Part 2 Chemical Composition, Market Survey, and Safety Assessment of Blue Lotus (Nymphaea caerulea Savigny) Extracts

2.4. Safety Assessment

N. caerulea is not GRAS classified, and no published safety data were found on the plant or the extracts as a whole. However, the plant has a long history of use. The safety data for all constituents present at 1% and above are presented in Table 3. The three main constituents detected in the concrete were 6,9-heptadecadiene (11.95 ± 1.65%), n-tricosane (8.03 ± 0.18%), and benzyl alcohol (7.58 ± 0.85%). The three main constituents identified in the absolute were 6,9-heptadecadiene (11.05 ± 1.53%), benzyl alcohol (10.46 ± 1.42%), and tetradecanol (5.32 ± 0.74%). Tsai et al. [22] studied the volatile compounds of N. caerulea (water lily) flowers using GC-MS and reported four main compounds: 6,9-heptadecadiene (40.1%), pentadecane (15.5%), 8-heptadecene (15.3%), and benzyl acetate (10.4%). This is different from our GC-FID results, except that the main compound, 6,9-heptadecadiene, was identified as the most abundant compound, although at a substantially lesser concentration in the flower extracts.

As these are absolute and concrete materials, they may contain an unknown and significant portion of nonvolatile compounds. As such, quantification through GC-FID may not be accurate and compounds present in the absolute and concrete may not be detected and therefore not evaluated as part of this assessment. Since the safety of unidentified compounds cannot be guaranteed, this presents an unknown safety risk.

Of the 28 compounds investigated (making up 85.43% of the concrete and 80.52% of the absolute), safety information was not found for 10 compounds (making up 27.29% of the concrete and 25.11% of the absolute) including 6.9-heptadecadiene, n-pentacosane, tetrapenol, 3-((8Z,11Z)-heptadeca-8,11-dien-1-yl)-5-methoxyphenol, 2-nonadecanone, heptacosane, benzyl linoleate, methyl cholesterol, benzyl linolenate, and benzyl hexadecanoate. We were able to gather safety information for the remaining 18 compounds (making up 58.69% of the concrete and 56.68% of the absolute) including n-tricosane, benzyl alcohol, tetradecanol, heneicasane, nonadecane, pentadecane, oleic acid, E-squalene, linoleic acid, γ-sitosterol, palmitic acid, phytol, E-β-farnesene, ethyl stearate, stigmasterol, hexadecyl acetate, ethyl linoleate, and ethyl palmitate. According to the data available from CIR, all of the assessed compounds, except two, were found to be at concentrations considered safe in accordance with current usage practices, as indicated in Table 3. Benzyl alcohol and tetradecanol are slightly above the maximum concentrations; however, when blue lotus extracts are used as part of a formulation, the concentration of these compounds will be reduced. For compounds with data on genotoxicity, there was no indication of genotoxicity risks. Very limited information was available on acute or chronic toxicity and phototoxicity or photoallergenicity. However, the data are not indicative of major safety risks.

3.1. Plant Material and Extraction

Authentic blue lotus absolutes and concrete samples were prepared using industrial extraction methods. Cultivated blue lotus plants were collected from Hainan and Guangdong, China (Figure 1). The plant prefers high temperatures, humidity, and sunlight. Fresh flowers were shredded with a flower-cutting machine. About 1000 Kg of the shredded material was extracted twice with hexane (1: 2, w/v) in an enamel extraction tank with continuous stirring for 12 h. After soaking, the hexane was discharged and filtered with 120 mesh stainless steel mesh. The collected extracts were allowed to settle for 4 h, then filtered. The solvent was then recovered by heating with jacketed steam. The extract was concentrated under atmospheric pressure with a spherical concentrator until all of the hexane was evaporated. The concentrated extract is called concrete. To prepare the absolute, the blue lotus concrete was dewaxed with 95% ethanol (1:5, w/v) in a stainless-steel barrel, stirred carefully, and placed in the freezer for more than 12 h. The resulting extract was filtered and the floral was separated. The filtrate was concentrated under low pressure in a spherical concentrator until all of the solvent was evaporated. Samples of both the concrete and absolute were tested for solvent residue. Eleven commercially available blue lotus oil products were purchased online (Amazon and Etsy). The product labels of these samples contained the information listed in Table 4.

3.2. Gas Chromatography−Mass Spectrometry (GC–MS) Analysis

Authentic and commercial samples were analyzed using a gas chromatograph coupled to a mass spectrometer QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) with electron impact (EI) mode with 70 eV, as previously described [55]. The components were identified by comparing the mass spectral fragmentation patterns (over 80% similarity match) and retention indices (RI) based on a series of homologous C8-C20 n-alkanes with those reported in databases (NIST database, and our in-house library) using the Lab Solutions GCMS post-run analysis software version 4.45 (Shimadzu Scientific Instruments, Columbia, MD, USA).

3.3. Gas Chromatography–Flame Ionization Detection (GC–FID) Analysis

Analysis of E. purpurea essential oil was carried out using a Shimadzu GC 2010 equipped with a flame ionization detector (Shimadzu Scientific Instruments, Columbia, MD, USA), as previously described [56], with a ZB-5 capillary column (Phenomenex, Torrance, CA, USA).

3.4. Detection and Quantification of Nuciferine and Apomorphine

LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nuciferine and apomorphine were purchased from Cayman Chemical (Ann Arbor, MI, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol. Nuciferine and apomorphine were quantified using a NEXERA UPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a mass spectrometer (Triple quadrupole, LCMS8060, Shimadzu, Kyoto, Japan) as previously described [18,25]. The detection was completed in multiple reaction monitoring mode (MRM) (Table 5). Samples were run in triplicate with external standards in between and the injection volume was 1 μL. The acquired chromatographic results were processed in LabSolutions Insight software version 3.2 (Shimadzu). For each compound, calibration curves (0.005–0.1 ppm) were created by linking the peak area and the concentration.

3.5. Safety Assessment

The safety assessment of blue lotus extracts was conducted by applying standard toxicology and risk assessment methods using the analytical results (Table 2), published safety data on the raw material as a whole plant, plant extract, and the constituents identified in the extracts. The information considered for the safety assessment included the historical use of the plant and extracts, safety and toxicology data on the plant and extracts, and safety and toxicology data of all constituents present at 1% and above. This safety assessment is based solely on the available literature. The documents collected and reviewed included scientific articles from books and scientific journals on botany and the safety of natural complex substances, fragrances, and flavors. Studies using different degrees of evidence from in vitro methods, pre-clinical models, clinical trials, and case reports were used as evidence of the safety or toxicity of the raw material as a whole. The sources of information used to evaluate the safety of individual constituents included the RIFM (Research Institute for Fragrance Materials, Inc.) Fragrance and Flavor Database, CIR (Cosmetic Ingredient Review) assessments, and ECHA (European Chemical Agency) REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) registrations. The main endpoints of interest included genotoxicity, developmental and reproductive toxicity, skin irritation and sensitization, photoirritation and photoallergenicity, as well as acute and chronic toxicity for oral, dermal, and inhalation routes of exposure.