1.1 Herbal Medicine.

Herbal medicines also known as home grown solutions are naturally happening plant-derived substances that are utilized to treat illness (Chen et al., 2014).The yield are intricate combination of natural synthetic compounds that may originate from any crude or prepared piece of a plant.
Traditional medication take its root in each nation across the world.There are numerous elective frameworks of antiquated drug, and furthermore the rationality and practices of each are affected by social conditions, environment and land area, that all concur on an all-encompassing way to deal with life. (Darmey et al., 2014).In spite of the fact that flavourer medicine has been polished for a large number of years, it keeps on being useful inside the popular, Western world (Ernst et al., 2011). the planet Health Organization (WHO) measurable that concerning eighty p.c of people worldwide have certainty flavourer drugs for two or three a piece of their essential medicinal services, and furthermore the overall yearly commercial center for these stock is moving toward $60 billion (World Health association, 1992). Individuals from the United State of America got a kick out of old solution attributable to the ascent inside the value of medication and furthermore the most prominent result of characteristic prescription.
Finish herbs comprises of the numerous constituents that ar used in the treatment of unwellness and reject signs. flavourer drug, utilizes the plant’s seeds, berries, roots, leaves, bark or blossoms for medicative capacities. The natural properties of those plants have helpful impacts. Elective elements ar responsible for their edges yet, and these epitomize the sort of surroundings amid which the plant developed, the way amid which it completely was reaped and the manner in which it totally was prepared. The plant is either oversubscribed crude or as concentrates, wherever it’s macerated with water, liquor or elective solvents to extricate some of the synthetic concoctions. The resulting stock contains many synthetic compounds, together with unsaturated fats, sterols, alkaloids, glycosides, saponis.
1.1.1 Historical Background of hrebal MedicinesArcheological confirmation shows that the use of medicative plants goes back to the period somewhere in the range of sixty, thousands years ago. In old Egypt, herbs are specified in Egyptian therapeutic papyri – imagined in topographic point representations, or on uncommon events found in restorative jugs containing follow measures of herbs (Atanasov et al., 2015). The documentation of herbs and their uses was a focal a piece of every Western and japanese restorative grant through to the 1600s, and these works contend an essential job inside the advancement of the study of natural science.
1.1.2 Advantages and Disadvantages of Herbal Medicines
There are fluctuated advantages and drawbacks of herbal medicine (Barnes et al., 2004). There are assortments of advantages identified with the work of herbal medicine as unfriendly universal pharmaceutical stock. Examples include the following
I. Decreased risk of side effects: Most herbal solutions are all around endured by the patient, with less happy results than pharmaceutical drug. Herbs for the most part have less side effects than orthodox medications, and ought to be more secure to use after some time.
ii. Effective in chronic conditions: Herbal Medicines have a tendency to be more effective for long-standing health dissensions that don’t react well to orthodox prescription (Burstein et al., 1999). One example is that the utilization of herbs and different solutions for the treatment of arthritics. Different medicines for arthritics, have few side effects. Example of such treatments include dietary changes like adding simple herbs, removing vegetables from the nightshade family and reducing white sugar consumption.

iii. Lower cost: Another advantage of herbal medicine is cost. Herbs cost much less than prescription. Research, testing, and marketing add greatly to the cost of prescription medicines. Herbs tend to be inexpensive compared to orthodox drugs.
iv. Widespread availability: yet one more preferred standpoint of herbal meds is their accessibility. Herbs are out there without a prescription. One will develop some clear herbs, similar to peppermint and camomile, at home. In a few edges of the world old medications is likewise the sole cure out there for a genuine assortment of people.
Herbs aren’t without inconveniences, and herbal drugs isn’t material on the whole things. These and heaps of a considerable measure of are the drawbacks of herbal meds:
I. Inappropriate for many conditions: Popular medications treat unexpected and overwhelming illness and accidents way more successfully than herbal or alternative medicines. A herbalist wouldn’t be prepared to treat serious injury, such to a broken leg, nor would he be prepared to heal an appendicitis or heart attack as effective as a convectional doctor using modern diagnostic tests, surgery, and drugs (Bishop et al., 2010).
ii. Absence of dosage instructions: Another detriment of herbal medicine is that the appallingly genuine dangers of completing oneself hurt through self-dosing with herbs. Though some will contend that a proportional factor will occur with ortodox pharmaceuticals, as inadvertently overdosing on cool cures, a few herbs don’t run with bearings or bundle embeds. Hence, there is a very risk of overdose.
iii. Toxic substance chance identified with wild herbs: Harvesting herbs inside the wild is hazardous, if not rash, anyway a few people endeavor to decide and choose wild herbs. They run an extremely genuine danger of harming themselves in the event that they are doing not appropriately decide the herb, or on the off chance that they utilize the off base a piece of the plant.
iv. Prescription activities: Herbal medicines will collaborate with solutions. About all herbs run with some notice, and many, much the same as the herbs utilized for tension like blossom and St. John’s Wort, will act with professionally prescribed solution like antidepressants.
v. Absence of control: Because of herbal products aren’t firmly directed, clients furthermore run the peril of looking for second rate quality herbs (Egede et al., 2002).

1.2 Mushrooms
Mushrooms describe a spread of branchiate fungi, with or while not stems, and therefore the term is employed even a lot of usually, to explain each the fleshing mature bodies of some basdiomycota, relying upon the content of definition. By extension, the term mushroom can even designate the whole flora once culture, the plant part (called a mycelium) of species forming the plant organ of a micro- flora. It’s the half seen growing higher than the soil on decaying logs of woods and tree stumps.Mushroom is a helpful supply of feed supplement and drugs to suppress the expansion rate of growth in patients (Borcher et al., 2008). Out of 2,327 helpful species of mushrooms listed worldwide, barely 100 will be cultivated and small analysis has been conducted in developing countries like Federal Republic of Nigeria on strictly native species. Qualitative and mensuration of mushroom show that they contain some biologically active compounds and nutrients like sugars and amino acids (Borcher et al. 1999). These bioactive compounds belong to the cluster of polysaccharides and antioxidants, that shield the body against free radicals that harm body cells to induce diseases. The presence of polysachharides (which ar polymers of sugar molecules) in mushrooms suggests that they will be helpful as natural health promoters against parasites, microorganism and viruses. Zhong et al., (2009) rumored that mushrooms will be accustomed stimulate the expansion of immune organs in Newcastle disease infected chickens. A mushroom was conjointly rumored to cut back severity of Marek’s malady in chickens. T-cell immune reaction characterised by secretion of antiviral, cyctokine stimulation and phagocyte activity were increased by mushroom (Borcher et al., 2008).1.2.1 Medicinal Properties of MushroomsMedicinal mushrooms are those that are utilized for or examined as potential medicines of diseases. Studies have demonstrated that mushrooms have potential antiviral, antimicrobial, anticancer, antihyperglycemic, cardioprotective and antiflammatory activities. Hentinan, Psk and Schizophyllan are mushroom extract nutraceuticals. Hentinan and Psk are dispatched doctor prescribed medications in bound nations. A few mushrooms produce mammoth measures of vitamin D once presented to bright ultraviolet light (Wasser and Weis 1999).
The potential healthful worth and wide satisfactoriness of mushrooms have attracted increasing interest within the rummage around for biologically active substances from them (Wasser et al., 2005). Nowadays, consistent with the confirmation and elucidation of its medical specialty activities and clinical effects, mushrooms are used not solely as food however conjointly for clinical medication for the hindrance and treatment of varied sorts of diseases, like cardiovascular disease, blood disease and cancer (Lin et al., 2001).

1.3 Toxicology of Lead
Lead harmfulness has been radiant to mankind since prior period and said in reports left by the Greeks, Romans and Arabs. one among the most seasoned destructive operators heavenly to mankind is lead (Nawrot et al 2002). Lead is of general wellbeing concern in view of its unhealthful consequences for fluctuated creating organs, and steadiness in pregnant and newborn child nourishing moms. Lead is elegant inferable from its thick, bendable, moldable and erosion safe properties (Nawrot et al 2002). Lead might be an inescapable, natural and modern waste issue that has been identified in each side of ecological and organic framework. Lead occurs in nature as oxides or salts. It might be found in water funnels, pesticides, covering of unit wherever erosion obstruction and malleability ar required in refinery, in development, projectiles of firearms, X-beam and nuclear radiation assurance and might be a noteworthy modern side-effect. Lead appear in range in a few structures, as lead channeling, paints, fired glasses, base metal utensils and installations. Additionally cream, powder, lipstick and hair colorants all have lead. Rural soil pollution could likewise be responsible for lead found in some seasoning pharmaceuticals and cigarettes. The destructive impacts of lead on physiological, natural science and social dysfunctions are recorded in creatures and people (Stewart et al., 2006).
1.3.1 Causes of Lead Exposure
All sources of saturnism square measure ecological; nevertheless, the sources of lead square measure moderately varied. Lead-based paint remains the one most vital supply of lead exposure to youngsters within the US. However, the presence of lead is known as a supply of toxin impact for decade, not till 1977 once the patron product safety committee ordered that lead mustn’t be intercalary to housing paint. However, this didn’t address problems with deteriorating paint in older homes and use of leaded paint for exterior surfaces.Chipping, cleaning, and peeling lead paint is much away the main offer of lead presentation in children. In any case, elective wellsprings of lead amid a kid’s environment like soil, drink could end in intense saturnism or add to relate degree effectively hoisted blood lead level. Work surroundings
Grown-ups could wind up uncovered or bring lead mud home from their activity on pieces of clothing, hands, hair, and shoes. Occupations with presentation to guide grasp exchange or decorating; home redesign, article of furniture restoring; lead refining or mining (Bartrem et al., 2013); guns guidance; car repair; battery delivering or work (Vander Kuijip et al 2013). SoilIn spite of the fact that lead was completely eliminated of gasoline by 1995; lead particles radiated in motor fumes still proceed with the dirt near major roadways. Also, weakening outside lead paint could taint the dirt around past homes. Youths who are enamored with soil effortlessly get presented to lead and connection can to get it from their work environment. CeramicsLead is utilized in some ceramics coats as an after effect of it creates beyond any doubt hues. Improperly released coatings and breaking down coatings may filter lead into food and beverages, especially if contact is drawn out or if the sustenance is hot or acidic. The America Food and Drug Administration (FDA) has built up movement constrains on economically made or outside item, however carefully assembled things aren’t controlled. Plates purchased in another nation and things not assumed for nourishment utilize discharge great measure of lead. Lead solder
Soldering produces minute fragments and dust-sized particles of lead, as well as lead fumes. Solders with differ concentrations of lead are used in the electronics industry and in production of stained glass. Some people have used them to make fishing tackle or in home plumbing projects, which is illegal. Homemade moonshine stills may be soldered with lead, which can result in lead leaching into the drink (Holstege et al., 2004). Drinking waterMost public water sources are habitually tested and don’t go beyond EPA lead limit of however fifteen parts per billion (ppb). For beverage, the limit may be a smaller quantity than 5 ppb. However, water may become polluted if it meets with previous lead-soldered pipes or lead-containing faucet at intervals previous buildings. Lead levels are highest in water left standing in pipes for quite variety of hours and in hot or acidic water. Fishing tackleLead weights and sinkers square measure little and swish and simply enveloped by curious youngsters, particularly once they square measure imitating adults UN agency use their teeth to govern the tackle. Clothing/ toy jewelryShabby jewelry marketed to kids, here and there sold-out in selling machines, has been the arrangement of the many archived instances of intense ailment. Kids immediately unintentionally ingest them. Toy adornments that has lead could likewise be a legitimate unsafe substance; notwithstanding, such things is moreover available .Foreign gems is unbelievably suspicious for disease. Window ornament weights
Some window ornament weights are imaginary from lead and are of swallow able size. They are sewn into the fix of shades or window hangings.
1.3.1. 9 Mexican candies
Studies have found high levels of lead in many Mexican candies, especially those with tamarind or chili powder as an ingredient. The ink used to print the wrappers has also been shown to contain dangerous amounts of lead (stewart et al., 2006). Projectiles
Lead has been acclimated make shots since the mid15th century. Its boundless accessibility, physical property, and high thickness still make it perfect for this reason. Today, most slugs for shotguns, handguns, and rifles square measure produced using a lead center enclosed by a copper or steel coat to shield the lead from dynamical shape at high speeds. Conservative strong lead projectiles additionally are open, as square measure old lead gag loader balls. (Zahran et al., 2013).1.3.2 Mechanisms of Lead Toxicity
Lead exerts varied adverse mechanisms of toxicity. Lead contains a high affinity for sulfhydryl teams. Several of the poisonous effects additionally results from its inhibition of cellular operate requiring metallic element. Lead binds to metallic element-activated proteins with abundant higher (105 times) affinity than calcium.The interaction of lead and metallic element with cellular sites depends on the concentration of free ions gift (i.e., Pb2+ and Ca2+). Pb2+ and Ca2+ contend at the cell wall for transport systems, that have an effect on their entry or exit (i.e., Ca2+ channels and therefore the Ca2+ pump.) living thing Ca2+ is buffered by proteins, endoplasmic reticulum, and mitochondria; Pb2+ disturbs this living thing Ca2+ physiological state.Pb2+ interacts with variety of Ca2+-dependent effector mechanisms like calmodulin (a Ca2+ receptor super molecule, that couples to many enzymes, e.g., phosphodiesterase, super molecule kinases), super molecule enzyme C, Ca2+-dependent K+ channels within the cell wall, and neurochemical unleash (Bressler et al., 1999).1.3.3 Acute Symptoms of Lead Poisoning1.3.3.1 Neurological diseaseThe development of neurological disease is taken into account the foremost harmful lead connected hazard. The microvasculature of a child’s developing brain is unambiguously at risk of high-level lead toxicity, characterised by neural structure hemorrhage, redoubled barrier permeableness, and vasogenic puffiness (Stewart et al., 2006). PathologyThe cellular, object, and molecular mechanisms of lead neurotoxicity square measure numerous. Lead affects many life activities at wholly totally different levels of management, at the side of the voltage-gated channels and additionally the initial, second, and third mortal systems.
One study found that chronic activity exposure junction rectifier to atrophy and redoubled substantia alba lesions years when termination of the exposure in an exceedingly cohort of employees (Stewart et al., 2006). Total brain volume, frontal and total nervous tissue volume, and membrane bone substantia alba volume were found to be reduced. Lead additionally impacts the sensory system systema nervosum. Lead exposure damages physical phenomenon within the distal cranial nerve and therefore the sensory system pathway within the lower brain stem. Refined impairments of sensory system process might have profound effects on learning (Bressler et al., 1999).Traditionally, the fibre bundle disorder related to illness has been strictly a motor pathology. However, patients may note sensory and involuntary neuropathic options. it’s been deduced that the normal motor syndrome related to acute illness is a lot of doubtless to be a style of lead-induced hereditary condition instead of a right away toxin result of lead (Spanier et al.,2013). Nephropathy
Lead poisoning inhibits the proximal cannular lining cells. Abnormalities that will occur with lead toxicity embrace symptom, phosphaturia, and symptom (Fanconi syndrome). These effects are unit reversible. Acute style of renal disorder is a lot of oft reportable in kids. Gouty arthritis secondary to lead-induced renal disorder is often a long complication of activity lead exposure (Batrem et al.,2013).Chronic lead renal disorder, a chronic tubulointerstitial nephrosis on diagnostic test, happens within the setting of long lead exposure and is generally related to high blood pressure and gouty arthritis. diagnosing of chronic lead renal disorder is tougher since the laboratory abnormalities seen with acute lead intoxication aren’t gift with chronic lead exposure (Spanier et al 2013). Compromised protein functionLead has a sway on haem biogenesis, inflicting anemia at high blood levels; but, at low levels, Pb2+ causes blood disease (i.e., diminished mean somatic cell volume MCV and mean somatic cell haemoprotein MCH) and an antagonistic increase within the range of RBCs (Bressler et al 1999).When the Lead irrevocably binds to the sulfhdryl cluster of enzymes it causes terrible injury beyond control. The enzymes delta-aminolevulinic acid dehydratase, that speed up the production of the porphobilinogen ring, and ferrochelatase, which enables the incorporation of iron into the protoporphyrin ring, area unit of each of them are affected by lead. The reticence of those enzymes could commence with lead level which is as low as 5µg/dL. If ferrochelatase is repressed (as with lead toxicity) or inadequate iron is given, Zn is replaced for iron and Zn protoporphyrin concentrations will go higher. The key result of this impact is that the decrease in current levels of haemoprotein. Stainability stippling of erythrocytes is also given. Increased blood pressure According to Nawrot et al. (2002), “meta-analysis focusing in an epidemiologic analysis of the relationship between force per unit area and blood lead, established that this relationship was identical in male and female. Within the combined studies, a 2-fold increase in blood lead concentration was related to a one pressure unit rise within the blood pressure and with a zero pressure unit increase within the blood pressure”. Altered fruitful operateLead toxicity has been related to diminished fertility (Bressler et al., 1999). Men with an increase lead level tend to have low sperm counts and decrease in sperm motility. In females, abortion, change in body structure and still birth are associated with lead toxicity. Bone deformation
The deposition of lead in bone could have hepatotoxic effects on bone integrity. Skeletal development and therefore the regulation of skeletal mass square measure ultimately determined by the four differing types of cells: osteoblasts, lining cells, osteoclasts, and osteocytes. These cells, that line and penetrate the mineralized matrix, square measure to blame for matrix formation, mineralization, and bone organic process, below the management of each general and native factor.Systemic parts of regulation embody thyroid hormone. Native devices embody various cytokines and growth factors. Lead intoxication directly and indirectly alters several aspects of somatic cell operate.First, lead could indirectly alter somatic cell operate through changes within the current levels of these hormones, notably 1, 25-dihydroxyvitamin D3, that modulate somatic cell operate. Second, lead could directly alter somatic cell operate by inhibiting the power of bone cells to reply to secretion regulation. For instance, the 1,25-dihydroxyvitamin D3–stimulated synthesis of osteocalcin, a calcium-binding macromolecule synthesized by osteoblastic bone cells, is inhibited by low levels of lead. Impaired osteocalcin production could inhibit new bone formation, likewise because the useful coupling of osteoblasts and osteoclasts. Third, lead could impair the power of cells to synthesize or secrete alternative parts of the bone matrix, like albuminoid. Finally, lead could directly have an effect on or substitute for atomic number 20 within the active sites of the atomic number 20 traveler system, leading to loss of physical regulation (Zahran et al., 2013). Other abnormalities
Lead might also cause different signs and symptoms. Colic could be a characteristic options of chronic sickness and is related to obstinate constipation. The Burton line or animal tissue sounding line could be a blueness line on the gums, signifying sickness. It happens generally once sickness is related to poor oral hygiene (Nawrot et al., 2002).Lead results in activation of macromolecule enzyme C (PKC) and binds to Phosphase kinase C additional keenly than Calcium ion, its physiological substance (Bressler et al.,1999). This additional compounds the matter with neurochemical unleash represented higher than. Modifications of PKC perform additionally conciliations second-messenger systems in a body cell, resulting in additional variations in organic phenomenon besides macromolecule production. When the blood level is increase, Lead ion interrupts the formation of epithelial tissue cells within the system. This could end in hurt nervous disorder, characterised by seizures and coma (Bressler et al.,1999).

1.3.4 Pharmacokinetics of Lead
The pharmacological medicine of lead in humans is advanced. Humans are in an exceedingly state of positive lead balance from birth. Within the us, the typical blood lead concentration has been rumored at zero mg/L in kids aged one and zero.11 mg/L in kids aged five years. Duct concentration
The amount of lead concentration through the duct (Gastro intestinal) tract is varies. Kids are more prone to lead adsorption than matured individual. Lead absorption is based on many parameters as well as size consumed, the period of consumption, and therefore the organic process standing of the person eaten it.

Lead intake is reciprocally directly to the magnitude, the little the particles the more lead consumed. Thus, exposure to guide dirt ends up in more intake than exposure to the equivalent quantity of lead from chips of lead paint. Lead absorption is increased within the presence of iron, zinc, and Ca deficiency. Lead absorption is additionally increased by deficiency disease, with lead absorption shriveled if phosphorus, riboflavin, vitamin C, and fat-soluble vitamin are within the diet. Low-energy (calorie) intake and high-fat intake are related to increase lead absorption. Lead intake is reciprocally directly to chronologic age. In all, almost half of the lead consumed is absorbed in relation to 100 percent of that eaten by adults. Skin concentration
Cutaneous absorption of lead is proscribed (typically so much but 1%). the quantity absorbed through the skin depends on the physical characteristics of the lead (i.e. organic versus inorganic) and therefore the integrity of the skin. though inorganic lead isn’t absorbed through intact skin, organic lead compounds (i.e. tetraethyl lead) are absorbed (Nawrot et al., 2002). Inhalational absorption
If inhaled in an exceedingly fine particulate state, lead could also be absorbed directly through the lungs or could also be carried by the mucociliary tree to the throat, wherever it’s enclosed and absorbed via the GI system. the quantity of absorption of particulate lead that happens through the system depends on the particle size, the patient’s metabolic process volume, the quantity of deposition, and therefore the mucociliary clearance of the lead inhaled. The bulk (nearly 100%) of lead inhaled as vapor or fumes is absorbed directly through the lungs (Bressler et al.,1999).

a) BloodAfter absorption, lead enters the blood compartment. Lead within the blood is primarily found inside red blood cells (RBCs). Though the blood typically carries solely a little fraction of the entire lead body burden, it is the initial receptacle of absorbed lead and distributes lead throughout the body, creating it offered to different tissues or for excretion. The elimination half-life of lead in adult human blood has been calculable to be one month, whereas in youngsters it should be as high as ten months.Approximately ninety nine of the lead in blood is related to RBCs; the remaining 1 Chronicles resides in plasm that transfers lead between the various compartments. Blood lead is additionally terribly important as a result of the blood lead level (BLL) is that the most generally used live of lead exposure. The less sensitive blood corpuscle protoporphyrin (EP) assay is additionally used as a live of blood lead (Batrem et al., 2013).

b) Soft tissue
Lead moves quickly in and out of soft tissues. The blood distributes lead to various organs and tissues. Animal studies indicate that the liver, lungs, and kidneys have the greatest soft-tissue lead concentrations immediately after acute exposure. The brain is a site of distribution as well. Children retain more lead in soft tissue than adults do. Selective brain accumulation may occur in the hippocampus. Lead in soft tissues has an approximate half-life of 40 days (Zahran et al., 2013).

c) Mineralizing tissueMost maintained lead within the figure is ultimately deposited in bones. The bones and teeth of adults contain over ninetieth of their total lead body burden, and people of kids contain or so seventy fifth. Lead in mineralizing tissues isn’t uniformly distributed, with accumulation in bone regions undergoing the foremost active calcification at the time of exposure.Bone is viewed as a double compartment, with a comparatively shallow labile compartment (trabecular bone), wherever the elimination half-life is ninety days, and a deep inert compartment (cortical bone,) wherever the elimination half-life could also be 10-30 years. Teeth are also thought of a part of the terminal compartment. The labile part promptly exchanges bone lead with the blood, whereas lead within the inert part could also be hold on for many years. In times of physiological stress, the body will mobilize lead stores in bone, thereby increasing the amount of lead within the blood.Bone-to-blood lead mobilization will increase during times of gestation, lactation, menopause, physiological stress, chronic unwellness, adenosis, uropathy, fractures, and advanced age, and is exacerbated by atomic number 20 deficiency. Consequently, the ordinarily inert pool poses a special risk as a result of it’s a possible endogenous supply of lead that may maintain BLLs long when exposure has all over (Nawrot et al., 2002).

Most of the lead absorbed into the body is excreted either through renal clearance or through biliary clearance in the feces. The percentage of lead excreted and the timing of excretion depend on a number of factors. Significant drops in a person’s BLL may take several months, or sometimes years, even after complete removal from the exposure sources. It is important for doctors evaluating a patient with potential lead poisoning to examine potential current and past lead exposures and look for other factors that affect the toxicokinetics of lead (e.g., poor nutrition) (Batrem et al., 2013).

Maternal-fetal kineticLead pronto crosses the placenta, with the foetus retentive lead cumulatively throughout gestation. Specific health issues, like deficiency disease, might end in higher lead absorption within the mother. Elevated maternal lead levels afterwards end in higher lead distribution to the foetus (Zahran et al., 2013)
1.3.5 Chelation TherapyOnce lead has entered the body, particularly bone, it’s terribly troublesome to get rid of. Consequently, bar is that the mainstay of treatment. However, chelations medical care is also accustomed decrease the blood lead concentrations acutely. The ultimate part of treatment is chelation medical care. Chelating agents bind metals at 2 or a lot of sites. Ideally, the chemical process metal would be excreted; but, the lead:chelate complicated might act tissues wherever the binding occurred or be decentralized to alternative tissues. An optimal chelating drug ought to increase lead excretion, be administered simply, and be cheap and safe. Lead removal ought to halt additional toxicity and reverse previous effects (Makrowitz , 2000).Several chelating agents area unit effective in lead excretion, however the chelator of alternative depends on the blood lead concentration, the patient’s symptoms and therefore the environmental lead burden. Symptomatic patients ought to be hospitalized and placed on chelation medical care commenced with Edetate metal Disodium (CaNa2EDTA). Edetate metal disodium, used alone, might irritate symptoms in patients with terribly high blood lead levels. Once clinical symptoms in keeping with saturnism or once blood lead levels area unit larger than seventy µg/dl, it’s suggested that edetate metal disodium be employed in conjunction with dimercaprol(Chisolm, 1971). Oral chelating agents area unit accessible for treatment of lead poisoned patients United Nations agency have elevated blood lead concentrations and area unit symptomless. Within the u. s., 2,3 dimercaptosuccinic acid (DMSA, Succimer) is that the drug most ordinarily used. Alternative oral agents that will be used are unit DMPS (Unithiol) and medicine.

1.3.6 Examples of Lead Chelating Agents 2,3 Dimercaptosuccinic Acid (DMSA, Succimer)Succimer is an orally active chelating agent that’s usually used for the treatment of blood lead concentrations higher than forty five µg/dl within the u. s.. It’s a water soluble analog of dimercaprol. However, it’s a large vary of therapeutic live and has benefits over dimercaprol and CaNa2EDTA.Succimer could be a four carbon molecule with 2 carboxyl teams and 2 sulfur teams. Lead and metallic element bind to connected sulfur and atomic number 8 atoms whereas arsenic and mercury bind to each sulfur atoms leading to a pH scale dependent soluble compound. The pharmacology of succimer has been assessed in primates and humans. In primates, the absorption has been shown to be speedy with the time to peak concentration occurring inside 1-2 hours.DMSA has been found to be, primarily, simple protein sure in plasma through a disulfide bond with amino acid with little remaining unbound. it’s unknown if macromolecule sure DMSA is ready to bind lead (Maiorino et al.,1990) whereas DMSA is primarily distributed within the extravascular area, mechanical primate models have shown that the degree of distribution is larger than plasma volume and calculable to be zero.4 L/kg (Maiorino et al.,1990). DMSA is metabolized in humans to mixed disulfides of amino acid. Solely 2 hundredth of the administered dose is eliminated unchanged within the pee once oral dosing compared to eightieth once blood vessel dosing however unclean elimination (nonabsorbed drug and biliary elimination) wasn’t assessed (McGown et al., 1984). Additionally, enterohepatic recirculation of the parent compound and its metabolites square measure suspected to occur (Asiedu et al., 1995). The bulk of the elimination happens inside twenty four hours and as DMSA-cysteine disulfide conjugates (Maiorino et al., 1990). Excretory organ clearance is larger in healthy adults than in youngsters or adults with unwellness (Dart et al., 1994). The elimination half-life in mechanical primates is thirty five and seventy minutes for the parent and parent and metabolites, severally (Asiedu et al., 1995).Utilization of DMSA for chelation treatments has resulted in few adverse effects. Varieties of studies have assessed the impact of DMSA on different metals. The sole essential metal that has systematically been found to be adversely laid low with DMSA is metal. Different adverse reactions related to the utilization of succimer embody popular rash, pruritis and body covering reaction.Use ought to be primarily reserved for kids with AN elevated blood lead concentration bigger than forty five µg/dl or in symptomatic children at lower concentrations. Hypersensitivity to DMSA could be a reason to its use (Guidotti ; Ragain, 2007). Racemic-2,3-dimercapto-1-propanesulfonic acid (DMPS)DMPS could be a chelating agent that’s associated with dimercaprol and DMSA. It water soluble and is reported to be less cytotoxic than dimercaprol. It’s accessible for oral, blood vessel and contractile organ use for the treatment of mercury, arsenic, lead, Cr and copper (Wilson’s disease) poisoning. Currently, it’s not government agency approved within the u. s., however is employed additional normally within the Union of Soviet Socialist Republics and Europe (Maiorino et al., 1996).The pharmacokinetic knowledge is out there because of the long-standing use of DMPS within the Union of Soviet Socialist Republics and Deutschland (Aposhian et al., 1992; Hurlburt et al., 1994; Maiorino et al., 1996). Also, DMPS is dispensed extracellularly and, to a smaller extent, intracellularly. It’s found to be bigger than eightieth sure by macromolecule, chiefly simple protein, within the plasma and is likely to be extremely stable prolonging the significant metal mobilizing activity. Oral DMPS seems to be less effective because the oral bioavailability is hr (Hurlburt et al., 1994). The elimination half-life is longer once blood vessel dosing (20 hours compared to nine.5 hours once oral dosing) and is likely to result to first-pass metabolism within the duct. DMPS undergoes excretory organ excretion with forty six to fifty nine of the dose detected within the pee once twenty four hours of dosing (Aposhian et al.,1992).Different dosing is needed betting on the significant metal toxicity. As DMPS is primarily used for the treatment of arsenic and/or poisoning. Oral doses of two hundred to four hundred mg in 2-3 divided doses increase the mercury excretion and cut back the body burden in adults. DMPS has been shown to be effective once copper levels square measure elevated and has been treated as single oral dose of three hundred mg daily or one hundred mg thrice daily for up to fifteen days in adults. Very little knowledge is out there concerning its use in youngsters. However, for the treatment of unwellness in youngsters, the oral daily dose of two hundred to four hundred mg per meter square of body extent has been used safely (Chisholm et al., 1971).The safety of DMPS has for the most part been assessed with blood vessel dosing. Common adverse reactions that have occurred in patients treated for significant metal poisoning embody nausea, vomiting, headache, fatigue, rash, and pruritis (Aposhian et al., 1983). Additional severe rash and hypersensitivity reaction reactions have occurred, however additional normally in patients with a history of aversions. No nephrotoxicity has been determined, however caution is usually recommended in patients with excretory organ impairment because the parent compound and significant metal complexes square measure eliminated within the pee. PenicillaminePenicillamine could be a D-?, ?-dimethylcysteine, an antibiotic drug degradation product. it’s a potent gold, lead, mercury, metal and copper chelator and is that the drug of alternative for treating Wilson’s sickness. it’s been in use since 1957 for the treatment of unwellness and was the sole oral chelator for lead till the provision of DMSA (Beattie et al., 1977). However, it’s not government agency approved within the u. s. Its sulfhydryl cluster combines with result in kind ring compounds thereby increasing elimination. Additionally, it’s been accustomed treat cystinuria and rheumatic disorders.Penicillamine is absorbed quickly, however has an oral bioavailability of forty to seventieth. It’s not dose dependent. Food, antacids, and iron decrease absorption. The height concentration happens inside one to three hours despite the dose. Cuprimine forms disulphide bonds with several proteins within the blood and tissues, making potential slow-release reservoirs of the drug (Hanna, 2005). Solely a tiny low portion of the parent compound is metabolized within the liver to S-methylpenicillamine. The first route of elimination is thru the kidneys. The elimination half-life of unchanged Cuprimine after single dosing ranges from 1.6 to 3.2 hours (Kuntzelman, 2005). Once a gradual state concentration is obtained, the elimination is prolonged (4 to six days) suggesting a slow unharness from deep tissues and skin.The dose for Cuprimine was, largely, established throughout the treatment of toxicity from different significant metals like arsenic and copper. AN early case report documented the effectiveness of D-penicillamine in 3 youngsters with arsenic poisoning treated with four daily doses of twenty five mg/kg/dose (Paterson et al., 2006). the quality dose for the treatment of unwellness used similar daily dosing at twenty five to thirty mg/kg/dose for many months (Shannon et al., 1988). Currently, the foremost normally used dose within the u. s. is thirty to forty mg/kg/day or 600 to 750 mg/square meter/day for one to six months, given a pair of hours before or three hours once meals.Since the introduction of Cuprimine, its use has been restricted because of the numerous adverse effects that result. This has crystal rectifier to the event of the thiol chelators (DMSA and DMPS) that square measure thought-about safer alternative. Early studies of Cuprimine within the treatment of Wilson’s sickness resulted in adverse reactions that were thanks to deficiency disease like skin lesions on pressure points, desquamations, delayed wound healing, phalacrosis and typically rubor, and redness (Kuntzelman, 2005).

1.4 GanodermaGanoderma may be a genus of exhausting, bitter mushroom that’s accustomed promotes health and longevity in ancient Chinese drugs. Proponents claim that Ganoderma will relieve fatigue, keep sterol under control, curb high pressure, tame inflammation, build stamina and support system. An increase in natural remedy, Ganoderma is employed as healthful mushroom (Wang et al., 2012).Ganoderma shows promise in reducing sterol level and easing hypersensitivity reaction connected inflammation of the airways, in step with preliminary proof from animal primarily based studies (Ajith et al., 2007). Some types of Ganoderma species embody Ganoderma lucidum, Ganoderma applanatum, and Ganoderma tsuage.

1.4.1 Ganoderma lucidumTaxonomyKingdom: FungiDivision: BasidiomycotaClass: AgaricomycetesOrder: PolyporalesFamily: GanodermataceaeGenus: GanodermaSpecies: G. lucidumGanoderma lucidum, is an oriental fungus. It’s an extended history of use for promoting health and longevity in China, Japan, and different Asian countries. It’s an outsized, dark mushroom with a shiny exterior and a woody texture. The Latin word lucidus suggests that “shiny” or “brilliant” and refers to the stained look of the surface of the mushroom. In China, G. lucidum is termed “lingzhi”, whereas in Japan the name for the Ganodermataceae family is “reishi” or “mannentake”.In Chinese, the name “lingzhi” represents a mixture of religious efficiency and essence of immortality, and is thought to be the “herb of religious efficiency,” symbolising success, well-being, divine power, and longevity. Among cultivated mushrooms, G. lucidum is exclusive in this its pharmaceutical instead of biological process price is predominant. a spread of economic G. lucidum product ar accessible in numerous forms, like powders, dietary supplements, and tea. These ar created from totally different components of the mushroom, together with mycelia, spores, and fruit body. the precise applications and attributed health advantages of “lingzhi” embody management of blood sugar levels, modulation of the system, hepatoprotection, organic process, and more. the assorted beliefs concerning the health advantages of G. lucidum ar primarily based for the most part on anecdotal proof, ancient use, and cultural mores. However, recent reports offer scientific support to a number of the traditional claims of the health advantages of “lingzhi.1.4.2 Ganoderma lucidum as an ethnomedicinal mushroomGanoderma lucidum has been recognized as a healthful mushroom for over 2000 years, and its powerful effects are documented in ancient scripts (Wasser et al., 2005). The mushroom is ascribed therapeutic properties, like tonifying effects, enhancing very important energy, strengthening viscus operate, increasing memory, and antiaging effects. in step with the state book of the People’s Republic of China (2000), G. lucidum acts to ease the mind, and relieve cough and respiratory illness, and it’s suggested for giddiness, insomnia, palpitation, and shortness of breath.Wild type of the mushroom ar rare, and within the years before it had been cultivated, solely the Nobles might afford it. it had been believed that the sacred flora grew within the home of the immortals on the “three aisles of the blest” off the coast of China (McMeekin, 2005). However, its name as a remedy might are earned a lot of by virtue of its irregular distribution, rarity, and use by the wealthy and privileged members of Chinese society than by its actual effects. notwithstanding, the Ganoderma species still be a well-liked ancient drugs in Asia and their use is growing throughout the planet (Sanodiya et al., 2009).

1.4.3 Botanial description of Ganoderma lucidum
The family Ganodermataceae describes pore mushroom fungus having a double-walled spore (Donk, 1964). Ganoderma species ar found everywhere the planet, and totally different characteristics, like form and color (red, black, blue/green, white, yellow, and purple) of the fruit body, host specificity, and geographical origin, ar accustomed determine individual members of the species (Zhao and Zhang, 1994). Some taxonomists additionally think about macromorphological options to be of restricted worth within the identification of Ganoderma species thanks to its high makeup physical property (Zhao and Zhang, 1994). Additional reliable morphological characteristics for Ganoderma species ar thought to incorporate reproductive structure form and size, context color and consistency, and also the microanatomy of the pilear crust. Spore production and form, catalyst studies and, to a lesser extent, the vary and optima of growth temperatures have additionally been used for differentiating morphologically similar species (Gottlieb et al., 1998).

Figure 1.1: The mushroom Ganoderma lucidum in its natural habitat.
1.4.4 Major bioactive components of Ganoderma lucidum
Most mushrooms area unit are made ninetieth water by weight. The remaining 100% consists of 10–40% supermolecule, 2–8% fat, 3–28% macromolecule, 3–32% fiber, 8–10% ash, and a few vitamins and minerals, with metal, calcium, phosphorus, magnesium, selenium, iron, zinc, and copper accounting for many of the mineral content (Borchers et al., 1999). Additionally to those, mushrooms contain a large sort of bioactive molecules, like terpenoids, steroids, phenols, nucleotides and their derivatives, glycoproteins, and polysaccharides. Mushroom proteins contain all the essential amino acids and area unit particularly made in essential amino acid and essential amino acid. The little fat and high quantity of unsaturated fatty acids relative to the whole fatty acids of mushrooms area unit thought of important health factor of mushroom. Polysaccharides, peptidoglycans, and triterpenes are 3 major physiologically active constituents in G. lucidum (Boh et al., 2007). However, the number and proportion of every element are often diverse in natural and business merchandise.1.4.5 Previous studies on pharmacotherapeutic uses of Ganoderma lucidumThe combination of advantages while not toxicity represents the specified consequence within the development of effective therapeutic interventions. G. lucidum has been used for many years as a health promotion and treatment strategy; there are currently several revealed studies that support animal and cell culture models and on in vitro evaluation of the health effects of G. lucidum, and there also are some reports of human trials within the field. However, there’s no cohesive body of analysis, and therefore the objective analysis of this ancient medical aid in terms of human health remains to be clearly established. G. lucidum exerts therapeutic impact on cancer cells, and is effective within the treatment of infective agent and microorganism infections moreover as polygenic disorder and liver injury (Yurkiv et al., 2005). CancerCancer may be a major global explanation for death; it remains a serious clinical challenge (WHO, 2008). As a part of checking out new chemopreventive and chemotherapeutical agents, many plant species, together with mushrooms, are evaluated. This has resulted within the isolation of thousands of bioactive molecules that were shown to own anticancer activity from various mushroom species, together with Ganoderma species. In G. lucidum, an oversized variety of chemical compounds are often extracted from the plant organ, mycelia, or spores. several polysaccharides and triterpenes, the 2 major teams of parts within the mushroom, exhibit chemopreventive and/or tumoricidal effects, as proved by various studies from in vitro experiments and animal and human in vivo studies. Tumor implanted animal models have shown restrictive effects on growing and metastasis. However, proof from well-designed human trials continues to be scarce. ImmunomodulationThere is hefty proof to support the immune stimulating activities of G. lucidum via induction of cytokines and improvement of immunologic effector (Wang et al.,1997). completely different parts from G. lucidum were proved to reinforce the proliferation and maturation of T and B lymphocytes, lienal mononucleate cells, NK cells, and nerve fiber cells in culture in vitro and in animal studies in vivo (Bao et al., 2001; Cao and sculptor, 2002). Wang et al., (1997) discovered that “a polysaccharide-enriched proportion from G. lucidum activated civilised macrophages and T-lymphocytes in vitro, that semiconductor diode to a rise of IL-1?, TNF-?, and IL-6 within the matter”. Moreover, serum-incorporated treatment with a carbohydrate amide fraction from G. lucidum markedly repressed the proliferation of human respiratory organ cancer cells, whereas the pure fraction by itself didn’t induce similar effects (Cao and sculptor, 2004). Ganoderma lucidum as an antioxidant
Consumption of antioxidant-rich plants could facilitate forestall cancer and different chronic diseases (Collins, 2005; Benzie and Wachtel-Galor, 2009). Antioxidants shield cellular elements from aerobic harm that is probably going to decrease the danger of mutations and carcinogenesis and conjointly shield immune cells, permitting them to take care of immune police investigation and response. Varied elements of G. lucidum, especially polysaccharides and triterpenoids, show inhibitor activity in vitro (Yuen et al., 2008; Chinese et al., 2009). Antioxidants from the mushroom were found to be absorbed quickly once bodily function, leading to a rise within the plasma total inhibitor activity of human subjects (Saito, 2003). Infectious agent and microorganism infectionsThe goal of analysis within the treatment of infectious agent and microorganism infections is that the discovery of agents that specifically inhibit infectious agent and microorganism multiplication while not poignant traditional cells. The unsought aspect effects of antibiotics and antivirals, and also the look of resistant associated mutant strains create the event of recent agents a pressing demand. This has light-emitting diode researchers to analyze the medicament and antiviral activity of medicative plants and fungi (Wasser and Weis, 1999). Some triterpenes from G. lucidum have conjointly been according to possess associate repressing result against human immunological disorder virus (HIV)-1 peptidase activity, with IC50 values starting from twenty to over a thousand ?M however not all of the examined triterpenes showed anti-HIV activity (Min et al., 1998). In further study, a ganoderic acid cut-off from G. lucidum showed repressing effects on the replication of serum hepatitis virus (HBV). Production of HBV surface matter (HBsAg) and HBV e-antigen (HBeAg) were, severally, two hundredth and four hundred and forty yards of controls once ganoderic acid treatment isn’t gift (Yurkiv et al., 2005 ). DMComponents of G. lucidum are evidenced to possess symptom result in primates. The administration of ganoderans A and B (dose of a hundred mg/kg), 2 polysaccharides isolated from fruit-body water extracts, by i.p. injection to traditional and alloxan-induced diabetic mice considerably shriveled (by up to 50%) the plasma aldohexose concentrations, and also the symptom result was still evident once twenty four hours (Hikino et al., 1985). employing a mouse model, ganoderan B was conjointly according to extend plasma hormone, decrease viscus animal starch content, and modulate the activity of glucose-metabolizing enzymes within the liver (Hikino et al.,1989). identical cluster according that a 3rd saccharide (ganoderan C) isolated from G. lucidum conjointly showed important symptom effects in mice, which ganoderan B hyperbolic plasma hormone levels in each traditional and glucose-loaded mice (Hikino et al., 1989; Tomoda et al., 1986).In one study, seventy one adult patients with confirmed kind two DM were supplemented with Ganopoly (polysaccharide fractions extracted from G. lucidum). The patients received either Ganopoly or placebo orally at 1800 mg, thrice daily for twelve weeks. Glycosylated hemoprotein (HbA1C) and plasma aldohexose shriveled considerably once twelve weeks, indicating a symptom result of the extract (Gao et al., 2004). Overall, the information from totally different studies counsel that G. lucidum intake helps in modulating blood sugar levels. However, the studies were performed largely in animals. additional support from well-planned human clinical studies is required with and while not combination with typical medicines. Liver and internal organ injuryHigh temp water and water–ether concentrates of the fruit body of G. lucidum were found to possess a potent hepato-protective result on liver injury evoked by perchloromethane (CCl4) given orally and intraperitoneally to rats (Lin et al., 1995; Kim et al., 1999). The measured markers for liver injury enclosed aspartate and aminoalkanoic acid transaminases (AST and ALT) and bottle-feed dehydrogenase (LDH). One active compound of the extract was separated and known as ganoderenic acid A. This was found to possess a potent repressing result on ?-glucuronidase, and also the authors counsel that this repressing result could have mediate the hepatoprotection seen once this isolated compound was given (Kim et al., 1999). Protection was conjointly according in an exceedingly study during which a predicament extract of G. lucidum was given orally to mice half-hour before administration of fermentation alcohol. The extract was found to possess associate repressing result against the formation of malondialdehyde (MDA), a degradation product of lipoid peroxides, in mouse liver and urinary organ stuff, with proof of a dose dependent (Shieh et al., 2001). The MDA result was conjointly according by Shi et al. (2008) once the extract was given orally to mice (at sixty, 120, and a hundred and eighty mg/kg/day) for two weeks before treatment with D-galactosamine, that evoked viscus injury. Additionally, pretreatment with G. lucidum maintained traditional values of AST, ALT, SOD (superoxide dismutase), and GSH (reduced glutathione) (Shi et al., 2008). Therefore, hepatoprotection may be mediate by the radical-scavenging properties of G. lucidum. Lin et al. (1995) discovered that predicament extracts of G. lucidum shows an important radical-scavenging activity against each superoxide and radical radicals.Further, G. lucidum methanolic extract was according to point out viscus protection. The extract was given orally to rats (500 mg/kg/day) for thirty days before viscus harm was evoked by benzo (a) pyrene (Lakshmi et al., 2006). The extract prevented the rise of body fluid AST, ALT, and alcalescent enzyme (ALP) activities that result from benzo(a)pyrene challenge, and increased the amount of GSH, SOD, GpX (glutathione peroxidase), CAT (catalase), and glutathione S-transferase (GST). The discharge of a hepatoprotective element from G. lucidum plant structure was conjointly according by Song et al. (1998). Polysaccharides extracted from G. lucidum and given orally to rats for twenty eight days were found to ameliorate cirrhosis of the liver evoked by biliary ligature (Park et al.1997). With the exception of its effects on chemically-induced liver injury, the results of the mushroom on internal organ injury have conjointly been investigated. internal organ ulcers were evoked in rats by carboxylic acid (Gao et al., 2004), and treatment with GL-PS fractions of zero.5 and 1.0 g/kg for fourteen days considerably accelerated the ulceration healing by four-hundredth and fifty six, severally. Treatment with one.0 g/kg extract considerably fixed up secretion and autocoid levels compared with the management cluster.

1.5.0 Rationale for the Study and Hypothesis
Heavy metal toxicity occurs often and the main mechanism of action is by inhibition of calcium through the binding of moieties and generation of free radicals
The current chelating agents are very toxic, costly and are often not available Ganoderma lucidum is available as an antioxidant nutracetical and it is possible that its consumption will prevent lead induced toxicity
It is hypothesized that antioxidant constituents present in the aqueous extract of the mushroom can prevent lead induced toxicity in rats.

1.6.0 Aim and Objectives
The aim of the study was to evaluate the consequences of liquid extract of G. lucidum in lead induced toxicity in rats.The objectives include to:1. determine the secondary metabolites within the extract2. Assess the intense toxicological profile of the extract3. Evaluate the consequences of the extract on lead induced toxicity4. Determine the possible mechanism for the observed effects.

2.1.1 Mushroom and Method of Extraction
Ganoderma lucidum was obtained from the Senior Staff Quarters of University of Benin, Benin City in September 2016. The mushroom was identified by Prof (Mrs) Angela Ejale. The identification of the mushroom was further authenticated by Prof John Okhuoya, the Director of Mushroom Research Center, University of Benin, Benin City, Edo State. Adulterants were carefully picked out and the mushroom was thoroughly rinsed in tap water, cut into smaller pieces and allowed to dry in the sun for five days. Thereafter, the mushroom was ground into powder. The aqueous extract of the mushroom was prepared by adding 1000 g of powdered mushroom to 1000 ml of water and boiled for 30 min. The mixture was filtered with a plain white handkerchief. The filtrate was allowed to cool, turned into a crucible and concentrated over a water bath to obtain the extract. The solid black concentrated extract was stored in a refrigerator and a known quantities were reconstituted in distilled water before administration according to the experimental procedure.
2.1.2 Animals
Adult Wistar rats (180-250 g) of either sex were obtained from the Animal Unit, Ambrose Alli University, Ekpoma, Nigeria. They were allowed time period adjustment before they were willy-nilly assigned to teams. They were put in commonplace (males break away females) and allowed access to rat pellets (Top Feeds and Flour Mills restricted, Nigeria) and water impromptu. Animals were exposed to natural light-weight and temperature (27.0 ± 2.0oC). The animals were handled consistent with commonplace protocols for the employment of laboratory animals (National Institutes Of Health USA; Public Health Service Policy On Human Care and Use of Laboratory Animals, 2002). The study was approved by institutional moral committee with reference EC/FP/018/24, University of Dahomey, Benin City, Nigeria.

2.1.3 Drugs and Chemicals
Chemicals and reagents were of analytical grade and were obtained from internationally known suppliers such as Sigma-Aldrich (USA) and BDH (UK). Only freshly prepared solutions and reagents were used. Lead acetate was procured by Nigerian-German Chemicals Plc (Nigeria). 2,3-dimercarptosuccinic acid ( succimer) was manufactured by Sigma-Aldrich (USA). Drug solutions were freshly prepared and stored in a refrigerator before administration.

2.2 Methods
2.2.1Phytochemical Screening
The whole extract (2 g) was dissolved in 20 ml of water. The resulting solution was subjected to preliminary phytochemical screening using conventional methods (Sofowora, 1993). Test for alkaloids (Wagner’s reagent)A fraction of extract was treated with 3-5 drops of Wagner’s chemical agent (1.27 g of iodine and a couple of g of iodide in one hundred milliliter of water) and sepia precipitate indicating the presence of alkaloids occured. check for internal organ glycosides (Keller-Kelliani’s test)The extract was treated with a pair of milliliter of glacial carboxylic acid in a very tubing and a drop of metal chloride resolution was additional thereto. This was fastidiously treated with one milliliter of focused oil of vitriol. A darker ring at the interface showed the presence of deoxysugar characteristic of cardenolides. check for flavonoids (Alkaline chemical agent test)The extract (2 ml) was treated with few drops of 2 hundredth hydroxide resolution on addition of dilute acid, there was no color modification to the extract that indicate the absence of flavonoids. check for phenols (Ferric chloride test)A volume of {two} milliliter of the extract was treated with two drops of binary compound five-hitter metal chloride and a blue deep coloration indicated the presence of synthetic resin compounds. check for saponins (Foam test)To a pair of milliliter of the extract was additional vi milliliter of H2O in a very tubing. The mixture was jolted smartly and therefore the look of persistent foam confirmed the presence of saponins. check for sterols (Liebermann-Burchard test)To one milliliter of the extract was additional 2 drops of chloroform, anhydride and conc. H2SO4 and green colored resolution was shaped that indicated the presence of sterols.

2.2.2 Oral acute toxicity study
Oral median lethal dose (LD50) of the extract was determined using Lorke’s (1983) method. In the first phase, 3 groups of 3 rats each were administered 10, 100, 1000 mg/kg of the extract orally. The second phase involved 3 groups of 1 rat each administered 1600, 2900 and 5000 mg/kg of the whole extract orally. In both phases the animals were monitored for gross toxicological symptoms such as locomotion, convulsion, piloerection, constipation, writhing, diarrhea, tremor and mortality continuously within the first two hours; then 30 min every 8 h for a period of 48 h; and for 30 min each day for 14 days.

2.2.3 Screening for preventive and curative effects of aqueous G. lucidum extract on lead toxicity
The treatment protocol for the preventive effect of G. lucidum aqueous extract is shown in Table 2.1. Thirty five rats were randomly allotted to seven groups of 5 animals each. Group 1 rats were given 2 ml/kg of distilled water. Group 2 was given lead acetate (100 mg/kg/day) for the period of 25 days. Groups 3, 4 and 5 were given lead acetate (100 mg/kg/day) and extract of G. lucidum at doses 100 mg/kg/day, 200 mg/kg/day and 400 mg/kg/day respectively for 25 days. Group 6 was administered lead acetate (100 mg/kg/day) and 50 mg/kg/day of 2, 3-dimecarptosuccinic acid (DMSA) (El-Tantaway, 2015) for the 25 days. Group 7 was given lead acetate (100 mg/kg/day), 400 mg/kg/day of the extract and 50 mg/kg/day of DMSA (Flora et al., 2007) for 25 days.
Table 2.1. Treatment protocol for preventive effect of G. lucidum aqueous extract
(mg/kg/day p.o.)
1 (Control) Distilled Water 25 5 –
2 Lead acetate 25 5 100*
3 Lead + Extract 25 5 100 + 100
4 Lead + Extract 25 5 100 + 200
5 Lead + Extract 25 5 100 + 400
6 Lead + Succimer** 25 5 100 + 50
7 Lead + Succimer + Extract 25 5 100 + 50 + 400
*El-Tantawy (2015); **Succimer =2, 3-dimercapto succinic acid, Flora et al (2007).

For curative result of the extract fifteen rats of either sex were haphazardly assigned into 3 teams of five animals every. The 3 teams were treated with one hundred mg/kg/day of acetate just for fourteen days. Thereafter, the animals were treated with G. lucidum extract two hundred and four hundred mg/kg/day for subsequent fourteen days. solely body fluid lead level was assayed. Changes in body weight
The percentage change in the weight of the treated animals was determined by using the formula:
Final weight – Initial weightx 100
Initial weight Tissues and blood sample collection
Under chloroform anaethesia, blood was obtained through the abdominal aorta by means of hypodermic needles and syringes and distributed into plain and EDTA-containing sample bottles. The EDTA sample bottles were for the evaluation of haematological indices while the plain sample bottles were for biochemical assays including alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP); electrolytes (Na+, K+, Cl-, HCO3-); urea and creatinine; lipids and antioxidants. Contents of plain bottles were allowed to clot before centrifuging at 3500 rpm for 10 min. Sera were decanted into new plain tubes.
Kidney, liver, and spleen were excised from the animals. The organs were weighed and homogenized and the homogenates were then centrifuged at 3500 rpm for 10 min. The resulting supernatants were stored in a freezer. Assay of lead concentration in tissues homogenate
Principle OF Flame Atomic Absorption Spectroscopy
Atomic Absorption Spectrophotometry is style to see the quantity (concentration) of AN object part in a very sample, utilizing the development that the atoms within the state absorb the sunshine of characteristic wavelength passing through AN atomic vapor layer of the part. The technique of flame absorption spectrographic analysis (FAAS) need a liquid sample to be aspirated, aerosolised and mixed with flamable gasses, like ethyne and air, or ethyne and gas. The mixture is enkindled in a very flame whose temperature ranges from 2100 to 28000C. throughout combustion, atoms of the part of interest within the sample ar reduced to free, unagitated state atoms, that absorb light-weight at characteristic wavelengths (Haswell, 1991).In their elemental kind, metals can absorb actinic ray once they ar excited by heat. every metal features a characteristic wavelength which will be absorbed. The sample of intrigue is aspirated into the fire. If that metal is gift within the sample, it’ll absorb a number of the sunshine, therefore reducing its intensity. a tool known as a photomultiplier detects the quantity of reduction of the sunshine intensity thanks to absorption by the analyte, and this may be directly associated with the quantity of the part within the sample. A pc information system converts the amendment in intensity into AN absorbance. The characteristic wavelengths ar part specific and correct to zero.01 – 0.1 nm. It measures the concentration of metals within the samples in mg/L vary (Haswell, 1991).

A volume (10 ml) of nitric acid and hydrogen perchloride in a ratio of 5: 2 was introduced into a chronical flask with 0.5 ml of the tissue homogenate. On heating in the fume chamber for 30 – 45 min, the coloured fumes gradually disappeared and the volume of the sample with the mixed acid reduced to about half of the original volume with the flask becoming clear. The digested sample was filtered using Whatman filter paper (No 1 size) into a 100 ml standard flask. It was finally marked up with distilled water before the lead assay was carried out using flame atomic absorption spectrophotometer (Haswell., 1991) and the data obtained was recorded in ppm. Assay of serum enzymes Serum Aspartate Aminotranferase (AST)
Serum salt – salt transferase assay (SGOT or AST) was done by the tactic of Reitman and Frankel’s, (1957). This methodology is predicated on the principle that salt is created from the reaction below:? – Oxoglutarate + L- aspartate L -glutamate + salt Aspartate transferase activity was measured by observance the concentration of salt hydrazone fashioned with two, four – dinitrophenyl reducer.Procedure The AST substrate (0.5 ml) was pipetted into each the chemical agent blank (B) and take a look at sample (T), take a look at tubes severally. The humour sample (0.l ml) was another to the take a look at sample (T) tube solely and mixed completely. Then 0.l cc of H2O was another to the chemical agent blank (B). the whole reaction medium was well mixed and incubated for 30minutes during a water tub at 37?C. at once once incubation, 2, four – dinitrophenyllydrazine chemical agent (0.5 ml) was another to the chemical agent blank (B) and therefore the take a look at sample (T) tubes, mixed completely and allowed to face for precisely twenty minutes at temperature. Finally, hydrated oxide answer (5.0 ml) was another to every of the chemical agent blank and therefore the sample take a look at tubes and mixed completely. The Absorbance of sample (Asample) was browse against the chemical agent blank once five minutes at 405nm. Serum alanine aminotransferase (ALT) Serum altitude assay per Reitman and Frankel (1957) methodology is predicated on the principle that pyruvate is created from the reaction below:? – Oxoglutarate + L- aminoalkanoic acid L -glutamate + Pyruvate ProcedureThe altitude substrate phosphate buffer (0.5 ml) was pipetted into 2 sets of take a look at tubes labelled B (Sample Blank) and T (sample test) severally. The humour sample (0.1 ml) was another to the sample take a look at (T) solely and mixed properly. The tubes were then incubated for precisely half-hour during a water tub at a temperature of 37?C. 2, 4-Dinitrophenylhydrazine (0.5 ml) was another to the take a look at tubes at once once the incubation. conjointly humour sample (0.1 ml) was another to the sample blank (B) solely. the whole medium was mixed completely and allowed to face for precisely 20min at temperature. afterward, 0.4N hydrated oxide (5.0ml) was another to the take a look at tubes and conjointly mixed completely and therefore the absorbance browse at 405nmc. Alkaline Phosphatase (ALP) AssayThis was supported King and type (1957) methodology that is predicated on the principle that humour alkalescent enzyme hydrolyses a colourless substrate of laxative monophosphate giving rise to oxyacid and laxative, that at alkalescent pH price turns into a pink color which will be photometrically determined. ProcedureDistilled water (1.0ml) was pipetted into 2 sets of take a look at tubes labelled militia (Sample) and ST (Standard) severally. Then one drop every of the compound substrate (2-Amino-2-methyl-1-propanol) was another to the H2O in every of the 2 sets of take a look at tubes. Their contents were mixed and incubated at 37?C for twenty min during a water tub. thenceforth one.0 cc of phenol was another to the quality tubing (ST) solely, followed by the addition of the humour sample (0.1 ml) to the sample tubing (SA). The contents of the tubes were conjointly mixed severally and incubated at 37?C for two min during a water tub. 5.0 cc of a color developer was another to every of the take a look at tubes. Absorbance of the sample against the blank (water) was browse at a wavelength of 550nm. The activity of alkalescent enzyme within the humour was obtained from the formula below.ALP (U/L) = Where SAOD = Sample optical density.STOD = customary optical density Assay of serum electrolytes.

a. Blood Urea Nitrogen (BUN)
The bodily fluid carbamide was measured by the changed Berthelot methodology (Young, 1995). This methodology is predicated on the principle that carbamide in bodily fluid is hydrolysed to ammonia within the presence of enzyme. The ammonia is then measured photometrically. ProcedureTo carry out this assay, duplicates of 3 take a look at tubes labeled T-test, S-standard and B-blank were used. Into the 3 take a look at tubes were pipetted a pair of.0 cc every of the mixed color chemical agent and mixed acid resolution severally. The solutions were mixed totally. To the T-test tube was pipetted bodily fluid (0.02 ml) and into the S-test tube was pipetted zero.02 cc of the operating customary. H2O (0.02 ml) was pipetted into the B-test tube. The contents in every tube were mixed totally and so stewed for ten min, then cooled in cold water. The absorbance of the sample and customary were scan against the blank at 540nm. From the formula below carbamide gas was calculated. Serum Chloride IonsThis was supported the strategy of Henry (1991)The PrinciplesHg(SCN)2 + 2CL- ?HgCl2 + 2SCN-3SCN + Fe32+ ?4Fe (SCN)3 red advancedThis methodology is predicated on the principle that chloride ions kind a soluble, non-ionized compound with metal ions and can displace salt ions from non-ionized metal salt. The free salt ions react with ferrous ions to create a color advanced that absorbs light-weight at 480nm. The intensity of the color made is directly proportional to the chloride concentration. ProcedureInto a collection of take a look at tubes labeled take a look at and customary were additional 50?L of bodily fluid. Then 3.5ml of H2O was additional, followed by 500?L of tetraoxosulphate IV. metal salt resolution (50?L), was pipetted into every of the take a look at tubes and mixed totally, and allowed to square for 5min. it absolutely was then centrifuged, thenceforth a pair of.0 cc of the supernatant was pipetted into a tube followed by the addition of 100?L of diphenylcarbozone (indicator). it absolutely was then titrated with metal nitrate five mmol/l employing a microburette. Faint blue violet color appeared as metal nitrate was additional drop wise. Chloride concentration was calculated thus: CL (mmol /L) = Where0.02 = Mmol chloride/ml of std0.2 = cc sample in volumetric analysis2. Serum Potassium IonsThe Assay was supported Milton Friedman (1997) methodology. This methodology is predicated on the principle that metal tetraphenyl atomic number 5 produces a sol during a specifically ready mixture. The muddiness of it’s a live of metal that is proportional within the vary of two – seven MEq/LPotassium Conc.(MEq/L) = ProcedureA set of take a look at tubes labeled ST-standard, T-sample and B-blank were severally started. Into every of the take a look at tubes, 1.0 cc of metal chemical agent was pipetted followed by zero.01ml of sample, then mixed totally and allowed to square for concerning three minutes. The absorbance was noted at 550 nm against blank. Sodium IonsThe method of Beckman (2007) was used, within which metal is precipitated because the triple salt, with metal uranyl radical acetate ions, with the surplus atomic number 92 thenceforth reacts with salt, manufacturing a chemical group whose absorbance varies reciprocally with the concentration of metal within the take a look at specimen.ProcedureA set of 3 take a look at tubes labeled T-Sample, B-blank and ST-standard were started severally. Into the take a look at tubes one.0ml of filtrate chemical agent followed by 50?L of the sample was pipetted into the sample tube, H2O into the blank and 50?L of the quality resolution into the tube labeled ST. The take a look at tubes were jolted totally and so centrifuged at 3500 rate for ten minutes. Thereafter, 50?L of supernatant were place into the several take a look at tubes, mixed totally. This was followed by adding the color chemical agent (5 ?L) to all or any the take a look at tubes and mixed totally. The absorbance was scan at 550nm against blank. The concentration of metal was calculated thus:Conc. Of metal (MEq/L) = Serum CreatinineThe bodily fluid creatinine was measured by the changed Jaffes kinetic methodology (Bowers, 1980). This methodology is predicated on the principle that creatinine in alcalescent resolution reacts with picrate to create a colored advanced. ProcedureSets of 3 take a look at tubes labeled S-sample, T-test and ST-standard were started. The bodily fluid and management were pipetted into the tube labeled S-sample and ST-standard severally, followed by adding zero.5 cc of sample and mixed totally. oxygen acid (4.5 ml) was additional into the take a look at tubes and mixed fastidiously and allowed to square for ten min. it absolutely was centrifuged at 3500rpm for ten min. In to a different set of take a look at tubes labeled T-test and ST-standard were pipetted three.0ml of the supernatant and customary severally. A chemical agent blank was additionally started into that three.0ml of H2O was pipetted. acid (1.0 ml) was additional to every tube and mixed totally. when thirty sec, 0.5ml of caustic soda was additional into every of the take a look at tubes and mixed totally. They were then allowed to square for 15minutes and also the absorbance scan at 550nm against blank, Concentration of creatinine in bodily fluid was calculated thus:= mg/dL Assay of lipid profile
a. High density lipoprotein-cholesterol
Low density lipoproteins (LDL), terribly density lipoproteins (VLDL), and particle fractions area unit precipitated quantitatively by the addition of phosphotungstic acid within the presence of metal ions. once action, the steroid alcohol concentration within the HDL fraction, that remains within the supernatant, is set (Christie 2003).A volume of a hundred µl of the sample was placed during a tube and a thousand µl of the chemical agent was another to that. The sample and therefore the chemical agent were mixed, allowed to incubate for ten min at twenty to 25oC. The absorbance of the mixture and therefore the commonplace were taken at 546 nm at intervals sixty min.b. Total cholesterolThe steroid alcohol is set once accelerator chemical reaction and oxidization. The indicator quinoneimine is made from peroxide and 4-aminoantipyrine within the presence of phenol and oxidase (Barter 2007).A volume ten µl was taken into the tube and a thousand µl of the chemical agent was another to that. The sample and therefore the chemical agent were mixed, allowed to incubate for ten min at twenty to 25oC. The absorbance of the mixture and therefore the commonplace were taken at 546 nm at intervals sixty min.(c) Triglyceride (TRIGS)A volume of ten µl of the sample was placed during a clean tube and a thousand µl of the chemical agent was another to that. The sample and therefore the chemical agent were mixed, allowed to incubate for ten min at twenty to 25oC. The absorbance of the mixture and commonplace were taken at 546 nm at intervals sixty min. Evaluationof haematological indicesWhite blood corpuscle (WBC), platelets count (PC), packed cell volume (PCV), haemoprotein concentration (HB), and therefore the white blood cell differential (lymphocyte (LYM) and neutrophils (NEU) were all analysed by the utilization of machine-controlled blood instrument ( QBC Autoread and , UK). The blood samples were initial pipetted into QBC capillary tubes and spun during a parafuge centrifuge (Becton Dickson, UK) for five min and browse by suggests that of AN autoread instrument. Evaluation of antioxidant statusa. Assay of superoxide levelThe catalyst SOD (SOD) change state the conversion of superoxide into peroxide and gas at a high reaction rate. Since its initial discovery by McCord and Fridovich in 1969, SOD has been found to be omnipresent in each aerobic organism from germ to human.The ability of SOD to inhibit the auto-oxidation of internal secretion at pH of ten.2 makes this reaction a basis for an easy assay for dismutase (Misra and Fridovich, 1972).Superoxide anion generated by organic compound enzyme reaction, causes the oxidization of internal secretion to adrenochrome, and therefore the yield of adrenochrome created per superoxide introduced, will increase with increasing pH, and conjointly decreases with increasing concentration of internal secretion (Sopjani et al, 2008).A one-in-ten dilution of the sample stuff was ready. AN aliquot of one.2 ml of 0.05 M phosphate buffer pH seven.8 was left to equilibrate during a photometer and therefore the reaction started by the addition of zero.1 metric capacity unit of freshly ready zero.3 nM internal secretion to the mixture that was quickly mixed by inversion. The reference cuvette contained one.0 metric capacity unit phosphate, 0.1 metric capacity unit of substrate (adrenaline) and zero.1 metric capacity unit of tissue stuff. the rise in absorbance at 480 nm was monitored each zero s, 30 s, and 60 s, severally to see the common.Calculation% Inhibition = (Absorbance of take a look at – Abs of reference ÷ Abs of test) × 100/1SOD activity = reciprocal inhibition ÷ weight × fiftyWhere;Weight = weight of tissue in volume (aliquot) of extract utilized in assay. Assay of nitric oxide level
The group commonplace reference curve was set by getting ready completely different concentrations of group answer. The sulpha answer and NED answer was allowed to equilibrate at temperature for 15-30 minutes. employing a micropipette, fifty µl of the liver material were placed in take a look at tubes, fifty µl of sulpha answer and fifty µl of the NED answer were superimposed to the tissue material. The mixture was incubated for 5-10 minutes, protected against lightweight. The absorbance was measured 530 nm inside thirty min (Chaea et al., 2004) Assay of malondialdehyde levelMalondialdehyde fashioned from the breakdown of unsaturated fatty acids, that is a convenient index for crucial the extent of peroxidation reaction.MDA, a product of lipoid peroxidation, once heated with 2-thiobabituric acid (TBA) beneath acid conditions, forms a pink coloured complicated, that absorbs maximally at 535 nm.(Guttride and Wilkins, 1982)ProcedureA volume of zero.5 cubic centimetre of every sample was mixed with one.0 cubic centimetre of acetic acid – Tribabituric acid – acid (15 g TCA in forty cubic centimetre of zero.25 N HCl + zero.375 g TBA in thirty cubic centimetre of zero.25 N HCl the answer was mixed and created up to a hundred cubic centimetre of zero.25 N HCl) stock chemical agent and therefore the mixture was heated for fifteen min in a very boiling water tub. when cooling, the precipitate was removed by activity at 3500 rate for ten min. The absorbance of the sample was measured at 535 nm against chemical agent blank. CalculationMDA (mol/g tissue)= (Absorbance × volume of reaction mixture)÷ (molar extinction × aliquot used).Where;Molar extinction constant =1.56 × a hundred and five m-1 cm-1 Volume of reaction mixture = one.5 mlAliquot used = zero. Histology of selected organs The harvested liver, kidneys and spleen were hold on in 100% formaldehyde-in-saline answer. The tissue specimens were dehydrated in alcohol, processed in xylol then embedded in paraffin before sectioning with a microton. Sections (5 small thick) of the tissues were stained with haematoxylin-eosin. All section were viewed beneath associate Mt. Olympus OMAX microscope and camera of ×100 magnification and photographed of the organs were taken.2.3 applied mathematics Analysis and knowledge PresentationData square measure bestowed as mean ± SEM (standard error of mean) and “n” represents the amount of rats per cluster. Comparisons were created between treated and management teams by the utilization of 1 method ANOVA followed by Newman-Keul’s post hoc ergo propter hoc. All knowledge were analysed mistreatment GraphPad Prism version five code (GraphPad Prism, UK).

3.1 Phytochemical Constituents of Aqueous Extract of G. lucidum
Phytochemical screening of the extract shows the presence of alkaloids, cardiac glycosides, tannins, saponins, phenolic compounds, and compounds with steroidal nucleus but flavonoids were absent (Table 3.1).

3.2 Oral Acute Toxicity of Aqueous Extract of G. lucidum
Table 3.2 shows that at doses of 10, 100 and 1000 mg/kg of the extract, no death occured. Also, in the second phase with relatively higher doses of 1600, 2900 and 5000 mg/kg of the extract, death did not occur in the animals. Physical observations revealed no changes in the eyes, fur, skin, paw and behavior and there were no signs of convulsion and constipation in the animals. The oral LD50 of the extract was evaluated to be greater than 5000 mg/kg.

3.3 Effects of Aqueous Extract of G. lucidum on Concurrent Lead Administration
3.3.1 Effect on whole body weight
Daily administration of the various doses for 25 consecutive days did not significantly alter the weight of the animals when compared with vehicle-treated controls. Although animals concurrently administered lead + 200 mg/kg of the extract and those administered lead + 50 mg/kg of DMSA, had mean weight loss, the change was not significantly different from other groups (Figure 3.1).
Table 3.1: Phytochemical constituents of the aqueous extract of G. lucidum
Phytochemical constituents Inference Values
Alkaloids + 33.5
Flavonoids – NQ
Cardiac glycosides + NQ
Phenols + 0.35
Saponis + NQ
Sterol + NQ
Tanins + NQ
Terpenoids + NQ
Polysaccharides + NQ
+ = Present; – = Absent; NQ = Not quantifiable
Table 3.2: Oral acute toxicity results of aqueous extract of G. lucidum in rats
( mg/kg) Convulsion Writhing Constipation Diarrhea Death Others
I 10 0/3 0/3 0/3 0/3 0/3 0/3
100 0/3 0/3 0/3 0/3 0/3 0/3
1000 0/3 0/3 0/3 0/3 0/3 0/3
II 1600 0/1 0/1 0/1 0/1 0/1 0/1
2900 0/1 0/1 0/1 0/1 0/1 0/1
5000 0/1 0/1 0/1 0/1 0/1 0/1
The denominator indicates the number of rats used while the numerator indicates the number of animals that experience the effect.

Figure 3.1: Change in body weight following the concurrent oral administration of aqueous extract of G. lucidum (GL) and lead (Pb) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. Changes are not significantly different between the groups. DMSA, dimercaptosuccinic acid. n = 5 per group.

3.3.2 Effects on lead levels in organs
In comparison with Pb-only treated rats, the liver contents of the lead was significantly reduced following the concurrent oral daily administration of 200 mg/kg (P ; 0.01) and 400 mg/kg (P ; 0.001) of the aqueous extract of G. lucidum. Succimer (DMSA); and the combination of DMSA with 400 mg/kg of the extract also significantly reduced liver Pb contents in the rats in comparison with Pb-only controls (Figure 3.2).

In the kidney of rats (Figure 3.3), the extract at all three doses significantly reduced Pb concentrations although not in a dose-related manner. The reducing effect of DMSA was superior to all the doses of the extract. The combination of DMSA and 400 mg/kg/day of the extract did not significantly reduce the concentration of Pb in the kidneys of the rats.
The concentration of Pb in the spleen of rats was not significantly reduced by the various treatments (Figure 3.4) although the mean concentration was lowest among the 200 mg/kg/day- of extract treated rats.

Among the controls, Pb concentration was highest in the liver (0.48 ± 0.05 µg/g) followed by the kidney (0.46 ± 0.02 µg/g) and lowest in the spleen (0.16 ± 0.04 µg/g) (Figures 3.2 – 3.4).
3.3.3 Effects on haematological indices
The haematological parameters following the concurrent oral daily administration of lead and aqueous extract of G. lucidum for 25 days in rats were largely comparable across the groups (Tables 3.3 and 3.4) except in the percentages of granulocytes which were significantly (P ; 0.05) reduced in all three groups treated with the extract only (Table 3.3). The standard drug DMSA also reduced the percentage of granulocytes. The mean corpuscular heamoglobin was significantly reduced in Pb-only; and extract-treated groups (100 and 400 mg/kg/ (Table 3.4).

Figure 3.2: Liver Pb concentration following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. **P ; 0.01; ***P ; 0.001 compared to Pb-only group. DMSA, dimercaptosuccinic acid. n = 4-5 per group.

Figure 3.3: Kidney Pb concentration following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. *P ; 0.05; **P ; 0.01; ***P ; 0.001 compared to Pb-only group. DMSA, dimercaptosuccinic acid. n = 4-5 per group.

Figure 3.4: Spleen Pb concentration following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. Values are not significantly different from control. DMSA, dimercaptosuccinic acid. n = 4-5 per group
Table 3.3: Effects of 25-day concurrent oral administration of aqueous extract of G. lucidum (GL) and lead (Pb) on white blood cell and platelet counts in rats.

GROUPS DOSE (mg/kg) WBC (×103/µl) LYM (×103/µl) MO
(%) GR
(%) PLT
CONTROL – 9.5±3.4 5.0±1.2 15.7±1.3 16.4±21.2 732.6±86.4
Pb ONLY 100 11.1±1.5 3.3±1.2 16.3±1.0 23.2±18.1 778.2±128.2
Pb + GL 100 + 100 9.7±0.6 4.5±0.3 16.2±0.8 19.7±16.9 677.8±90.6
Pb + GL 100 + 200 12.7±2.8 7.3±0.9 14.4±0.7 9.4±4.4* 746.2±63.7
Pb + GL 100 + 400 7.4±0.5 5.3±1.7 18.4±1.0 6.0±1.2** 381.4±78.2
Pb + DMSA 100 + 50 8.0±0.1 5.1±1.2 13.7±0.9 9.9±4.2* 797.8±133.6
Pb+GL+DMSA 100 + 400+50 9.6±0.8 4.2±0.9 15.9±0.4 27.8±18.2 514.4±55.9
*P ; 0.05; **P ; 0.01 versus control. WBC: White blood cells; LYM: Lymphocytes; MO: Monocytes; GR: Granulocytes; PLT: Platelets; DMSA: Dimercaptosuccinic acid. n = 5 per group.

Table 3.4: Effects of 25-day oral administration of aqueous extract of G. lucidum on red blood cell parameters in rats
(mg/kg) Hgb
(g/dl) HCT
(%) RBC (×106/µl) MCHC (g/dl)
CONTROL – 15.1± 0.7 43.9±1.6 7.6±0.3 29.7±6.9
Pb ONLY 100 12.4± 0.4 39.5±1.9 7.5±0.5 26.1±6.5
Pb + GL 100 + 100 14.5±0.3 42.8±1.3 8.1±0.2 34.0±0.9
Pb+ GL 100 + 200 11.5±2.3 28.9±13.2 6.4±1.3 28.0±7.0
Pb+ GL 100 + 400 14.0±0.3 40.1±1.2 7.8±0.2 35.0±0.8
Pb+ DMSA 100 + 50 14.0±0.4 39.4±1.0 7.2±0.3 35.3±0.5
Pb+ GL+ DMSA 100 + 400 + 50 14.0±0.3 39.8±0.9 7.7±0.2 36.6±0.2
*P ; 0.05, ***P ; 0.001 versus Control. Hgb: Hemoglobin; HCT: Hematocrit; RBC: Red blood cell. n= 5 per group
3.3.4 Effects on serum enzymes
Daily Pb administration for 25 consecutive days significantly (P ; 0.001) increased the serum level of alanine transaminase (ALT) in rats when compared with their untreated counterparts (Figure 3.5). Although there were lower mean values of the enzyme in the extract-treated groups, only the dose of 200 mg/kg/day caused a significant (P ; 0.001) reduction. The standard drug DMSA, also significantly (P ; 0.01) reduced the level of the enzyme.

Figure 3.6 shows that the extract of G. lucidum significantly and dose-dependently reduced the serum levels of aspartate transaminase (AST) in Pb-administered rats but not to values comparable with untreated controls. The standard drug DMSA was not as effective as the extract. The combination of the extract and DMSA did not reduce Pb concentration significantly.

In Figure 3.7, Pb administration led to a significant (P ; 0.001) increase in the serum level of alkaline phosphatase (ALP) in comparison with untreated rats. The mean level of the enzyme significantly decreased dose-dependently across the extract-treated groups. Also DMSA alone or in combination with 400 mg/kg/day of the extract significantly (P ; 0.001) decreased ALP level. Aside from 100 mg/kg/day of the extract, all other treatment brought the enzyme to levels comparable with that of the untreated controls.

Figure 3.5: Alanine transaminase (ALT) levels following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. #P ; 0.0001 versus Pb-only; **P ; 0.01 versus Pb-only; ***P ; 0.001 versus control. DMSA, dimercaptosuccinic acid. n = 5 per group.

Figure 3.6: Aspartate transaminase (AST) levels following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. *P ; 0.05 **P ; 0.01; ***P ; 0.001 versus Pb-only. DMSA, dimercaptosuccinic acid. n = 5 per group.

Figure 3.7: Alkaline phosphatase (ALP) levels following concurrent oral administration of Pb and aqueous extract of G. lucidum (GL) to rats for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. The extract significantly reduced ALP levels in comparison with Pb-only. DMSA, dimercaptosuccinic acid. n = 5 per group3.3.5 Effects on serum electrolytes, urea and creatinine
Table 3.5 shows that 25-day consecutive administration of Pb did not significantly alter the serum concentrations of sodium, potassium, bicarbonate and chloride when compared with values obtained from untreated rats. The renal function biomarkers (urea and creatinine) were also not significantly altered. Treatment with the extract or its combination with succimer (DMSA) did not significantly alter these serum analytes.
3.3.6 Effects on serum lipids
The mean values of lipids in the group treated with Pb only were not significantly different from that of the untreated control group although values were lower in all the parameters (Table 3.6). Doses of 100 and 200 mg/kg/day concurrently administered with Pb resulted in significant (P;0.01, P;0.05 respectively) reduction in serum LDL levels. Succimer (DMSA) administered alone or in combination with the extract significantly reduced triglyceride (TG, P;0.001) and low density lipoproteins (LDL, P;0.05) in the Pb-poisoned rats.

Table 3.5: Effect of 25-day concurrent administration of aqueous extract of G. lucidum (GL) and lead (Pb) on serum electrolytes, urea and creatinine
GROUPS DOSE (mg/kg) Urea (mg/dl) Cr (mg/dl) Na+ (mmol/l) K+ (mmol/l) HCO-3
(mmol/l) Cl-
CONTROL – 59.6±3.4 0.7±0.1 135.6±1.7 7.0±0.7 24.4±2.3 103.2±1.8
Pb ONLY 100 47.2±1.9 0.5±0.1 142.2±1.9 7.3±0.8 17.4±1.9 107.0±0.8
Pb + GL 100 +100 58.4±2.7 0.6±0.1 136.6±1.0 6.5±0.4 18.2±1.9 107.6±0.8
Pb + GL 100 +200 47.6± 1.2 0.6±0.1 135.0±1.3 6.6±0.4 19.6±1.3 104.8±0.9
Pb + GL 100 +400 54.4±2.0 0.7±0.1 136.2±1.8 6.5±0.5 20.0±2.4 106.0±0.6
Pb + DMSA 100 +50 57.6±1.6 0.6±0.1 135.6±0.9 5.7±0.4 18.8±1.6 105.6±0.9
Pb +GL+DMSA 100 +400 +50 55.0±0.8 0.7±0.1 133.4±1.2 5.4±0.1 22.5±0.5 103.4±0.7
Values are not significantly different from the control. Cr: Creatinine; DMSA, dimercaptosuccinic acid. n = 5 per group.

Table 3.6: Effect of 25-day concurrent oral administration of aqueous extract of G. lucidum (GL) and lead (Pb) on serum lipids
(mg/kg) TC
(mg/dl) TG
(mg/dl) HDL
(mg/dl) LDL
Control – 64.8±2.6 59.4±3.8 36.0±3.8 26.0±5.9
Pb only 100 56.0±5.5 48.8±8.5 28.8±3.9 20.0±1.6
Pb + GL 100+100 57.2±2.4 59.8±3.5 27.2±2.9 13.8±1.2**
Pb + GL 100+200 61.4±2.0 59.4±4.1 32.8±1.9 14.8±1.1*
Pb + GL 100+400 60.2±3.1 67.4±6.7 29.0±2.1 17.4±1.9
Pb + DMSA 100+50 71.4±5.1 153.6±15.6*** 36.6±4.0 15.4±1.3*
Pb + GL + DMSA 100+400+50 71.0±2.8 146.4±12.8*** 38.6±2.8 15.2±1.2*
*P;0.05; **P;0.01; ***P;0.001 compared to the control. TC: Total cholesterol; TG: Triglycerides; HDL: High density lipoproteins; LDL: Low density lipoproteins; DMSA: Dimercaptosuccinic acid n= 5 per group.

3.3.7 Effects on oxidative stress
Figure 3.8 shows that liver superoxide dismutase (SOD) was significantly (P;0.001) elevated in Pb-administered groups when compared with the untreated control group. Concurrent administration of the extract or DMSA with Pb did not significantly alter liver SOD levels.
The mean nitric oxide (NO) concentration in the liver of the untreated control group was higher than in other groups but only significant (P;0.05) in Pb-only and Pb + DMSA groups. The extract did not significantly alter NO levels (Figure 3.9).

In Figure 3.10, Pb administration resulted in significant (P;0.001) elevation of malondialdehyde (MDA) levels in the liver of rats. In groups treated with extract of G. lucidum, the levels of MDA were reduced significantly (P ;0.01). Succimer (DMSA) alone and in combination with the extract also significantly reduced MDA levels.

Figure 3.8: Liver superoxide dismutase (SOD) levels in rats concurrently administered Pb and aqueous extract of G. lucidum (GL) for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. #P;0.0001, ***P ;0.001 compared with control. DMSA, dimercaptosuccinic acid. n = 5 per group

Figure 3.9: Liver nitric oxide (NO) levels in rats concurrently administered Pb and aqueous extract of G. lucidum (GL) for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. *P;0.05 versus control. DMSA, dimercaptosuccinic acid. n = 5 per group

Figure 3.10: Liver malondialdehyde (MDA) levels in rats concurrently administered Pb and aqueous extract of G. lucidum (GL) for 25 consecutive days. Values tagged with abbreviations are doses in mg/kg. **P;0.01, #p;0.0001 versus Pb-only. DMSA, dimercaptosuccinic acid. n = 5 per group.

3.3.8 Effects on organ histology
Figures 3.11, 3.12 and 3.13 show the photomicrographs of sections of the liver, kidney and spleen of rats respectively from selected groups. The liver micrograph shows periportal inflammation in the rat treated with Pb only. In the micrograph of the liver from the rat given Pb + 400 mg/kg G. lucidum, central vein congestion can be seen.
The micrographs of the kidneys, and spleen are essentially normal across the groups.
028023440011719773889419004939048656824003267075000990600-1348336004798770-4969002005574030-2794000Pb + DMSA
0Pb + DMSA
2317750-2778760Pb + 400GL
0Pb + 400GL
5556885-5626735Pb Only
00Pb Only
Figure 3.11: Representative photomicrographs of the liver samples from rats. Control = vehicle treated; Pb-only = administered 100 mg/kg/day Pb acetate for 25 consecutive days; Pb + 400 GL = Pb + 400 mg/kg/day G. lucidum (GL); and Pb + DMSA = Pb + 50 mg/kg/day DMSA. Treatment was by the oral route. There is periportal inflammation in Pb-only; central vein congestion in Pb + 400 mg/kg/day GL. H ; E, ×100.

2967355-8255002974975311467500-75565310642000-81915-22860005149215-3105785Pb + DMSA
0Pb + DMSA
2057070-3096260Pb + 400GL
00Pb + 400GL
5328920-6239510Pb Only
0Pb Only

Figure 3.12: Representative photomicrographs of the kidney samples from rats. Control = vehicle treated; Pb-only = administered 100 mg/kg/day Pb acetate for 25 consecutive days; Pb + 400 GL = Pb + 400 mg/kg/day G. lucidum (GL); and Pb + DMSA = Pb + 50 mg/kg/day DMSA. Treatment was by the oral route. Micrographs are essentially normal. H ; E, ×100.5458460-2923870Pb + DMSA
0Pb + DMSA
2177415-2926410Pb + 400GL
0Pb + 400GL
3130550315150500-1835153144520005634990-6008675Pb Only
0Pb Only
Figure 3.13: Representative photomicrographs of the spleen samples from rats. Control = vehicle treated; Pb-only = administered 100 mg/kg/day Pb acetate for 25 consecutive days; Pb + 400 GL = Pb + 400 mg/kg/day G. lucidum (GL); and Pb + DMSA = Pb + 50 mg/kg/day DMSA. Micrographs are essentially normal. (H ;E, ×100 ).

3.4 Curative Effects of Aqueous Extract of G. lucidum on Lead-Administered Rats
A 14-day administration of 200 and 400 mg/kg/day of G. lucidum extract to rats that had been given oral doses of Pb for 14 days resulted in a significant reduction in the concentration of Pb in the kidney (Figure 3.14). In the liver (Figure 3.15), this protocol did not result in a significant depletion of the concentration of Pb.

Figure 3.14: Depleting effect of 14-day oral administration of G. lucidum extract on kidney Pb content of rats. The rats were first given 100 mg/kg/day of Pb acetate for 14 days. Values tagged with abbreviation are doses in mg/kg/day. n = 5 per group.

Figure 3.15: Effect of oral administration of G. lucidum extract on liver Pb content of rats. The rats were first given 100 mg/kg/day of Pb acetate for 14 days. Values tagged with abbreviation are doses in mg/kg/day. n = 5 per group.

4.1 Acute Toxicity Study
The presence of tannins, cardiac glycosides, saponins, in the aqueous extract of Ganoderma lucidum is believed to be responsible for the wide arrays of pharmacological and biological actions. The phytochemical results from the present study were similar to the one obtained by Nigatu (2004). Oral acute toxicity test of the extract G. lucidum has shown that the mushroom may be relatively safe since the LD50 is greater than 5000 mg/kg. Any substance with LD50 greater than 5000 mg/kg by oral route is assigned a Class 5 status which is the least toxicity class (OECD, 2001). Kennedy et al. (1986) stated that any substance with LD50 greater than 5000 mg/kg by the oral route can be said to be safe or particularly harmless. The absence of death indicates that the extract may be very safe in acute doses and is unlikely to cause fatality in humans (Hodge and Sterner, 2005).

4.2 Haematological Effects
Haematological evaluations are useful in the diagnosis of disease states. They are therefore important in toxicological studies (Onyeyili et al., 1992; Togun et al., 2007) because blood acts as a pathological reflector of the status of the animals exposed to toxicant or other conditions (Olafedehan et al., 2010). In this study, haematological parameter were normal in both the test and the control groups. White blood cells produce and distributes antibodies in immune response. By phagocytosis, these cells defend the body against foreign invasion by microorganisms or xenobiotics. The significant increase observed in granulocytes following 25 days of extract administration suggest the absence of either central vein congestion that is reported to occur due to prolonged intake of G. lucidum extract or inflammatory conditions in experimental rats (Shamaki et al., 2014).. This agrees with the reports by Zhang et al. (2002) that G. lucidum extract decreases low density lipoproteins and inhibits adhesion of monocytes to endothelium.
4.3 Serum Enzymes effects
The aminotransferases (ALT and AST) describes hepatocellular integrity; alkaline phosphatase (ALP) describes relationship of the liver with the biliary tract while albumin and protein levels describe its functionality (Adeoye and Oyedapo, 2004). The highest levels of aspartate aminotransferase (AST) are present in the liver, cardiac tissue, and skeletal muscle, while smaller amounts are present in the kidneys, pancreases and erythrocytes. Alanine aminotransferase (ALT), however is predominantly present in the liver. Hence, it is said to be liver specific (Esani, 2014). Biochemical test of serum enzymes are frequently used to assess patients with symptomatic and asymptomatic liver diseases. Lead (Pb) toxicity is one of the major causes of liver disease worldwide. It accounts for a large proportion of deaths from liver disease (Signal et al., 2013; Gao et al., 2011). The study has established the distruptive effect of Pb as seen in the group treated with Pb only. The extract of G. lucidum was able to protect against the hepatoxicity of Pb. This may be due to the presence of polysaccharide which was able to protect the liver cells from oxidative damage as reported by Ooi and Lui (2000). It also significantly increase the superoxide dismutase (Sun-Hee Jang et al.,2014).

4.4 Serum Lipid Effects
The belief that low density lipoprotein (LDL) cholesterol causes atherosclerosis and subsequent heart diseases is a fundamental precept of modern medicine (Colpo, 2005). The primary function of high density lipoprotein (HDL) is to mobilize excess cholesterol to the liver for it to be metabolized into bile salt. This function of cholesterol removal from the tissue underlies the inverse relationship between the plasma concentration of HDLs and the incidence of heart diseases (Miles, 2003). Ganoderma lucidum significantly decreased the serum content of triglyceride and low density lipoprotein (P ; 0.05 and P ; 0.01 respectively) showing the protective effect of the extract and markedly increased the level of serum high density lipoprotein (HDL). Mean level of blood lipoprotein in the experimental groups treated by G. lucidum at different dosages were much lower than those in lead acetate group. Thus, G. lucidum can enhance the antioxidant and reduce the lipid peroxidation in the rats.

4.5 Antioxidant Effects
Lead induces in hepatic microsomal membrane (Lowton et al.,1991).It also causes increased expression in the liver (Mudipali, 2007). This prompted the determination of level of lipid peroxidation, as indicator of Pb-induced oxidative stress in the liver tissue. Consumption of antioxidant-rich mushroom could facilitate stop cancer and alternative chronic diseases (Collins, 2005). Antioxidants shield cellular elements from aerophilous injury, that is probably going to decrease the chance of mutations and carcinogenesis and additionally shield immune cells, permitting them to keep up immune police investigation and response. numerous elements of G. lucidum above all polysaccharides and triterpenoids, show inhibitor activity in vitro (Lee et al, 2001). Ooi and Lui (2000) according that protein-bound saccharide (PBP) and saccharide amide were able to mimic the endogenous inhibitor enzyme (SOD) in cancer-bearing animals in vivo. These polysaccharides were additionally according to shield the immune cells from aerophilous injury (Ooi and Lui, 2000) .The protecting effects of G. lucidum on desoxyribonucleic acid strand cut induced by a metal-catalyzed Fenton reaction, ultraviolet irradiation, and hydroxyl attack are documented by Lee et al. (2001). The extract of G. lucidum considerably protected cells from peroxide (H2O2)-induced desoxyribonucleic acid injury (Shi et al., 2002). plight extract additionally protected human white cell desoxyribonucleic acid solely at low doses. The binary compound extract protected cellular desoxyribonucleic acid from aerophilous injury, whereas the alcohol extract broken cellular desoxyribonucleic acid, with redoubled H2O2 production and important cell-killing effects ascertained. This study indicates that oral administration of binary compound G. lucidum at the same time with lead (Pb) considerably reduced the cell damaging result of lead within the excretory organ and liver however has very little important result within the spleen.

4.6 Effects on serum electrolytes
Serum electrolytes, urea and creatinine are reliable indicators of renal function (Aliyu et al., 2006). This study has shown that serum level of sodium, potassium, urea and creatinine were not significantly affected by the treatment. This suggests that the disorders associated with the abnormalities in the values of electrolytes and substance may not occur. For example, changes in serum levels of these parameters may be suggestive of some underlying renal pathology (Bonomini et al., 2010). That is, G. lucidum was able to protect the abnormal increase in sodium level which might precipitate high blood pressure and some other chronic diseases.

4.7 Histological Effects of Liver, Kidney and Spleen
Histomorphorlogical changes in target organs are first hand indication of toxicity of a chemical or biological substance (Kwan et al., 2013). Histological examination of the kidney and spleen showed no abnormality nor structural changes in the cells of the organs in both the treated group and the control group. However, in the liver, there are central vein congestion and peripotal inflammation which showed that the hepatocytes have been damaged.

5.1 Conclusion
Current drugs used in the management of toxicological conditions have worrisome effects. The paucity of scientific information on the therapeutic potentials of Ganoderma lucidum extract in management of lead poisoning formed the basis of this study. This study shows that:
The aqueous extract of G. lucidum contains alkaloid, cardiac glycosides, saponins, tanins and phenol.

The extract appears safe as its LD50 is greater than 5000 mg/kg in rats
The extract may protect against Pb toxicity by:
Reducing Pb accumulation in liver, kidney but not in spleen because the spleen were essentially normal
Reducing ALT, AST and ALP levels which are marker enzymes for liver and kidney test.
Increasing SOD levels
Protecting against lipid peroxidation
The curative effect appears more pronounced in the kidney than the liver.
5.2Contribution to dataThe study has contributed to data within the following ways:
It has shown level of effectiveness in the use of G. lucidum in management of liver and kidney diseases
It has established the deleterious effects of lead (Pb) on soft tissues such as liver and kidney.

It has established the phytochemical properties present in Ganoderma lucidum
It has revealed the antioxidant constituents present in the extract
It has provided some information on the acute toxicity profile of aqueous extract of Ganoderma lucidum.

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