Chemicals and reagents

Andrographolide (≥ 98.0%) was obtained from TCI (Tokyo, Japan), while all the other active substance standards (standard analytical grade) were purchased from Sigma-Aldrich, Merck (St. Louis, MO, USA). Formic acid (LiChropur, 97.5–98.5%) and ethanol (LiChrosolv, gradient grade) were supplied by Supelco (Bellefonte, PA, USA). Acetonitrile (HPLC grade) was purchased from Avantor (Radnor, PA, USA). Ultrapure water was produced using an Ultra Clear UV water purifying system (SG Water, Barsbuttel, Germany), resistivity >18 MΩ/cm at 25 °C and total organic carbon <5 µg/mL. Excipients for the selectivity testing were hydroxypropyl methylcellulose Methocel K100M Premium CR (Colorcon, Harleysville, PA, USA), stearic acid, lactose monohydrate, wheat, rice and corn starch (Kemig, Zagreb, Croatia) and magnesium stearate (Acros Organics, Princeton, NJ, USA).

Samples

In total, 54 samples (raw material, food and dietary supplements), of which 35 preparations contained extracts, while 19 contained processed botanical forms and botanical products. From local pharmacies 13 samples were procured, 15 from food health stores and 26 were purchased from the Internet (products available online only in Croatia). The samples were coded with letter S followed by the corresponding sample number. All samples were analyzed prior to the stated expiry date. A detailed description of all analyzed samples, including mode of acquisition, manufacturer origin, sample type and label, is given in Table S1.

Instrumentation

Weighing of masses below 100 mg was done on an MX5 Microbalance with a readability of 1 µg, while those above 100 mg were weighed using an AG245 balance, both from Mettler Toledo (Columbus, OH, USA). Extraction procedure was done using an Elmasonic xtra TT ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany). Centrifugation was conducted on a mini G centrifuge (IKA, Staufen im Bresgau, Germany) and a centrifuge with temperature control Z 326K (Hermle, Gosheim, Germany) at 1200×g. Analyses were carried out on an Agilent 1260 series chromatograph equipped with a binary pump, degasser, autosampler, column oven and diode array detector operated using Chemstation OpenLab CDS rev. C01.10 (Agilent Technologies, Santa Clara, CA, USA).

Chromatographic analysis

Separation was conducted on an HSS Cyano column, 150 mm×3.0 mm, 3.5 µm particle size (Waters Corporation, Milford, MA, USA) thermostated at 40 °C. Ultrapure water and acetonitrile, both acidified with final content of 0.1% formic acid, were used as mobile phase components A and B, respectively. Gradient elution at a flow of 1 mL/min was applied as follows: 0–6 min isocratic 40% B, 6–16.5 min linear gradient 40–70% B, 16.5–17.5 min linear gradient 70–100% B, and 17.5–18 min isocratic 100% B. Pure organic phase was applied for two more minutes and the column was equilibrated to starting conditions with the total method run time of 25 min. Injection volume was set to 5 µL and the needle was washed prior to each injection with methanol . Autoinjector temperature was 15 °C. Detection wavelengths were set to 206 (for ABA, BBA, AABA, ABBA and NANDR), 230 (for ANDR), 256 (for 14-DANDR, KBA and AKBA), 340 (for PIP) and 422 nm (for BDMC, DMC and CUR), bandwidth 4 nm, no reference wavelength.

Identification of AABA and ABBA using mass spectrometry

AABA and ABBA identification in real samples was conducted using Synapt G2-Si ESI-QTOF-MS system, controlled using MassLynx v. 4.1 software (Waters Corporation). MS conditions were as follows: sampling cone voltage 60 V, source temperature 120 °C, desolvation temperature 350 °C, capillary voltage 3 kV, and desolvation gas flow 600 L/h. Spectra were acquired in positive ion mode. MS/MS experiments were done using m/z=499.4 as precursor and collision energy of 5 V, scanning from 100 to 500 m/z.

Optimization of extraction procedure using response surface methodology

Ultrasound-assisted extraction of active substances was optimized using response surface methodology approach, more specifically a three-factorial Box-Behnken design. The methodology was applied to a mixture of processed botanical forms (m(powdered green chiretta leaf):m(powdered Indian frankincense resin):m(powdered turmeric rhizome):m(powdered black pepper fruit)=20:10:2:1). A mass of 25 mg of mixture was suspended in 10 mL of solvent and subjected to ultrasonic extraction at designated temperature and duration. Independent variables were ethanol ratio in the hydroethanolic extraction solvent (40–100%), extraction temperature (30–80 °C) and sonication time (10–30 min). Considering the physicochemical similarities within compounds of the same botanical source, the responses were the sums of extraction yields of all analytes for the respective herbal substance.

Sample preparation

Samples were extracted using optimized conditions predicted via Box-Behnken design. Firstly, the contents of six dosage forms (tablets or capsules) were individually weighed and thoroughly homogenized in a ceramic mortar. For samples in bulk, this step was omitted. A mass of 25 mg of sample was accurately weighed and suspended in 25 mL of 81.5% ethanol (V/V), sonicated for 30 min at 60 °C and centrifuged at 1200×g. The supernatants were diluted, if necessary, and injected into the HPLC system. Liquid samples were injected post centrifugation and appropriate dilution.

Method validation

The method was validated according to International Council on Harmonization (ICH) guidelines (23). The examined parameters included selectivity, linearity, limits of detection (LOD) and quantification (LOQ), precision, accuracy, stability and robustness. The used model samples were previously mentioned processed botanical mixture (for composition, vide supra) and dry extract mixture (m(S31):m(S26):m(S21):m(S4)=20:5:5:1).

Statistical analysis

Statistical analyses were done using Microsoft Excel v. 16.0.14026.20270 (24). Plackett-Burman robustness testing and Box-Behnken extraction optimization were conducted using Design Expert v. 7.0.0 (25).

Chromatographic method development

Before starting method development, distribution coefficients (log D) of analytes were examined to gain insight into their chromatographic behaviour at different pH of mobile phase. Higher log D values for all analytes but PIP were observed at low pH, subsequently leading to stronger retention in nonpolar stationary phases. Application of low pH mobile phases would allow for better separation of hydrophilic matrix components from the analytes, enhancing selectivity. Thus, an acidic mobile phase modifier with 0.1% formic acid was chosen. Regarding stationary phases, multiple column packings were tested: C4 (Kromasil, 150 mm×4.6 mm, 3.5 µm particle size, Nouryon, Amsterdam, the Netherlands), C8 (Kinetex, 150 mm×4.6 mm, 2.6 µm particle size, Phenomenex, Torrance, CA, USA), C18 (Hypersil GOLD, 150 mm×4.6 mm, 3 µm particle size, Thermo Fisher Scientific, Waltham, MA, USA), phenyl (CORTECS Phenyl, 150 mm×4.6 mm, 2.7 µm particle size, Waters Corporation) and cyano (HSS Cyano, 150 mm×3.0 mm, 3.5 µm particle size, Waters Corporation). The use of methanol as organic modifier yielded no resolution between the three curcuminoids, which coeluted within a single peak. Additionally, the stated peak severely fronted, up to two minutes prior to its apex. The use of acetonitrile improved the fronting, as well as produced adequate resolution between curcuminoids, although the resolution between CUR and PIP proved unsatisfactory on all columns (lower than 0.89) but HSS Cyano. Moreover, the use of nonpolar columns required 100% organic modifier in the mobile phase to elute the highly lipophilic boswellic acids. Using HSS Cyano column and acetonitrile as a modifier, all peaks were successfully resolved. Boswellic acids also eluted fairly rapidly (under 16 min) and used less organic modifier than in other columns owing to higher polarity of the stationary phase, improving time- and cost-efficacy of the method. After the optimization of flow rate, gradient (mobile phase components A and B were ultrapure water and acetonitrile acidified with 0.1% formic acid, respectively), column temperature and wavelength, the final method was developed with a total run time of 25 min. A representative chromatogram of the standard solution is shown in Fig. 1a. The resolution of the peaks in the standard chromatogram was higher than 1.53 (ABA-BBA pair). In real samples (representative chromatogram of processed botanical mixture is shown in Fig. 1b), resolution between the ABA-BBA pair, as well as among AABA, ABBA and matrix components was observed to be lower than 1.5 (0.90, 1.35 and 1.23 for ABA-BBA, AABA-matrix component and ABBA-matrix component pairs, respectively); however, this was not expected to alter the results markedly.

Fig. 1

Representative chromatograms of: a) standard solution, and b) botanical processed mixture. ANDR=andrographolide, NANDR=neoandrographolide, 14-DANDR=14-deoxy-11,12-didehydroandrographolide, PIP=piperine, BDMC=bisdemethoxycurcumin, DMC=demethoxycurcumin, CUR=curcumin, KBA=11-keto-β-boswellic acid, AKBA=3-O-acetyl-11-keto-β-boswellic acid, ABA=α-boswellic acid, BBA=β-boswellic acid, AABA=3-O-acetyl-α-boswellic acid, ABBA=3-O-acetyl-β-boswellic acid

Extraction optimization

Active substance extraction using ultrasound was optimized. As the representative sample, a mixture of processed botanical forms as the more complex sample type to extract was chosen. Seeing that all active substances are moderately to highly lipophilic, the choice of organic solvent is crucial. Therefore, methanol, acetonitrile and ethanol in various volume ratios in hydro-organic extraction solvent (20, 60 and 100%, V/V) were tested. The lowest yields of all compounds were in methanol. Acetonitrile showed similar yields for curcuminoids, PIP and andrographolides to ethanol, but was deemed inferior in the extraction of boswellic acids. Therefore, ethanol was chosen as the organic modifier in the extraction solvent. Afterwards, a three-factorial Box-Behnken design was employed to maximize the active substance extraction yield from the samples. As the independent variables (factors), volume ratio of ethanol in the hydroethanolic extraction solvent (40–100%), extraction temperature (30–80 °C) and sonication time (10–30 min) were selected. The dependent variables (responses) were extraction yields of active substances (in mg/g of processed botanical mixture). Table 1 shows the experimental design, as well as factors and responses.

Upon analyzing the data, it was deduced that the reduced quadratic models best describe the extraction yields. The model equations are as follows:

Y(andrographolide)=11.527–0.011X₁**+0.048X₂**–0.026X₃**+1.210·10^-3 X₁X₂**+1.097·10^-3 X₁X₃–7.673 ·10^-4 X₁²**–9.829·10^-4 X₂²** /1/

Y(piperine)=–0.276+0.030X₁**+0.008X₂+0.022X₃–3.194·10^-4 X₂X₃–1.934 ·10^-4 X₁²** /2/

Y(curcuminoid)=–2.789+0.101X₁**+0.032X₂**+0.068X₃**–3.109·10^-3 X₁X₃*–4.349·10^-4 X₂X₃–6.182·10^-4 X₁²**–1.385·10^-4 X₂² /3/

Y(boswellic acid)=–195.758+4.924X₁**+0.620X₂**+3.066X₃**–0.019X₁X₃**–0.023X₂X₃**–0.025X₁²** /4/

where X₁ is the volume ratio of ethanol in the hydroethanolic extraction solvent, X₂ is the extraction temperature and X₃ is the sonication time. Terms denoted with one asterisk (*) are statistically significant at the 10% level, while those with two asterisks (**) are significant at the 5% level.

Analysis of variance (ANOVA) of the models is shown in Table 2. All models were deemed significant at the 5% level (p<0.012) with insignificant lack of fit (p>0.293). Determination coefficients for all models except PIP were higher than 0.934, indicating very good description of the models. PIP model showed somewhat lower, but still acceptable determination coefficient of 0.698. Adjusted and predicted determination coefficients were in good agreement with each other (within 0.2), while adequate precision of the models was higher than 6, signalling they can be used to navigate the design space.

Fig. 2 shows the response surface plots for selected factors and responses. The figures confirm the model equations showing that the increase of ethanol ratio in the extraction solvent mostly positively influenced the extraction yields. This is especially evident regarding boswellic acids. Such results are not surprising since the examined analytes are comprised of multiple rings and show moderate to high lipophilicity (log P higher than 2.33) (26). As for the extraction temperature and time, both factors were positive as linear terms. Their quadratic and interaction terms demonstrated negative influence on the yield, albeit were deemed markedly less significant than the linear terms. This demonstrates the enhancement of extraction with increasing temperature and sonication time, as well as the stability of the analytes during the procedure. The models were used to assess the optimal parameters for maximum yield of active substances. Firstly, each model was considered individually. Then, all models were considered simultaneously to ascertain if there is a considerable difference between the maximum yields predicted individually and simultaneously. Differences between the yields were smaller than 4.02%, so a universal extraction procedure was chosen for all samples in favour of simplicity and high throughput. The predicted optimal extraction parameters were 81.5% ethanol in the extraction solvent, extraction temperature of 60 °C and sonication time of 30 min with overall desirability of 0.927. Lastly, the prediction was validated by extracting the sample in pentaplicate at the predicted optimal parameters. Normalized bias between the observed and predicted yields was less than -3.98%, indicating excellent predictive capability of the model.

Fig. 2

3D response surface plots for: a and b) andrographolide, c) piperine, d and e) curcuminoid, and f and g) boswellic acid yields

To ensure that the extraction solvent does not saturate when analyzing samples with high amounts of analytes, thus leading to falsely low results, solvent-to-sample testing was conducted. Varying amounts of dry extract mixture (10, 50 and 200 mg) were subjected to the extraction procedure in 10 mL of solvent and adequately diluted. A decrease in the determined concentration in each of the tests was not observed, signalling all the analytes were successfully dissolved even at extreme amounts. Therefore, concentrations of 1 mg/mL (as stated in the protocol) can be easily achieved.

Validation of the methods

Identification of acetylated boswellic acids

As six boswellic acids constitute the majority of Indian frankincense resin and the acid content of its extracts, quantification of only four of them could lead to falsely low results. Being unable to procure two boswellic acid standards (AABA and ABBA), we decided to quantify them using calibration curves of ABA and BBA, respectively, since the literature search revealed similar calibration slopes of acetylated and non-acetylated forms using spectrophotometric detection at 200–210 nm (28, 29). Mass spectrometry was used to identify AABA and ABBA peaks in the frankincense processed botanical form sample. MS scans revealed that peaks at 14.84 and 15.32 min contain peak m/z=499.385, which are likely quasi-molecular ions of the analytes of interest. Monoisotopic masses of AABA and ABBA after the addition of a proton (m/z=499.379) are in high accordance with this value. MS/MS analyses were further carried out with the peak m/z=499.4 as the precursor. Peaks at 14.84 and 15.32 min showed the same fragmentation pattern with fragment ions at m/z=481.380 (loss of hydroxyl moiety of the carboxyl group), 453.380 (cleavage of the carboxyl group), 439.365 (cleavage of the acetoxy group) and 393.360 (loss of both acetoxy and carboxyl groups) (Fig. S3). The combination of chromatographic behaviour, relative abundance in the samples compared to other boswellic acids, UV-Vis spectra and mass spectra all imply that the peaks at 14.84 and 15.32 min are AABA and ABBA, respectively.

Sample analysis

Analysis of the samples revealed large differences in the content of active substances (Table 5), as well as discrepancies between the found and declared contents (Table 6).

Firstly, in the extract samples S1-S35, the mass fraction of curcuminoids varied the most, ranging from 0.1 (S16) to 894 mg/g of sample (S9). USP monograph (10) prescribes a certain curcuminoid distribution for turmeric extracts, viz. 70-80% CUR, 15-25% DMC and 2.5-6.5% BDMC. Seven of mainly turmeric-based preparations did not satisfy this criterion. Among the most striking ones are S1, which contains above 99% CUR, and S6 and S13, in which the relative content of BDMC is 30%. These results point to possible adulteration with synthetic CUR (S1 and S14) and adulteration with synthetic BDMC or Curcuma species other than C. longa (S6, S13), raising concerns (30). Mass fraction of PIP ranged from undetectable to a high 190.2 mg/g. Andrographolide mass fraction in sample S30 was estimated at 912.0 mg/g, although it consisted of only ANDR and virtually no other andrographolides, which could imply adulteration with the pure substance. Samples S31, S33 and S34 contained markedly lower mass fractions of andrographolides (below 159.8 mg/g), but with a profile more characteristic of the herbal constituent. Sample S32, even though claiming to contain both the processed botanical form and extract in appreciable amounts, showed no andrographolides above the detection limit, while sample S35 contained trace amounts of andrographolides (0.6 mg/g). Indian frankincense-based samples contained from 1.6 (S28) to 636.7 (S26) mg/g of boswellic acids. Most pharmacologically active keto derivatives were in the range from below LOQ to 128.3 mg/g (S26). Interestingly, one product (S25), purchased from an online supplier, claiming to be pure frankincense extract standardized to 65% boswellic acids only displayed 12.9 mg/g of all six boswellic acids and a miniscule 3.0 mg/g of keto derivatives. As for the found vs. declared content, about half of the turmeric-containing samples with stated content (12 of 19) conformed to the USP criteria of 90 to 110% declared content. A few samples (namely S6, S13 and S16) showed contents lower than 20% declared. It should be pointed out that these products were purchased from an online retailer. A recent survey of products on the American market revealed good agreement of declared and found content in the products from local suppliers, similar to the findings for the locally bought products in Croatia (30), which points to the questionable quality of products bought from dubious Internet sources. PIP content conformed to the product declaration in 4 out of 9 samples. Samples containing equal to or less than 20% declared content (S3, S13 and S21) can be presumed to be underdosed in recommended dosing regimens, especially sample S21, which consists of only black pepper extract. Sample S30 was the only green chiretta sample to have unambiguously declared content, also conforming to the USP requirements (93.1% of declared content). As for the Indian frankincense samples, USP criteria could not be applied here since most manufacturers do not declare the content of keto derivatives, only total boswellic acids. The content of boswellic acids spanned from 2.0 (S25) to 176.7% (S14) of the declared values, although the extracts are standardized to minimum rather than absolute content of boswellic acids. Two of 11 products contained more than 90% of declared boswellic acids, while only one product exhibited KBA below 1% of total boswellic acids (S5); however, it contained elevated amounts of AKBA, which is the more potent component.

Materials such as herbal substances and their processed forms can also be taken as dietary supplements or be used as raw material in the manufacture of various preparations. Therefore, the content of active substances in the processed botanical forms (S36-S54) is also of paramount importance for the quality of the final product. Active substance content is again expressed as yield (mg/g of sample) (Table 5), as well as a mass ratio with regard to the mass of the corresponding herbal component in the sample (Table 6). Content of curcuminoids varied from 14.7 to 102.3 mg/g of sample, while the mass fractions varied from 2.7 to 10.2%. Similar results have been obtained by other research groups (31, 32). Ph. Eur. (11) prescribes 2% curcuminoids as the lowest limit, which all samples satisfy. Only one sample did not conform to the USP criterion of 3%, albeit barely (2.7%, S46). Two of the samples (S42 and S43) contained an unusually high content of curcuminoids (8.8 and 10.2%, respectively). Upon examining the curcuminoid profile, amounts of BDMC comparable to CUR are observable, which is not typical of C. longa and could point to substitution or adulteration. Green chiretta samples contained from 12.1 (S53) to 44.4 mg/g (S54) of andrographolides (1.2 to 4.4%), conforming to the Ph. Eur. and USP limits of 0.8 and 1%, respectively. Sample S54, from Thailand, contained the most andrographolides, which is consistent with the higher amounts found on the Thai market (33). Frankincense resin contained 27.8 to 30.5% boswellic acids, complying with the literature (34). All samples containing above 1.5% KBA and 7.0% AKBA conform to both Ph. Eur. and USP (10, 11) criteria for keto derivatives.

The estimated daily intake of each active substance group per sample is shown in Table 6. Drastic differences in the highest and lowest estimated intakes of active substance groups between different samples can be observed (up to 9000 times, curcuminoids in S4 and S16). As is expected, active substance intake in the processed botanical form samples is lower than in extracts (the exception being andrographolides in S54, 301.9 mg). As for the preparations, few demonstrated drastically low intakes of curcuminoids without any adjuvants (S6, S15, S16 and S20, below 30 mg/day). A few samples also demonstrated lower values, although they were coupled with PIP or formulated in lipid vesicles, which enhances the bioavailability of curcuminoids. Samples S20, S25, S28 and S33 showed very low content of boswellic acids (estimated daily intake lower than 12.4 mg/day). Apart from S20, S25 and S33, which are combination products, it can be presumed that sample S25 has little to no therapeutic value. On the other hand, high daily intakes were also observed in multiple products (up to 872.0 mg of curcuminoids in S4, 21.8 mg of PIP in S1, 482.6 mg of boswellic acids in S22 and 301.9 mg of andrographolides in S54). Curcuminoids are shown to be safe up to 12 g a day in healthy individuals, while doses of around 500-1000 mg daily (or less with absorption enhancement) demonstrated therapeutic efficacy in various inflammatory diseases (35). Boswellic acids improved inflammatory bowel disease and arthritis symptoms in doses of 100–500 mg and higher with a good safety profile, even more so when AKBA was present in higher amounts (5, 32), which would imply that S5 and S26 were the most desirable in terms of boswellic acid content. However, daily intake of andrographolides and PIP when using products S54 and S1, respectively, raises concerns. Although andrographolides show good safety and efficacy in doses up to 100 mg/day, studies have shown doses of 5 mg/(kg·day) and higher can cause various side-effects such as allergic reactions, diarrhoea, heartburn, etc. (36). In addition, in vitro and ex vivo studies have suggested that 14-DANDR could have a hypotensive effect in higher doses (37). An intake of 301.9 mg of andrographolides daily using S54 could, thus, lead to possible adverse effects. As for PIP, doses of 1–10 mg daily are sufficient for bioavailability enhancement; however, simultaneous use of doses higher than 10 mg with CYP3A or P-glycoprotein substrates could affect their pharmacokinetics, leading to impaired safety and efficacy (38). As supplementation with S1 can provide a single dose of 21.8 mg PIP, there is a potential for interaction with concomitantly used drugs. Although there are no exact guidelines for maximum dosages of these substances, care should be taken with older patients on polytherapy or with liver disease.