Literature Review on Growth Promoters in Animal Feed

CHAPTER-II

REVIEW OF LITERATURE

Growth Promoter – Growth promoters are agents added to animal feeds in order to enhance the feed conversion efficiency in terms of increased formation of muscles, fats and body growth in feed-lot animals, including poultry. (Vani Prasad and Koley, 2006)

Pharmacology of Growth Promoters in general

Probiotics

The word “probiotic” comes from the Greek words “pro” and “biotic,” meaning “for life” (Gibson and Fuller, 2000). Probiotics have been defined as “live microorganisms of non-pathogenic and non-toxic nature, which when administered through the digestive route, are favorable to the host’s health” (Kabir, 2009). The joint Food and Agriculture Organization of the United Nations (FAO) and World Health Organization (WHO) Working Group defined probiotics as “live micro-organisms which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). Certain species of bacteria, fungi and yeasts belong to group of probiotics. Existing probiotics can be classified into colonizing species (Lactobacillus sp., Enterococcus sp. and Streptococcus sp.) and free noncolonizing species (Bacillus and Saccharomyces cerevisiae). The definite mechanism through which probiotics may improve the defence and performance of chickens remains unclear, but some possible mode of actions have been proposed:

(1) maintaining a healthy balance of bacteria in the gut by competitive exclusion (the process by which beneficial bacteria exclude potential pathogenic bacteria through competition for attachment site in the intestine and nutrients) and antagonism (inhibit the growth of pathogenic bacteria by producing for example lactic acids), (2) promoting the gut maturation and integrity,(3) modulating the immune system and preventing inflammation (4) improving the metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production, (5) improving feed intake and digestion (as a result from the improved microbial balance in the gut), and (6) neutralizing enterotoxins and stimulating the immune system (Khaksefidi and Rahimi, 2005; Haghighi et al., 2006; Kabir, 2009; Brisbin et al., 2010; Sugiharto, 2016). In terms of immune responses, different species and/or strains of probiotics may have different immunomodulatory activities due to the ability of probiotics to induce cytokine production, which leads to modulation of innate and adaptive immune responses (Brisbin et al., 2010; Sugiharto, 2016).

Their effect on production results in improvement of the function of the immune system (Kabir et al., 2004)and by exhibiting significant influence on morpho-functional characteristics of intestines. These effects lead to growth of broiler chickens,improvement of feed conversionand reduced mortality (Mohan et al., 1996). Schneitz et al. (1990) developed the first successful commercial probiotic (under the tradename Broilact), which was a mixture of 22 anaerobic bacilli and 10 facultative anaerobes isolated from healthy adult hens. Broilact was shown to prevent colonization and infection of chicks by S. Enteritidis, S. Typhimurium, Campylobacter jejuni, and E. coli O157:H7 (Edens, 2003; Singh et al., 2015). Kalavathy et al. (2003) found that a supplementation of twelve Lactobacillus strains in broiler diets improved the body weight gain, feed conversion rate and was effective in reducing abdominal fat deposition.Wishing to explain in a scientific way, inconsistent results which they obtained in their studies, majority of authors concluded that the effect of probiotics depend on the combination of bacterial strains contained in the probiotic preparation, level of its inclusion in the mixture, composition of mixture, quality of chickens and conditions of the environment in the production facility (Patterson and Brukholder, 2003).

Prebiotics

Prebiotics are defined as “Nondigestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity ofone or a limited number of bacteria in the colon” (Gibson and Roberfroid, 1995). Prebiotics are non-digestible feed ingredients that beneficially affect the host by selectively altering the composition and metabolism of the gut microbiota (Huyghebaert et al., 2011; Das et al., 2012). For a dietary substrate to be classed as a prebiotic, at least three criteria are required: (1) the substrate must not be hydrolysed or absorbed in the stomach or small intestine, (2) it must be selective for beneficial commensal bacteria in the large intestine such as the bifidobacteria, (3) fermentation of the substrate should induce beneficial luminal/systemic effects within the host. (Manning and Gibson, 2004). The most significant compounds which belong to group of prebiotics are oligosaccharides: fructo-oligosaccharides (FOS), gluco-oligosaccharides and mannan-oligosaccharides (MOS). The most common prebiotics used in poultry are oligosaccharides, including inulin, fructooligosaccharides (FOS), mannanoligosaccharides (MOS) galactooligosaccharides (GOS), soya-oligosaccharides (SOS), xylo-oligosaccharides (XOS), pyrodextrins, isomalto-oligosaccharides (IMO) and lactulose (Huyghebaert et al., 2011; Kim et al., 2011; Alloui et al., 2013). Their advantage compared to probiotics is that they promote the growth of useful bacteria which are already present in the host organism and are adapted to all conditions of the environment. As they are non-digestible, these prebiotics provide substrates for commensal bacteria in the gastrointestinal tract and in consequence help to balance microfloral populations in favor of beneficial organisms and to the exclusion of pathogens (Callaway et al.,2008; Hajati and Rezaei, 2010). Similar to probiotics, results of the effects on broiler performance are contradictory. In analysis of the effects of implementation of FOS on broiler performances it was established that improvement of gain was by 5-8% and improvement of feed conversion by 2-6%. It has been reported that increased production of SCFAs in the gastrointestinal tract due to prebiotics may benefit the host by recovering some of the lost energy from competition with bacteria (Ganguly, 2013). Owing to this, dietary prebiotic supplementation is attributable to the improved bird performance and energy utilization (Choct, 2009; Nabizadeh, 2012). This proves that effect of application of prebiotics depends on the condition of animals, environment conditions, composition of food and level and type of prebiotic included in the mixtures.

Synbiotics

This is relatively recent term among additives used in poultry nutrition. Synbiotics may be defined as a mixture of probiotics and prebiotics that beneficially affects the host by improving the survival and implantation of live microbial dietary supplements in the gastrointestinaltract (Gibson and Roberfroid, 1995). This combination can work synergistically to exclude pathogens from the gastrointestinal tract and reduce disease (Callaway et al., 2008; Collins and Gibson, 1999). Bailey, Blankenship and Cox (1991) reported that diets containing 0.75% FOS in addition to probiotic flora were able to reduce Salmonella colonization rate to 12% fewer birds than the control. Investigations showed that combinations used in synbiotics are often more efficient in relation to individual additives (Li et al., 2008). The acquisition of data on the efficacy of synbiotic products as feed additives in livestock and poultry needs further investigation. However, results on in vivo trials are promising, showing a synergistic effect coupling probiotics and prebiotics in the reduction of food-borne pathogenic bacterial populations (Bomba et al., 2002).

Enzymes

Supplementation of mixtures for broiler with enzymes is applied in order to increase the efficiency of production of poultry meat. Various exogenous enzymes including β-glucanase, xylanase, amylase, carbohydrase, α-galactosidase, protease, lipase, phytase, etc. have been supplemented in poultry diets for decades (Adeola and Cowieson, 2011; Bedford and Cowieson, 2012). The use of exogenous enzymes is of importance in poultry given that chicken diets are composed primarily of corn and soybean meal, which contain varying levels of different anti-nutritive factors (e.g., non-starch polysaccharides [NSP] and protease inhibitors) that can impede normal digestion and absorption processes of nutrients in the digestive tract (Yegani and Korver, 2013). The tendency to use non-conventional ingredients (containing anti-nutritional factors and fibre) in poultry diet to reduce the cost of feeding may also encourage the use of exogenous enzyme, as these materials cannot be fully digested and utilized by the chickens (Costa et al., 2008). In this context, the exogenous enzymes are used to correct the lack of specific endogenous enzymes for digesting certain nutrients in various feedstuffs or to hydrolyse anti-nutritional factors in feed ingredients (Ao, 2005). Enzyme supplementation is also important for environmental issues, as it may reduce the pollutant potential of excreta (Costa et al., 2008). Exogenous enzymes are also beneficial to prevent the horizontal transmission of Salmonella infection between the birds (Amerah et al., 2012). Yang et al. (2008) reported that the crypt depth of jejunum was reduced by xylanase and this was associated with the increased growth of chicken fed xylanase. Concomitantly, Adeola and Cowieson (2011) revealed that carbohydrase supplementation improved villi length and supported the growth of chicken. Furthermore, reduction in the viscosity of intestinal contents is associated with the growth-promoting effects of enzymes, as with reduced viscosity, enzymatic action on intestinal content is more efficient (Costa et al., 2008; Adeola and Cowieson, 2011).

To exert their functions, the activity of enzymes must not be affected by processing or by the low pH (<4) or endogenous digestive enzymes in the digestive tract of chickens (Ao, 2005). To obtain the maximum benefit from the enzymes, the use of multiple enzymes is recommended as the combination of the enzymes may target different anti-nutritive compounds in the feedstuffs (Adeola and Cowieson, 2011). However, it should be noted that the beneficial effect of enzyme combination may be dependent on the diet composition (Meng et al., 2005).

Acidifiers

Acidifiers have been used in poultry nutrition for long time, in different forms and combinations which are constantly changing. Organic acids, such as lactic, acetic, tannic, fumaric, propionic, caprylic acids, etc., have been shown to exhibit beneficial effects on the intestinal health and performance of birds (Adil et al., 2010; Saki et al., 2012; Menconi et al., 2014). Saki et al. (2012) reported that supplementation of organic acids in the diet increased LAB (i.e., Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium spp.) counts in the ileum and caecum of broiler chicken. This treatment also significantly decreased Enterobacteriaceae and Salmonella counts in the intestine of birds (Cengiz et al., 2012; Saki et al., 2012; Sugiharto Sugiharto, 2014). In terms of performance, feeding organic acids resulted in improved body weight gains and feed conversion ratio (Adil et al., 2010). Antimicrobial property of acids has been suggested to play a crucial role in controlling the population of pathogenic bacteria in the gut of birds (Partanen and Mroz, 1999; Sugiharto Sugiharto, 2014). The mechanism of action of organic acids probably reflects their antibacterial nature, such as decreasing the pH of drinking water and reducing the buffering capacity of the feed with subsequent effect on the physiology of the crop and proventriculus (Van Immerseel et al., 2006; Huyghebaert et al., 2011). Once in the cell, where the pH is maintained near 7, the acid will dissociate and suppress bacterial cell enzymes (e.g., decarboxylases and catalases) and nutrient transport systems (Huyghebaert et al., 2011).Result of this is improved consumption of food, better feed conversion and increased body weight gain. Favourable effect of supplementation of individual organic acids to mixtures was established relatively long time ago for formic acid (Kirchgessner et al., 1991). Supplementation of organic acids may facilitate the nutrient absorption and that in turn growth performance in broiler chicken (Adil et al., 2010). The potential of organic acids in lowering chyme pH is another property of this compound to support the growth, as this feature may increase protein digestion (Gauthier, 2002). Although supplementation of organic acids has been evidenced to support the health and performance of birds, the use of organic acids must be administrated in low dosage, as excessive dosage may result in growth depression in intestinal villus height and width, as well as crypt depth (Smulikowska et al., 2010). It has been suggested that combinations of organic acids are more effective than supplements that contain only one type of acid. This is because different types of organic acids diffuse through the bacterial cell wall and membrane and into the cell cytoplasm at different rates. These acids dissociate to form a conjugate base and a free hydrogen ion at different rates and respective pKa values (Novus International Inc., 2006). However, in utilization of acidifiers it should always be taken into consideration that these are substances which can exhibit negative effect on humans, animal and equipment and it is necessary to carefully select adequate preparations, in sense of combinations of acids, their doses and forms.

Antioxidants

To this group belong substances which act as antioxidants, such as vitamin E, selenium, carotinoids, etc. Selenium is component of enzyme glutathione peroxidase (GSHPx) which prevents forming of free radicals which are very harmful to cells in the way that they disrupt their integrity. Therefore, selenium and other antioxidants have favourable effect on quality of broiler meat (Tomovic et al., 2006). These authors established better protective effect of organically bound selenium compared to inorganic selenium forms, based on production and some bio-chemical parameters including activity of GSHPx. One of the most accepted approaches to preservation of sensory properties of meat is addition of antioxidants such as selenium or vitamin E directly to livestock food or during technological procedure of processing (Surai, 2002).

Bacteriophages

Bacteriophages, also known as phages, have the ability to infect bacteria and most bacterial species have their own unique bacteriophages (Carrillo et al., 2005). Adoption of phage therapy has proven to be effective (Sulakvelidze et al., 2001); however, widespread use has not been adopted in the poultry industry. Reducing the bacteria shed by broilers before they enter a commercial processing facility is the most ideal approach to meeting USDA-FSIS performance standards. One such approach involves the use of lytic bacteriophages to reduce the gut colonization of poultry-borne pathogens. Carrillo et al. (2005) and Wagenaar et al. (2005) separately demonstrated that in an experimental situation this methodology was able to achieve between 2 and 3 log 10 reductions in Campylobacter shedding.

Phytobiotics

Phytobiotics are plant-derived natural bioactive compounds that can be added to the feed to improve the performance and well-being of animals (Vidanarachchi et al., 2005; Windisch et al., 2008). Phytogenic additives influence improvement of consumption and conversion of food, digestibility and body weight gain of broiler chickens (Ertas et al., 2005). Based on the biological origin, formulation, chemical description and purity, Yang et al. (2009) classified phytobiotics to: (1) herbs (product from flowering, non-woody and non-persistent plants), (2) botanicals (entire or processed parts of a plant, e.g., root, leaves, bark), (3) essential oils (hydro distilled extracts of volatile plant compounds), and (4) oleoresins (extracts based on non-aqueous solvents). Antimicrobial activity and immune enhancement probably are the two major properties belonging to phytobiotics which are essential for the health and well-being of the chicken (Yang et al., 2009; Fallah et al., 2013). The mechanisms by which the phytobiotics exert their benefits on the gut remain unclear, but possible mechanisms could be proposed as follows: (1) modulating the cellular membrane of microbes leading to membrane disruption of the pathogens, (2) increasing the hydrophobicity of the microbial species which may influence the surface characteristics of microbial cells and thereby affect the virulence properties of the microbes, (3) stimulating the growth of favourable bacteria such as lactobacilli and bifidobacteria in the gut, (4) acting as an immunostimulatory substance and (5) protecting the intestinal tissue from microbial attack (Vidanarachchi et al., 2005; Windisch and Kroismayr, 2007). Studies have shown that phytobiotics improved the growth performance of broiler chickens, similar to those of AGP (Windisch and Kroismayr, 2007; El-Ghany and Ismail, 2013). The phytobiotics especially those from the group of essential oils have been reported to improve flavour and palatability of feed and may thus improve the feed intake and performance of chickens (Windisch et al., 2008; Grashorn, 2010). The potential of phytogenic bioactive compounds to stimulate the proliferation and growth of absorptive cells in the gastrointestinal tract (resulting in greater villus height and deeper crypt, Jamroz et al., 2006), and to influence the production and/or activity of the digestive enzymes, e.g., increasing the activities of amylase and protease (Vidanarachchi et al., 2005; Jang et al., 2007), have also been thought to improve the growth performance of birds. Overall, it should be noted that the efficacy of phytobiotics as feed additives and their impact on the gut health and growth performance may vary as a result of the variation in their composition due to biological factors (plant species, growing location, and harvest conditions), and manufacturing (extraction/distillation, stabilization) and storage conditions (light, temperature, oxygen tension and time) (Huyghebaert et al., 2011).

DESCRIPTION AND HABITAT OF THE PLANT

Distribution:

Spinacia oleracea is an edible flowering plant in the family of Chenopodiaceae, common name is spinach or in Hindi known as Palak. It is an annual plant, which grows to a height of up to 30 cm. Spinach may survive over winter in temperate regions. Spinacia oleracea Linn. (SO) is an annual plant having medicinal property native to central and southwestern Asia. It is cultivated for the sake of its succulent leaves and was introduced in Europe in the 15 th century. It is the favorite food among Indians in winter season (Guha and Das,2008). It’s Ayurvedic name is ‘Paalankikaa’, in ‘Unani’ it is called as ’Paalak’, where as in ‘Siddha’ it is known by ‘Vasaiyila-keerai’( Khare,2007).

Botanical Name

Spinacia oleracea Linn.

Other Names

Sanskrit – Chhurika

Hindi – Palak

Tamil – Pasalai

Telugu – Mathubucchali

Oriya – Palanga

Chhattisgarhi – Palak Bhaji

Bengali – Palang

 Classification of Spanacia oleracea Linn. plant

Botanical characteristic

The leaves are alternate, simple, and ovate to triangular-based, very variable in size from about 2-30 cm long and 1-15 cm broad, with larger leaves at the base of the plant and small leaves higher on the flowering stem. The flowers are inconspicuous, yellow-green, 3-4 mm diameter, maturing into a small, hard, dry, lumpy fruit cluster 5-10 mm across containing several seeds (Boswell,1949).The leaves of Spinacia oleracea (Family: Chenopodiaceae) commonly known as Palak/Spinach. Spinacia oleracea useful in diseases of blood and brain, asthma, leprosy, biliousness; causes kapha (Ayurveda). It has been used in the treatment of urinary calculi and has poglycemic properties. Leaves are cooling, emollient, wholesome, antipyretic, diuretic, maturant, laxative, digestible, anthelmentic, useful in urinary stone, inflammation of the lungs and the bowels, sore throat, pain in joints, thirst, lumbago, cold and sneezing, sore eye, ring worm scabies, leucoderma, arrest vomiting, biliousness, flatulence and have been used in the treatment of febrile conditions. Seeds are useful in fevers, leucorrhoea, urinary discharges, lumbago and diseases of the brain and of the heart. They have been used in the treatment of difficulty in breathing, inflammation of the liver and jaundice (Chopra et al., 1956; Kirtikar and Basu, 2005).

Stem: Erect from 30-60 cm high, round, smooth, piped, succulent, sometime reddish (Singh et al., 2015).

Leaves: Alternative, the lower ones very long petioled, variously lobed with lobes of an acute triangular shape, smooth on both the side (Singh et al., 2015).

Flowers Male: Flowers on long terminal glomerate spikes and on shorter ones from the axial, very numerous, sessile, calyx 4-parted, stamen 4, anthers twin, very large (Singh et al., 2015).

Female: Flowers axillary, sessile, crowded. Calyx 2-tipped with a projecting horn in each side, growing into spines when the seed is ripe. Styles generally 4, white tapering. Capsule 1-celled, 1-valved, armed, with 2 opposite short horns, and crowned with the small remaining calyx (Kirtikar and Basu, 2005).

ETHANOBOTANICAL USES

Plant:

It is sweet, cooling, carminative, laxative, alexipharmic; useful in diseases of blood and brain, asthma, leprosy, biliousness; causes “kapha” (Ayurveda). It has been used in the treatment of urinary calculi. In experiments it has been shown to have hypoglycemic properties (Singh et al., 2015).

Leaves:

These are cooling, emollient, wholesome, antipyretic, diuretic, maturant, laxative, digestiblle, anthelmentic, useful in urinary concretion, inflammation of the lungs and the bowels, sore throat, pain in joints, thirst, lumbago, cold and sneezing, sore eye, ring worm scabies, leucoderma, arrest vomiting , biliousness, flatulence and have been used in the treatment of febrile conditions (Singh et al., 2015) .

Seeds:

Seeds are useful in fevers, leucorrhoea, urinary discharges, lumbago, and diseases of the brain and of the heart (Yunani). Seeds are laxative and cooling. They have been used in the treatment of difficulty in breathing, inflammation of the liver and jaundice. The green plant is given for the urinary calculi (Chopra and Nayar, 1956; Kirtikar and Basu, 2005).

Chemical Consituents:

Flavonoids: Spinacia oleracea is very rich in the flavonoids. Various flvonoids reported to be present are querecetin; myricetin; kampeferol (Gupta and Singh, 2006); apigenin; luteolin; patuletin; spinacetin; jaceidin; 5,4’-dihydroxy-3.3’- dimethoxy-6:7-methylene dioxyflavone-4’-glu-curonide (Longnecker et al.,1997), 5,4’-dihydroxi-3,3’-dimithoxi-6,7-methylene dioxiflavone (C18H14O8.); 3,5,7,3’,4’pentahydroxi-6-methoxiflavone (Annonymus, 2004; Singh et al., 2015).

Phenolic Compounds: The polyphenols isolated from the plant are para-coumaric acid, ferulic acid, orthocoumaric acid (Andjelkovic et al., 2008).

Carotinods: Spinach shows presence of different carotinoids like lutein, β-carotene, violaxanthin and 9’-(Z)- neoxanhin (Guha and Das, 2008).

Glycolipids: It also contain mainly three glycolipids: monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol (Maeda et al., 2008).

Two antifungal peptides: They are designated as alpha- and betabasrubrins (Wang, 2004).

Vitamins: Spinacia oleracea contains high concentration of vitamin A, E, C, and K and also folic acid, oxalic acid (Guha and Das, 2008).

Minerals: Magnesium, manganese, calcium, phosphorus, iron, zinc, copper and potash (Singh et al., 2015).

The pharmacological activities of different parts of Spinacia oleracea have been investigated by several workers.

Nyska et al. (2001) reported that topical and oral administration of the natural water-soluble antioxidant from spinach  (NAO) reduces the multiplicity of papillomas in the Tg.AC mouse model. This study evaluated the efficacy of the non-toxic, water soluble antioxidant from spinach, natural antioxidant (NAO), in reducing skin papilloma induction in female hemizygous Tg.AC mice treated dermally five times over 2.5 weeks with 2.5 µg 12-tetradecanoylphorbol-13-acetate (TPA). The TPA only group was considered as a control; the other two groups received, additionally, NAO topically (2 mg) or orally (100 mg/kg), 5 days/week for 5 weeks. Papilloma counts made macroscopically during the clinical observations showed a significant decrease in multiplicity (P<0.01) in the NAO topically treated group. According to histological criteria, papilloma multiplicity were lower in both topical-NAO and oral-NAO groups, but significantly so only in the oral-NAO mice (P<0.01). The beneficial effect of NAO in the Tg.AC mouse is reported.

Verma et al. (2003) investigated the radioprotective efficacy of spinach against γ-radiation induced oxidative stress in the brain of swiss albino male mice as the antioxidants present in spinach leaf extract reduces the lipid peroxidation values of brain by quenching free radicals. Healthy Swiss albino male mice of 6-week-old age group were selected from an inbred colony; maintained on standard mice feed and water ad libitum. For the experiments, mice were divided in four groups. Group I (normal) it did not received any treatment. Group II (drug treated) was orally supplemented spinach extract (SE) once daily at the dose of 1100 mg/kg.b.wt. /day for 15 consecutive days dissolved in double distilled water. Group III (experimental) was also administered orally spinach extract at the dose of 1100 mg/kg.b.wt./day for 15 consecutive days thereafter exposed to single dose of 5 Gy of gamma radiation at the dose rate of 1.07 Gy/min. Group IV (control) received distilled water orally equivalent to spinach extract for 15 days thereafter it was exposed to 5 Gy of gamma radiation. After the exposure mice were sacrificed at different autopsy intervals viz. 1,3,7,15 and 30 days. Brain was removed and processed to estimate lipid peroxidation (LPO). The levels of LPO products in brain of spinach extract supplemented mice activates antioxidant enzymes in brain suggesting that spinach leaf extract reduces LPO values by quenching free radicals. The protection rendered with SE in LPO value of brain in the present study indicates the possible role of Spinach as radioprotector to some extent if taken continuously which might be due to synergistic effect of antioxidant constituents present in the spinach.

Maeda et al. (2008) reported Anti-tumor effect of orally administered spinach glycolipid fraction on implanted cancer cells, Colon-26, in mice. They were succeeded in purifying a major glycolipid fraction from a green vegetable, spinach. This fraction consists mainly of three glycolipids: monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), and sulfoquinovosyl diacylglycerol (SQDG). They found that the glycolipid fraction inhibited DNA polymerase activity, cancer cell growth and tumor growth with subcutaneous injection. They  clarified oral administration of the glycolipid fraction, suppressing colon adenocarcinoma (colon-26) tumor growth in mice. A tumor graft study showed that oral administration of 20 mg/kg glycolipid fraction for 2 weeks induced a 56.1% decrease in the solid tumor volume (P<0.05) without any side-effects, such as loss of body weight or major organ failure, in mice. The glycolipid fraction induced the suppression of colon-26 tumor growth with inhibition of angiogenesis and the expression of cell proliferation marker proteins such as Ki-67, proliferating cell nuclear antigen (PCNA), and Cyclin E in the tumor tissue. From these results, he suggested that the orally administered glycolipid fraction from spinach could suppress colon tumor growth in mice by inhibiting the activities of neovascularization and cancer cellular proliferation in tumor tissue.

Islam et al. (2009) evaluated the efficacy of spinach against arsenic (As) induced toxicity in rats during the period between July to October 2008. Thirty six female Long Evans rats (age about 120days; average body weight at day 0 = 154.5g) were randomly divided into three equal groups (n=12) and marked as T0, T1 and T2 groups. Rats of T0 group were given normal feed and water and kept as control. Rats of T1 and T2 groups were given 5mg Sodium arsenite/kg body weight (BW) and 5mg Sodium arsenite/kg (BW) plus spinach extract 100 mg/kg body weight respectively daily for 30 days orally. Four rats from each group were sacrificed at 10 days interval in order to quantitatively determine the As content in liver, lungs and kidney by using Hydride Generation Atomic Absorption Spectrophotometer. Serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and serum creatinine were determined by Autoanalyser. No visible clinical sign were observed in any group of experimental rats except loss of body weight in the spinach treated group. Tissue (lung, liver and kidney) concentration of As was significantly (p<0.01) higher in T1 group rats compared to that of T0 and T2 groups and the highest concentration of As was found in kidney followed by lung and liver in T1 group rats. After 30 days of feeding, spinach significantly (p<0.01) decreased As from lung, liver and kidney. As intoxication significantly (p<0.01) increased SGOT values but insignificantly decrease SGPT values and spinach treatment improve these condition. There was no significant effect found in serum creatinine level.

Dande et al. (2010) reported the pharmacognostical studies of leaves of Spinacia oleracea Linn. They stated its use as a traditional medicinal plant with high nutritional value, used as anti diabetic, anti bacterial and hepatoprotective agent.

Al-Dosari (2010) reported hepatoprotective activity of the ethanolic extract of the leaves of spinach (EESO) against carbon tetrachloride (CCl4)-induced oxidative stress (OS) and liver injury in rats. He found that pretreatment of rats with EESO, at 250 and 500 mg/kg body weight for 21 consecutive days significantly prevented the CCl4 -induced hepatic damage as indicated by the serum marker enzymes (SGOT, SGPT, ALP and GGT) and bilirubin levels. Parallel to these changes, the leaves extract also prevented CCl4-induced OS in rat liver by inhibiting lipid peroxidation (LPO) and restoring the levels of antioxidant non-enzymatic biomarker, such as total protein (TP) and non-protein sulfhydryl (NP-SH) in liver tissue. The biochemical changes were consistent with the histological findings of the liver tissue suggesting marked hepatoprotective effect of the leaves extract in a dose-dependent manner; besides, a significant reduction was also observed in pentobarbital-induced sleeping time in mice. The results of spinach extract were comparable to that of silymarin. The protective effect of the EESO against CCl4-induced acute hepatotoxicity could be attributed to the potent antioxidant constituents of the spinach.

Bigoniya et al. (2011) evaluated the anti-infl ammatory activity of the methanolicaqueous fraction (MAF and water extract (WE) of Spinacia oleracea leaf in an acute inflammation model. The water extract of Spinacia oleracea and its methanolic aqueous fraction at 600 mg/kg dose showed significant (P < 0.001) inhibition of inflammation in both acute and chronic anti-inflammatory models. The potency of the extracts was compared with the standard diclofenac sodium (5 mg/kg). The methanolic aqueous fraction and water extract showed a dose-dependent increase and decrease in catalase content. The highest protein content was found in the water extract. The significant ameliorative activities of the extracts (MAF and WE) and the standard drug (Diclofenac sodium) were observed in the present study. The WE, at a dose of 600 mg/kg, suppressed the second phase of carrageenan-induced inflammation in an extremely significant manner, with 73.52% suppression of edema volume compared to 58.82% of MAF and 82.35% of diclofenac sodium. This clearly shows the presence of the dose-dependent, anti-infl ammatory activity in the MAF and WE of the S. oleracea leaf.She also reported that rats treated with water extract and methanolic aqueous fraction at a dose of 600 mg/kg showed a signifi cant increase in the activity of SOD in the granulation tissue when compared with the control.This enzyme is known to quench the superoxide radical, and thus, prevent the damage of cells caused by free radicals.

Giri (2012) mentioned the hypolipidemic activity of Spinacia oleracea L. in atherogenic diet induced hyperlipidemic rats. They found that rats receiving Spinacia oleracea powder showed significant reduction in total cholesterol, triglycerides, total protein and elevation of high density lipoprotein cholesterol. Spinacia oleracea was found to possess significant hypolipidemic activity. The results also suggested that Spinacia oleracea powder at 200mg and 400 mg/kg b.wt. concentrations are an excellent lipid-lowering agent and the levels of total serum cholesterol, triglyceride and total protein which are actually raised in atherogenic diet, can be lowered significantly with S. oleracea (spinach).

Jain and Singhai (2012) investigated the in vitro and in vivo protective effects of Spinacia oleracea (Chenopodiaceae) seeds on carbon tetrachloride (CCl4)-induced hepatic toxicity. In the in vitro studies, different extracts (i.e. petroleum ether, ethanol and aqueous) and fractions derived from ethanol extract (i.e. chloroform, ethyl acetate and n-butanol) of Spinacia oleracea seeds were screened at a concentration of 100 µg/mL against carbon tetrachloride (CCl4)-toxicity in rat hepatocyte culture. Hepatoprotective activity was assessed in vivo in rats intoxicated with CCl4. Level of biochemical markers along with histological changes were monitored to evaluate the extent of hepatoprotection. Silymarin was taken as reference drug. In the in vitro screening, n-butanol fraction of Spinacia oleracea seeds was found to be more potent than other screened plant samples, hence selected further for phytochemical and in-vivo studies. In the in vivo studies, the n-butanol fraction of Spinacia oleracea showed significant protection against CCl4-induced hepatotoxicity as evident by restoration of biochemical and histological changes caused by CCl4 intoxication. HPTLC fingerprinting of the n-butanol fraction of Spinacia oleracea confirmed the presence of 20-hydroxyecdysone (20-HE) besides other phytochemicals, which partially may explain the effects.

Otari et al., (2012) assessed the protective effect of aqueous extract of Spinacia oleracea leaves (AESO 250, 500, and 1,000 mg/kg, p.o.) in inflammatory bowel disease using acetic acid and ethanol-induced colitis in mice and indomethacin-induced enterocolitis in rats. The preliminary phytochemical analysis and further high performance thin layer chromatographic (HPTLC) analysis and phytochemical tests of HPTLC bands confirmed the presence of flavonoids and tannins in AESO. In acute oral toxicity study, administration of AESO (5,000 mg/kg, p.o.) did not show any sign of toxicity and mortality. The treatment with AESO significantly increased body weight, decreased diarrhea with bloody stools, increased blood hemoglobin and plasma total protein, and decreased serum and ileum or colon malondialdehyde content and attenuated the extent of lesions and ameliorated the histological injury of mucosa in all paradigms. The most prominent effects were evident for AESO 1,000 mg/kg. The results of the present study revealed that AESO was effective in attenuating almost all the symptoms of IBD in experimental paradigms. The effect might be due to the antioxidant activity of the flavonoids present in the AESO.

Das and Chatterjee (2013) evaluated the antibacterial potential of Spinacia oleracea L. against urinary tract pathogens. They evaluated the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for all solvent extracts of Spinacia oleracea against each pathogen that recorded antibacterial activity and found the significant bactericidal MIC ranged between 1 to 40 mg/ml and MBC ranged between 10 to 50 mg/ml against UTI causing bacteria by well diffusion method. The methanolic extract of Spinacia oleracea was found to be highly active against UTIpathogens because might be the presence of largenumber of bioactive compounds such as steroids, tannins,alkaloids, flavonoids, terpenoids, saponins, glycosides,carbohydrates, amino acids, carbonyl compounds,phenolic compounds compared to other solvent extracts.The inhibitory effect of phenolic compounds could beexplained by adsorption to cell membranes, interactionwith enzymes, substrate and metal ion deprivation. It was seen that methanolic extract of Spinacia oleracea has lowest MIC and lowest MBC valuesfor each test organisms showing that it is most effectiveas compared to other extracts. Thus Spinacia oleracea can be used as non-toxic, cheaper and natural antimicrobial agents.

Metha and Belemkar (2014) contributed the complete overview of pharmacological activity of Spinacia oleracea Linn.. They stated that Spinach is packed with vitamins such as vitamin C, vitamin A and vitamin E and minerals like magnesium, manganese,iron, calcium and folic acid. Spinach is also a good source of chlorophyll, which is known to aid in digestion. Spinach is also rich in the carotenoids beta-carotene and lutein. It is a good source of the bioflavonoid quercetin with many other flavonoids which exhibits anti-oxidant, ant proliferative, anti-inflammatory, antihistaminic, CNS depressant, protection against gamma radiation, hepatoprotective properties in addition to its many other benefits. Spinach is also used to prevent the bone loss associated with osteoporosis and for its anti-inflammatory properties in easing the pain of arthritis. Spinach is good for the heart and circulatory system and has energy-boosting properties. Spinach is truly one of nature’s most perfect foods.

Ko et al. (2014) revealed antioxidant effects of spinach (Spinacia oleracea L.) supplementation in hyperlipidemic rats. They have found that increased consumption of fresh vegetables that are high in polyphenols has been associated with a reduced risk of oxidative stress-induced disease. For measurement of in vitro antioxidant activity, spinach was subjected to hot water extraction (WE) or ethanol extraction (EE) and examined for total polyphenol content (TPC), oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA), and antigenotoxic activity. The in vivo antioxidant activity of spinach was assessed using blood and liver lipid profiles and antioxidant status in rats fed a high fat-cholesterol diet (HFCD) for 6 weeks. The TPC of WE and EE were shown as 1.5±0.0 and 0.5±0.0 mg GAE/g, respectively. Increasing the concentration of the extracts resulted in increased ORAC value, CAA, and antigenotoxic activity for all extracts tested. HFCD-fed rats displayed hyperlipidemia and increased oxidative stress, as indicated by a significant rise in blood and liver lipid profiles, an increase in plasma conjugated diene concentration, an increase in liver thiobarbituric acid reactive substances (TBARS) level, and a significant decrease in manganese superoxide dismutase (Mn-SOD) activity compared with rats fed normal diet. However, administration of 5% spinach showed a beneficial effect in HFCD rats, as indicated by decreased liver TBARS level and DNA damage in leukocyte and increased plasma conjugated dienes and Mn-SOD activity. Thus, the antioxidant activity of spinach may be an effective way to ameliorate high fat and cholesterol diet-induced oxidative stress.

Rao et al. (2015) studied the preliminary phytochemical screening of Spinacia oleracea L. on the fresh and dried leaves after being subjected to cold maceration.

Singh et al. (2016) reported pharmacognostic evaluation and quantitative phytochemical screening of different extracts of Spinacia oleracea leaves which justified the pharmacological activities of particular chemical constituents in different extracts.