Introduction

The jamaica Hibiscus sabdariffa L. is native to tropical Africa, from Egypt and Sudan to Senegal, including Malawi, Mozambique, Zambia, Zimbabwe.For its medicinal properties, nutraceúticas and rusticidad of the plant has been distributed to other tropical regions of the world, as they are: Mexico, Central America, South America and Southeast Asia; in these places has reached great economic importance. To the American Continent was brought by black slaves, in the seventeenth century arrived in Brazil and the country of Jamaica in the year 1707; it is believed that the entrance to Mexico was at the time of the colony (^{Morton, 1987}). According to ^{Augstburger et al. (2000)}, the main producing countries of jamaica are: Egypt, Sudan, Mexico, Thailand and China; but China and Thailand are the largest exporters and control supply (^{FAO, 2010}).

In Mexico, the jamaica name is known to the plant and dry calyxs of H. sabdariffa L. During 2010 were harvested, 18 416 (ha) that produced 4 878 (t) of calyxs (^{SAGARPA, 2013}). The producing states are Guerrero and Oaxaca, since 85% of the national production is obtained (^{SAGARPA, 2013}). Possibly, it was domesticated in Sudan for 6 000 years, at first it was for its seeds and later it was made for the production of leaves and calyxs. Apparently wild plants Hibiscus sabdariffa L., were collected in Ghana, Niger, Nigeria and Angola (^{Shamsuddin and Van Der Vossen, 2003}).

The calyxs of jamaica have become important mainly for the medicinal properties, and attributed as a diuretic (^{Márquez et al., 2007}), to lower cholesterol (^{Agpreyo et al., 2008}), to control blood pressure (^{Ojeda et al., 2009}), antibacterial (^{Olaleye, 2007}); to regenerate tissues affected by degenerative diseases (^{Liang-Chih et al., 2009}); to reduce obesity (^{Alarcón et al., 2007}), has antiviral effect (^{Omilabu et al., 2010}), decreases calculi or stones (^{Woottisin et al., 2011}) and is purifying water (^{Yongabi et al., 2011}).

Some plants synthesized saponins, alkaloids, glycosides and tannins, which have been documented to exhibit various biological activities, such as antiinflammatory, antiarteroescleróticos, antitumor, antimutagenic, anticarcinogenic, antibacterial and antiviral (^{Mbaebie et al., 2012}). However, most plants have not been investigated for their antioxidant potential. The Jamaica H. sabdariffa L., of the Malvaceae family, is a plant that measures 2.0 m in height, is shrubby, stems from green to red, the leaves are green and red and green ribs; the difference is in the color of the calyx of flowers, which are yellow, pink and red (^{Serrano et al., 2011}; ^{Alarcón et al., 2012}).

The production depends on a criollo genotype with low yield; the plant is more sensitive to photoperiod and is grown during the months of july to december, its production is concentrated in the month of december and causes a low sale price. Ignorance of genotypes and their intrinsic properties make their use impractical for consumers and per capita consumption is low (^{Serrano et al., 2011}). The production of the dehydrated calyxs of the plant is mainly intended to prepare a refreshing beverage. In seeds they have been no studies of content and nutritional properties 25% protein and 21% oil (^{Abu-Tarboush et al., 1997}); however, studies of the flower’s calyx are scarce.

The importance of jamaica is not only limited to the medicinal aspect; also, the use of calyces in the feeding of chickens has been explored (^{Onibi and Osho, 2007}); the seed is used as a substitute in diets of sheep (^{Beshir and Babikers, 2009}), tilapia (^{Fagbenro et al., 2004}) and broilers (^{Jínez et al., 1998}), mainly. The seed is an excellent source of protein (30%) and oil (22%) (^{Abu-Tarboush et al., 1997}; ^{Yagoub et al., 2004}), which is used as cooked food and fermented in African countries. In Mexico, the Sudan variety has been distributed, which was introduced by the international market: this is of large calyces and red;however, the yields obtained do not exceed three varieties recent generation (^{Serrano et al., 2011}).

The jamaica has great economic potential for its nutritional value and various uses for the consumer. Likewise, most by-products are made by passing through a cooking process, so that their biochemical properties are not maintained and their nutraceutical properties may decrease in consumers; therefore it requires improving manufacturing processes of the products to be used by the population and increasing per capita consumption (^{Serrano et al., 2011}). In Mexico, there have been four new varieties of jamaica: Alma Blanca (VAB), Cotzaltzin (VC), Rosalíz (VR) and Tecoanapa (VT) (^{Serrano et al., 2011}), to drive the food and nutritional use the calyces of flowers, it was considered important to know the phytochemical and antioxidant characteristics of these new varieties and their comparison with the Sudan (VS) variety, which is of import.

Materials and methods

The plant material. The hubiscus calyxes of the new varieties Alma Blanca, Cotzaltzin, Rosalíz and Tecoanapa, and the Sudan variety introduced in Mexico were obtained from the Germplasm Bank of the Central Vales Experimental Field of Oaxaca of the National Institute of Forestry, Agriculture and Livestock Research (INIFAP) ).The colors of the dehydrated calyxs are of Alma Blanca (yellow), Rosaliz (roses), Cotzaltzin, Tecoanapa and Sudan (red). The calyces were dehydrated at 38 °C for three days.

Proximal analysis. The proximal analysis was determined following the ^{AOAC (2000)} methods.Proteins were quantified through nitrogen content (960.52), ethereal extract (920.85) and ash (923.03).The conversion factor for total protein was 6.25 (N*6.25).The dietary fiber was determined according to the method of ^{Prosky et al.(1998)}. The percentage of carbohydrates was obtained by difference.The evaluations were done in quadruplicate.

Aminoacids. The aminoacid content was obtained using of the methodology ^{AOAC (2000)}. The sample was hydrolyzed in an acid medium (HCl 6N and phenol, 1g L^-1 HCl and water 1:1, v/v) for 24 and 48 hours, both samples were made separately to free aminoacids. The methionine and cysteine were determined from a separate sample, which was performed by oxidation with performic acid.The content of the amino acids was obtained by mixing in reversed phase high performance liquid chromatography (HPLC), compared by their retention time and spectrum; a sample of 2 mL min^-1 was taken and placed in the HPLC flow (Agilent Technologies), with a Zorbax Eclipse AAA column (4.6 x 75 mm, 3.5 μm, PN 966400-902) and mobile phase A of 40 mM Na₂PO₄, NaOH 10 N to pH 7.8 water. The evaluations were performed on four replicates.

Total phenol content.The total phenolic content were obtained by the Folin-Ciocalteu described by ^{Singleton et al.(1999)}.The evaluations were performed on four replicates.

Determination of tannins.To 200 mg of dehydrated calyxs were added 10 ml of methanol, stirred for 20 min, centrifuged and the supernatant recovered (^{Deshpande and Cheryan, 1985}).To 1 mL of the supernatant was added 5 mL of freshly prepared vanillin reagent consisting of 1% vanillin in methanol and 8% HCl in methanol in the ratio 1:1.The sample was allowed to stand for 20 min at 30 °C and read in a spectrophotometer (6405 UV/Vis, JENWAY) at 500 nm.To determine the tannin content a calibration curve was prepared with (+) catechin;The final concentration is expressed as equivalent milligrams of (+) catechin/100 g of sample (mg EC/100 g).The evaluations were done with four replicates.

Determination of anthocyanins.The content of anthocyanins in each variety was obtained by the method developed by ^{Abdel and Hucl (1999)}.The total anthocyanins content was expressed in mg kg^-1 and was determined as cyanidin 3-glucoside; according to the equation:

Where: C= concentration of total anthocyanins (mg kg^-1); A= maximum absorbance;ε= molar absorptivity of cyanidin 3-glucoside (25 965 cm^-1 M^-1); Vol= total volume of the anthocyanin extract;PM= cyanidin 3-glucoside molecular weight of 449. The evaluations were done with four replicates.

Determination of simple phenols. The method used was based on the report ^{Ramamurthy et al. (1992)}. The identification and quantification of the simple phenolic compounds were performed through comparison with the retention time, commercial standard absorption spectra and calibration curves thereof. The evaluations were done with four replicates.

Statistic analysis. The data were statistically analyzed using ^{SAS (2004)}, by analysis of variance and mean comparison Tukey (p< 0.05); are expressed as averages and their standard deviation.

Results

Proximal analysis

Statistical differences were found among varieties for protein, carbohydrate, lipid, ash and fiber contents (Table 1).The calyx of the jamaica flower has a high lipid content of 17.7 to 22.8% of the varieties;vere lower in the VAB, VC and VR, and were higher in the VT and VS.The protein content is higher in the VT (5.8%) and is statistically equal to the VS (6%), intermediate in the VAB (4.2%) and lower in the VR (3.6%) and VC (3.4% ).The VAB and VR have a high fiber content with 43.9% and 40.1%, respectively, the intermediate is VS (38.5%) and are lower in VT (34%) and VC (33.1%).Also, they present high ash content, being higher in the VAB and VC, followed by the VR, VS and VT, values ranged from 9% to 6.2%. In carbohydrates are higher for VC (37.5%), intermediate and similar in VR and VT with 31.2% and are low in VS and VAB.

Table 1 Biochemical composition (%)^a extracts of calyxs of five varieties of jamaica.  

^a= Los datos son expresados como promedios ± desviación estándar (n= 4). Promedios con letras iguales en el mismo renglón son estadísticamente iguales (Tukey,p< 0.05).

Aminoacid content. The essential and non-essential aminoacid content of the calyces of the jamaica flower with is presented in Table 2. We demonstrate statistically differences for jamaica varieties in the amino acid content. Six of the ten essential amino acids were detected in the jamaica flower, these are: isoleucine, leucine, lysine, threonine and methionine + cysteine. The content of isoleucine and threonine in all varieties were greater than the minimum daily requirement defined by ^{FAO/WHO/UNO (1985)}.

Table 2 Amino acid content (mg 100 g^-1 of protein)^a extracts calyxs five varieties of jamaica. 

nd= no determinado; FAO/QHO= requerimiento mínimo necesario diario definido por estos organismos públicos de la salud; ^a= los datos son expresados comopromedios ± desviación estándar (n= 4). Promedios con letras iguales en el mismo renglón son estadísticamente iguales (Tukey, p< 0.05).

The isoleucine values in all varieties pass the minimum requirements required by infants, pre-school and adult children, as well as threonine for pre-school and adult children.The VT has a higher lysine content, however, they do not exceed those required by preschoolers and adults recommended by ^{FAO/WHO/UNO (1985)}.

The methionine + cysteine exceeds the requirements for adults in the VAB, VC, VR and VS, while it is lower in the VT.The contents of leucine do not exceed those required by children of infancy and preschoolers in the five varieties; however, they all have the values required by adults.Were not detected in the calyces of the jamaica flower the essential amino acids phenylalanine + tryptophan, histidine and valine, as well as the non-essential amino acids asparagine, glutamine and serine.

Total phenols. In the Table 3 shows the content values of tannins, total phenols and anthocyanins. Statistical differences were found between the varieties. With respect to total phenols, all varieties showed approximately 50% less compared to VS. On the other hand, the VAB presented the lowest tannin content (65.5 mgEC/100 g), with respect to the rest of the varieties. It is reported that tannins confer color on plants; the VC and VT presented almost twice the tannin content, than the VS; this is interesting because of the high antioxidant capacity attributed to the tannins.

Table 3 Contents of phenolic compounds of dry basis of extracts of calyxs of five jamaica varieties.  

^a= equivalentes de acido gálico; ^b= equivalentes de catequina; ^c= equivalentes de cianidin 3 glucosido. ^d= los datos son expresados como promedios ± desviación estándar(n= 4). Promedios con letras iguales en el mismo renglón son estadísticamente iguales (Tukey, p< 0.05).

Regarding the content of anthocyanins, VS presents the highest content, when compared to the four new varieties.Probably, the lowest concentration of tannins in this variety is compensated by the synthesis of anthocyanins.

Phenolic acids. Twelve phenolic acids were detected (Table 4), as well as vanillin. The statistically significant differences were found between varieties for the content of phenolic acids and showed differences between means, for each of the varieties.

Table 4 Content of phenolic acids on a dry basis of extracts calyxs five varieties of jamaica.  

nd= no detectado; ^a= los datos son expresados como promedios ± desviación estándar (n= 4). Promedios con letras iguales en el mismo renglón son estadísticamente iguales (Tukey, p< 0.05).

The vanillic acid was the most abundant in VR and VT with 276.64 to 206.04 mg 100 g^-1, respectively; however, this acid was not detected in the VS. ehe 4-hydroxybenzoic acid is higher in VC with 70.25 mg 100 g^-1, lower in VT with 49.5 mg 100 g^-1 and was not detected in VS.

Discussion

The content of protein in the flower’s calyx is low;whereas, ^{Abu-Tarboush et al.(1997)} report that the seed has 26.48% protein, 20.1% crude oil, 43.21% carbohydrates and 4.83% ash.The fiber content in jamaica is considered important in the production of jams and other by-products, since the calyx is used as the main product. For this reason, the VAB, VC and VR varieties showed higher ash content, indicating the presence of high mineral content.The variation in carbohydrate content of the four new varieties and the VS were relatively low.

The seed flower jamaica, ^{Abu-Tarboush et al.(1997)}, report lower contents of these aminoacids, compared to those reported in this study of flower buds of the five varieties.From the nutritional point of view, the aminoacids present in products derived from the jamaica flower, such as soft drinks and jams, these can contribute to human health.Also, seven of the eleven non-essential aminoacids in the jamaica flower were detected, which from a nutritional point of view are not important;as it happens with the essential aminoacids, since the human can synthesize them and contribute to maintain a better health.

The phenolic compounds are known for their antioxidant capacity.The presence of these compounds, such as tannins, flavonoids, anthocyanins and phenols of jamaica calyx extract, can give credibility in the focused use for the management of food-induced oxidative stress.The tannins have been used for the treatment of diarrhea, bleeding and detoxification (^{Afolayan and Mahebie, 2010}).The antioxidant activity of anthocyanins proves to be 50 times greater than vitamin C and 20 times more than vitamin E (^{Majo et al., 2008}), anthocyanins have been shown to help protect against tissue damage, prevent risks cancer and clogging of blood circulation in the capillaries, arteries and veins (^{Letelier et al., 2011}). Therefore, the concentration of these compounds contributes synergistically to enhance the antioxidant capacity of this plant for treatments and local uses related to diseases.

With regard to the content of phenolic acids, protocatecuic acid was found to be high in VR and VT, compared to VS;this acid is attributed anticancer property consumers as occurs in some citrus (^{Sawsen et al., 2012}).The salicylic acid is high in VAB;the cloragénico acid is high in the VAB and VS, similarly as presented in citrus (^{Sawsen et al., 2012}). The 4-Hydroxy-3-benzoic acid is very high in VS, followed by VAB.Other phenolic acids detected in all varieties are ferulic, cumaric, gallic and caffeic;these acids are reported with antioxidant capacity in various foods (^{Zavaleta et al., 2005}).The VAB presented more types of phenolic acids and in different amounts, which makes it very suitable for human consumption.

The antioxidant activity of extracts from jamaica calyces are significantly high, compared to those presented in the drugs.The study demonstrates a high content of phenolic acids and phenolic compounds of the extract; however, these compounds are detached from the calyces good electron donors may terminate the reaction and the radical change and become more stable free radical products (^{Mbaebie et al., 2012}).This type of compounds are attributed anti-cancer, anti-oxidants and the prevention of heart disease medicinal properties (^{Agpreyo et al., 2008}; ^{Ojeda et al., 2009}), among others.

Conclusions

The calyxes of the four new varieties of jamaica are high in protein, as well as lipids and fiber, mainly. The aminoacids are high and some varieties exceed minimum nutritional requirements for humans of different ages. Alone, three of the new varieties present anthocyanins, but all have a high content of tannins, which give them antioxidant capacity.The varieties are high in phenolic acids, highlighting the variety Alma Blanca with salicylic acid, while Rosaliz and Teconoapa varieties with protocatecuic acid, a compound that is attributed anticancer properties. Thus, it is shown that jamaica calyx extract exhibits a high natural antioxidant capacity, for therapeutic and traditional medicine use by phytochemical components, as it can be used for the treatment of radical-related diseases free and age-related diseases. However, studies are required to know the phenolic components, to establish their pharmacological properties and to serve as model trials.