Hibiscus are garden plants within the USA. However, their potential as a new source of edible flowers, seed, food colorants, and renewable fiber elevates them to the status of a new crop. There are 17 native Hibiscus species grown within USDA Zones 4-10, though they are wetland plants in the wild. These species include Hibiscus aculeatus, H. coccineus, H. grandiflorus, H. laevis, H. moscheutos and H. striatus lambertianus. Other hardy species H. mutabilis, H. syriacus, H. hamabo and H. tileaceous, are suitable for USDA Zones 8-10. Potential applications from these Hibiscus are many, i.e., fresh flowers in salads and garnishes, or dried and used in processed food products, nutraceuticals, dietary supplements and natural food colorants The development of Hibiscus species and hybrids suitable for sustained cultivation here in the US and the commercialization of a wide variety of products prepared with their flowers and seed would add an important new crop to the US agricultural base. This proposal focuses on exploiting this opportunity by the commercialization of teas, vinaigrettes, bulk dried petals, and concentrated petal pigment extracts, derived from Hibiscus species & The Village Botanica Inc.'s (VBI) proprietary Hibiscus hybrids. VBI has complete formulation protocols for a number of value-added product lines, complementing multiple edible products, all from our organically-grown Malvaceae genomes. OBJECTIVES: Our four objectives follow: 1.) To establish the colorant content of the Hibiscus flower pigment liquid formulations. From the chemical and spectroscopic analyses of previous Phase I and Phase II projects, we have information on the flavonoid aglycone and glycoside contents of the flowers. While this data is essential, it does not necessarily indicate the flavonoid content that will be found in the liquid formulations. This is due to the use of a methanol solution containing t-butylhydroquinone (Puckhaber et al., 2002). This protocol will not be used in the formulation of the edible formulations. Instead, we will extract dried flower petals at room temperature in aqueous ethanol containing ascorbic acid. Preliminary tests have shown that ethanol is effective in extracting pigments from Malvaceae petals; however, we do not know quantitatively how effective it is. Therefore, we will prepare our line of liquid color concentrates and analyze them by HPLC (Puckhaber et al., 2002) for their flavonoid aglycone and glycoside identities and concentrations. 2.) We also do not know how effectively hot water and vinegar extract the flavonoids from Hibiscus petals. Thus, we have no information on the concentration of Hibiscus flavonoids in our tea brews and our vinegars. We will be determining the flavanols that will originate from the Hibiscus petals in these products by preparing and analyzing hot water brews of dried Hibiscus petals used in our teas, and vinegar extracts of fresh petals used in our vinegars. 3.) The third objective of this proposal is to determine the stability of the colorants in dried Hibiscus petals. This is necessary to establish shelf lives for the use of dried petals in the color concentrates, teas, and powdered petals. To achieve our objective, fresh petals will be dried and then stored at room temperature. Immediately after drying, and then at one-month intervals during the grant period, petals will be brewed in hot water or extracted with ethanol containing ascorbic acid. The brews and extracts then will be analyzed by HPLC for flavonoid content. 4.) Not only are shelf-lives important, but acceptable in product appearance is, too. Flavonoids are known to be unstable. It is quite probable that the proposed formulations will show changes in color and clarity that are dependent on the storage conditions. Therefore, the last objective of the work proposed herein is to evaluate, based on color and clarity, the shelf life of our color concentrates, teas and vinegars. We will formulate, package and store at room temperature the teas. Immediately after packaging, and then at intervals throughout the grant period, samples from the teas will be brewed in hot water and the teas evaluated for color and clarity. We also will evaluate over time the color and clarity of the bottled formulations of the color concentrates and the vinegars. Storage conditions for the vinegars will mimic those most likely employed in stores and homes including a wide variety of temperature and lighting conditions. From this data, we will establish shelf lives for our products under various real world conditions. APPROACH: 1.) Measurement of the flavonoid contents of color concentrates is achieved by harvesting the flowers of Hibiscus genomes then drying under forced air at 40 deg C 1.0 gm of tissue from a dried petal color suite (selections of flowers segregated into 7 distinct color groups) will be extracted in 120 mL of 95% ethanol containing 0.1% ascorbic acid in a sealed flask at 22 deg C. Aliquots will be removed at 4, 8 & 12 hrs, centrifuged, subjected to HPLC analysis for flavonoid aglycone and glycoside content on a Hewlett-Packard 1050 Series system equipped with an 1100 Series diode array detector and a Kayak computer with 3D-ChemStation software for instrument operation and data analysis. For the flavonoid aglycones, separation is with a Hewlett-Packard (Agilent) Zorbax Eclipse XDB-C18 column (250 x 4.6mm) kept at 22 deg C & a mobile phase gradient of water, methanol (MeOH) and acetonitrile (ACN) [each containing 0.05% trifluoroacetic acid] run at 1.00mL/min. The gradient set points are as follows: 0 min at 90% H2O, 6% MeOH and 4% ACN; 5 min at 90% H2O, 6% MeOH and 4% ACN; 30 min at 85%, 9% and 6%; 60 min at 71%, 17.4% and 11.6%; 61 min at 0%, 85% and 15%; and 66 min at 90%, 6% and 4%. For the anthocyanidins, the chromatogram signal is monitored at 520 nm (20 nm bandwidth) with reference to 570 nm (60 nm bandwidth) while, for all other flavonoid aglycones, the chromatogram signal is monitored at 210 nm (20 nm bandwidth) with reference to 550 nm (100 nm bandwidth). In addition to the chromatogram signals, spectra are collected over 190-600 nm. Along with peak retention times, peak spectra are compared to those of standards for identification of compounds present. Glycoside separation is achieved with a Phenomenex Hypersil-ODS-5 m (250 x 4.6mm) column held at room temperature and a mobile phase gradient of 16.5% aqueous acetic acid (HOAc) and 100% acetonitrile (ACN) run at 1.00mL/min. The gradient set points are as follows: 0 min at 100% HOAc; 12.5 min at 100% HOAc; 17.5min at 80% HOAc and 20% ACN; 25min at 80% HOAc and 20% ACN; and 26min at 100% HOAc. The chromatogram signal and spectra are collected and used to identify sample compounds in the same manner as that for the aglycone analysis described above. Replicates performed similarly to achieve mean flavonoid concentrations in the color concentrates. 2.) The teas will be similarly treated as the color concentrates. 3) The vinegars, similarly, but with 2.5 gm of fresh tissue extracted in 50 mL of 10% vinegar in sealed flasks (22 deg C) w/o agitation. 4.) To quantify dried Hibiscus petals flavonoid stability over time, samples will be dried, packaged & stored at 22 deg C in light and dark conditions. At drying & at 2-month intervals, extractions and brews will be analyzed for flavonoid content by HPLC. 5.) To measure color loss over time of the 3 products, samples taken at packaging & at 1-month intervals quantified using a Helios Delta Visible Spectrophotometer (for transmittance, reflectance hue, value and chroma data). CLELAB (L*a*b*) values obtained for color evaluation, & transmittance for clarity. The means of the 6 replicate per product are base line values
