Ecdysteroid-containing food supplements from Cyanotis arachnoidea on the European market: evidence for spinach product counterfeiting

Phytoecdysteroids like 20-hydroxyecdysone (“ecdysterone”) can exert a mild, non-hormonal anabolic/adaptogenic activity in mammals, and as such, are frequently used in food supplements. Spinach is well-known for its relatively low ecdysteroid content. Cyanotis arachnoidea, a plant native in China, is among the richest sources of phytoecdysteroids, and extracts of this plant are marketed in tons per year amounts via the internet at highly competitive prices. Here we report the investigation of a series of food supplements produced in Germany and claimed to contain spinach extracts. Twelve ecdysteroids including two new compounds were isolated and utilized as marker compounds. A comparative analysis of the products with Cyanotis and spinach extracts provides evidence that they were manufactured from Cyanotis extracts instead of spinach as stated. Based on the chromatographic fingerprints, 20-hydroxyecdysone 2- and 3-acetate are suggested as diagnostic markers for related quality control. This case appears to represent an unusual type of dietary supplement counterfeiting: undeclared extracts from alternative plants would supposedly ‘guarantee’ product efficacy.

Scientific RepoRts | 6:37322 | DOI: 10.1038/srep37322 ca. 0.005-0.08% of ecdysteroids (primarily 20E) per fresh weight depending on the time of harvest 7 , which means that even a "heavy-consumer" of spinach could hardly take more than a hundred milligrams of 20E per day from this source. On the other hand, sportsmen (most typically body-builders) are usually advised to take up to several grams of 20E per day, which dose has never seriously been studied for long-term safety.
In contrast with spinach, Cyanotis arachnoidea, a plant native in China, is undoubtedly among the richest sources of 20E; depending on the time and area of harvest, the levels of 20E in the roots can reach as high as 4-5% 8 . A simple internet search reveals dozens of companies offering several tons per year (some even claiming to be able to supply over a ton per week) which is available for internet-based purchase with a worldwide delivery. The scale of this market can well be demonstrated by the fact that some of these companies have a minimum order limit of 200 kg of "20E" or rather C. arachnoidea extract, and, depending on the purity, the price can be as low as around 10-20 USD per kg. Based on information obtained from the manufacturers, these extracts are offered for various uses: health benefits (including some questionable ones, e.g. antitumor properties) of ecdysteroids are typically advertised, and the use of such extracts for increasing the biomass production in agriculture and aquaculture is apparently also widespread.
Concerning any dietary uses, C. arachnoidea and its preparations have not been used in traditional herbal medicines or foodstuffs in Europe and it is unclear whether they can be legally marketed for human consumption 9 . In Hungary, high ecdysteroid containing plants, including a related species, Cyanotis vaga (together with Achyranthes aspera, Cyathula capitata, Pfaffia paniculata and Polypodium virginianum), are banned by the National Institute for Food and Nutrition Science (OÉTI) and, as such, cannot be marketed as food supplements 10 . It is also important to mention, that C. arachnoidea is neither a foodstuff nor a valued medicinal plant in China. Even though there are two ecdysteroids containing plant species in the Chinese Pharmacopoeia, such as Cyathula officinalis (Chuan Niuxi) and Achyranthes bidentata (Huai Niuxi), no Cyanotis species are recorded (Prof. De-an Guo; personal communication, 04.09.2015).
Due to decades-long experience of our group with phytoecdysteroids including the discovery of ecdysteroids' presence in spinach 6 , the appearance of spinach-containing products marketed for their ecdysteroid content on the European market attracted our attention. Accordingly, our aim was to study the ecdysteroid composition of some of these products and to investigate the possibility of adulteration.

Results and Discussion
Analysis of ecdysteroid-containing food supplements. A quick analysis of the purchased products by thin-layer chromatography (TLC) immediately revealed such a complex-pattern ecdysteroid content, which seemed extremely unlikely for spinach. Following this, the HPLC fingerprints of the food supplements were compared with those of two independent samples of C. arachnoidea extracts purchased from China (CA1 and CA2), as well as with that of an authentic spinach extract prepared by us. Selected chromatograms for the product FS1 are presented in Fig. 1.
It can well be seen from Fig. 1A, that not only the main accompanying ecdysteroids but also numerous minor constituents of C. arachnoidea are perfect match to those of FS1, only the relative amounts are different. Additionally, no ecdysteroids of FS1, other than 20E, were detectable within the spinach extract, nor could any characteristic constituents of spinach be observed within FS1 except for 20E itself (Fig. 1B). One could certainly argue that FS1 might still be originated from spinach, and the differences we observe come from a different extraction procedure or different processing techniques, e.g. solid-phase extraction (SPE) or column chromatography that could have been applied in order to enrich its originally low ecdysteroid content. There are simple techniques to separate ecdysteroids from phenolic constituents, e.g. on polyamide the two flavonoid-like compounds observed close to the peak of 20E within spinach could likely be removed 11 , and the large amount of highly polar constituents of spinach could also be removed by SPE on (quite expensive) reversed-phase silica. No such processing would, however, explain why not even trace amounts of any of the minor or accompanying major ecdysteroids of FS1 are detectable in an authentic sample of spinach. This, together with the matching overall chromatographic fingerprint of FS1 to those of two independent samples of Cyanotis extracts, provides evidence that the food supplements were manufactured using Cyanotis arachnoidea instead of spinach.
Isolation and structure elucidation of ecdysteroids from food supplement FS1. Following the above observations, our next aim was to isolate the major and possibly the minor ecdysteroids of C. arachnoidea by utilizing FS1, the product which was proven to be a rich raw material for such an initiative. By processing 5 g of FS1 with the consecutive use of various chromatographic methods of different selectivity, twelve ecdysteroids other than 20E were successfully isolated including two new compounds (4 and 7). Chemical structures of the two new isolates were elucidated by comprehensive one-and two-dimensional NMR methods allowing a complete signal assignment. 1 H and 13 C NMR data of the new ecdysteroids 4 and 7 are presented in Table 1, along with those of shidasterone (6), the non-acetylated reference compound for 7. The new NMR data obtained in methanol-d 4 for compounds 11 and 12 are compiled in Table 2. Chemical structures of the isolated compounds are presented in Fig. 2.
The 1 H and 13 C NMR spectra of compounds 1-3, 5, 6, and 8-12 confirmed that these compounds are identical with 20E 2-acetate (1), 20E 3-acetate (2), ajugasterone C (3), ajugasterone C 3-acetate (5), shidasterone (6), dacryhainansterone (8), rubrosterone (9), 5α -20-hydroxyecdysone (10), and 25R-and 25-S-20,26-dihydroxyecdysone (11 and 12).  In case of compound 4, twenty-nine 13 C signals were discernible in the DEPTQ spectrum, indicating the presence of six methyl, seven methylene, and nine methyne groups, in addition to one quaternary sp 2 (δ 165.7) and four quaternary sp 3  In case of compound 7, the molecular formula of C 29 H 46 O 7 was established by HRMS. The NMR analysis revealed twenty-nine 13 C signals, six methyl, eight methylene, seven methyne groups, furthermore five quaternary sp 3 and one quaternary sp 2 carbon (δ 168.4) atoms, and one conjugated C= O (δ 205.6) and one O-C= O (δ 172.6) by moiety. Integration of the signals in the 1 H NMR spectrum summed up to 41H atoms. Considering the molecular formula, the molecule contains three -OH groups. Thus, compound 7 contains five rings and three double bonds. As also demonstrated in Table 1, the 1 H and 13 C chemical shifts of this steroid skeleton were nearly identical with those of shidasterone 12 13 ; these two compounds were distinguished from each-other by their 1 H and 13 C NMR data taken in pyridine-d 5 . In the present work, our comprehensive one-and two-dimensional NMR study performed in methanol-d 4 resulted a complete 13 C NMR and 1 H signal assignment also including the differentiation of the H α and H β methylene signals. A comparison of the 1 H and 13 C chemical shifts of the C-25 epimers revealed that they were almost identical. The only characteristic difference in the spectra was observed at the signals of the CH 2 -OH group, namely in compound 11 the δ H 2 -27 signal appeared as a singlet at 3.37 (2H) and δ C-27 appeared at 70.8 ppm, whereas in case of 12 the δ H 2 -27 signal appeared in form of dublets at 3.39 (1H) and 3.35 (1H), and δ C-27 appeared at 70.2 ppm. It is noteworthy that the NMR results do not permit the evaluation of the absolute configuration of both epimers. Representative NMR spectra of compounds 11 and 12 are available as Supplementary Information.
Compounds 4 and 7 are new ecdysteroids. However, no sound judgment concerning their natural origin or artefact nature can be made at this point considering that the source was an ecdysteroid containing product not directly the plant itself. The presence of several ecdysteroid acetates, mainly those of 2-acetyl substituted ones, might be of chemotaxonomical interest: according to the regularly updated online ecdysteroid database 12 , only five ecdysteroid 2-acetates have been isolated from plants till now, and three of them from Cyanotis species. Ajugasterone C 2-acetate (4) might be the fourth in this series, assuming that its natural (i.e. not artefact) origin will be proven. Anyhow, its presence in the marketed spinach products, together with that of 20-hydroxyecdysone 2-acetate (1), is further evidence supporting that these products originate from a Cyanotis plant. Considering these phytochemical aspects and that compounds 1 and 2 appear as major peaks in the chromatographic fingerprints of both CA1 and CA2 but not in that of spinach (see Fig. 1B), 20E 2-acetate (1) and 20E 3-acetate (2) are hereby suggested as chemical marker compounds for a diagnostic identification of Cyanotis extracts within ecdysteroid-containing products.
Quantitative analysis of food supplements FS1-5. The amounts of the major isolated ecdysteroids within products FS1-5 were determined by RP-HPLC-DAD, results of this study are compiled in Table 3.
Based on our results, the following approximate doses are ingested with a recommended daily intake of 2 capsules. 20E: 2-24 mg; 1: ≤ 9 mg; 2: ≤ 11.7 mg; 4: 0.2-0.8 mg; 6: 0.1-0.9 mg; 8: 0.3-1.8 mg; 10: ≤ 0.3 mg; 11 + 12: ≤ 0.7 mg. These doses are in fact well within the limit generally considered safe for 20E per os. On the other hand, it must be emphasized that neither European traditional knowledge nor safety studies are available for Cyanotis species. The roots of Cyanotis arachnoidea are not considered dietary anywhere, and practically nothing is known on the other constituents of the plant or their safety, in contrast with the case of spinach. We believe that mentioning the tragic story that has become well-known as the "Chinese herb nephropathy", exerted by the aristolochic acid content of certain supposedly safe herbal remedies 14 , is sufficient to highlight the potential danger in marketing the extracts of uninvestigated plant species for human consumption on the sole basis of the known safety of one single constituent group (ecdysteroids).

Conclusions
Our results provide evidence that the investigated food supplements contain Cyanotis extract instead of spinach as claimed. This case appears to represent an unusual type of dietary supplement counterfeiting: the alleged pharmacological activities are supposedly enhanced by the addition of extracts from undeclared plants that contain higher amounts of the same active ingredient(s). Although this may be to "guarantee" product efficacy, such an act may bypass European dietary supplement regulations (correct declaration of composition, dosage, safety, etc.).
It needs to be highlighted that the current study investigated only one single product family from a single provider. Large quantities of ecdysteroid-enriched Cyanotis extracts are available on the market at inexpensive prices, but at this point it is difficult to make a sound judgment on how much this type of counterfeiting is spread within the EU. Since neither C. arachnoidea nor other Cyanotis species have a history of human consumption in Europe, any products containing such extracts may need to be subject to proper authorization. At the same time, our results highlight the urgency of in-depth safety studies on Cyanotis extracts.

Methods
Plant material and sample preparation. Two independent preparations of C. arachnoidea have been purchased: CA1 (90% claimed purity of 20E by means of HPLC, Shaanxi KingSci Biotechnology Co., Ltd., Shanghai, China) and CA2 (50% claimed purity of 20E by means of UV absorbance, Xi'an Olin Biological Technology Co., Ltd., Xi'an, China). CA1 was re-crystallized from ethyl acetate-methanol (2:1, v/v). The mother liquid of this process, enriched in minor constituents, was utilized together with CA2 for comparison with the food supplements. Fresh spinach leaves were purchased from the local food market in Szeged, Hungary in July, 2014. Extraction was performed by sonication in methanol, and the re-dissolved crude extract was utilized for analysis after evaporation under reduced pressure at 40 °C.
Ecdysteroid containing food supplements. A series of ecdysteroid-containing products claimed to contain spinach extract were purchased by ordering from the producer, VerdeVital Beratungs-, Importund Vertriebsgesselschaft mbH (Bovenden, Germany) in 2013. These products were VerdeFit (L13761012), VerdeKlimEx (L09760711), VerdeDry (L09810711), VerdeOs (L09800711) and VerdeArthroSan (L00250113), and hereby they are referred to as FS1-5, respectively. Herbal constituents of FS1-5 per capsules were claimed as follows.   Table 3. Amounts of the individual compounds in the investigated products by means of calibration with 20E (R 2 = 0.9999). The structures of 10 and 11 could not be unambiguously assigned by NMR, hence their peaks are considered as interchangeable; *: trace amounts. The indicated amounts are results of a single experiment: since the exact composition of a food supplement of unknown origin can greatly vary from batch to batch due to many unknown factors (extraction and processing methods, storing conditions during transportation, etc), presenting data with two decimals accuracy from a single experiment was considered sufficient.

Analytical HPLC and quantitative determination of ecdysteroids in FS1-5. Products FS1-5,
Cyanotis samples CA1 and CA2, and spinach extract were compared by analytical scale RP-HPLC-DAD on the above-mentioned Jasco HPLC system by using two columns of different selectivity, a Zorbax Eclipse XDB-C8, 150 × 4.6 mm, 5 μ m (Agilent Technologies, Santa Clara, CA, USA), and a Kinetex XB-C18, 250 × 4.6 mm, 5 μ m (Phenomenex, Torrance, CA, USA), both with aqueous MeOH and aqueous ACN as solvents. For quantifying compounds 1, 2, 4, 6, 7 and 10-12 within the five products, a single point experiment was performed on the above-mentioned Kinetex column, with a gradient system of 17% aqueous ACN increasing to 30% in 15 min, then to 55% in 2 min which composition stayed for 2 min and subsequently returned to 17% of ACN. The flow rate was 1 mL/min. Six-point calibration was performed by injecting 10 μ L of standard solutions containing 500, 125, 31.25, 7.81, 1.95 or 0.49 μ g/mL of 20E, prepared by four-times serial dilutions from a 2 mg/mL stock solution hence the results are expressed in equivalents of 20E, and integration was performed automatically at λ = 245 nm at a slope sensitivity of 100.00 μ V/sec.