> ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® CURCUMINOIDS
> ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® VOLATILE BISABOLANE SESQUITERPENES
ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® CURCUMINOIDS
OxiTurmeric® curcuminoids are analyzed by HPLC-DAD coupled to ESI-tandem mass spectrometry. The accurate quantitation of the curcuminoids curcumin (CUR), demethoxycurcumin (DMC), and bis-demethoxycurcumin (BDMC) of OxiTurmeric® is based on the AOAC Official Method 2016.16. After extraction with methanol and due dilution, the extract is analyzed by HPLC by setting the detector to 425 nm, the C18 (2.6 μm, 2.1 × 30 mm) column to 55°C temperature, using an injection volume of 0.8 μL and a flow rate of 1.4 mL/min. The actual concentration of the curcuminoids is then calculated based on calibration curves obtained on pure standards. The identification of the curcuminoids is carried out by HPLC-DAD-ESI-MS / MS.
Very often we are used to see claims of 95-98% curcuminoids. Here we report an example of a turmeric sample claiming 98% curcuminoids that eventually was found to possess a real percentage of 63% total curcuminoids. In a few step we will show how a typical analysis of curcuminoids is done.
The typical chromatogram obtained from a commercial turmeric extract that claims 98% curcuminoids is reported in Figure 1
Figure 1. Typical chromatogram from a turmeric sample claiming 98% curcuminoids
By reading at a fixed wavelength (425 nm) it is expected that only curcuminoids appear in the chromatogram. Therefore, if we calculate the total percentage of the curcuminoids shown in Figure 1 we obtain the results of Table 1, where actually the total curcuminoids percentages is even higher than the declared 98%.
Table 1. Total percentage of peak areas from Figure 1
|Compound||Retention Time (min)||Calculated Area||Area Percentage|
A common mistake is to consider the percentages of Table 1 as a quantitative measure of the turmeric curcuminoids. In order to quantify the real content of curcuminoids quantitation is performed with external calibration against reference standards for CUR, DMC, and BDMC, according to the AOAC Official Method 2016.16. In order to obtain these curves, the analyte concentration (x-axis) is plotted versus individual integrated peak area (y-axis) for CUR, DMC, and BDMC. The least-squares analysis is used to determine the slope, intercept, and determination coefficient (R2) of the best-fit line for each analyte.
Figures 2-4 show the typical calibration curves obtained on pure CUR, DMC, and BDMC standards, respectively.
Figure 2. calibration curve obtained with pure Curcumin standard.
Figure 3. calibration curve obtained with pure Demethoxycurcumin standard.
Figure 4. calibration curve obtained with pure Bis-demethoxycurcumin standard.
Based on these external calibration curves, the amount of curcuminoids in the original sample is calculated as follows:
where C = concentration of the analyte from the standard curve; V = extract volume; W = weight of the test portion (g); and D = dilution factor.
Taking in consideration the above calculation, the data of Table 1 are then transformed to the quantitative data of Table 2
Table 2. Quantitative analysis of data from Table 1 and Figure 1 based on calibration curves of Figures 2-4.
|Compound||Retention Time (min)||Calculated Area||Area Percentage||μg from calibr. curve||mg/g in sample||% in sample|
Therefore, the correct use of external calibration curves shows that the total percentage of curcuminoids in the sample claiming a 98% curcuminoids it is actually 62.4%
A further confirmation of the identity of curcuminoids is given by coupling the HPLC with an electrospray source ionization and tandem mass spectrometry. The typical mass spectra of CUR, DMC, and BDMC are depicted in Figures 5-7.
Figure 5. Mass fragmentation of Curcumin
Figure 6. Mass fragmentation of Demethoxycurcumin
Figure 7. Mass fragmentation of Bis-demethoxycurcumin
ANALYTICAL METHODS FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF OXITURMERIC® VOLATILE BISABOLANE SESQUITERPENES
The bisabolane sesquiterpens (e.g., ar-turmerone, α-turmerone, β-turmerone) present in OxiTurmeric® are identified by gas chromatography coupled to mass spectrometry (GC-MS) and quantified by gas chromatography coupled to Flame Ionization Detector (GC-FID). These are the only reliable methods for the analysis of turmeric volatile bioactive compounds. Other methods (including TLC, UV-Vis, etc.) do not provide neither the precise identification of compounds (only possible through GC-MS) nor their precise quantification (which is obtained by using GC-FID with external calibration curves of pure standards and internal standards).
The typical GC-FID profile of OxiTurmeric® is depicted in Figure 8.
Figure 8. GC-FID chromatogram of OxiTurmeric® that shows the presence of several sesquiterpenes. This analysis is used for the quantitative determination of the bioactive volatile sesquiterpens of OxiTurmeric® by using both external calibration curves with pure standards and internal standards (e.g., trans-nerolidol).
The identification of the main sesquiterpenes of OxiTurmeric® is provided by GC-MS as shown in Figure 9.
OXITURMERIC® IS BIOAVAILABLE AT THE CELLULAR LEVEL – AS DEMONSTRATED IN HUMAN HEPG2 CELLS
The antioxidant capacity of foods and food supplements is often measured by chemical reaction of the plant extract or ingredient with ROS sources. The most widely used methods are: ORAC, TRAP, FRAP, TEAC, DPPH, etc. These assays are traditional chemical tests that do not inform about the physiological functions of antioxidants and have been discredited for years and strongly discouraged by the main health monitoring authorities (e.g., EFSA and USDA).
This is why we tested the bioavailability and capacity of OxiTurmeric® to activate responses at the cellular level by using the ultimate technology based on LUCS (Light-Up Cell System) a patented (EP2235505 and US20110008783) approach developed by the Anti Oxidant Power Company, Toulouse, France (for more details, see OxiP® facts).
To demonstrate that OxiTurmeric® is bioavailable at the cellular level, we performed the ARE/Nrf2 live cell assay, which is a reporter gene approach that measures the ability of the sample to activate ARE (Antioxidant Response Element) DNA promoter sequence following the nuclear release of Nrf2 transcription factor from the Keap1/Nrf2 cytosolic complex. This genomic pathway, also known as “the natural antioxidant cell defense” has the capability to increase cell capacity to adapt to oxidant stress or aggression (Ma, Annu. Rev. Pharmacol. Toxicol. 2013, 53:401-426). Nrf2-ARE pathway has been shown dynamic changes and examined for its neuroprotective role (Gan & Johnson, Biochim. Biophys. Acta-Mol. Basis Dis. 2014,1842:1208-1218). In HepG2-Nrf2 cells, ARE promoter sequence is coupled to the expression of luciferase enzyme which, in presence of luciferin, catalyses the production of the luminescent oxyluciferin compound.
OXITURMERIC® WORKS AS A CYTOPROTECTOR VIA ARE/NRF2 PATHWAY PROMOTION
OxiTurmeric® has the ability to act at the cellular level, thus showing a clear bioavailability. The interaction of OxiTurmeric® prompts a clear effect on natural antioxidant human HepG2 cell defense (ARE/Nrf2 pathway). Different concentrations were used to evaluate the maximum fold change gene expression. The maximum fold change (5.11) is obtained with a 12.5 mg/ml concentration, whereas the minimal concentration able to activate the gene expression is 6.25 mg/ml (Figure 10).
Figure 10. Effect of OxiTurmeric® on ARE/Nrf2 pathway. Fold change represent the ability of OxiTurmeric ® to increase the natural antioxidant human HepG2 cell defense. At 12.5 mg/ml, OxiTurmeric® activates 5 times more the antioxidant cell self-defenses with respect to controls.
The absence of external additives and the pure “all natural” characteristic of OxiTurmeric® show an EC50 of 5.60 mg/mL, with no cytotoxic effect observed after 17 h of treatment at any assayed concentrations (Figure 11). Therefore, the bioavailability and gene activation capacity of OxiTurmeric® is granted by the natural presence of a high level of curcuminoids along with the volatile turmerones, that increase bioavailability without the addition of other synthetic molecules.
Figure 11. Dose-response graphical representation of OxiTurmeric® obtained on triplicate experiments. The data show that OxiTurmeric® is not cytotoxic and that the EC50 (that is 50% efficacy concentration, calculated on the dose-response sigmoid fit) is 5.60 mg/ml with a high coefficient of determination (R2=0.984). Bars represent standard deviation.
In conclusion, OxiTurmeric® shows a proved bioavailability as demonstrated at the cellular level in human HepG2 cells. OxiTurmeric® activates the natural antioxidant cell defense and shows no cytotoxic effects at any tested concentration. OxiTurmeric®, by acting on the ARE/Nfr2 pathway, has the potential to increase the cells’ ability to adapt to oxidative stress or cell damage, and to stimulate the natural cell’s neuroprotection.
OXITURMERIC® DOES NOT CONTAIN ZEDERONE
Occurrence of the hepatotoxic compound ZEDERONE in Curcuma extracts
Several studies showed that some sesquiterpenes (e.g., zederone, Figure 12) exhibited liver toxicity, which is mainly based on reactive metabolites formation, increased concentration of reactive oxygen species and impaired antioxidant defense (Zarybnicky et al., 2018, Archives of Toxicology, 92(1): 1-13).
Figure 12. Mass spectrum and chemical structure of the toxic compound zederone
Sofistication of Turmeric, for example with C. comosa Roxb. widely cultivated in Thailand as a source for the traditional medicine wan chak motluk, commonly used for the treatment of menopausal symptoms (contains estrogenic diarylheptanoids), or with Curcuma elata, whose rhizome contains a high proportion of the sesquiterpene zederone (Pimkaew et al. 2013, Toxicol In Vitro 27:2005–2012) are sometimes occurring. The hepatotoxicity of the sesquiterpene zederone isolated from C. elata was demonstrated (Pimkaew et al. 2013, Int J Toxicol 32:454–462). Zederone is also a typical sesquiterpene found in C. kwangsiensis (Liu et al, 2016, Chin Med 11:21), C. zedoaria (Hamdi et al, 2014, Scientific World Journal, Article Number: 321943) and in C. amada Roxb (Akter, et al, 2019, Asian Pacific Journal of Tropical Medicine, 12(3):123-129 ), where it shows a strong cytotoxicity against KG1a cells (Anuchapreeda et al, 2018, Natural Product Communications, 13(12):1615-1618).
OxiTurmeric® does not contain zederone as demonstrated by our GC-MS analyses
OXITURMERIC® IS 100% NATURAL AS DEMONSTRATED BY 14C ANALYSES
We assessed the presence of artificial substances in OxiTurmeric® through 14C radiocarbon analysis. Natural Turmeric completely from biomass has a known 14C level of 100% biobased. On the other hand, turmeric wholly made from petroleum-derived components have 14C level of 0% and will be 0% biobased. A product made of biomass (plant extracts) and petroleum-based chemicals or additioned with synthetic molecules will have a biobased content between 0% and 100% proportional to the quantity of each component in the product.
OxiTurmeric® 14C value was 99.9% ± 3.7, indicating the pure natural origin of the extract