The structures of the identified compounds, mass spectra, and calibration curves are provided as Supplementary material . The first method is based on the scavenging of the ABTS radical 2,2′ – azino-bis and the radical cation was produced by reacting 3.5 mM ABTS with 1.2 mM K2S2O8 in H2O. Before use, the mixture was stored in darkness at room temperature for 12–16 h and then diluted with EtOH to a working solution with an absorbance of 0.700 at 734 nm. Vitamin C standards were freshly prepared in EtOH for the calibration. After the addition of diluted ABTS working solution to diluted samples or standard , the absorbance was recorded after 30 min . The test for ferric reducing antioxidant power, or FRAP, is based on the reduction of Fe3+ to Fe2+ by the antioxidant compound, which forms a coloured complex with an absorption maximum at 593 nm with 2,4,6- tripyridyl-s-triazine in acetate buffer at pH 3.6. The working solution was freshly prepared with 25 mL of 300 mM acetate buffer , 2.5 mL of 10 mM 2,4,6-tripyridyl-s-triazine in 40 mM HCl, and 2.5 mL 20 mM FeCl3⋅6H2O in deionized water . Between 30− 100 μL hemp root extract was mixed with 2 mL FRAP working solution and the absorbance was recorded after 30 min. In vitro antioxidant activity assays were carried out on a Lambda 25 UV/VIS spectrometer . The calibration was performed with vitamin C, which was purchased from Sigma Aldrich . The results are expressed in vitamin C equivalent mg per 100 g dried hemp roots. Cellular antioxidant activity was determined with Saccharomyces cerevisiae ZIM 2155 as a model system following the procedures described in Slatnar et al. , which estimates intracellular oxidation by fluorometric measurements using the ROS-sensitive dye 2′ , 7′ -dichlorofluorescin . The ethanolic hemp root extracts were incubated with 10 mL yeast suspension at their stationary phase in phosphate buffered saline at a density of 108 cells/mL at 28◦C and 220 rpm for 2 h.
Thereafter, 2 mL of yeast suspension was centrifuged at room temperature for 5 min at 14,000 x g. The resulting pellet was washed three times with 50 mM potassium phosphate buffer and was finally resuspended in 9 volumes 50 mM potassium phosphate buffer . The suspension was kept for 10 min at 28◦C and 220 rpm in the dark before addition of 10 μL H2DCF . After incubation for further 30 min at 28◦C and 220 rpm, the fluorescence of the yeast cell suspension was measured on a GloMax® Multi Microplate Reader using excitation and emission wavelengths of 490 and 520 nm, respectively. Values of fluorescence intensity were compared with a control, in which the sample was replaced with ethanol. Data are expressed as relative fluorescence intensity according to the control, where the values obtained with the control are defined as 1. Values lower than 1 indicate a higher antioxidant activity than the control, values above 1 indicate prooxidant behaviour .In total, 20 secondary metabolites were identified and were numbered from 1-20 according to their retention times and the corresponding mass spectrometric data is presented in Table 1. This includes five triterpenes and ten phytosterols, of which two triterpenoids and four phytosterols were identified for the first time in hemp root extracts. In addition, five aliphatic compounds were identified, of which one was a novel compound in C. sativa extracts , whereas the others were putative artefacts. All identified structures and the corresponding mass spectra are shown in the Supplementary Material. Several triterpenes are known to be present in C. sativa roots, in particular friedelin , epifriedelinol or the recently discovered β-amyrin. Besides β-amyrin, another oleanane skeleton based triterpenoid, namely β-amyrone was firstly discovered in this study in the root’s extracts. Furthermore, the presence of the pentacyclic triterpenoid glutinol can be reported. Glutinol and amyrone based pentacyclic triterpenoids are known to be accumulated in root barks of Maytenus cuzcoina . In addition to the group of triterpenoids, ten phytosterols were identified in the extracts. Slatkin et al. described the steroids, campesterol , stigmasterol , and β-sitosterol , as well as the steroid ketones 4-campestene-3-one , stigmasta-4,22-dien-3-one , and stigmast-4-ene-3-one in cannabis grow system roots. Furthermore, two yet undescribed phytosterols in the roots, stigmastanol and fucosterol , were extracted in this study. Both sterols were identified in other roots, stigmastanol has been reported in D. cinnabari and fucosterol was found in Hordeum vulgare L. .
An additional new group of steroids can be reported herein. Thus, two steroid hydrocarbons, namely stigmasta-3,5-diene and stigmasta-3,5,22-triene were extracted and identified by GC–MS analysis. Compounds 6 and 7 have been isolated in other roots before, respectively in Cordia rothii and Moringa oleifera roots . Further investigation of the mass spectrometric data led to the identification of five aliphatic lipophilic compounds. As a result, oleamide could be determined in the roots of C. sativa for the first time. The derived oleic acid amid has been reported in ethanolic root extracts of Arctium lappa L. . In addition, the fatty acid ethyl esters ethyl palmitate , ethyl linoleate , ethyl elaidate , and ethyl stearate were identified. It can be assumed that compounds 1 to 4 are derived from their respective fatty acids and possibly resulted during the extraction with EtOH. Fatty acids have been isolated from hemp roots before . The triterpenoids friedelin and epifriedelinol are the prevalently present compounds and have been associated with a broad spectrum of health-related effects . Although largely neglected so far, this makes hemp roots an interesting additional product for exploitation by the hemp industry. Utilization of further plant parts would increase the income of growers and the virtual absence of phytocannabinoids would overcome legal hurdles. Therefore, the amounts of friedelin and epifriedelinol in three chemovars were determined and compared within the studied varieties. The alcoholic extraction of sample A–E yielded 0.100− 0.709 mg/g DW of friedelin, with the lowest yield in sample C and the highest yield in sample E. In addition, the highest yield of epifriedelinol was 0.205 mg/g DW in sample B and the lowest amount of 0.059 mg/g DW was observed in sample C . Slatkin et al. reported 150 mg of column chromatographically purified friedelin and 100 mg epifriedelinol from ethanolic extracts of 4.7 kg air dried hemp roots. In another study the author V. Sethi isolated 20 mg and 49 mg of crystallized friedelin and epifriedelinol, respectively, from dried hemp roots, extracted with petrolether . Hence, both studies reported significantly lower yields of the investigated triterpenoids, due to the purification with column chromatography and subsequent recrystallization. A recent study described higher contents of friedelin and epifriedelinol, ranging from 0.083− 0.135 mg/mg% and 0.033− 0.092 mg/mg%, respectively, in ethyl acetate extracts from hemp roots of three different chemovars, which resulted in slightly higher amounts of both triterpenoids, compared with this study. In this case, the roots were air dried for 24 h and medicinal varieties were analysed, which predominantly accumulate THC with a CBD:THC ratio of 1:2 .
In contrast, herein, the roots were freeze dried until a consistent weight was achieved before subsequent analysis, and industrial varieties were used, which have a low THC limit. The three analysed chemovars, Futura 75, Felina 32 and Uso 31, are commonly used industrial varieties, from the approved list of industrial hemp in the EU according to article 17 of the guideline 2002/53/EC. Futura 75 and Felina 32 are French varieties, largely used by industrial hemp growers in Central Europe for production of grain and fibers. Uso 31 is of Dutch origin with a particularly low THC content and especially suitable for grain production with a shorter vegetation period . For comparison, these chemovars were harvested at the same time and extracted with EtOH, which led to no significant difference in the yields of epifriedelinol. However, from Felina 75 roots significantly more friedelin was extracted compared with Futura 75 as well as Uso 31 . Thus, Felina 32 accumulated the highest total levels of triterpenoids and demonstrates potential for yield optimization of bioactive compounds in hemp roots, by careful selection of varieties. Flavonoids and cannabinoids could not be detected, as reported before. However, both triterpenoids have been described in the stem bark, indicating that the analysed triterpenoids accumulate in the outer tissue layer of the roots and stem . Depending on the growth conditions, root harvest techniques and sample preparation, the results differ in the amount of fine root structures, which mainly contribute to the root surface, correlating with the amount of bark per weight. Currently, a possible positive correlation between the concentrations of cannabinoids and terpenes, particularly mono and sesquiterpenes, is a matter of debate . The comparable rates of triterpenoids of the industrial varieties herein and the medicinal varieties used by Jin et al. do not point to a correlation of the concentrations of triterpenoids and cannabinoids. For chemovar Futura 75 the effects of harvest time as well as theinfluence of subsequent drying were analysed. No significant reduction of friedelin was observed between sample A and B, however for sample A, significantly lower amounts of epifriedelinol were obtained , which was harvested one month earlier than sample B. In addition, sample C was dried at 45◦C for 30 h directly after the harvest, which correlates with the significant reduction from 0.434 to 0.100 mg/ g DW friedelin and from 0.205 to 0.059 mg/g DW epifriedelinol . Therefore, it can be assumed that drying at higher temperature and storage over several weeks have an impact on the targeted triterpenoids. Additionally, the reduced overall concentration of both triterpenoids could be attributed to the seasonal difference in the harvesting time points. With the harvest in October, as in sample C, the marijuana grow plants are already in the process of senescence with starting degradation processes and rearrangement of the metabolome. For comparison of extraction efficiency, sample E was extracted with ethanol, n-hexane and supercritical CO2 combined with ethanol as a modifier . No significant difference between the conventional extractions for the target triterpenoids was observed. Significantly less friedelin and slightly less epifriedelinol were yielded by SFE. However the lower yields of extracted epifriedelinol were insignificant.
It can be assumed that the supercritical CO2 extraction conditions at 60◦C, 20 MPa and 10 vol% of EtOH are sufficient for epifriedelinol, but not for friedelin, due to the structural difference and can be further optimized for a dual extraction. Supercritical fluid extraction of triterpenes is reported to be favoured at high pressure, high temperature and with EtOH as a modifier from Alnus glutinosa Gaertn. . Furthermore, conventional extractions herein provided higher extraction yields with smaller amounts of solvent compared with SFE. Therefore, EtOH seems the best choice for extracting friedelin and epifriedelinol due to its extraction efficiency, environmental benignity and experimental simplicity. In addition, the values of the intracellular oxidation ranged between 0.809–1.024 for the investigated ethanolic extracts. In agreement with the in vitro assays, sample A exhibited the highest cellular antioxidant activity. Compared with the control, whose oxidation was nominally defined as 1.000, a 20% decrease in intracellular oxidation was observed. Treatment of cells with other samples did not show any changes compared with the control. The latter could be a result from a limited uptake of the relevant compounds by the yeast cells or a low aqueous solubility . In general, antioxidant and other biological activities of compounds depend on the rate of their incorporation into cells, which is related to the balance between their lipophilicity and hydrophilicity . Furthermore, when bioactive compounds enter a cell, their antioxidative activity is influenced by such factors as their reduction potential and the antioxidant defence activity endogenous to the cell itself . Thus, while in vitro assays can be used as a first step in screening compounds for possible antioxidative activity , subsequent testing in a cellular environment provides insight into the actual effects in a natural physiological setting. The simple, yet eukaryotic nature of S. cerevisiae presents a good model for these preliminary tests before moving on to more complex organisms .