Sample collection

Ten different samples of commercially available dietary supplements labeled as Ashwagandha root extracts, which are according to legal classification over-the-counter products, were obtained randomly from drug stores, pharmacies, and online stores. Moreover, only mono-herbal Ashwagandha products were used but no poly-herbal preparations. To avoid potential conflicts, information about the manufacturers of products that do not meet their stated claims is deliberately omitted.

Ashwagandha root was procured from a pharmaceutical wholesaler (Kottas Pharma GmbH) who supplies Austrian pharmacies with medicinal plants. The specimen with the batch number W23201745, taken in accordance with GMP regulations, was checked and confirmed for identity by botanically trained experts. Another Ashwagandha root was obtained from Kräuterkontor (Berlin, Germany). Table 1 gives an overview of the investigated samples.

Preparation of sample and stock solution

100 mg of each Ashwagandha dietary supplement was suspended in 15 mL of methanol and subjected to extraction at 70°C for 30 min in a closed vial using an ultrasonic bath. This procedure was repeated twice. The supernatants from these extractions were combined, and the total volume was adjusted to 50 mL in a volumetric flask. In summary, the samples (roots and dietary products labeled as root extracts) were extracted three times with methanol at elevated temperatures. In a fourth extraction only negligible amounts of the investigated compounds were detected, indicating that triple extraction is clearly sufficient. WS IV and withaferin A from Merck (Darmstadt, Germany) were obtained in the form of a stock solution in a concentration of 100 µg/mL methanol.

Reagents and standards

Water with a resistivity of 18 MΩ·cm was produced using a Milli-Q water purification system (Millipore). Analytical-grade acetonitrile and methanol were sourced from VWR (Fontenay-sous-Bois, France), ammonium formate from Sigma-Aldrich (St. Louis, MO, USA), and hydrochloric acid, phloroglucinol, iodine, potassium iodide, and WS IV and withaferin A from Merck (Darmstadt, Germany). Various types of starch were acquired from an Austrian pharmacy. Lugol’s solution, which was used for the microscopic staining of starch granules, was prepared with 2% iodine and 4% potassium iodide in water. The Wiesner reagent, employed for the microscopic staining of lignified cell parts, comprised 1% phloroglucinol in ethanol mixed with 12 M HCl (1:1, v:v).

Instrumentation

Analytical and chromatographic conditions of HPLC

The chosen UV detection wavelength was 227 nm. Chromatographic separation was achieved using a YMC-Triart C18 column (100 × 3 mm ID, 3 μm particle size) supplied by YMC (Kyoto, Japan). The injection volume was set at 5 μL. The mobile phase [13] comprised acetonitrile (solvent A) and water (solvent B), with a gradient program starting at 25% solvent A and 75% solvent B. This ratio shifted to 55% solvent A and 45% solvent B at 10.0 min, then to 95% solvent A and 5% solvent B at 13.0 min, which was maintained until 16 min. The conditions returned to their initial conditions at 16.1 min, completing the run within a total time of 20 min. The flow rate was maintained at 0.5 mL/min.

The chromatographic conditions for HPLC-MS, including the column, run time, flow rate, and acetonitrile gradient, were identical to those of the HPLC-UV method, with the sole modification of replacing 5% of solvent B with a 10 mM ammonium formate solution in water containing 0.1% formic acid (solvent C).

Assay validation

Regarding the specificity of the method, the individual WSs and WLs were identified using high-resolution mass spectrometry. For linearity 10 different concentrations in the range of 0.2–10 µg/mL for WS IV and withaferin A were analyzed. The precision of the method was tested by a triplicate determination of a dietary supplement labeled as Ashwagandha root extract. The limit of quantitation was set as the concentration corresponding to one-fifth of the lowest standard of the calibration for WS IV and one-tenth for withaferin A.

Microscopically detected plant parts in dietary supplements

Microscopic examinations were conducted to compare dietary supplements of Ashwagandha root extracts with pulverized authentic Ashwagandha root. These comparisons revealed a substantial issue at first glance: plant fragments were visible in the dietary supplements, which contradicts the expectation that properly prepared extracts should be devoid of plant parts. The supplements were labeled explicitly as extracts and not as mixtures of extract and plant material, creating a discrepancy between the observed presence of plant fragments and the product labeling as “extract”. Figure 1 shows the microscopic identification of the xylem fragments described and depicted in prior literature [18, 19]. These lignified xylem parts were stained red with Wiesner reagent (Figure 1, right image).

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Figure 1. 

Microscopy of xylem parts identified in a dietary supplement (sample K) labeled as Ashwagandha root extract. Transmitted light microscopy of xylem parts unstained (left) and stained with phloroglucinol (right), magnified 500×

Xylem parts were found in all examined Ashwagandha root extracts. A remarkably large xylem fragment of about 800 µm is shown in Figure S1 in the supplementary data. Even small xylem fragments are clearly identified due to their characteristic shape, see Figure S2 (supplementary data).

Microscopy of starch grains

The starch found in the root appears as roundish to oval single grains, as well as in conglomerates of two, three, or, more rarely, four grains [18]. The fissure shapes emanating from the mostly centric hila exhibit varying shapes, ranging from longitudinal to asymmetrical stellate in both single grains and conglomerates. Figure 2 shows the starch grain aggregates, each comprising two grains, stained with diluted Lugol’s solution. The starch grains of authentic Ashwagandha roots measure up to approximately 30 µm, as shown in Figure 2.

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Figure 2. 

Starch aggregates composed of two starch grains stained with diluted Lugol’s solution of Ashwagandha root (500×), sample A

Characteristic starch grains as described above were not found in any of the analyzed dietary supplements. Instead, in certain samples, the starch grains measured up to 50 µm, approximately double the size of those in pulverized Ashwagandha root. Their shape resembled that of deformed, gelatinized starch grains, with a typical bulge at the edge, as shown in Figure 3. The deformation of starch granules, which often results from thermal or pressure effects, is a recognized phenomenon.

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Figure 3. 

Deformed gelatinized starch grains stained with diluted Lugol’s solution in a dietary supplement labeled as Ashwagandha root extract (500×), sample K

The gelatinization of starch grains was investigated using authentic roots. The resulting gelatinized starch grains bore a striking resemblance in shape and size to those found in some analyzed capsule contents labeled as Ashwagandha root extract.

It should be noted that starch (from wheat, rice, and maize) may be used as an excipient in preparing these capsule-based Ashwagandha extracts. Therefore, careful attention to the shape and size of starch grains is crucial when identifying the presence of starch using Lugol’s solution.

HPLC-UV

HPLC-UV is a pivotal analytical method that is widely acclaimed for its versatility and broad applicability in areas such as food control. In the present case, specificity was confirmed by unambiguous detection of both the molecular ion [M⁺H]⁺ and the ammonium adduct [M⁺NH4]⁺. Concerning linearity, the calibration curve for withaferin A yielded an equation of y = 100.39x – 0.9197 with a coefficient of determination R² of 0.9999. For WS IV, the equation was y = 36.375x + 0.3797 with an R² of 0.9999. The precision from a triplicate determination showed a relative standard deviation of 2.1 % for withaferin A and 4.6% for WS IV. The limit of quantitation was 0.04 µg/mL for WS IV and 0.02 µg/mL for withaferin A. The developed chromatographic method was used for comparison between Ashwagandha root extracts and authentic Ashwagandha root.

The integration of HPLC with high-resolution mass spectrometry was solely for the accurate peak identification of principal WLs and WSs throughout the HPLC-UV method’s development. The principal signals were identified by high-resolution mass spectrometry and correlated with their respective UV signals. Table 2 organizes the individual compounds by their retention times.

Figure 4 shows a chromatographic comparison between the dietary supplements labeled as Ashwagandha root extracts and the authentic Ashwagandha root used as a reference. The chromatograms of the dietary supplements and the authentic root are almost identical in terms of peak pattern and signal intensity. The concentration of WLs in the dietary supplements agrees with that of the purchased Ashwagandha root. This finding corroborates the microscopic observations, indicating that the capsules tested do not contain a properly prepared concentrated Ashwagandha extract. It may be an interesting question if leaf material has been added to the dietary supplements labeled as root extracts. For this purpose, the signal intensities of withaferin A and WL A were compared. As can be seen from Figure 4, withaferin A exhibits a signal intensity similar to WL A. However, a previous study investigating a WL-rich fraction prepared from roots and leaves showed a significantly different peak pattern in chromatography. The chromatogram of this rich fraction from roots and leaves demonstrates a signal ratio of Withaferin A to WL A exceeding 10 times [20]. Therefore, it is not expectable that the samples used in our study are contaminated or adulterated with leaves.

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Figure 4. 

Chromatographic comparison between authentic Ashwagandha roots and dietary supplements labeled as Ashwagandha root extracts. A: Crushed Ashwagandha root; B: pulverized Ashwagandha root; C–E: dietary supplements containing Ashwagandha extracts. Peaks 1–12 are detailed in Table 2

WS IV and withaferin A were selected as standard substances for quantifying the principal components of WSs and WLs, respectively. Using the response of a standard substance for the quantitative characterization of a group of ingredients is a common approach for secondary plant metabolites [21, 22]. The UV response of WS IV facilitated the quantification of WS IV (peak 1), WS VII (peak 2), and WS V (peak 4). The quantities of WSs determined in this way were summed and reported as the WS content in percentage. Similarly, the response of withaferin A was employed to estimate the content of dihydrowithaferin A (peak 3), withaferin A (peak 5), 12-deoxywithastramonolide (peak 6), withanone (peak 7), WL A (peak 8), WL B (peak 12), and its isomers (peaks 9, 10, 11). The cumulative results of the WLs quantified using this approach were also summed and expressed as the WL content in percentage.

Table 3 lists the content for both summed WSs and WLs of all coded samples. The detected main WL content in all tested Ashwagandha root extracts was on average 0.13%, and therefore is considerably lower than the claimed 5% median content, yet it agrees with our measurements of Ashwagandha root and corroborates published data on this matter.

The presence of only 0.13% of the main WLs in all tested Ashwagandha root extracts, which is considerably lower than the stipulated 5% median content, aligns with published data regarding Ashwagandha root.

Aspect of manufacturing dietary supplements

The microscopic detection of plant fragments in dietary supplements labeled as Ashwagandha root extracts prompts several concerns. One possibility is that the plant material was intended as a filler but was not appropriately disclosed on the supplement packaging. Alternatively, there could have been a mix-up in the manufacturing process, where the pulverized root was used instead of the extract. Another reason may be a highly inadequate extraction procedure which could explain the presence of xylem fragments and the much too low concentrations of the main ingredients.

Given that multiple batches of some supplements exhibited this issue, a systematic processing error may be at play. For instance, supplement manufacturers might be sourcing from a central wholesaler whose “extract” is used without proper verification.

Regardless of the underlying reason, plant fragments raise serious concerns about the proper preparation and the integrity of the claimed concentrations. HPLC-UV analyses further validate these concerns, demonstrating that the Ashwagandha root extracts in dietary supplements do not meet the manufacturers’ claimed median WL content of 5%. The concentration of selected main WLs detected falls within the range typical of the herbal raw material, suggesting that the efficacy of the tested supplements is similar to that of the raw plant rather than an enhanced extract.

Lack of testing

The pressing issue is the universal failure of tested dietary supplements labeled as containing Ashwagandha root extract to live up to their claims. The discovery that none of the samples from 10 different manufacturers contained the advertised pure Ashwagandha extracts signals a dire need for regulatory oversight and quality testing in the food industry. The reasons behind this widespread lack of compliance could be manifold, but a notable factor might be the intrinsic limitations of current testing methodologies. Determining the total WL content, even with advanced techniques, yields only approximate values. Consequently, adequate testing protocols may not have been established or enforced. Furthermore, there has been a decline in the application of simple pharmacognostic tests like microscopy, likely due to the emphasis on sophisticated instrumental analysis in recent decades.

The testing methods introduced in this study, employing simple and accessible tools such as microscopes and HPLC-UV, demonstrate that it is feasible to conduct a valid evaluation of dietary supplements containing Ashwagandha root extracts. These proposed methodologies not only negate the need for the labor-intensive isolation of standard compounds but also considerably reduce the costs associated with advanced analytical equipment. In addition, the quantitative estimates produced by these methods align with published data on the WL and WS content in Ashwagandha root, using more expensive and sophisticated equipment. The comparison of these results with those obtained from HPLC-MS analyses demonstrates the reliability of the procedure presented herein. This comparison highlights the advantages of the proposed approach in terms of cost efficiency and simplicity without compromising performance.

Overall, this approach represents a notable advancement in the evaluation of natural products by introducing a straightforward and rapid quality check for Ashwagandha root extracts in dietary supplements. This procedure offers a reliable estimate of the main WL content.