THE EFFECT OF AQUEOUS EXTRACT OF HIBISCUS SABDARIFFA CALYX ON MALE RAT REPRODUCTIVE HORMONES

ABSTRACT

Hibiscus sabdariffa Linn (Roselle) is an annual shrub commonly used to make jellies, jams, beverages and used traditionally as a medicinal plant for the treatment of various ailments. The present study was undertaken to investigate the effect of aqueous extract of Hibiscus sabdariffa calyx on male rat reproductive hormones. Twenty (20) male wistar rat weighing 0.25±0.2 kg   were grouped into one water control group and three experimental groups. While the water control group received 1.0 ml of water for 28 days, the experimental group were administered 250 mg/kg oral doses of aqueous extract of H.sabdariffa calyx. The effect on the basal levels of follicle stimulating hormone, testosterone, prolactin, luteinizing hormone and estradiol were conducted in experimental animals. The 28 days oral administration of aqueous extract of H.sabdariffa L. is associated with a decreased circulating plasma levels of follicle stimulating hormone, Testosterone and prolactin in male wistar rat compared with the control group. The study did not show a change in the plasma levels of both circulating luteinizing hormone and estradiol following 28 days oral administration of H.sabdariffa calyx to the experimental animal. Moreover, Marked histological changes were detected on testes of the experimental animals after 28 days of administration. This may indicate that H.sadariffa calyx extract can cause both morphological and physiological testicular damage. The study concluded that H. sabdariffa calyx extract at a dose of 250 mg/kg caused mild effects on rat reproductive hormones.

 

 

 

 

 

TABLE OF CONTENT

CHAPTER ONE

1.0    INTRODUCTION

1.1     Literature Review

1.1.1. Botanical description

1.1.1.2. Morphology

1.1.1.3   Ecology/cultivation

1.1.1.4. Karyotype

1.1.2    Economic importance of Hibiscus sabdariffa

1.1.2.1. Traditional culinary use

1.1.2.2. Use in local and traditional food and medicine

1.1.2.3. Source of fibre

1.1.2.4. Animal feed

1.1.2.5. Cosmetic

1.1.3. Phytochemistry

1.1.3.1. Nutritional value

1.1.3.2. Bioactive constituents

1.1.3.3. Organic acids

1.1.3.4. Hydroxycitric acid

1.1.3.5. Hibiscus acid

1.1.3.6. Flavonoids

1.1.3.7. Anthocyanins

1.1.3.8. Mucilage, pectin and carbohydrates (polysaccharides)

1.1.3.9. Volatile compounds

1.1.4 Biological and pharmacological activities

1.1.4a     Effects on smooth muscles

1.1.4b Antibacterial, antifungal and antiparasitic activity

1.1.4c. Antipyretic, antinociceptic and anti-inflammatory activities

1.1.4d   Antioxidant activity

1.1.4e Hepatoprotective activity

1.1.4f Nephroprotective activity

1.1.4gRenal effects/diuretic effect (incl. clinical studies)

1.1.4h Cancer-preventive activity                                                                                                

1.1.4i Lipid metabolism – anticholesterol effects/effects on lipid metabolism

1.1.4j Anti-obesity activity

1.1.4k Lactating activity

1.1.4l Anti-diabetic activity

1.1.4m Delayed puberty activity

1.1.4n Anti-hypertensive activity

1.1.4o Anti-anaemic activity

1.1.5   Safety

1.1.6. Toxicology

1.1.7. Interactions

1.1.8. Anatomy of the male rat testes

1.1.8.1. Brief anatomy of the rat testes

1.1.8.2. Structure-Function relationship of the male rat testes

1.1.9. Reproductive hormones

1.1.9.1. Follicle stimulating hormone (FSH)

1.1.9.2. Luteinizing hormone (LH)

1.1.9.3. Testosterone

1.1.9.4.   Estradiol

1.1.9.5     Prolactin

 

CHAPTER TWO

MATERIALS AND METHODS

2.1         MATERIALS

2.1.1a   Chemicals and scientific kits.

2.1.1b List of Equipments

2.1.2    Plant Material

 

2.1.3   Experimental Animals

2.2       METHODS

2.2.1    Preparation of whole extract of Hibiscus sabdariffa calyx

2.2.2    Experimental Design

2.2.4      Blood collection.

2.2.5      Histological Examination

2.2.5         Hormonal Assay

2.2.5.1       Assay for Reproductve hormones

2.2.5.1a     Assay for Plasma FSH and LH

2.2.5.1b      Principles of Assay

2.2.5.1c      Assay Procedure

2.2.5.1d    Calculation of FSH Concentration

2.2.5.1e        Calculation of LH Concentration

2.2.5.2    Assay for Plasma Estradiol

2.2.5.2a       Principle of the Assay

2.2.5.2b      Assay Procedure

2.2.5.2c.       Calculation of Estradiol concentration

2.2.5.3        Assay for Testosterone

2.2.5.3a      Principle of Assay

2.2.5.3b       Assay Procedure

2.2.5.3c           Calculation of Testosterone Concentration

2.2.5.4a.     Assay for Prolactin

2.2.5.4b.        Principle of Assay

2.2.5.4b        Assay Procedure

2.2.5.4c          Calculation of prolactin Concentration

2.2.6 Statistical Analysis

                                                       

CHAPTER THREE

3.0                                                            RESULTS

3.2 Histological Examination of Male rat testes                                   

3.2.1 Histological examination of rat testes of the control group.

3.2.2 Histological effect of H.sabdariffa calyx extract on rat testes 0f H.sabdariffa

administered group. (Group 2)

 

CHAPTER FOUR

DISCUSSION AND CONCCLUSION

CONCLUSION

REFERENCE

APPENDIX I

APPENDIX II

APPENDIX III

 

 

CHAPTER ONE

1.0    INTRODUCTION

Endocrine disrupting compounds (EDCs) are natural or synthetic compounds that have the ability within the body to alter endocrine functions often through mimicking or blocking endogenous hormones (James et al., 2013). These actions on the endocrine system have resulted in developmental deficits in various invertebrate and aquatic species (Crain et al., 2007; Elango et al., 2006) and mammals (Christopher et al., 2012). Exposures in adulthood have consequences but fetal and early life exposures appear to have more severe effects that persist through life (Rubin and Soto, 2009). Among these classes of chemicals are phytoestrogens that show effects suggestive of estrogenicity, such as binding to the estrogen receptors, induction of specific estrogen-responsive gene products, stimulation of estrogen receptor(s) and positive breast cancer cell growth (James et al., 2013). Through these interactions by acting as agonists or antagonists, EDCs are able to alter the activity of response elements of genes, block natural hormones from binding to their receptors, or in some cases increase the perceived amount of endogenous hormone in the body by acting as a hormone mimic to its receptor (Ze-hua et al., 2010).

Hibiscus sabdariffa Linn (Roselle) is an annual shrub commonly used to make jellies, jams and beverages (Sirag et al., 2013). The brilliant red colour of its calyx makes it a valuable food product, a part from its multitude of traditional medicinal uses (Sirag et al., 2013). Infusions of the calyces are considered as diuretic, cholerectic, febrifugal and hypotensive, decreasing the viscosity of the blood and stimulating intestinal peristalsis (Salleh et al., 2002). Roselle calyx extract is a good source of antioxidants from its anthocyanins and associated with antitumor and inhibitory effects on the growth of several cancer cells (Ajiboye et al., 2011).

Extracts of H.sabdariffa calyces have been reported to be rich in phytoestrogens (Adigun et al., 2006; Orisakwe et al., 2004; Brian et al., 2009; Omotuyi et al., 2011) and some reports indicated that H.sabdariffa calyces have estrogenic effects, although exact estrogen-like ingredient is not determined (Ali et al., 1989).

This study was undertaken to determine to which extent H.sabdariffa calyces extract alters the basal levels of selected reproductive hormones: Follicle stimulating hormone, testosterone, prolactin, estradiol, and luteinizing hormone as well as the histological features male wistar rat testes.

 

1.2     Literature Review

H.sabdariffa L.

Hibiscus sabdariffa L. also known as roselle, is an ideal crop for developing countries as it is relatively easy to grow, can be grown as part of multi-cropping systems and can be used as food and fibre (Da-costa-Rocha et al., 2014). In China the seeds are used for their oil and the plant is used for its medicinal properties, while in West Africa the leaves and powdered seeds are used in meals (Da-costa-Rocha et al., 2014). Additionally, it is used in the pharmaceutical and food industries (Da-costa-Rocha et al., 2014).

A limited number of reviews on H.sabdariffa have been conducted. Only one detailed review on the phytochemical, pharmacological and toxicological properties of H.sabdariffa ( Ali et al., 2005) and two more focused, later reviews are available: One on the effectiveness of H.sabdariffa in the treatment of hypertension ( Wahabi et al., 2010) and another on the treatment of hypertension and hyperlipidemia ( Hopkins et al., 2013).

 

1.1.1. Botanical description

The genus Hibiscus (Malvaceae) includes more than 300 species of annual or perennial herbs, shrubs or trees (Wang et al., 2012). H.sabdariffa (syn.: Abelmoschus cruentus (Bertol.) Walp., Furcaria sabdariffa Ulbr., Hibiscus cruentus Bertol., Hibiscus fraternus L., Hibiscus palmatilobus Baill. and Sabdariffa rubra Kostel ( The Plant list, 2010) is commonly known as roselle, hibiscus, Jamaica sorrel or red sorrel (English) and in Arabic, karkadeh ( Ali et al., 2005 ; Ross, 2003). Its native distribution is uncertain, some believe that is from India or Saudi Arabia ( Ismail, Ikram, and Nazri, 2008), while Murdock ( Murdock, 1959) showed evidence that H.sabdariffa was domesticated by the black populations of western Sudan (Africa) sometime before 4000 BC. Nowadays, it is widely cultivated in both tropical and subtropical regions (Morton, 1987 and USDA, 2007) including India, Saudi Arabia, China, Malaysia, Indonesia, The Philippines, Vietnam, Sudan, Egypt, Nigeria and México (Chewonarin et al., 1999; Dung et al., 1999; Eslaminejad and Zakaria, 2011; Ismail et al., 2008; Mahran et al., 1979; Rao, 1996; Sharaf, 1962 and Yagoub Ael et al., 2004).

There are two main varieties of H.sabdariffa, the first being H.sabdariffa var. altissima Wester, cultivated for its jute-like fibre and the second is H.sabdariffa var. sabdariffa. The second variety includes shorter bushy forms, which have been described as races: bhagalpuriensi, intermedius, albus and ruber. The first variety has green, red-streaked, inedible calyces, while the second and third race have yellow-green edible calyces (var. ruber) and also yield fibre (Morton, 1987).

1.1.1.2. Morphology

H.sabdariffa var. sabdariffa ruber is an annual, erect, bushy, herbaceous subshrub that can grow up to 8 ft (2.4 m) tall, with smooth or nearly smooth, cylindrical, typically red stems. The leaves are alternate, 3 to 5 in (7.5–12.5 cm) long, green with reddish veins and long or short petioles. The leaves of young seedlings and upper leaves of older plants are simple; lower leaves are deeply 3 to 5 or even 7 lobed; the margins are toothed. Flowers, borne singly in the leaf axils, are up to 5 in (12.5 cm) wide, yellow or buff with a rose or maroon eye, and turn pink as they wither at the end of the day. At this time, the typically red calyx, consisting of 5 large sepals with a collar (epicalyx) of 8 to 12 slim, pointed bracts (or bracteoles) around the base, begins to enlarge, becomes fleshy, crisp but juicy, 1 1/4 to 2 1/4 in (3.2–5.7 cm) long and fully encloses the velvety capsule, 1/2 to 3/4 in (1.25–2 cm) long, which is green when immature, 5-valved, with each valve containing 3 to 4 kidney-shaped, light-brown seeds, 1/8 to 3/16 in (3–5 mm) long and minutely downy. The capsule turns brown and splits open when mature and dry. The calyx, stems and leaves are acid and closely resemble the cranberry (Vaccinium spp.) in flavour (Morton, 1987; Ross, 2003).

Fig.1. H.sabdariffa calyx

1.1.1.3   Ecology/cultivation

H.sabdariffa is easy to grow in most well drained soils but can tolerate poor soils. It requires 4-8 montH.sabdariffa growth with night-time temperatures with a minimum of 20 °C, as well as 13 h of sunlight and a monthly rainfall ranging from 5–10″ (130–250 mm) during the first few montH.sabdariffa to prevent premature flowering. Rain or high humidity during the harvest time and drying process can downgrade the quality of the calyces and reduce the yield. The quality of H.sabdariffa is determined by seed stock, local growing conditions, time of harvest, post-harvest handling and mainly the drying step. Most of the time it grows as a supplement crop and it is susceptible to fungi, viral and bacterial attack and also to insects. A single plant produces about 1.5 kg of fruit, approximately 8 t/ha. Yields of leaves may be about 10 t/ha ( EcoCrop, 2007; Plotto, 2004).

1.1.1.4. Karyotype

2n = 36 (Huang et al., 1989; Menzel and Wilson, 1961) and 72 (Chennaveeraiah and Subbarao, 1965; Wilson and Menzel, 1964) were observed. Somatic tissue showing diploid and tetraploid segments were also occasionally noticed (Tjio, 1948). In a karyomorphological study conducted in India, both root and flower segments showed great similarity in the types of chromosomes in the complement. This indicates that the tetraploid tissue must have arisen in an autotetraploid manner (Bhatt and Dasgupta, 1976). Later, this species was reported to be tetraploid (2n = 72) (Hiron et al., 2006).

1.1.2    Economic importance of Hibiscus sabdariffa

1.1.2.1. Traditional culinary use

Fresh or dried calyces of H.sabdariffa (cH.sabdariffa) are used in the preparation of herbal drinks, hot and cold beverages, fermented drinks, wine, jam, jellied confectionaries, ice cream, chocolates, flavouring agents, puddings and cakes (Bako et al., 2009; Bolade et al., 2009; Esselen and Sammy, 1975; Ismail et al., 2008; Okoro, 2007; Plotto, 2004; Rao, 1996; Tsai et al., 2002; Wilson and Menzel, 1964). In Egypt, the fleshy calyces are used in making “cacody tea” and fermented drinks (Kochhar, 1986), while in Sudan and Nigeria, the calyces are boiled with sugar to produce a drink known as “Karkade” or “Zoborodo” (Gibbon and Pain, 1985). In Mexico this drink is called Jamaica or “agua de Jamaica’” or “té de Jamaica”. In the West Indies the calyces can also be used as colouring and flavouring ingredient in rum (Ismail et al., 2008).

The seeds are eaten roasted or ground in meals, while the leaves and shoots are eaten raw or cooked, or as a sour-flavoured vegetable or condiment (Wilson and Menzel, 1964). In Sudan, the leaves are eaten green or dried, cooked with onions and groundnuts, while in Malaysia the cooked leaves are eaten as vegetables (Ismail et al., 2008). In Africa, the seeds are roasted or ground into powder and used in meals, suh as oily soups and sauces. In China and West Africa, the seeds are also used for their oil (Atta and Imaizumi, 2002). Another use for the seed is as a substitute for coffee (Morton, 1987).

1.1.2.2. Use in local and traditional food and medicine

H.sabdariffa has been widely used in local medicines. In India, Africa and Mexico, infusions of the leaves or calyces are traditionally used for their diuretic, cholerectic, febrifugal and hypotensive effects, decreasing the viscosity of the blood and stimulating intestinal peristalsis. It is also recommended as a hypotensive in Senegal (Morton, 1987). In Egypt, preparations from the calyces have been used to treat cardiac and nerve diseases and also to increase the production of urine (diuresis). In Egypt and Sudan, an infusion of “Karkade” calyces is also used to help lower body temperature (Leung, 1996). In Guatemala it is used for treating drunkenness (Morton, 1987). In North Africa, calyces preparations are used to treat sore throats and cougH.sabdariffa, as well as genital problems, while the emollient leaf pulp is used for treating external wounds and abscesses (Neuwinger, 2000). In India, a decoction from the seeds is used to relieve pain in urination and indigestion. In Brazil, the roots are believed to have stomachic and emollient properties. In Chinese folk medicine, it is used to treat liver disorders and high blood pressure (Morton, 1987). In Iran, sour hibiscus tea is reportedly a traditional treatment for hypertension ( Burnham et al., 2002), while in Nigeria the decoction of the seeds is traditionally used to enhance or induce lactation in cases of poor milk production, poor letdown and maternal mortality ( Gaya et al., 2009).

1.1.2.3. Source of fibre

H.sabdariffa is one of the most important species grown commercially as a fibre plant and became increasingly important in India after independence and partition with Pakistan, where the most important jute (Corchorus capsularis L. or Corchorus olitorius L.) growing areas are. It is used as a jute substitute in making clothing, linen, fishing nets, ropes and similar items (Clydesdale et al., 1979). Despite the fact that this species is slow growing, as it requires about 180 days to produce a satisfactory yield of fibre, there is still interest in the plant as some varieties of H.sabdariffa (not edible but fibre type) have a high degree of genetic resistance to root-knot nematodes.

However, H.sabdariffa fibres are subject to ongoing research showing promising technical properties when used as a substitute for synthetic or mineral fibres in composite materials, as well as a source material for high quality paper production (Dutt et al., 2010; Kumar et al., 2013; Singha and Kumar, 2008).

1.1.2.4. Animal feed

The leaves are used for animal fodder and fibre (Plotto, 2004). The seeds can be used to feed poultry as well as sheep and the residue from the seeds oil extraction can also be used to feed cattle and chicks (Al-Wandawi et al., 1984; Elamin et al., 2012; Morton, 1987; Mukhtar, 2007).

 

1.1.2.5. Cosmetic

In Malaysia the oil is used to produce scrubs and soaps (Ismail et al., 2008).

1.1.3. Phytochemistry

1.1.3.1. Nutritional value

The nutritional composition of fresh calyx of H.sabdariffa varies between studies, probably due to different varieties, genetic, environmental, ecology and harvest conditions of the plant. Early studies reported that cH.sabdariffa contains protein (1.9 g/100 g), fat (0.1 g/100 g), carbohydrates (12.3 g/100 g) and fibre (2.3 g/100 g). They are rich in vitamin C (14 mg/100 g), β-carotene (300 μg/100 g), calcium (1.72 mg/100 g) and iron (57 mg/100 g) (Ismail et al., 2008).

The leaves contain protein (3.3 g/100 g), fat (0.3 g/100 g), carbohydrate (9.2 g/100 g), minerals (phosphorus (214 mg/100 g), iron (4.8 mg/100 g) thiamine (0.45 mg/100 g), β-carotene (4135 μg/100 g), riboflavin (0.45 mg/100 g) and ascorbic acid (54 mg/100 g) (Ismail et al., 2008).

The seeds contained crude fatty oil (21.85%), crude protein (27.78%), carbohydrate (21.25%), crude fibre (16.44%) and ash (6.2%). In terms of minerals, the most prevalent is potassium (1329 ± 1.47 mg/100 g), followed by sodium (659 ± 1.58 mg/100 g), calcium (647 ± 1.21 mg/100 g), phosphorus (510 ± 1.58 mg/100 g) and magnesium (442.8 ± 1.80 mg/100 g). The major saturated fatty acids identified in the seed oil are palmitic (20.84%) and stearic (5.88%) acids and the main unsaturated fatty acids are linoleic (39.31%) and oleic acid (32.06%) (Nzikou et al., 2011).

1.1.3.2. Bioactive constituents

The main constituents of H.sabdariffa relevant in the context of its pharmacological are organic acids, anthocyanins, polysaccharides and flavonoids (Eggensperger and Wilker, 1996; Müller and Regensburg, 1990).

1.1.3.3. Organic acids

H.sabdariffa extracts contain a high percentage of organic acids, including citric acid, hydroxycitric acid, hibiscus acid, malic and tartaric acids as major compounds, and oxalic and ascorbic acids as minor compounds (Da-costa-rocha et al., 2014). Based on previous studies, the percentage of organic acids in “hibisci flos” varies; hibiscus acid accounts for 13–24%, citric acid 12–20%, malic acid 2–9%, tartaric acid 8% and 0.02–0.05% of ascorbic acid (vitamin C) (Eggensperger and Wilker, 1996 and Schilcher, 1976).

In the late 1930s, citric and malic acids were first reported in aqueous extracts of the calyx (Buogo and Picchinenna, 1937, Indovina and Capotummino, 1938 and Reaubourg and Monceaux, 1940) and also in five different strains (from Egypt, Senegal, India, Thailand and Central America) of H.sabdariffa var. sabdariffa (Khafaga, Koch, El Afry, and Prinz, 1980). Ascorbic acid is also present in cH.sabdariffa but its content varies dramatically between fresh (6.7–14 mg/100 g (Ismail et al., 2008; Morton, 1987)) and dried calyces (260–280 mg/100 g (Ismail et al., 2008)). The amount of ascorbic acid in the latter report being much higher than the ones previously reported in the literature. The differences observed might be due to different varieties, genetics, and environment, ecology and harvest conditions.

1.1.3.4. Hydroxycitric acid

(Fig. 2) has an additional hydroxyl group at the second carbon of citric acid. This acid has four stereoisomers, (2S, 3S), (2R, 3R), (2S, 3R) and (2R, 3S), and their lactone forms. The principal organic acid found in the cH.sabdariffa is the (2S, 3R)-hydroxycitric acid (Hida et al., 2007). It is the principal organic acid found in the calyces of H.sabdariffa. It is worth noting that, (2S, 3R)-hydroxycitric acid from Hibiscus is different from the more commonly known (2S,3S)-hydroxycitric acid (HCA) extracted from, e.g., Garcinia sp., thus raising the question as to whether both diastereomers have identical or partially different pharmacological profiles.

1.1.3.5. Hibiscus acid

(Fig. 2) is the lactone form of (+)-allo-hydroxycitric acid. It compromises a citric acid moiety with an additional hydroxyl group at the second carbon and has two diastereomers due to the existence of two chiral centers in the molecule (Boll et al., 1969; Eggensperger and Wilker, 1996; Griebel and Lebensm, 1939; Griebel and Lebensm, 1942).

Hydroxycitric acid, hibiscus acid and it derivatives as the major organic acids in the leaves and calyces extracts of H.sabdariffa ( Beltran-Debon et al., 2010; Herranz-Lopez et al., 2012; Peng et al., 2011;Ramirez-Rodrigues et al., 2011a; Ramirez-Rodrigues et al., 2011b ; Rodriguez-Medina et al., 2009).

 Fig.2 citric acid and its derivatives

1.1.3.6. Flavonoids

H.sabdariffa contains polyphenols of the flavonol and flavanol type in simple or polymerised form. The following flavonoids have been described in H.sabdariffa extracts: hibiscitrin (hibiscetin-3-glucoside), sabdaritrin, gossypitrin, gossytrin and other gossypetin glucosides, quercetin and luteolin ( McKay, 2009; Williamson et al., 2013); as well as chlorogenic acid, protocatechuic acid, pelargonidic acid, eugenol, quercetin, luteolin and the sterols β-sitosterol and ergosterol ( McKay, 2009 ; Williamson et al., 2013).

Earlier the flowers of H.sabdariffa were recorded to contain 3-monoglucoside of hibiscetin (hibiscitrin) (Rao and Seshadri, 1942; Rao and Seshadri, 1942; Rao and Seshadri, 1948), 7-glucoside of gossypetin (gossypitrin) and sabdaritrin, which on acid hydrolysis yielded an hydroxyflavone named sabdaretin (Rao and Seshadri, 1942; Rao and Seshadri, 1942). The presence of these flavonol glycosides was low, with hibiscitrin being the major compound followed by gossypitrin and sabdaritrin (Rao and Seshadri, 1942; Rao and Seshadri, 1942). In 1961, gossypetin-3-glucoside (gossytrin) was isolated (Seshadri and Thakur, 1961). The petals of H.sabdariffa var. altissima also contain gossypetin-8-glucoside (0.4%) and gossypetin-7-glucoside (Subramanian and Nair, 1972).

From the leaves of H.sabdariffa, β-sitosteryl-β-d-galactoside (Osman et al., 1975) and from the seeds ergosterol (Salama and Ibrahim, 1979) were reported. β-sitosterol and ergosterol were also reported in H.sabdariffa extracts ( McKay, 2009; Williamson et al., 2013).

The methanolic extract of the flowers also contains quercetin, luteolin and its glycoside (Salah et al., 2002). Quercetin had already been identified in H.sabdariffa (Takeda and Yasui, 1985). One study reported that the amount of quercetin present in cH.sabdariffa WE (calyces of H.sabdariffa water extract) was 3.2 mg/g while rutin was 2.1 mg/g (Alarcon-Alonso et al., 2012). Quercetin and its conjugated glycosides (quercetin-3-glucoside), as well as, rutin (quercetin-3-rutinoside; Fig. 3) were frequently identified in cH.sabdariffa WE, alongside with kaempferol ( Beltran-Debon et al., 2010; Herranz-Lopez et al., 2012; Peng et al., 2011; Ramirez-Rodrigues et al., 2011a ; Ramirez-Rodrigues et al., 2011b).

 Fig.3. Quercetin-3-rutinoside

1.1.3.7. Anthocyanins

The anthocyanins are a group of flavonoid derivatives and natural pigments present in the dried flowers of H.sabdariffa and their colour varies with pH.

Delphinidin and cyanidin-based anthocynins, include delphinidin-3-sambubioside (hibiscin), cyanidin-3-sambubioside (gossypicyanin), cyanidin-3,5-diglucoside, delphinidin (anthocyanidin) and others (Williamson et al., 2009).

The first anthocyanin from the calyx of H.sabdariffa to be isolated was “hiviscin”, also known as “hibiscin”, later named delphinidin-3-sambubioside and assigned the structure of cyanidin-3-glucoside (Yamamoto and Osima, 1932), which was later renamed as delphinidin-pentoside-glucoside (Yamamoto and Osima, 1936). From the pigments of cH.sabdariffa, three different anthocyanins were isolated: delphinidin-3-sambubioside (hibiscin), delphinidin-3-glucoside and cyanidin-3-glucoside (chrysanthenin) using material from Taiwan and Trinidad (Du and Francis, 1973; Shibata et al., 1969). The last study also identified cyanidin-3-sambubioside (gossypicyanin). Later, the presence of cyanidin-3,5-diglucoside and cyanidin-3-(2G-glucosylrutinoside) in the flower pigments of H.sabdariffa var. altissima ( Subramanian and Nair, 1972) was reported. A study conducted with 5 different strains of H.sabdariffa var. sabdariffa reported cyanidin-3-sambubioside and cyanidin-3-glucoside as the major compounds present in this plant (khafaga et al., 1980). In one of the strains (Senegalese strain), delphinidin glycosides were absent. In this study, the anthocyanin content reached 1.7% to 2.5% of the dry weight in all strains. A similar anthocyanin content was observed in another study where their amount was about 1.5 g per 100 g of dry weight of cH.sabdariffa, in terms of delphinidin-3-sambubioside (Du and Francis, 1973).

Several studies have identified delphinidin-3-sambubioside (delphinidin-3-O-(2-O-β-d-xylopyranosyl)-β-d-glucopyranoise) and cyanidin-3-sambubioside (cyanidin-3-O-(2-O-β-d-xylopyranosyl)-β-d- glucopyranoside) as the major anthocyanins present in extracts from cH.sabdariffa ( Alarcon-Aguilar et al., 2007; Alarcon-Alonso et al., 2012; Beltran-Debon et al., 2010; Degenhardt et al., 2000; Herranz-Lopez et al., 2012; Peng et al., 2011) and leaves ( Rodriguez-Medina et al., 2009).

 Fig.4. Chemical structures of main anthocyanins

1.1.3.8. Mucilage, pectin and carbohydrates (polysaccharides)

Polysaccharides are another key group of compounds present in large quantities in the cH.sabdariffa WE (calyces of H.sabdariffa water extract). In one study, the ethanol-precipitated water extract yielded 10% of reddish polysaccharides. The following compounds were identified in two different fractions, arabinose, galactose, glucose, rhamnose and smaller amounts of galacturonic acid, glucuronic acid, manose and xylose ( Müller et al., 1989ss Similar results were obtained in two other studies ( Brunold et al., 2004; Müller and Franz, 1992).

The mucilage content was determined in the calyces of five strains of H.sabdariffa var. sabdariffa, reaching 24–28% in strains from Central America and Egypt but only 15% in an Indian strain. This amount was only reached at a later stage of development in the strains from Senegal and Thailand. The pectin content only accounted for 2–4% while the sugars reached a maximum of 3–5% in these five strains. Mucilage and pectin consisted of 60–80% anhydrouronic acid (Afry et al., 1980).

The petals of H.sabdariffa yielded 65% of dry weight of mucilage, which on hydrolysis produced galactose, galacturonic acid and rhamnose, while the leaves only yield 10% (El-Hamidi et al., 1967; Sengupta and Banik, 2011).

1.1.3.9. Volatile compounds

Volatile compounds are responsible for the aroma of H.sabdariffa. In a study conducted in 1992, more than twenty-five volatile compounds (accounting for less than 8% of total H.sabdariffa seeds composition) were reported in seed oil of H.sabdariffa. They were mainly unsaturated hydrocarbons, alcohols and aldehydes from C8 to C13. (Jirovetz et al., 1992) Subsequently, thirty-seven volatile compounds from five different groups from the cH.sabdariffa WE were characterised. These compounds included fatty acid derivatives (such as 2-ethylfuran and hexanal), sugar derivatives (furfural and 5-methyl-2-furaldehyde), phenolic derivatives (eugenol), terpenes (such as 1,4-cineole, limonene) and miscellaneous compounds (e.g. acetic acid) (Chen et al., 1998). In another study, the volatile profile was examined in four aqueous extracts from fresh and dried calyx using two different, time–temperature extraction conditions by GC–MS. A total of thirty-two compounds were identified and could be divided into five chemical groups: aldehydes (fourteen compounds), alcohols (ten compounds), ketones (five compounds), terpenes (two compounds) and acids (one compound) (Ramirez-Rodrigues et al., 2011a; Ramirez-Rodrigues et al., 2011b). A total of seven aromatic volatiles were common to all four samples tested (hexanal, 3-octanone, octanal, 1-octen-3-one, nonanal, 2,4-nonadienal (E,E), and geranylacetone).

 

1.1.4 Biological and pharmacological activities

A detailed review of the pharmacological effects of H.sabdariffa extract is presented below.

1.1.4a     Effects on smooth muscles

Early studies showed that the alcoholic extract of H.sabdariffa flowers had an antispasmodic effect by relaxing the uterus and intestine stips in vitro (Sharaf, 1962). This was also observed in rabbit aortic smooth muscle (Obiefuna et al., 1994). Interestingly, from various isolated muscle preparations, the extract of H.sabdariffa inhibited the tone of rabbit aortic strip, rhythmically contracting rat uterus, guinea-pig tracheal chain and rat diaphragms, but it stimulated the tone of solated quiescent rat uterus and frog rectus abdominis (Ali et al., 1991).

More recently, the H.sabdariffa WE (1–100 mg/kg) was found to inhibit rat bladder and uterine contractibility in a dose dependent manner, but via a mechanism unrelated to local or remote autonomic receptors or calcium channels ( Fouda et al., 2007) as previously suggested by Salah ( Salah et al., 2002).

Later, it was shown that H.sabdariffa crude extracts mainly induced the endothelium-dependent relaxant effect in the isolated thoracic aorta of rats, via stimulation of NOS enzyme by the Pi3-K/Akt pathway. It was suggested that this was due to polyphenols. The non-endothelium dependent relaxation is a direct smooth muscle activation and results in the activation of smooth muscle potassium channels (Sarr et al., 2009).

 

1.1.4b Antibacterial, antifungal and antiparasitic activity

The calyx of H.sabdariffa WE and protocatechuic acid (5 mg/ml) inhibited the growth of methicillin-resistant Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii (Liu et al., 2005). Moreover, protocatechuic acid (in a dose dependent manner) showed greater antimicrobial activity against these pathogens in broth than in human plasma. The study also revealed that the antibacterial effect was independent from temperature, as shown by a heat treatment. Hibiscus extract also demonstrated antibacterial effect against Streptococcus mutans, cariogenic bacteria from the oral cavity, with a minimum inhibitory concentration of 2.5 mg/ml ( Afolabi et al., 2008) and Campylobacter species (Campylobacter jejuni, Campylobacter coli and Campylobacter fetus) that contaminates meat like poultry, beef and pork at a concentration range of 96–152 μg/ml (Yin and Chao, 2008). This time, the aqueous-methanol extract of dried cH.sabdariffa also showed an in vitro inhibitory effect against several bacterial strains, such as S. aureus, Bacillus stearothermophilus, Micrococcus luteus, Serratia mascences, Clostridium sporogenes, Escherichia coli, K. pneumonia, Bacillus cereus and Pseudomonas fluorescence, but did not effect the growth of fungus Candida albicans (Olaleye, 2007). The fresh cH.sabdariffa WE, ethanol extract and protocatechuic acid (20 mg/ml) was effective in inhibiting the growth of food spoilage bacteria such as Salmonella typhimurium DT104, E. coliO157:H7, Listeria monocytogenes, S. aureus and B. cereus. Again the antibacterial effect was not affected by heat treatment and the ethanolic extract showed greater antimicrobial effect than the aqueous extract. The study further suggests that both, ethanolic extract and protocatechuic acid, might be potent agents for use as food additives to prevent contamination from these bacteria (Chao and Yin, 2009; Yin and Chao, 2008).

A methanol-water extract of H.sabdariffa was effective against E. coli O157:H7 isolates from food, veterinary and clinical samples (Fullerton et al., 2011), with the highest concentration (10%) being the most effective.

The crude extracts of H.sabdariffa seeds (200 mg/l) also showed antimicrobial effect against three types of Gram-negative bacteria. The extract exhibited higher activity against Salmonella followed by Shigella and Enterobacter (Nwaiwu et al., 2012).

1.1.4c. Antipyretic, antinociceptic and anti-inflammatory activities

Despite the claims that H.sabdariffa is effective in the relief of pyrexia in popular medicines, limited studies are available. The antipyretic and anti-inflammatory potential of the cH.sabdariffa extract were studied in vivo. The ethanol (more potent) and aqueous extracts showed antipyretic effects by significantly reversing yeast-induced fever in rats. The mechanism is different from the one of aspirin, a prostaglandin inhibitor. Nevertheless, fever entails enhanced formation of cytokines such as interleukins (IL), interferons and tumour necrosis factor-α (TNF-α). The cH.sabdariffa extract may be involved in the inhibition of some of these substances, resulting also in an anti-inflammatory effect (Reanmongkol and Itharat, 2007). Similar results were obtained by Dafallah (Dafallah and al-Mustafa, 1996), suggesting that the flavonoids, polysaccharides and organic acids might be the compounds responsible for the pharmacological activity. In a more recent study the ethanolic extract from the calyces also showed antinociceptive effect in a rat model (Ali et al., 2011).

Another in vivo study showed that the two fractions of the crude aqueous-ethanolic extract of the dried cH.sabdariffa exhibited impressive immunostimulatory activity by increasing the production of IL-10 and decreasing the production of TNF-α (Fakeye, 2008). Another mechanism in which the polyphenol extract exhibit its anti-inflammatory activity is by impairing cyclooxygenase-2 induction by down-regulating JNK and p38 MAPK (Kao et al., 2009).

1.1.4d   Antioxidant activity

Several studies both in vitro (Duh and Yen, 1997; Farombi and Fakoya, 2005; Hirunpanich et al., 2005; Mohd-Esa et al., 2010; Sayago-Ayerdi et al., 2007; Steenkamp et al., 2004; Tseng et al., 1997) and in vivo (Farombi and Fakoya, 2005; Mossalam et al., 2011; Olalye and Rocha, 2007; Usoh et al., 2005) have shown that extracts of H.sabdariffa have a potent antioxidant effect.

The antioxidant activity of the extract is due to its strong scavenging effect on reactive oxygen and free radicals (Farombi and Fakoya, 2005; Mohd-Esa et al., 2010; Olalye and Rocha, 2007; Sayago-Ayerdi et al., 2007;Tseng et al., 1997; Usoh et al., 2005), inhibition of xanthine oxidase activity, protective action against tert-butyl hydroperoxide (t-BHP)-induced oxidative damage ( Tseng et al., 1997), protection of cell from damage by lipid peroxidation ( Duh and Yen, 1997; Farombi and Fakoya, 2005; Olalye and Rocha, 2007), inhibition in Cu²⁺-mediated oxidation of LDL and the formation of thiobarbituric acid reactive substances (TBARs) ( Hirunpanich et al., 2005; Ochani and D’Mello, 2009; Olalye and Rocha, 2007), inhibition of the formation of malondialdehyde content (100–300 mg/kg) ( Farombi and Fakoya, 2005; Usoh et al., 2005), reduction of glutathione depletion, increase of the liver and decrease blood activity of superoxide dismutase and catalase ( Usoh et al., 2005) while in the liver it increased superoxide dismutase, catalase and glutathione and decreased malondialdehyde ( Mossalam et al., 2011). The effects were observed for both water and ethanolic extracts from flowers of H.sabdariffa, as well as from the seeds or leaves (Mohd-Esa et al., 2010).

1.1.4e Hepatoprotective activity

cH.sabdariffa WE (100–800 mg/kg) showed hepatoprotective effects in a range of models based on toxin-induced hepatitis including, tert-butylhydroperoxide, lipopolysaccharides, azathioprine, carbon tetrachloride, cadmium, ammonium chloride, acetaminophen and irradiation in vivo ( Adaramoye et al., 2008; Ajiboye et al., 2011; Ali et al., 2003; Amin and Hamza, 2005; Asagba et al., 2007; Essa et al., 2006; Lin et al., 2003; Liuet al., 2002; Liu et al., 2010; Liu et al., 2006; Olaleye and Rocha, 2008; Tseng et al., 1996; Wang et al., 2000) and in vitro ( Ajiboye et al., 2011; Lee et al., 2012; Yin et al., 2011).

This effect is due to a strong antioxidant activity, which reduces cellular damage by reducing oxidative stress and by attenuating mitochondrial dysfunction through decreasing Bax and tBid expression in the liver (Lee et al., 2012). The extract also increased the activity of superoxide dismutase (SOD), catalase (CAT), gluthathione peroxidase (GPx), and d-aminolevulinate dehydratase (d-ALA-D) enzymes while decreasing lipid peroxidation in induced models of liver damage (Adaramoye et al., 2008; Ajiboye et al., 2011; Olaleye and Rocha, 2008), and decreased liver marker enzymes such as aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) in experimental hyperammonemia ( Essa et al., 2006). An Hibiscus anthocyanin extract also induced phase II drug-detoxifying enzymes, such as glutathione S-transferase, NAD(H):quinone oxidoreductase, and uridyl diphosphoglucuronosyl transferase in an induced liver damage model (CCl₄-mediated toxicity model) ( Ajiboye et al., 2011).

The anthocyanins present in the extract seem to be the ones responsible for this effect (Ajiboye et al., 2011; Ali et al., 2003; Wang et al., 2000). Another compound that has been identified to have this effect was protocatechuic acid, a phenolic compound present in the H.sabdariffa extract ( Lin et al., 2003; Liu et al., 2002; Tseng et al., 1996).

1.1.4f Nephroprotective activity

Two studies were reported on the nephroprotective activity of H.sabdariffa extracts on diabetic nephropathy in streptozotocin-induced type 1 diabetic rats (Lee et al., 2009; Wang et al., 2011). Nephropathy may progress to end-stage renal disease. A study was conducted to investigate the effect of the polyphenol extract of H.sabdariffa (100 and 200 mg/kg/day) in streptozotocin-induced diabetic nephropathy in rats. The extract revealed beneficial effects as the kidney mass was reduced and the hydropic change of renal proximal convoluted tubules was improved, it reduced serum triglyceride, total cholesterol and LDL as well as increased the activity of catalase and glutathione and reduced lipid peroxidation in the kidney (Lee et al., 2009). It was found that the extracts reduced kidney mass and improved hydropic change of renal proximal convoluted tubules in this rat model. The positive effect shown by the extracts might be via improving oxidative status and regulating Akt/Bad/14-3-3γ signalling (anti-apoptotic mechanisms). Another in vivo study also revealed that its nephroprotective effect is a result of the protection of the kidney from the oxidative stressed (Mossalam et al., 2011).

1.1.4gRenal effects/diuretic effect (incl. clinical studies)

The renal effect of H.sabdariffa has been characterised pharmacologically both in clinical trials (Herrera-Arellano et al., 2004; Kirdpon et al., 1994; Prasongwatana et al., 2008) and in pre-clinical experiments in rats (Aguwa et al., 2004; Laikangbam and Damayanti Devi, 2012).

A two-phase study in Thailand with thirty-six healthy men was conducted to evaluate the changes in urine after consumption of H.sabdariffa juice (16 g/day and 24 g/day) to determine its effect on the treatment and prevention of renal stones. Despite the fact that the consumption of H.sabdariffa caused a decrease in creatinine, uric acid, citrate, tartrate, calcium, sodium, potassium and phosphate it did not affect the concentration of oxalate in urinary excretion. The authors suggested that there was no beneficial effect in preventing renal stone formation and that long term and higher doses should be investigated (Kirdpon et al., 1994). Another intervention study carried out in Thailand with eighteen subjects with or without history of renal stones revealed that H.sabdariffa tea drinking, at a dose of 3 g/day for 15 days, did not show evidence for antilithiatic or diuretic effects. No significant difference in serum sodium and urinary volume were observed during this study. However, H.sabdariffa tea consumption produced a uricosuric effect (Prasongwatana et al., 2008). Similar results were observed in albino rats when given a decoction of dried calyces at an oral dose of 1 g/kg (Caceres et al., 1987).

However, in vivo an antilithiatic effect was observed. In Wistar rats, which were given extract of H.sabdariffa orally at a dose of 3.5 mg daily, the oxalate retention in the kidney decreased with increased excretion into urine and decreased calcium crystal deposition in the kidneys (Woottisin et al., 2011). The cH.sabdariffa WE (250, 500 and 750 mg/kg body weight) also effectively prevented the development of urolithiasis (stone-disorder) in male albino rats (Laikangbam et al., 2012). A decrease in renal Ca²⁺ andMg²⁺ATPase activity and unaltered renal calcium handling in rats after administration of cH.sabdariffa WE at 25 and 50 mg/kg was shown. Renal function was also enhanced by reduction of serum urea and creatinine concentrations (Olatunji et al., 2012). In another pre-clinical study in rats, cH.sabdariffa WE produced diuretic and natriuretic effects at the dose range of 500 to 2500 mg/kg b.w. with a potassium-sparing effect (Alarcon-Alonso et al., 2012). This diuretic effect is in accordance with previous studies in experimental animals (Aguwa et al., 2004; Caceres et al., 1987; Onyenekwe et al., 1999; Ribeiro Rde et al., 1988) and one clinical trial (Herrera-Arellanoet al., 2004). In this single clinical trial, assessing a chemically characterised extract of H.sabdariffa (9.6 mg of total anthocyanins) in patients with mild to moderate hypertension, the treatment demonstrated a natriuretic effect with no effects on chloride, potassium and pH ( Herrera-Arellano et al., 2004).

1.1.4h Cancer-preventive activity                                   

H.sabdariffa is rich in phenolic compounds, such as protocatechuic acid. This compound demonstrated in vitro protective effects against cytotoxicity and genotoxicity of hepatocytes induced by tert-butylhydroperoxide (t-BHP), through inhibiting action on DNA repair synthesis caused by t-BHP and by showing radical quenching effect (Tseng et al., 1996). It also inhibited 12-O-tetradecanolyphorbol-13-acetate (TPA)-induced skin tumour formation in CD1-mice (Tseng et al., 1998) and inhibited the survival of human promyelocytic leukaemia HL-60 cells (Tseng et al., 2000). The mechanism by which it exerted anticancer properties might be through antitumour promotion by reducing reactive oxygen species (ROS), DNA fragmentation, G1 arrest and apoptosis. The apoptosis-inducing activity was associated with the phosphorylation and degradation of RB and the suppression of Bcl-2 protein. Similar effects were observed in human gastric carcinoma (AGS) cells in which the apoptotic effect may be mediated via p53 signaling and p38 MAPK/FasL cascade pathway (Lin et al., 2005). Another group of compounds present in cH.sabdariffa extracts are anthocyanins such as delphinidin-3-sambubioside. They induced apoptosis against human leukaemia cells ( Chang et al., 2005; Hou et al., 2005) via the p38-FasL and Bid pathway and ROS-mediated mitochondrial dysfunction pathway and against smooth muscle cells (SMC) via p38 and p53 pathway ( Lo et al., 2007).

Recently, the anti-cancer activity of H.sabdariffa leaf extracts were assessed against human prostate cancer cells in vitro and in vivo (Lin et al., 2012). The study showed the anti-apoptotic effect to be mediated via both intrinsic (Bax/cytochrome c-mediated caspase 9) and extrinsic (Fas-mediated caspase 8/t-Bid) pathways, as well as by inhibiting the growth of prostate tumour xenograft in athymic nude mice. The extract from leaves instead of calyces represented a possible source of greater polyphenolic compounds.

1.1.4i Lipid metabolism – anticholesterol effects/effects on lipid metabolism

Several studies have showed that extracts of H.sabdariffa have a lipid lowering activity, which could prevent diseases like hyperlipidemia and cardiovascular diseases (atherosclerosis and coronary heart disease) (Carvajal-Zarrabal et al., 2005;Chang et al., 2006; Chen et al., 2003; Chen et al., 2004; el-Saadany et al., 1991; Gosain et al., 2010; Hirunpanich et al., 2006; Ochani and D’Mello, 2009; Yang et al., 2010).

The extracts (water and ethanolic extracts of dried calyces or leaves) were able to decrease low-density lipoprotein cholesterol (LDL-c), triglycerides (TAG), total cholesterol (TC) and lipid peroxidaxion in vivo. A few of them even reported that the extract was also able to reduce very-low density lipoprotein cholesterol (VLDL-c) (Farombi and Ige, 2007; Ochani and D’Mello, 2009) along with an increase in serum level of high density lipoprotein cholesterol (HDL-c) levels (Ochani and D’Mello, 2009; Yang et al., 2010). Additionally, it also reduced foam cell formation and inhibited smooth muscle cell migration and calcification in blood vessels of treated rabbits. A possible explanation for the decrease in LDL-c could be related to the inhibition of the triacylglycerol synthesis or other hypolipidemic effects, through the antioxidant activity against LDL-c oxidation and hepatic liver clearance. Several groups of compounds in the extract, such as anthocyanins and protocatechuic acid, have been implicated as responsible for these effects (Chang et al., 2006; Lee et al., 2012; Tseng et al., 1997).

1.1.4j Anti-obesity activity

Pre-clinical data from Brazil indicates a potential role in the control of certain conditions associated with obesity, such as hyperlipidemia. However, further studies were suggested (Dickel et al., 2007).

A report showed that a standardised (33.64 mg of total anthocyanins per each 120 mg) water extract of cH.sabdariffa was able to reduce weight gain in obese mice while at the same time it increase the liquid intake in healthy and obese mice (Alarcon-Aguilar et al., 2007). This effect is probably achieved through the modulation of PI3-K/Akt and ERK pathway, which play pivotal roles during adipogenesis (Kim et al., 2007).

In vitro and in vivo studies showed that Hibiscus extract (or tea) inhibited the activity of α-amylase, blocking sugars and starch absorption, which may assist in weight loss (Hansawasdi et al., 2000; Hansawasdi et al., 2001; Preuss et al., 2007). A study conducted in Mexico using an ethanol extract of H.sabdariffa concluded the extract could be considered as a possible anti-obesity agent due to its effects on fat absorption-excretion and body weight of rats (Carvajal-Zarrabal et al., 2009).

The therapeutic use of the extract, possibly due to polyphenols, was also evaluated in patients with metabolic syndrome, an obesity-associated collection of disorders (Perez-Torres et al., 2012). Meanwhile a study showed that the aqueous extract was more efficient in inhibiting triglyceride accumulation when devoid of fibre and polysaccharides, but when polyphenols were fractionated and isolated, the benefits of the whole extract was greater than the sum of its parts (Herranz-Lopez et al., 2012).

 

1.1.4k Lactating activity

The ethanolic seed extract of H.sabdariffa (200–1600 mg/kg) increased the serum prolactin level (p < 0.01) when compared to the control in a dose-dependent manner in lactating Albino Wistar rats (Gaya et al., 2009).

1.1.4l Anti-diabetic activity

Diabetes mellitus can be defined as an endocrine and metabolic disorder characterised by chronic hyperglycaemia, dyslipidemia, and protein metabolism that results from defects in both regulations of insulin secretion and/or insulin action.

The protective effect of a polyphenol extract of H.sabdariffa was studied in a type II diabetic rat model (high fat diet model). At a dose of 200 mg/kg, the extract demonstrated anti-insulin resistance properties as it reduced hyperglycaemia and hyperinsulinemia. It decreased serum triacylglycerol, cholesterol and the ratio of low-density lipoprotein/high-density protein (LDL/HDL), as well as reduced the plasma advanced glycation end products (AGE) formation and lipid peroxidation (Peng et al., 2011).

The currently accepted therapeutic strategy for the control of postprandial hyperglycaemia is based on the inhibition of α-glucosidase and α-amylase. This results in an aggressive delay of carbohydrate digestion to absorbable monosaccharide. With this in mind, a study was conducted to determine the effect of H.sabdariffa extract on intestinal α-glucosidase and pancreatic α-amylase activity in vitro. As a result, H.sabdariffa extract was shown to be a potent pancreatic α-amylase inhibitor (Adisakwattana et al., 2012). Similar results were found for hibiscus acid (hibiscus-type (2S,3R)-hydroxycitric acid lactone) (Yamada et al., 2007), which inhibited pancreatic α-amylase and intestinal α-glucosidase enzyme (Hansawasdi et al., 2000; Hansawasdi et al., 2001).

Diabetes mellitus is a risk factor for coronary heart diseases as well as atherosclerosis. An ethnobotanical study conducted in the Caribbean for urinary problems and diabetes mellitus revealed that H.sabdariffa is traditionally used to ‘clean’ the liver and blood within a group of plants used for “cooling”, high cholesterol and urinary problems. When the respondents were asked which medicinal plants were used for high blood pressure, diabetes and jaundice, H.sabdariffa was referred to hypertension (Lans, 2006). A study in alloxan-induced diabetic rats showed that an ethanolic extract of H.sabdariffa flowers (200 mg/kg) had a strong hypolipidemic as well as antioxidant effect. Thus, H.sabdariffa extract showed therapeutic promise in decreasing and preventing the development of atherosclerosis and possible related cardiovascular pathologies linked with diabetes. The authors suggest that this activity might be linked to polyphenolic compounds and dihydrobenzoic acids, like protocatechuic acids, but further identification of the active compounds is warranted (Farombi and Ige, 2007). A similar effect was reported (Huang et al., 2009) with the extract suppressing the high-glucose-induced migration in a vascular smooth muscle cell model.

1.1.4m Delayed puberty activity

A few studies with rats have shown that consumption of H.sabdariffa WE during pregnancy and lactation resulted in increased postnatal weight gain, delayed onset of puberty and elevated body mass index at onset of puberty in the female offsprings (Iyare and Adegoke, 2008a; Iyare and Adegoke, 2008b; Iyare and Adegoke, 2008c; Iyare and Nwagha, 2009). The consumption of the extract during pregnancy and lactation caused decrease of maternal fluid and food intake with increased plasma Na⁺ and corticosterone concentration, while the accelerated growth and delayed puberty observed in the offspring could be due to increased corticosterone and decreased leptin delivery through breast milk (Iyare and Adegoke, 2008b; Iyare et al., 2010). These studies however require confirmation as no observations have been reported in the literature up to-date pointing to the presence of respective effects in humans.

1.1.4n Anti-hypertensive activity

Decoctions of H.sabdariffa have been used traditionally in West Africa and Mexico as an anti-hypertensive remedy. Several in vitro (Jonadet et al., 1990;Obiefuna et al., 1994) and in vivo studies have shown that the extract of the calyces (ranging from 125 to 500 mg/kg) indeed reduce both the systolic and diastolic pressures, lowering heart rate and working as a vasodilator (Adegunloye et al., 1996; Ajay et al., 2007; Inuwa et al., 2012; Mojiminiyi et al., 2007; Onyenekwe et al., 1999;Shehata and El Menoufy, 2008). The anti-hypertensive activity might be through: inhibition of angiotensin-converting enzymes (ACE) ( Jonadet et al., 1990; Ojeda et al., 2010), acetylcholine-like and histamine-like mechanisms ( Adegunloye et al., 1996), diuretic effect ( Mojiminiyi et al., 2000), reduction in the diffusion distance between capillaries and myocytes, as well as new vessel formation (Inuwa et al., 2012) and direct vaso-relaxant effects ( Adegunloye et al., 1996; Ajay et al., 2007; Obiefuna et al., 1994; Adegunloye et al., 1994). The relaxant effect might be partially endothelium independent and possibly mediated by endothelium-derived nitric oxide (EDNO)-dependent action. Endothelium-dependent vasodilator component results through activation of the endothelium-derived nitric oxide/cGMP-relaxant pathway, whereas the endothelium-independent component could be due to inhibition of Ca²⁺ influx (Ajay et al., 2007).

Additionally, H.sabdariffa showed antiplatelet but no thrombolytic activity in vitro (Yamamoto et al., 2005). One in vivo study reported that despite the beneficial effect as an anti-hypertensive, H.sabdariffa might produce undesirable effects on gonadal activity (Shehata and El Menoufy, 2008).

1.1.4o Anti-anaemic activity

A preliminary study on the use of H.sabdariffa decoctions as an alternative source of iron for the treatment of anaemia and some other mineral deficiency diseases was conducted and showed that dry fermented calyces of hibiscus exhibited a very low pH value which enhanced mineral availability. Another reason for enhancing mineral (iron, zinc, calcium and magnesium) bioavailability is the high concentration of ascorbic acid (Falade et al., 2005).

The effect of cH.sabdariffa extract (200 to 1000 mg/kg body weight) on some haematological parameters in rats was studied to determine its medicinal usefulness in the treatment of anaemia. The study suggested that at a comparatively high dose range of 200 to 400 mg/kg, the extract had a beneficial effect on the red cells, but this was not sustained at even higher doses (Adigun et al., 2006). Another study using a rat model of infection with Trypanosoma congolense showed that the use of H.sabdariffa WE (equivalent to 9.61 mg/100 g/day of ascorbic acid for 3 weeks) prevented the disease-induced anomalies with increase of serum creatinine and urea levels. It was concluded that consumption of the extract ameliorated the pathological changes in blood as well as hepatic and renal structures of T. congolense-infected rats. The observed effects might be due to the ascorbic acid component or other antioxidants present, which presumably kept the free radical load in infected rats low as well as preventing the disease-associated depletion in systemic antioxidants. Nevertheless, further studies are needed to determine the long-term effects and the mechanism of action before recommendations could be made (Umar et al., 2009).

1.1.5   Safety

H.sabdariffa preparations, predominantly the infusion and aqueous extracts, have a long standing traditional use both in food and in medicine, and in general are considered to be safe. The available toxicological data, however limited, are in support of this assessment. The literature search for this review did not reveal any case reports of adverse reactions following oral consumption of H.sabdariffa preparations.

 

1.1.6. Toxicology

In a bioassay for screening plant extracts for their biological activity, the lethal dose (LD₅₀) of three different types of H.sabdariffa extract was assessed in the brine shrimp toxicity assay. Aqueous H.sabdariffa extract (i.e., infusion) produced an LD₅₀ of 9.59 μg/ml, while for the dichloromethane extract it was 24.51 μg/ml and 4.75 μg/ml for the ethanolic extract (Serrano et al., 1996). Given the very limited value of the brine shrimp assay for complex mixtures like plant extracts (Manilal et al., 2009) and the incomplete information on the mode of preparation of the extracts, this work is mentioned here for completeness only.

The LD₅₀ in mice (b.w. 30 g) was reported to be about 0.4–0.6 ml on intraperitoneal administration of a 30% aqueous H.sabdariffa decoction (20 min in distilled water) ( Sharaf, 1962). The same authors observed a lowered blood pressure in dogs (b.w.: 7 kg) with no side effects after administering (i.p.) 10 ml of a 10% solution of the H.sabdariffa decotion.

According to a study of Onyenekwe and coauthors, no deatH.sabdariffa were observed in Albino mice after fourteen day’s administration (i.p.) at doses of 1000–5000 mg/kg b.w./d., thus the calculated LD₅₀ of cH.sabdariffa aqueous extract was >5000 mg/kg b.w. The same authors assessed the effect of the extract on blood pressure in spontaneously hypertensive and normotensive Wistar-Kyoto rats. As part of this study it was observed that between the seventh and the twenty-first day after extract administration, the highest dose of 1000 mg/kg resulted in spontaneous deatH.sabdariffa in hypertensive but not in normotensive rats. With reference to the well known increased risk of sudden cardiac death in patients receiving non-potassium sparing diuretics, the authors speculate that the death of the animals may have been due to a diuretic effect of the extract (Onyenekwe et al., 1999), however, the dose found to be active is excessively high. Although Kirdpon and co-authors report a decrease of potassium and sodium in 36 healthy young men after successive administration of 16 g/d Hibiscus “Juice” (while surprisingly no such effect was seen in the high dose group with 24 g/d), this interpretation remains questionable in view of a much larger and well-documented controlled clinical study in which Hibiscus extract showed a natriuretic effect with no effects on chloride, potassium and pH in 171 men with mild to moderate hypertension (Herrera-Arellano et al., 2004). Lack of acute toxicity with calculated LD₅₀ values >5000 mg/kg b.w. was reported for a methanolic dried flower extract in adult albino mice on a herb-drug interaction study after 24 h administration (i.p.) (Ndu et al., 2011) and for an ethanolic extract of H.sabdariffa seed in albino Wistar rats while studying the effect of the extract on lactogenic activity (Gaya et al., 2009).

Administration of the water-soluble fraction of a concentrated cH.sabdariffa extract (extraction solvent: Methanol 80%) given orally at up to 15 successive doses of 250 mg/kg/d to Wistar albino rats showed no pathological features in both liver and heart after 24 h (Akindahunsi and Olaleye, 2003). The authors observed a dose-related increase in the levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) when compared to the control group. However, levels of the same enzymes decreased slightly in the liver. In view of the extraordinarily high serum AST value in the control group as compared to reference values from the literature (Boehm et al., 2007), the conclusions of the authors regarding a putative cardio- or liver toxicity of the tested extract have to be read with caution.

In recent years the effect of Hibiscus extracts on reproduction and development in rats has been subject to in vivo studies by several authors. In female rats, addition of hibiscus extracts to drinking water resulted in a dose-dependent reduction of fluid and feed intake. The effects observed in the pups included increased weight gain and delayed onset of puberty. As the authors rightly state, these effects have likely been caused by the reduced fluid and feed intake in accordance with earlier publications. Rather than representing specific toxicologic properties of hibiscus, the decreased fluid and feed intake and subsequent adverse effects observed in this study may be the result of the animals dislike for the flavour of hibiscus ( Iyare and Adegoke, 2008a; Iyare and Adegoke, 2008b; Iyare et al., 2010). Despite the fact that earlier studies showed the LD₅₀ to be above 5000 mg/kg and doses as high as 4600 mg/kg were administered to rats in drinking water for 12 weeks with no increase in mortality, the extract induced testicular toxicity (reduced sperm counts and spermatogenesis with evidence of marked degenerative histological changes) at all concentrations tested (1150–4600 mg/kg) (Orisakwe et al., 2004). Additionally, deleterious effects on the testis and spermatozoa and an adverse influence in the male reproductive fertility of albino mice were also reported after a cH.sabdariffa WE was administered daily for 4 weeks in a dose of 200 mg/kg (Mahmoud, 2012). In contrast to these studies, long term administration of H.sabdariffa WE for 10 weeks and hibiscus anthocyanins (50–200 mg/kg b.w.) for 5 days did not affect the male reproductive system in rats (Ali et al., 2012).

Fakeye and co-authors assessed toxicological effects of a 90-day oral administration of aqueous, ethanol or 50% ethanol extracts of dried cH.sabdariffa at doses of 300 and 2000 mg/kg b.w., respectively, in male Charles Foster rats. Strikingly contrasting with previous studies by various other authors, they observed a strong toxicity with total mortality in all 2000 mg groups until day 28, and in the aqueous and 50% ethanol groups at 300 mg, until day 60 and 40, respectively. (Fakeye et al., 2009). At a dose of 300 mg/kg (b.w.) all extract types produced a significant increase of plasma creatinine after 30 days administration of the extracts. High creatinine blood levels may be associated with muscular dystrophy, loss of kidney function or even mortality. However, since the ethanol extract produced the strongest increase in creatinine but no mortality at the 300 mg dose level, it is unlikely that the elevated creatinine levels have been the cause of death in the other groups (Fakeye et al., 2009). Overall, the results of this study remain highly questionable. The possible reasons considered by the authors, including differences in anthocyanin content, are not convincing in view of the still moderate anthocyanin doses provided with the extracts.

A similar effect was observed on rat testis in a cisplatin (CIS)-induced rodent model of reproductive toxicity when ethanol extracts of H.sabdariffa was administered for 26 days (1 g/kg per day) (Amin and Hamza, 2006). The extract also revealed anti-mutagenic activity in vitro against 1-nitropyrene, a potent mutagen (Olvera-Garcia et al., 2008) and reduction of micronuclei in polychromatic erythrocytes (PCEs) (Adetutu, et al., 2004). The administration of ethanolic H.sabdariffa extracts (200 mg/kg and 300 mg/kg, orally) in rabbits over a period of eight weeks did not show any toxic effect when dyslipidemia and oxidant stress associated with prolonged excessive intake of cholesterol was studied in these animals ( Ekor et al., 2010).

Based on the data presented above, dosages up to 200 mg/kg should be safe and not show signs of toxicity, but further studies, most importantly with chemically well characterised extracts, are warranted.

1.1.7. Interactions

While there is no particular ground for suspicion of a relevant interaction potential of Hibiscus, several preclinical and one clinical study have addressed this issue. The sub-acute effect of an aqueous cH.sabdariffa extract on CYP450 activity, clinical blood chemistry and haematology was investigated in male Wistar rats at 250 and 1000 mg/kg/day for 30 days. The authors found no effect on hepatic phase I enzymes (CYP 1A1, 1A2, 2B1/2, 2E1 and 3A) and the extract did not significantly affect blood chemistry and haematology. (Prommett et al., 2006). More recently, the interaction of a methanolic H.sabdariffa flower extract and hydrochlorothiazide (a diuretic drug) was examined in adult albino mice, albino rats and healthy adult rabbits. Co-administration of the extract (20–40 mg/kg b.w.) with hydrochlorothiazide (10 mg/kg) caused a significant increase in the volume of urine excreted, as well as a significant decrease in the pH of urine and the concentrations of sodium, bicarbonate and chloride ions. It also increased and prolonged the plasma concentration, the mean area under the concentration–time curve and the volume of distribution of the diuretic drug over a 24 h sampling period (Ndu et al., 2011). Regarding the type of preparation (methanol extract) and mode of administration (intraperitoneal), the relevance of these findings for the use of traditional hibiscus preparations remains highly questionable.

The pharmacokinetics of chloroquine (600 mg) and a freshly prepared H.sabdariffa beverage, similar in taste and concentration to the one usually consumed by Sudanese people, were studied in healthy males. The study showed a statistically significant reduction in the area under the plasma concentration versus time curve and the peak plasma concentration of chloroquine (Mahmoud et al., 1994). In view of the very small group size (N = 6) and the poor information on the mode of preparation and dosage of the hibiscus beverage, the results of this study need to be interpreted with caution.

The effect of so-called Zobo Drink (sweetened aqueous extract prepared from 30 g dried hibiscus calyces/l) on acetaminophen pharmacokinetics was studied in healthy young men (N = 6, crossover design). No significant kinetic changes were observed when the extract was administered concomitantly with acetaminophen, except for a slight elevation of the clearance by Ca. 11%. Given the poorly described study protocol and the small sample size of N = 6, the conclusions of the authors on a possible interaction potential of hibiscus should be read with caution (Kolawole and Maduenyi, 2004).

Taken together, the data available today from preclinical and clinical studies does not provide substantiated evidence of any therapeutically relevant interaction potential of commonplace teas or beverages containing hibiscus and its preparations. This complements the evidence based on the complete absence of drug interaction case reports involving hibiscus in the scientific literature.

1.1.8. Anatomy of the male rat testes

1.1.8.1. Brief anatomy of the rat testes

Grossly, the normal adult rat testis is a paired organ that lies within the scrotum suspended by the spermatic cord. The average weight of each testis is 1–2g, the right usually being 10% heavier than the left. The organ is covered by a capsule composed of three layers: the outer serosa or tunica vaginalis (covered by a flattened layer of mesothelial cells), the tunica albuginea, and the inner tunica vasculosa. The posterior portion of the capsule, called the mediastinum, contains blood and lymph vessels, nerves, and the mediastinal portion of the rete testis.

Fig.5.   Structure of male rat testes

Histologically, The parenchyma is divided into approximately 250 lobules, each lobule containing up to four seminiferous tubules The seminiferous tubules are bound by a limiting membrane made up of basal lamina and alternating layers of cells of Leydigand collagen fibers. These tubules contain germ cells in various stages of development and Sertoli cells

Fig. 6: Spermatogenic series

1.1.8.2. Structure-Function relationship of the male rat testes

Testes is the male gonad in rat and other animals. Like the ovaries to which they are homologous, testes are components of both the reproductive system and the endocrine system. The primary functions of the testes are to produce sperm (spermatogenesis) and to produce androgens, primarily testosterone.

Both functions of the testicle are influenced by gonadotropic hormones produced by the anterior pituitary. Luteinizing hormone (LH) results in testosterone release. The presence of both testosterone and follicle-stimulating hormone (FSH) is needed to support spermatogenesis. It has also been shown in

animal studies that if testes are exposed to either too high or toolow levels of estrogens (such as estradiol ; E2) spermatogenesis can be disrupted to such an extent that the animals become infertile.

Within the seminiferous tubules, germ cells develop into spermatogonia, spermatocytes, spermatids and spermatozoon through the process of spermatogenesis. The gametes contain DNA for fertilization of an ovum.

Sertoli cells – the true epithelium of the seminiferous epithelium, critical for the support of germ cell development into spermatozoa. Sertoli cells secrete inhibin.

Peritubular myoid cells surround the seminiferous tubules.

Between tubules (interstitial cells), Leydig cells are localized between seminiferous tubules and they produce and secrete testosterone and other androgens important for sexual development and puberty, secondary sexual

Characteristics like facial hair, sexual behavior and libido, supporting spermatogenesis and erectile function. Testosterone also controls testicular volume. Also present are - Immature Leydig cells, interstitial macrophages and epithelial cells.

The accessory glands of the male reproductive system are the seminal vesicles, prostate gland, and the bulbourethral glands.These glands secrete fluids that enter the urethra.

Seminal Vesicles

The paired seminal vesicles are saccular glands posterior to the urinary bladder. Each gland has a short duct that joins with the ductus deferens at the ampulla to form an ejaculatory duct, which then empties into the urethra. The fluid from the seminal vesicles is viscous and contains fructose, which provides an energy source for the sperm; prostaglandins, which contribute to the mobility and viability of the sperm; and proteins that cause slight coagulation reactions in the semen after ejaculation.

Prostate

The prostate gland is a firm, dense structure that is located just inferior to the urinary bladder. It is about the size of a walnut and encircles the urethra as it leaves the urinary bladder. Numerous short ducts from the substance of the prostate gland empty into the prostatic urethra. The secretions of the prostate are thin, milky colored, and alkaline. They function to enhance the motility of the sperm.

Bulbourethral Glands

The paired bulbourethral (Cowper's) glands are small, about the size of a pea, and located near the base of the penis. A short duct from each gland enters the proximal end of the penile urethra. Inresponse to sexual stimulation, the bulbourethral glands secrete an alkaline mucus-like fluid. This fluid neutralizes the acidity of the urine residue in the urethra, helps to neutralize the acidity of sthe vagina, and provides some lubrication for the tip of the penis during intercourse.

Seminal Fluid

Seminal fluid, or semen, is a slightly alkaline mixture of sperm cells and secretions from the accessory glands. Secretions from the seminal vesicles make up about 60 percent of the volume of the semen, with most of the remainder coming from the prostate gland. The sperm and secretions from the bulbourethral gland contribute only a small volume.

1.1.9. Reproductive hormones

1.1.9.1. Follicle stimulating hormone (FSH)

Follicle-stimulating hormone (FSH) is a glycoprotein hormone found in humans and other animals. It is synthesized and secreted by gonadotropes of the anterior pituitary gland, and regulates the development, growth, pubertal maturation, and reproductive processes of the body. FSH and luteinizing hormone (LH) act synergistically in reproduction .FSH is a 35.5 kDa glycoprotein heterodimer, consisting of two polypeptide units, alpha and beta. The alpha subunits of the glycoproteins FSH consist of about 96 amino acids, while the beta subunit  consists of 111 amino acids (FSH β),which confers its specific biologic action, and is responsible for interaction with the follicle-stimulating hormone receptor(Jang et al., 2012) .In males, FSH induces Sertoli cells to secrete androgen- binding proteins (ABPs), regulated by inhibin 's negative feedback mechanism on the anterior pituitary .

In females, FSH initiates follicular growth, specifically affecting granulosa cells .GnRH has been shown to play an important role in the secretion of FSH, with hypothalamic-pituitary disconnection leading to a cessation of FSH. GnRH administration leads to a return of FSH secretion.

FSH is subject to oestrogen feed-back from the gonads via the hypothalamic pituitary gonadal axis. FSH stimulates the growth and recruitment of immature ovarian follicles in the ovary. In early (small) antral follicles, FSH is the major survival factor that rescues the small antral follicles (2– 5 mm in diameter for humans) from apoptosis (programmed death of the somatic cells of the follicle and oocyte). In the luteal-follicle phase transition period the serum levels of progesterone and estrogen (primarily estradiol) decrease and no longer suppress the release of FSH, consequently FSH peaks at about day three (day one is the first day of menstrual flow). The cohort of small antral follicles is normally sufficiently in number to produce enough Inhibin B to lower FSH serum levels.

In addition, there is evidence that gonadotrophin surge-attenuating factor produced by small follicles during the first halfof the follicle phase also exerts anegative feedback on pulsatile luteinizing hormone (LH) secretion amplitude, thus allowing a more favorable environment for follicle growth and preventing

premature luteinization (Fowler et al., 2003). As a woman nears perimenopause, the number of small antral follicles recruited in each cycle diminishes and consequently insufficient Inhibin B is produced to fully lower FSH and the serum level of FSH begins to rise. Eventually the FSH level becomes so high that downregulation of FSH receptors occurs and by postmenopause any remaining small secondary follicles no longer have FSH nor LH receptors (Vihkko, 1996).

When the follicle matures and reaches 8–10 mm in diameter it starts to secrete significant amounts of estradiol. Normally in humans only one follicle becomes dominant and survives to grow to 18–30 mm in size and ovulate, the remaining follicles in the cohort undergo atresia. The sharp increase in estradiol production by the dominant follicle (possibly along with a decrease in gonadotrophin surge-attenuating factor) cause a positive effect on the hypothalamus and pituitary and rapid GnRH pulses occur and an LH surge results.

The increase in serum estradiol levels cause a decrease in FSH production by inhibiting GnRH production in the hypothalamus (Dickerson et al., 2008). The decrease in serum FSH level causes the smaller follicles in the current cohort to undergo atresia as they lack sufficient sensitivity to FSH to survive. Occasionally two follicles reach the

10 mm stage at the same time by chance and as both areequally sensitive to FSH both survive and grow in the low FSH environment and thus two ovulations can occur in one cycle possibly leading to non-identical (dizygotic ) twins.

FSH stimulates primary spermatocytes to undergo the first division of meiosis, to form secondary spermatocytes.

FSH enhances the production of androgen-binding protein by the Sertoli cells of the testes by binding to FSH receptors on their basolateral membranes, and is critical for the initiation of spermatogenesis (Boulpaep and boron, 2005).

1.1.9.2. Luteinizing hormone (LH)

Luteinizing hormone (LH), also known as lutropin and sometimes lutrophin (Ujihara et al., 1992) is a hormone produced by gonadotropic cells in the anterior pituitary gland. In females, an acute rise of LH (" LH surge") triggers ovulation and development of the corpus luteum. In males, where LH had also been called interstitial cell- stimulating hormone (ICSH ), it stimulates Leydig cell production of testosterone. It acts synergistically with FSH (louvet et al., 1975).

LH is a hetero dimeric glycoprotein. Each monomeric unit is a glycoprotein molecule; one alpha and one beta subunit make the full, functional protein. Its structure is similar to that of the other glycoproteinhormones, follicle-stimulating hormone (FSH), thyroid- stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The protein dimer contains 2 glycopeptidic subunits, alpha and beta subunits, that are non-covalently associated (Jiang et al., 2014). The alpha subunits of LH, contain 92 amino acids in human but 96 amino acids in almost all other vertebrate species.  The beta subunits vary. LH has a beta subunit of 120 amino acids (LHB) that confers its specific biologic action and is responsible for the specificity of the interaction with the LH receptor. The biologic half-life of LH is 20 minutes, shorter than that of FSH (3–4 hours).

LH supports theca cells in the ovaries that provide androgens and hormonal precursors for estradiol production. At the time of menstruation, FSH initiates follicular growth, specifically affecting granulosa cells (Bowen, 2004).  With the rise in estrogens, LH receptors are also expressed on the maturing follicle, which causes it to produce more estradiol. Eventually, when the follicle has fully matured, a spike in 17-hydroxyprogesterone production by the follicle inhibits the production of estrogens, leading to a decrease in estrogen-mediated negative feedback of GnRH in the hypothalamus, which then stimulates the release of LH from the anterior pituitary (Mahesh, 2011).  LH is necessary to maintain luteal function for the first two weeks of the menstrual cycle. If pregnancy occurs, LH levels will decrease, and luteal function will instead be maintained by the action of hCG (humanchorionic gonadotropin), a hormone very similar to LH but secreted from the new placenta. LH acts upon the Leydig cells of the testis and is regulated by GnRH. The Leydig cells produce testosterone (T) under the control of LH, which regulates the expression of the enzyme 17- β hydroxysteroid dehydrogenase that is used to convert androstenedione, the hormone produced by the gonads, to testosterone, an androgen that exerts both endocrine activity and intratesticular activity on spermatogenesis.

LH is released from the pituitary gland, and is controlled by pulses of gonadotropin-releasing hormone (GnRH). When T levels are low, GnRH is released by the hypothalamus, stimulating the pituitary gland to release LH. As the levels of T increase, it will act on the hypothalamus and pituitary through a negative feedback loop and inhibit the release of GnRH and LH consequently. Androgens (T, DHT) inhibit monoamine oxidase (MOA) in pineal, leading to increased melatonin and reduced LH and FSH by melatonin-induced increase of GnIH synthesis and secretion.

1.1.9.3. Testosterone

Testosterone is a steroid hormone from the androgen group and is found in humans and other vertebrates. In humans and other mammals, testosterone is secreted primarily by the testicles ofmales and, to a lesser extent, the ovaries of females. Small amounts are also secreted by the adrenal glands. It is the principal male sex hormone and an anabolic steroid .

In men, testosterone plays a key role in the development of male reproductive tissues such as the testis and prostate as well as promoting secondary sexual characteristics such as increased muscle, bone mass, and the growth of body hair .  In addition, testosterone is essential for health and well-being (Bassil et al., 2009) as well as the prevention of osteoporosis (Tuck and Francis, 2009).

On average, in adult males, levels of testosterone are about 7–8 times as great as in adult females (Torjesen and sandnes, 2004).  As the metabolic consumption of testosterone in males is greater, the daily production is about 20 times greater in men (Southren et al., 1967).Females are also more sensitive to the hormone (Dabbs and Dabbs, 2000).Testosterone is observed in most vertebrates.

Testosterone is necessary for normal sperm development. It activates genes in Sertoli cells, which promote differentiation of spermatogonia. Regulates acute HPA (Hypothalamic–pituitary–adrenal axis) response under dominance challenge, Regulator of cognitive and physical energy, Maintenance of muscle trophism.

Testosterone regulates the population of thromboxane A 2receptors on megakaryocytes and platelets and hence platelet aggregation in humans.

High androgen levels are associated with menstrual cycle irregularities in both clinical populations and healthy women.

Testosterone does not cause deleterious effects in prostate cancer. In people who have undergone testosterone deprivation therapy, testosterone increases beyond the castrate level have been shown to increase the rate of spread of an existing prostate cancer.

Recent studies have shown conflicting results concerning the importance of testosterone in maintaining cardiovascular health.

Nevertheless, maintaining normal testosterone levels in elderly men has been shown to improve many parameters that are thought to reduce cardiovascular disease risk, such as increased lean body mass, decreased visceral fat mass, decreased total cholesterol, and glycemic control.

1.1.9.4.   Estradiol

Estradiol, or more precisely, 17β-estradiol, is a steroid and estrogen sex hormone, and the primary female sex hormone. Itis named for and is important in the regulation of the estrous and menstrual female reproductive cycles. Estradiol is essential for the development and maintenance of female reproductive tissues (Ryan, 1982) but it also has important effects in many other tissues including bone. While estrogen levels in men are lower compared to women, estrogens have essential functions in men as well. Estradiol is found in most vertebrates as well as many crustaceans, insects, fish, and other animal species (Ozon, 1972).

Estradiol derives from estra, literally meaning "verve or inspiration") and -diol , a chemical name and suffix indicating that this form of steroid and sex hormone is a type of alcohol bearing two hydroxyl groups .

Estradiol is produced especially within the follicles of the female ovaries, but also in other endocrine (i.e., hormone-producing) and non-endocrine tissues (e.g., including fat, liver, adrenal, breast, and neural tissues). Estradiol is biosynthesized from progesterone (Saldanha et al., 2011).

In the female, estradiol acts as a growth hormone for tissue of the reproductive organs, supporting the lining of the vagina, the cervical glands, the endometrium, and the lining of the fallopian tubes. It enhances growth of the myometrium. Estradiol appears necessary to maintain oocytes in the ovary. During the menstrual cycle, estradiol produced by the growing follicle triggers, via a positive feedback system, the hypothalamic-pituitary events that lead to the luteinizing hormone surge,inducing ovulation. In the luteal phase, estradiol, in conjunction with progesterone, prepares the endometrium for implantation.

During pregnancy, estradiol increases due to placental production. In baboons, blocking of estrogen production leads to pregnancy loss, suggesting estradiol has a role in the maintenance of pregnancy.

The development of secondary sex characteristics in women is driven by estrogens, to be specific, estradiol. These changes are initiated at the time of puberty, most are enhanced during the reproductive years, and become less pronounced with declining estradiol support after the menopause. Thus, estradiol produces breast development, and is responsible for changes in the body shape, affecting bones, joints, and fat deposition. Fat structures and skin composition are modified by estradiol.  The effect of estradiol (and estrogens in general) upon male reproduction is complex. Estradiol is produced by action of aromatase mainly in the Leydig cells of the mammalian testis, but also by some germ cells and the Sertoli cells of immature mammals (Carreaus et al., 2003).  It functions (in vitro) to prevent apoptosis of male sperm cells (Pentikainen et al., 2000).

Several studies have noted sperm counts have been declining in many parts of the world, and estrogen exposure in the environment has been postulated to be the cause (Sharpe and Skakkebaek, 1993).

Suppression of estradiol production in a subpopulation ofsubfertile men may improve the semen analysis (Raman and Schlegel, 2002).

Males with certain sex chromosome genetic conditions, such as Klinefelter's syndrome, will have a higher level of estradiol (Visootsak and Graham, 2006).

1.1.9.5     Prolactin

Prolactin (PRL), also known as luteotropic hormone or luteotropin, is a protein that in humans is best known for its role in enabling female mammals to produce milk; however, it is influential over a large number of functions with over 300 separate actions of PRL having been reported in various vertebrates (Bole-Feysot, et al., 1998). Prolactin is secreted from the pituitary gland in response to eating, mating, estrogen treatment, ovulation, and nursing. Prolactin is secreted in a pulsatile fashion in between these events. Prolactin also plays an essential role in metabolism, regulation of the immune system, and pancreatic development. Prolactin is a Peptide hormone, encoded by the PRL gene. Although often associated with human milk production, prolactin plays a wide range of other roles in both humans and other vertebrates. (For example, in fish—the oldest known vertebrates — an important function is probably related to control of water andsalt balance.Prolactin also acts in a cytokine -like manner and as an important regulator of the immune system. It has important cell cycle related functions as a growth-, differentiating- and anti- apoptotic factor. As a growth factor, binding to cytokine like receptors, it also has profound influence on hematopoiesis, angiogenesis and is involved in the regulation of blood clotting through several pathways. The hormone acts in endocrine, autocrine, and paracrine manner through the prolactin receptor and a large number of cytokine receptors.

Prolactin has a wide range of effects. It stimulates the mammary glands to produce milk (lactation): increased serum concentrations of prolactin during pregnancy cause enlargement of the mammary glands of the breasts and prepare for the production of milk. Milk production normally starts when the levels of progesterone fall by the end of pregnancy and a suckling stimulus is present.

Sometimes, newborn babies (males as well as females) secrete a milky substance from their nipples known as witch's milk. This is in part caused by maternal prolactin and other hormones. Prolactin also has been found to play an important role in maternal behavior (lucas et al., 1998).

Prolactin provides the body with sexual gratification after sexual acts: The hormone counteracts the effect of dopamine, which is responsible for sexual arousal. This is thought to cause the sexual refractory period. The amount of prolactin can be an indicator forthe amount of sexual satisfaction and relaxation. Unusually high amounts are suspected to be responsible for impotence and loss of libido.

Prolactin within the normal reference ranges can act as a weak gonadotropin but at the same time suppresses GnRH secretion.

The exact mechanism by which it inhibits GnRH is poorly understood although expression of prolactin receptors (PRL-R) have been demonstrated in rat's hypothalamus, the same has not been observed in GnRH neurons (Grattan et al., 2007). Physiologic levels of prolactin in males enhance luteinizing hormone -receptors in Leydig cells, resulting in testosterone secretion, which leads to spermatogenesis (Hair et al., 2002).

Prolactin also stimulates proliferation of oligodendrocyte precursor cells. These cells differentiate into oligodendrocytes, the cellsresponsible for the formation of myelin coatings on axons in thecentral nervous system (Gregg et al., 2007).

Prolactin promotes neurogenesis in maternal and fetal brains (Larsen and Grattan, 2012).

Prolactin delays hair regrowth in mice (Craven et al., 2006)

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