Essay: Karkadeh

1.INTRODUCTION

Hibiscus sabdariffaL. (family Malvaceae), commonly known as roselle or red sorrel in English and as karkadeh in Arabic, is widely grown in tropics and subtropics of both hemispheres and has become naturalized in many areas of Central and West Africa, South East Asia, many areas of America and elsewhere (Pukclai and Kato-Noguchi, 2011).It is considered as one of the most important exporting crops beside its local consumption in Sudan.

1.1Origin

Karkadeh may have been domesticated in Western Sudan before 4000 BC;(Wilson and Menzel, 1964). It was first recorded in Europe in AD 1576. It seems to have been carried from Africa to the New World by slaves for use as food plant.

1.2 Producer countries

China and Thailand are the major producers, and control much of the world’s supply. Thailand has invested heavily inroselle production and their product is of superior quality, whereas China’s, with less stringent quality control practices, is less reliable and reputable. The world’s best comes from the Sudan, but the quantity is low, and poor processing hampers quality. Mexico, Egypt, Senegal, Tanzania, Mali and Jamaica are also important suppliers but production is mostly used domestically (Mohamadet al., 2002).

Sudan is presently the major producer of roselle; however, farmers regard it as a famine food. When drought is expected, farmers prefer to cultivate roselle rather than cereals because of its hardiness under adverse conditions (Mohamadet al., 2002).

1.3Sudanese Karkadeh

Karkadeh is grown in various parts of the Sudan, particularly Kordofan and Darfur. It is one of the cash crops cultivated by traditional farmers in Kordofan and Darfur States, where large quantities are produced both for local consumption and for export (see map below).Roselle is an important cash crop and a source of income for small farmers in Western Sudan, especially in North Kordofan State.The crop is grown mainly by traditional farming methods, exclusively under rain-fed conditions (El Naim and Ahmed, 2010). In Sudan, it is collected by goat-herding nomadic tribes, but the product is frequently inferior because of poor processing conditions. Nevertheless, the Sudanese product is attractively bright red, very acidic, and it is extremely popular in Germany, which imports most of the crop (Mohamed et al., 2012).

Map of Sudan showing the areas of roselle cultivation (colored green). (Source, Mohamed et al., 2012).

2LITERATURE REVIEW

2.1Chemical composition of calyx

Studies demonstrated that the calyx of H. sabdariffa (Mourtzinoset al., 2008) was rich in phenolic compounds, anthocyanins and protocatechuic acid. The dried calyces contain flavonoids such asgossypetin, sabdaretine, hibiscetine and anthocyanins (Pietta, 2000). Flavonoids are phenolic substances and they act in plants as antioxidants. Previous reports showed that the extracts from the red calyces ofH. sabdariffa contain potent antioxidant principles (Ologunduduet al., 2010).

2.2Chemical composition of seeds
Some investigations on the nutritional value of plant seeds as unconventional sources of proteins have been done by many researchers[Ingale and Shrivastava, 2011;Narsing et al., 2011].The seed is used for its oil in China and eaten in West Africa (Robert, 1996); in Sudan, it is used for edible oil production and poultry feeding (Al-Wandawi et al., 1984). Roselle seeds are a good source of lipid and soluble antioxidants, particularly ??-tocopherol.

2.3 General uses
Roselle is a multi-use plant, whose outer floral leaves (calyx), is frequently used in the production of jelly, jam, juice, wine, syrup, gelatin, pudding, cake, ice cream and flavoring. Its brilliant red colour and unique flavor make it a valuable food product (Tsai and Ou, 1996). The juice from the calyces is claimed to be a health-enhancing drink due to its high content of vitamin C, anthocyanins and other antioxidants. In Sudan, the dry calyx is used to produce a flavorsome and healthy drink and dried calyces are used for tea, jelly, marmalade, ices, ice cream, sorbets, butter, pies, sauces, tarts, and other desserts. Sudanese roselle tea is well known as an organic product and is highly valued for its beneficial effects.
Roselle seeds, which until now do not have any commercial applications, are a source of a vegetable oil that rich in phytosterols and tocopherols, particularly ??-sistosterol and ??-tocopherol(Mohamed etal., 2002). The global characteristics of roselle seed oil allow important industrial applications for this oil. The seeds have also been used as an aphrodisiac coffee substitute.These characteristics represent an added value for the culture of this plant (Mohamed etal., 2007).

2.4Medicinal applications
Many medicinal applications of Hibiscus sabdariffaL.have been described in several studies (Alarcon-Aguilar et al., 2007).The biological activities of roselle, such as protection effects from atherosclerosis (Kao et al., 2009), anti-carcinogenic activities (Chen et al., 2003; Tseng et al., 2000) especially in the field of leukemia (Chang et al., 2005), cyclooxygenase inhibitory activities (Christian et al., 2006), chemo-preventive properties (Liu, 2006), hepatoprotective effects and antihypertensive effects (Liu et al., 2002) have been reported. Due to the biological activity of anthocyanins and their potential benefit for human health, roselleanthocyanins could be a good source of antioxidants as well as a natural colourant. Taken as a drink made from the calyx, it is a mild diuretic and purgative, among many other effects.
2.5General objective
The overall objective of this study is to screen biochemical and molecular properties of selected Sudanese karkadeh genotypes (Hibiscus sabdariffa). The expecting findings could help researchers for releasing a new genotype of high crop quality.

2.5.1Specific objectives
1- Extraction of the target compounds using different solventsof different polarities.
2- Determination of antioxidant capacity of the extracted compounds from the calyx.
3- Evaluation of the extracted compounds as anti-fungal and anti-bacterial agents.
4- Determination of protein, oil and fatty acids contents in the seeds.
5- Evaluation of genetic diversity among the examined genotypes using biochemical and molecular markers.
3MATERIALS AND METHODS
3.1 Plant materials
Nineteen new genotypes of Hibiscus sabdariffaL. having different calyx colours [colorless,red, yellow, blue, violet, orange etc.] will be examined in this study. These genotypes were derived by the supervisor of this work, DrAbd El Wahab H AbdAlla, Department of Agronomy, Faculty of Agriculture, University of Khartoum, within breeding programmes at Faculty of Agriculture for the improvement of crop quality. The study will be carried out on the dried calyces, young leaves and seeds.

3.2Extraction
Compoundsfrom calyx will be extracted using:
1 ‘ Water extract or watermethanol extract (4:1 v/v).
2 ‘ Sequential extracts, hexane, ethylacetate/ and acidified methanol (0.1% HCl in methanol).

3.3Chemical analysis of calyces
Dry calyces will be subjected to the following analysis:
1- Determination of antioxidant capacities using different standard methods.
2- Estimation or analyzingtotal phenol (TP) contents:
A – Hydroxyl cinnamate as caffeic acid absorbance at 316 nm.
B -Anthocyanin as delphinidin absorbance at 520 nm.
C -Flavanols as catechin absorbance at 280 nm.
D – Flavanols as rutin absorbance at 365 nm.
Others, like ??-carotene, sterols, etc. will be detected.Gallic acid,catechin, caffeic acid etc. will be used as standards when usingHigh Performance Liquid Chromatography(HPLC) or Gas Chromatography Mass Spectroscopy (GC MS) apparatus.
3- Proximate analysis (Ash, moisture, fibre, total soluble solid, monosaccharides, etc.) will be determined according to the standard methods.
4- Total minerals: Minerals were determined in samples’ extracts prepared by the dry-ashing method.
5- Evaluation of vitamins (A, C, and E).
6- Determination of organic acid (Oxalic, citric hibiscus acid, etc.)
7- Biological activities: anti-fungal and anti-bacterial activities, etc’will be detected.

3.4Molecular analysis
1 – Genomic DNA extraction: A modified CTAB (hexadecyltrimethylammonium bromide) procedure will be adopted for obtaininggood quality total DNA.
2 – Evaluation of genetic variability among the studied new derived genotypes of Hibiscus sabdariffaL.using different moleculartechniques such as Randomly Amplified Polymorphic DNA (RAPD).
3 – DNA sequencing using DNA sequencer machine.

3.5Chemical analysis of the seed
The powdered seeds will be subjected to the following analysis:
1 – Protein extraction and determination using spectrophotometeric and electrophoresis techniques.
2 – Estimation of oil content and fatty acids profile using Gas Chromatography Mass Spectroscopy (GC /MS) apparatus.
3.6 Statistical analysis
The collected data will be subjected to statistical analysis using different soft ware.

4Plan of work
Activity Duration (Month)
Collection and preparation of the plant materials. 3
Evaluation of the antioxidant capacities of the different extracts. 4
Quantification of phenolic compounds. 4
Proximate analysis (ash, moisture, fibre, saccharides, etc.) 5
Estimation of vitamins andtotal minerals. 2
Antimicrobial and antifungalactivities of ethanol extracts. 2
Molecular analysis techniques: Genomic DNA extraction, RAPD-PCR Reactions, DNA sequencing. 6
Protein extraction and determination. 3
Determination of oil content and fatty acids profile. 3
Statistical analysis, writing and submission of the thesis. 4
Total 36

Budget
Description Cost (SDG)
1- Field work (planting, watering and harvesting the crop) 650
2- Glassware (columns, flasks, beakers, test tubes, cylinders, etc.) 800
3- Solvents: Methanol, Ethanol, Ethyl Acetate, Hexane, Absolute ethanol, Chloroform, Isopropanol etc. 1150
Description Cost (SDG)
4- Reagents: Folin-Ciocalteu,coomassie Brilliant Blue, ninhydrin, etc. 950
5- General chemicals:mineral acids, salts, resins, buffers, etc. 1150
6- Specific chemicals:
(a) DPPH(2, 2-diphenyl-1-pycrilhydrazil hydrate) 700
(b) ABTS (2, 2??-azinobis (3-ethylbenzothiazoline- 6-sulfonic
acid) 450
(c) Standers phenolic compounds (caffeic acid, delphinidin,
catechin,rutin, 1100
(d) CTAB (hexadecyltrimethyl ammonium bromide) 400
(e) Oligonucleotides (primers) and standards fatty acids 850
7-Other consumables 700
Total 8900

REFERENCES

Alarcon-Aguilar, F. J., Zamilpa, A., Perez-Garcia, M. D., Almanza-Perez, J. C., Romero-Nu??nez, E., Campos-Sepulveda, E. A., Vazquez-Carrillo, L. I. and Roman-Ramos, R. (2007). Effect of Hibiscus sabdariffa on obesity in MSG mice.J. Ethnopharm. 114: 66-71.

Al-Wandawi, H., Al-Shaikhly, K. and Abdul-Rahman, M. (1984). Roselle seed: A new protein source. J. Agric. Food Chem.32: 510-512.

Chang, Y. C., Huang, H. P., Hsu, J. D., Yang, S. F. and Wang, C. J. (2005). Hibiscusanthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells.Toxicol. Appl. Pharm. 205: 201-212.

Chen, C. C., Hsu, J. D., Wang, S. F., Chrang, H. C., Yang, M. Y., Kao, E. S., Ho, Y. O. and Wang, C. J. (2003). Hibscussabdariffaextrac inhibit the development of atherosclerosis in cholesterol-fed rabbits. J. Agric. Food Chem.51(18): 5472-5477.
Christian, K. R., Nair, M. G. and Jackson, C. J. (2006).Antioxidant and cyclooxygenase inhibitory activity of sorrel (Hibiscus sabdariffa).J. Food Compos. Anal.19: 778-783.

El Naim, A. M. and Ahmed, S. E. (2010).Effect of weeding frequencies on growth and yield of two roselle (Hibiscus sabdariffaL.) varieties under rain fed. Aust. J. Basic Appl. Sci.4(9): 4250-4255.

Ingale, S. and Shrivastava, S. K. (2011).Amino acid profile of some new varieties of oil seeds.Adv. J. Food Sci. Technol.3(2): 111-115.

Kao,E. S., Tseng, T. H., Lee, H. J., Chan, K. C. and Wang, C. J. (2009). Anthocyanin extracted from Hibiscusattenuate oxidized LDL’mediated foam cell formation involving regulation of CD36 gene. Chem-Biol. Interact.179: 211-218.

Liu, C. L., Wang, J. M., Chu, C. Y., Cheng, M. T. and Tseng, T. H. (2002). In vivo protective effect of protocate-chuic acid on tert-butyl- hydroperoxideinduced rat hepatotoxicity. Food Chem. Toxicol.40: 1415-1422.

Liu, J. Y., Chen, C. C., Wang, W. H., Hsu, J. D., Yang, M. Y. and Wang, C. J. (2006). The protective effects of Hibiscus sabdariffa extract on CCl4-induced liver fibrosis in rats. Food Chem. Toxicol.44: 336-343.

Mohamad, O., Mohd. Nazir, B., Abdul Rahman, M. and Herman, S. (2002). Roselle: A new crop in Malaysia. Bio: A grand international biotechnology event. Bulletin PGM. Kuala Lumpur.

Mohamed, B. B. Sulaiman, A.A and Dahab, A. A. (2012).Roselle (Hibiscus sabdariffaL.) in Sudan, cultivation and their uses.Bull. Environ. Pharmacol. Life Sci.1: 48-54.

Mohamed, R., Fernadez, J., Pineda, M. and Aguilar, M. (2007). Roselle (Hibiscus sabdariffa) seed oil is a rich source of ??-tocopherol. J. Food Sci. 72: 207-211.

Mourtzinos, I., Makris, D. P., Yannakopoulou, K., Kalogeropoulos, N., Michali, I. and Karathsnos, V.T. (2008). Thermal stability of anthocyanin extract of Hibiscus sabdariffa L.in the Presence of ??-Cyclodextrin. J. Agric. Food Chem. 56: 10303-10310.

Narsing, R. N. G., Prabhakara, R. P. G. and Govardhana, R. D. (2011).Preparation of wood apple seed protein concentrate and evolution of its nutritional and functional characteristics. Inter. Food Res. J.18(3): 949-955.

Ologundudu, A., Ologundudu, A. O., Oluba, O. M., Omotuyi, I. O. and Obi, F.O. (2010). Effect of Hibiscus sabdariffaanthocyanins on 2, 4-dinitrophenylhydrazine-induced tissue damage in rabbits.J. Toxicol. Envir. Health Sci.2(1): 1 – 5

Pietta, P. G. (2000). Flavonoids as antioxidants.J. Nat. Prod.63 (7): 1035-1042.

Pukclai, P. and Kato-Noguchi, H. (2011).Evaluation of allelopathic activity of Hibiscus sabdariffaL. Advan.Bio. Res.5 (6): 366-372.

Roberts, S. M. (1996). Roselle production manual (Hibiscus sabdariffa).http://www.herbs.org/africa/hibiscus_production_manual.html

Tsai, P. J. and Ou, A. S. M. (1996).Colour degradation of dried roselle during storage. Food Science.23(5): 629-640.

Tseng, T. H., Kao, T. W., Chu, C. Y., Chou, F. P., Lin, W. L. and Wang, C. J. (2000). Induction of apoprosis by Hibiscusprotocatechuicacid in human leukemia cells via reduction of retinoblastoma (RB) Phosphorylation and Bcl-2 Expression. Biochem.Pharm.60: 307- 315.

Wilson, F. D. and Menzel, M. Y. (1964).Kenaf (Hibiscus cannabinus), roselle (Hibiscus sabdariffa).Econ. Bot.18: 80-91.

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