1 Introduction

Flower is an important part of plant which contains a great variety of natural antioxidants, such as phenolic acids, flavanoids, anthocyanins and many other phenolic compounds (Kaur et al., 2006). Due to their appealing and desirable aesthetic aspects edible flowers are gaining renewed interest as rich source of bio active compounds, globally. Proliferating awareness among consumers as resulting from global intent has contributed to the comeback of the early lifestyles, in which edible flowers played an important role (Kopec and Balik, 2008). Besides their anti-oxidative properties and anti-carcinogenic effect, phenolic acids and flavanoids have long been recognized to possess anti-allergic, anti-inflammatory and antimicrobial activities as well (Robard et al., 1999). A high nutritional value, antioxidant capacity and attractive appearance pre determines edible flowers to be a new and promising foodstuff species for a wider use in human nutrition. Flowers produce a wide array of secondary metabolites, serving both as food stuffs and medicinal products which are of interest for several Parma and nutraceutical industries (Bungihan and Matias, 2013).

Phytochemicals are naturally occurring and biologically active plant compounds that have potential disease inhibiting capabilities due to their antioxidant effects. The high antioxidant capacity of these flowers is correlated with the level of flavanoids which corresponds to the medicinal properties of these flowers (Mato et al., 2000). Studies have demonstrated a positive correlation between the qualities of phenolic content to antioxidant properties. Oxidative stress in human results from imbalance of antioxidant in the body caused by factors as pollution, diet, chronic infections and so on (Agarwal et al., 2005). Antioxidants play an essential role in the prevention of disease and have capacity to reduce oxidative stress by chelating trace elements or scavenging free radicals and protecting antioxidant defenses (Banerjee et al., 2005). Now days, the application of plant based anti-oxidant or natural antioxidants is replacing synthetic molecules because of toxicities associated with the latter (Cruz et al., 2001).

As described in ancient literature, the use of edible flowers for dish preparation has been an atavistic custom. Besides their use for fresh consumption and garnishing, edible flowers can also be consumed dried, in cocktails, canned in sugars and preserved in distillated etc. (Neugtebauerova and Vabkova, 2009). Thus a drift towards natural sources for mitigating these problems has gradually come into vogue. The usage of plants in the system of medicine as antimicrobial, antiseptic and as cure for infection and sores (Bungihan and Matias, 2013), is widely accepted by nearly 80 percent of the people throughout the world. Hence, research of indentifying specific plants for their bioactivity and bio efficiency, is being conducted widely. However, the toxicity of the flower extracts with high antioxidant activity needs to be tested, so as to determine the daily intake limits (Kaisoon et al., 2011).

Generally, flowers are nearly neglected or rarely studied for their biological properties. Thus analyses for the presence of these considerable secondary metabolites, antioxidants and anti-bacterial profiles of ornaments plants is worthwhile for the establishment of their potentials as nutraceutical, cosmeceutical and pharmaceutical ingredient / additive (Bungihan and Matias, 2013). Keeping in view the importance of Phytochemicals, the present study was planned to examine and extract various Phytochemicals viz. lycopene, phenols, caroteniods etc. present in flowers crops grown under Indian condition and their antioxidant potential with specific Phytochemicals composition. In this study, a total of 14 flowering species, grown locally, were evaluated for their antioxidant potency and phytoconstituents.

2 Material and Methods

2.1 Basic experimental material and laboratory analysis

Flower samples belonging to 14 different genera were collected fresh from the common plant present and grown in and around Katrain region of H.P. (India). The collected samples were carries to the lab within a maximum of 6 hours for experimentation. Their names are present in Table 1. True to type representative samples from, each replication were collected at appropriate stages of harvesting. These were chopped, homogenized and a fresh sample of 5 g of each was stored immediately under refrigerated conditions (-20°C) until assay. The 5 g sample was further homogenized in 15 ml absolute ethanol to prepare the ethanol extract, which was further centrifuged at 10,000 rpm for 15 mins at 4 degree to obtain the supernatant, which is then stored at -20°C.

2.2 Antioxidant capacities and total phenolic assay

Measuring the antioxidant activity / capacity level of plants is carried out for the meaningful comparison of the antioxidant content of several plants. The parent CUPRAC (Cupric Reducing Antioxidant Capacity) method of antioxidant measurement, is based on the absorbance measurement of the Cu (I) - neucoproine (Nc) chelate formed as a result of the redox reaction of chain breaking antioxidants with the CUPRAC reagent, Cu (II) Nc where absorbance is recorded at the maximal light absorption wavelength of 450 nm; thus this is an electron transfer (ET) based method. The method described by Apak et al. 2006 was followed with minor modifications. For CUPRAC analysis, 100 µl samples were mixed with 4ml of CUPRAC reagent (1 ml of neucoproine; 1 ml ammonium acetate; 1 ml copper chloride and 1 ml distilled water; pH 7.4). Then the absorbance was recorded at 450 nm in spectrophotometer.

Similarly, FRAP (Ferric Reducing Ability of Plasma) was performed based on the procedure described by Benzie and Strain (1999) with slight modifications. For this, 100 µl of the distilled sample was added to 3 ml of the FRAP reagent and the reaction was monitored after 4 mins at 593 nm. The results were expressed as µmol Fe (II)/g fresh weight of the sample.

Total phenolic contents were determined with Folin–Cicalteau method (Singleton and Rossi, 1965). Modifications were done accordingly for the amount of sample present. Briefly, 0.50 ml extract was mixed with 2.5 ml of 1:10 diluted Folin–Cicalteau reagent. After 4 min, 2 ml of saturated sodium carbonate solution was added. The mixture was incubated in dark for 2 h at room temperature. The resulting complex was measured at 760 nm at the spectrophotometer for absorbance. Gallic acid was used as a standard for the calibration, and the results were expressed as mg of Gallic acid equivalents (mg GAE) per 100 g fresh weight (FW) of sample.

2.3 Plant pigment content

Lycopene: it is an important phytonutrient and consumers are becoming increasingly aware of the health benefits of its consumption. For determination of Lycopene content 5 g of sample was taken and crushed with Acetone until the residue becomes colourless. Filtrate was transferred in the separating funnel containing 20 ml of petroleum ether. 2-3 drops of Sodium sulphate was added in separating funnel. Then 20 ml of petroleum ether was added to make 2 separate phases. Lower phase was re extracted with additional petroleum ether till it became colourless. Final volume was made up to 50 ml and absorbance was taken at 503 nm and 452 nm using petroleum ether as blank.

Absorbance (1 unit) = 3.1206 µg Lycopene/ml

Statistical Analysis:

Statistical analyses were conducted using OP Stat software and SPSS. The variability estimates were worked out through Analysis of Variance (ANOVA) in a completely randomized Design, while correlation coefficients were determined by co variance and variance between the traits.

3 Results and Discussion

Phenolic compounds are widely distributed in fruits, vegetables and cereals. Plants vary widely both in their phenolic composition and content which are controlled both genetically and environmentally (Awika and Rooney, 2004). These components have received considerable attention due to their antioxidant activities and free radical scavenging capacity, which potentially have beneficial implications in human health (Imeh and Khokar, 2002). The Analysis of Variance revealed highly significant difference amongst the flower species for all the Phytochemicals under study, almost all the species contained good amount of antioxidants, Phenol content ranging CUPRAC 4.98→32.33 µM trolox/ g and FRAP 0.22→2.07 µM trolox/ g, while TPC varied from 973.59→2282.54 µg Gallic acid/ gfw. Vinca Red flower exhibited highest antioxidant capacity viz. CUPRAC 32.33 µmol trolox/g followed by Vinca Pink 30.46 while, least was observed in Gomphrina.4.983 µmol while its counterpart FRAP was highest (2.07 µmol /g) in poppy as well as in Vinca pink and least (0.22) in wild salvia. The total phenolic content (TPC) was found to be highest in African Marigold cv.Pusa Narangi (2282.54 µg GAE/gfw). Kaisoon et al. (2011) also reported high antioxidant activities from the edible flowers from Thailand. Similarly Rop et al. (2012) consider edible flower to be a new and promising food stuff species for a wider user in human nutrition, due to high antioxidant capacity.

From Table 2, a positive significant correlation was found among the phenol content and the antioxidant capacity viz. CUPRAC (0.262= r) and FRRAP (0.027= r). Similar results were reported by Zeng et al. (2014) who reported a statistically significant relationship between polyphenolic content and the antioxidant capacity of 19 Chinese edible flowers. Also previous reports that phenolic compound were major antioxidant constituents in medicinal herbs, vegetables, fruits and spices gave similar results (Cai et al., 2004; Huang et al., 2010). Caroteniods, which have a polyisoprenoid structure, are generally found in plants, algae, photosynthetic bacteria, yeast and moulds. They plays an important role in human nutrition, by acting as a precursor of vitamin A. lycopene and β- carotene are among the caroteniods, popular to consumers. β- Carotene belongs to their carotene class, which is one of the most abundantly found in diet and is used as food colourant.

The proposed method was applied for the analysis of total caroteniods, Lycopene and β- carotene content from different edible flowers. The amount of total caroteniods varied from 3.24 µg/100g in Morning Glory to 66.64 µg/100g in Calendula Orange. Similarly the Lycopene content was found as low as 0.55 mg/100g in Gomphrina up to 41.94 mg/100g in Calendula Orange whereas Coreopsis had highest amount of β- carotene content (10.97 µg/100g) while Vinca Pink contained least(4.09 µg/100g).

In the correlation study among the caroteniods, a highly statistically significant correlation was found between Lycopene and total caroteniods. A similar correlation was found amongβ- carotene and total caroteniods. A negative correlation was found between phenols and three carotenes. β- Carotene was found to have negative correlation with the three antioxidant factors (CUPRAC, FRAP and Phenols) while lycopene and total caroteniods showed a positive correlation with CUPRAC and FRAP. Prakash et al. (2015) in their study also found out Lycopene, β- carotene and Total caroteniods having Positive Association with each other. Kaulmann et al. (2014) also reported positive correlation between FRAP and phenols.

Grouping of Genotypes into various clusters

A hierarchical cluster analysis has been shown in the dendrogram constructed in Figure 1. The variations observed in the cluster means also points out to the degree of variability and divided the 14 species into two major groups A and B. Group A was further divided into two subgroups viz. A1 and A2. The subgroup A1 comprised of three species i.e. 1, 8 and 2, while subgroup A2 accommodated seven species i.e. 3, 10, 13, 4, 11, 12 and 7. In the meanwhile, group B was subdivided into subgroups B1 and B2 which comprised of 2 species each viz. 5 and 9 & 6 and 4 respectively.

In the present study two species of group A2 viz. 11 and 12 were found to be genetically most similar in regards to the six quality traits considered. These are the closest substitute present to each other in the nutrient composition present in them.

Result of PCA indicated that first two components having Eigen value greater than one retained in the analysis because of the substantial amount of variation amongst them. The two components had a variance of 42.07 and 30.62 percent aggregating to total of 72.69 percent of the total variation explained (Table 3).

The first factor (PC1) had the highest positive values for lycopene (0.959), caroteniods (0.958) and β- carotene (0.399) content; while the second factor was found superior for CUPRAC (0.798), FRAP (0.707) and Phenols (0.456). The positive values of different traits in three components indicated its importance in divergence among the 14 different species of edible flowers whereas, negative value showed the least contribution to divergence.

Further loading of different traits based on first two principle components indicated that CUPRAC, FRAP and Lycopene are the main components of divergence among the 14 species of edible flowers, whereas, Phenols, caroteniods and carotenes had lesser contributions, comparatively in the divergence. Banerjee et al. (2013) had also applied the unweighted pair group clustering through dendrogram showing inter-relationships between food flowers species and grouped them into two high level clusters based on activity.

4 Conclusions

The current study on the antioxidants ability and phytoconstituents of 14 species of edible flowers provided the results supporting their usage as nutrient rich raw material. The results revealed the presence of antioxidants, phenols and caroteniods in most of the flowers confirming flowers as a potential source of these phytonutrient. However, there stand needs to assess their toxicological and pharmacological effects and establish their safety limits. Furthermore, there are still lots of flowers which could be given consideration. The obtained results should contribute to the popularization of edible flowers as a new and prospective source for food, pharma and nutraceutical industry; as well as a promising object of human nutrition.

Refrences

Agarwal A., Gupta S., and Sharma R.K., 2005, Role of oxidative stress in female reproduction, Reproductive Biology and Endocrinology, 3: 28

https://doi.org/10.1186/1477-7827-3-28
PMid:16018814 PMCid:PMC1215514

Apak R., Güçlü K., Özyürek M., Esin Karademir S., and Erçağ E., 2006, The cupric ion reducing antioxidant capacity and polyphenolic content of some herbal teas, International Journal of Food Sciences and Nutrition, 57(5-6), 292-304

https://doi.org/10.1080/09637480600798132
PMid:17135020

Awika J.M. and Rooney L.W., 2004, Sorghum phytochemicals and their potential impact on human health, Phytochemistry, 65: 1199-1221

https://doi.org/10.1016/j.phytochem.2004.04.001
PMid:15184005

Banerjee A., Dasgupta N., and De B., 2005, In vitro study of antioxidant activity of Syzygium cumini fruit, Food chemistry, 90(4), 727-733

https://doi.org/10.1016/j.foodchem.2004.04.033

Banerjee A., and De B., 2013, Comparative study of antioxidant activity of the food flowers of West Bengal, India, International Journal of Food Properties, 16(1), 193-204

https://doi.org/10.1080/10942912.2010.535188

Benzie I.F., and Strain J.J., 1999, Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration, Methods Enzymol, 299: 15-27

https://doi.org/10.1016/S0076-6879(99)99005-5

Bungihan M.E., and Matias C.A., 2013, Determination of antioxidant, phytochemical and antibacterial profiles of flowers from selected ornamental plants in Nueva Vizcaya , Philippines, Journal of Agriculture Science and Technology, 3: 833-841

Cai Y., Luo Q., Sun M., and Corke H., 2004, Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer, Life sciences, 74(17), 2157-2184

https://doi.org/10.1016/j.lfs.2003.09.047
PMid:14969719

Huang W.Y., Cai Y.Z., Corke H., and Sun M., 2010, Survey of antioxidant capacity and nutritional quality of selected edible and medicinal fruit plants in Hong Kong, Journal of Food Composition and Analysis, 23(6), 510-517

https://doi.org/10.1016/j.jfca.2009.12.006

Imeh U. and Khokar S., 2002, Distribution of conjugated and free phenols in fruits: antioxidant activity and cultivar variation, Journal of Agricultural and Food Chemistry, 50(22), 6301-6306

https://doi.org/10.1021/jf020342j
PMid:12381107

Kaisoon O., Siriamornpun S., Weerapreeyakul N., and Meeso N., 2011, Phenolic compounds and antioxidant activities of edible flowers from Thailand, Journal of functional foods, 3(2), 88-99

https://doi.org/10.1016/j.jff.2011.03.002

Kaulmann A., Jonville M.C., Schneider Y.J., Hoffmann L., and Bohn T., 2014, Carotenoids, polyphenols and micronutrient profiles of Brassica oleraceae and plum varieties and their contribution to measures of total antioxidant capacity, Food chemistry, 155, 240-250

https://doi.org/10.1016/j.foodchem.2014.01.070
PMid:24594181

Kaur G., Alam M.S., Jabbar Z., Javed K., and Athar M., 2006, Evaluation of antioxidant activity of Cassia siamea flowers, Journal of ethnopharmacology, 108(3), 340-348

https://doi.org/10.1016/j.jep.2006.05.021
PMid:16846707

Kopec K. and Balik J., 2008, Kvalitologie zahradnickych produktu, Brno. MZLU., 34-40

Mato M., Onozaki T., Ozeki Y., Higeta D., Itoh Y., Yoshimoto Y., ... and Shibata M., 2000, Flavonoid biosynthesis in white-flowered Sim carnations (Dianthus caryophyllus), Scientia Horticulturae, 84(3), 333-347

https://doi.org/10.1016/S0304-4238(99)00140-5

Moure A., Cruz J.M., Franco D., Domı́nguez J.M., Sineiro J., Domı́nguez H., ... and Parajó J.C., 2001, Natural antioxidants from residual sources, Food chemistry, 72(2), 145-171

https://doi.org/10.1016/S0308-8146(00)00223-5

Neugebauerova J. and Vabkova J., 2009, Jedle kvety soucast food styling, Zahradnictvi, 83: 22-24

Parkash C., Dey S.S., Kumar R., Dhiman M.R., Dhiman M., Kumar S., and Kumar V., 2015, Comparative analysis for antioxidant capacities, important plant pigments and total phenolic contents in different Brassica vegetables, Molecular Plant Breeding, 6

https://doi.org/10.5376/mpb.2015.06.0010

Robards K., Prenzler P.D., Tucker G., Swatsitang P., and Glover W., 1999, Phenolic compounds and their role in oxidative processes in fruits, Food chemistry, 66(4), 401-436

https://doi.org/10.1016/S0308-8146(99)00093-X

Rop O., Jurikova T., Mlcek J., Kramarova D., and Sengee Z., 2009, Antioxidant activity and selected nutritional values of plums (Prunus domestica L.) typical of the White Carpathian Mountains, Scientia Horticulturae, 122(4), 545-549

https://doi.org/10.1016/j.scienta.2009.06.036

Singleton V.L., and Rossi J.A., 1965, Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents, American journal of Enology and Viticulture, 16(3), 144-158

Zeng Y., Deng M., Lv Z., and Peng Y., 2014, Evaluation of antioxidant activities of extracts from 19 Chinese edible flowers, SpringerPlus, 3(1), 315

https://doi.org/10.1186/2193-1801-3-315
PMid:25013750 PMCid:PMC4082252