, whose content in this material is not quantified. Positive-mode mass spectrometry and a methoxyflavone assay on the same batch would settle this, and are planned.

Lipoxygenase inhibition was broadly similar (about 50% at 12.5 mg/mL) across most ingredients, so this assay discriminated poorly between them at the concentrations tested. The constant 23–25% inhibition by elderberry across a 20-fold concentration range suggests an assay artefact, possibly related to its intense anthocyanin colour, rather than a true dose–response.

Limitations. No reference anti-inflammatory drug was included, so the anti-inflammatory assays support relative ranking only. Several samples could be tested only at a single concentration because of solubility or colour interference, and turmeric could not be evaluated above 5 mg/mL in the albumin assay, so its activity may be underestimated. Ingredients were commercial materials without reported marker content. All assays were cell-free. The ingredients tested are the authors’ own commercial materials or materials sourced by their company, and are reported as sold rather than matched for active loading: extraction ratios range from 10:1 to 100:1 and declared actives from no stated marker to at least 15% total tocotrienols and tocopherol. No specification sheet is held for the colostrum.

5. Conclusions

Across four in vitro assays, liposomal vitamin C and tocotrienol were the most potent radical scavengers, and most botanical ingredients inhibited lipoxygenase moderately. Black ginger (Kaempferia parviflora) extract almost completely prevented heat-induced albumin denaturation at 25 mg/mL despite weak radical scavenging, indicating a non-radical, protein-stabilising mechanism. Black ginger therefore merits targeted follow-up, including positive-mode mass spectrometry of its methoxyflavones, comparison with diclofenac sodium, and cell-based inflammation models; these are planned for the next phase of testing.