CHAPTER II
REVIEW OF RELATED LITERATURE
Infectious diseases represent a serious health problem today and account for one third of all deaths worldwide (Perumal et al., 2012), and herbal medicine has been used in many parts of the world as a rich tradition for the treatment of these infectious diseases (Titilope et al., 2012), both infectious and non-infectious. Attah et al. (2012) makes similar claims with the evaluation and development of compounds from similar claims with the evaluation and development of compounds from medicinal plants for the treatment of diseases which is one of the areas that is gaining grounds and acceptability world-wide (p.1). According to the World Health Organization, medicinal plants would be the best source to acquire different effective drugs and, therefore, those plants should undergo investigation to have a better understanding of their properties, safety and efficacy (Rajeh et al., 2010; Nascimiento et al., 2000). There are some published reports describing the antimicrobial activity of various crude plant extracts (Igoli et al., 2005; Alzoreky et al., 2003). It is estimated that there are about 2.5 million species of higher plants and the majority of these have not yet been examined for their pharmacological activities (Ram et al., 2003). In relation with the applications of herbal plants, weed, which is a plant that grows out of place and is competitive, persistent and pernicious (James et al., 1991), have been a part of civilization and many ancient documents speak of humans battling weeds in the crops they grow and also found to be resistant to most of the microbial disease that made us interested to know the potency behind (Ibrahim et al., 2012). Furthermore, according to Ibrahim et al. (2012), there is increasing documentation to strengthen the hypothesis that weeds are relatively high in bioactive secondary compounds and are thus likely to held promise for drug discovery (p. 113).
Euphorbia hirta Linn., a frequently seen herb occupying open waste spaces and grasslands, roadsides and pathways, belongs to the family Euphobiaceae (Abubakar, 2009). It is commonly known by many vernacular names such as: euphorbia, dudhi, dudhi khurd, cat’s hair, asthma weed and pill bearing spurge (Khare, 2007). It is commonly erect, slender-stemmed; spreading up to 45cm tall, though sometimes can be seen lying down (Burkill, 1994). It cannot grow on the shade and requires dry or moist soil (Kuta et al., 2013). It is an annual herb common to tropical countries (Sofowora, 1982). In the Philippines, this is most commonly known as Tawa-tawa plant (Arceo et al., 2007).
E. hirta is commonly called asthma weed in Asia and Australia because of its widely usage as traditional medicine in treating a variety of diseased conditions including asthma, coughs, diarrhea and dysentery (Ogbolie et al., 2007), which is also same with the report of Kuta et al. (2013), wherein, in Asia this plant has been traditionally used to treat bronchitic asthma, laryngeal spasm and various other lung complaints. In India, which is stated by Jyothirmayi et al., (2012), it is used to treat worm infections in children and for dysentery, gonorrhea, jaundice, pimples, digestive problems and tumors (p. 335). In east, central and west Africa, a decoction of the herb is used to treat asthma, oral thrusth, boils, sores, skin and wound infections, in addition to its been used as an antispasmodic, antipuritic, carminative, depurative, diuretic, febrifuge, galactogogul, purgative and vermifuge (Darwish et al., 2002; Abubakar, 2009). In Nigeria, exudates of the stem is used to treat eye and ear infections (Igoli et al., 2005), and Abubakar et al. (2009) stated that while a decoction of the plant is used to treat enteric infections including diarrhea and dysentery, constipations and other stomach problems, asthma, bronchitis,eczema,athlete’s foot and scorpion bits pains (p. 499). In malay medicine, this plant has been used widely as a treatment for skin problems, gastrointestinal disorders, particularly intestinal parasitotosis, amoebic dysentery, diarrhea and ulcer (Perumal et al., 2012). According to Arceo et al. (2007), researchers believed that this weed has properties that help increase platelet count and regulate blood flow (p. 9). In past studies, researchers have found metabolites such as alkaloid, flauonoids, coumarine and terpenes, a number of substances as tannins, Gallic acid, quercetin, phenols, phyto-sterols and alcohols, etc. (Upadhyay et al., 2010) that serves as the factor for E.hirta’s biological activity. Previous study done by Rajeh et al. (2010) was the first study that performed the separation of the parts of the plants to evaluate the potential antimicrobial activity of the different parts of E. hirta (p. 6010). This is not a disadvantage for the present study because if the whole plant will be included for the extraction, then the antimicrobial activity of the different parts are merge in the extracts that will be obtained. In Jyothirmayi et al.’s (2012) report, it is asserted that the white latex found abundantly in this plant is used in treating warts and whitlows since it has revealed 1-inositol, pyrrogallic and catechuic tannins and the alkaloid, xanthoramnine. Whereas the roots of the plant can help in treatment of sprains, maggots in wounds, irregular growth of teeth, miscarriage, epilepsy and inflammation (Jyothirmayi et al., 2012). E. hirta is also documented for its diverse biological activities such as antihelmintic, lactagogue, antipyretic, anti-inflammation, antioxidant, antibacterial, antifungal and anticancer (Hussain et al., 2014), which is also stated by Perumal et al. (2012 and recounted of additional biological activities of E. hirta such as antixiolytic, antihypertensive, antimalarial and antidiarrhoeial (p. 69).
The target microorganisms of antimicrobial activity of the plant related studies often develop new genetic variants which subsequently become resistant to available antimicrobial agents and the effective lifespan of any antibiotic is thus limited (Rajeh et al., 2010). Same as with the study of Hussain et al. (2014) wherein he stated that survey study has revealed that almost all the microbes have developed resistance against all introduced antibiotics, such microbes are methicillin resistant Staphylococci, vancomycin resistant Enterococci, penicillin resistant Pneumococci and gram-negative microbes having multi-drug resistant, are the prominent examples of the drug resistance (p. 546). One of the bacteria that show an increase in the incidence of antibiotic-resistant clinical surveillance of antimicrobial resistance patterns (Camara et al., 2013).
According to Camara et al. (2013), respiratory tract infections, such as acute sinusitis, acute otitis media, pharyngitis, community-acquired pneumonia, and acute bronchitis, are widespread and represent a major health concern particularly in low resource settings and one of the major causes of these is Streptococcus pyogenes (p. 71). S. pyogenes is a gram positive coccus bacterium that is extremely common bacteria and is part of Group A streptococci, which meant that it has a certain type of polysaccharide antigen on its cell surface and produces over twenty exotoxins (Febvre, n.d.). It is an aerobic bacterium and made-up of non-motile, non-sporing cocci that form chains and large colonies greater than 0.5 mm in size (Murray et al., 2007; Kilian, 1998). According to Dr. Fevre (n.d.), Streptococci pyogenes is a common bacterium that is seen everywhere and this bacteria falls under Group A streptococcus because of its cell surface. The cell contains M protein, a protein that is a major virulence factor for the bacteria. M protein and lipoteichoic acid are both surface receptors that aid in the binding of the bacteria to the host cell. A major characteristic of Group A streptococci is ß-haemolysis on blood agar plates. S.pyogenes produces many extracellular products; they are streptococcal pyrogenic exotoxins and are classified into three serotypes, A-C. SPE B is the most unique of the three and is responsible for multiple diseases, including toxic shock syndrome (Febvre, n.d.). S.pyogenes is an exclusively human pathogen (Bessen, 2009; Cohen et al., 2005). The incubation period of S. pyogenes is usually 1-3 days (Vincent et al., 2004). According to Bessen et.,al (2005) and Carapetis et al (2005), this bacterium is responsible for a wide array of infections. It can cause streptococcal sore throat which is characterized by fever, enlarged tonsils, tonsillar exudate, sensitive cervical lymph nodes and malaise (Broch et al., 2000; Vincent et al., 2004). Scarlet fever (pink-red rash and fever) as well as impetigo (infection of the superficial layers of skin) and pneumonia are also caused by this bacterium (Fleming&Hunt, 2006). Septicalmia, otitis media, mastitis, sepsis, cellulitis, erysipelas, myositis, osteomyelitis, septic arthritis, meningitis, endocarditis, pericarditis, and neonatal infections are all less common infection to S. pyogenes (Murray et al., 2007). Streptococcal toxic shock syndrome, acute rheumatic fever (joint inflammation, carditis and CNS implications), post- streptococcal glomerulonephritis (inflammation, hematuria, fever, edema, hypertension, urinary sediment abnormalities and severe pain) and necrotizing fasciitis (rapid and progressive infection of subcutaneous tissue, massive systematic inflammation, and hemorrhagic ballal, crepitus and tissue destruction) are some of the more serious complications involving S.pyogenes infections (Aenningham, 2008; Carapetis et al., 2005; Cohan et al., 2005). Transmission via respiratory droplets, hand contact with nasal discharge and skin contact with impetigo lesions are the most important modes of transmission (Bessen, 2009). The pathogen can be found in its carrier state in the anus, vagina, skin and pharynx and contact with these surfaces can spread the infection (Bessen, 2009; Rasi & Pour-Heidari, 2009; Mead & Winn, 2000).
Camara et al. (2013) stated with their study of the antibiotic susceptibility of Streptococcus pyogenes that the treatment of respiratory streptococcal infections is difficult and there are many factors to consider when choosing an antibiotic regimen (p.74). Susceptibility to antibiotics of any isolated strain should be evaluated as this is the only guarantees and prompt and effective treatments (Camara, et al., 2013). S. pyogenes infections are susceptible to a variety of drugs: Beta-lactams such as penicillin, as well as erythromycin, clindamycin, imipenem, rifampin vancomycin, macrolides and lincomycin; however, certain strains of the bacterium have been found to resistant to macrolides, lincomycin, chloramphenicol, tetracyclines and cotrimoxazole (Bessen et al., 2009; Cohen et al., 2005). In Camara et al’s (2013) previous study of the antibiotic susceptibility of S. pyogenes, the results showed that all strains of the S. pyogenes were susceptible to penicillin G (p. 73). Penicillin is used for respiratory tract infections (pharyngitis) and macrolides or lincosamides are used if there are allergies (Bessen, 2009). Penicillin is any one of a number of antibiotics derived from Penicillium molds are used to treat infections caused by a wide variety of bacteria and there are two forms of penicillin that are used today: penicillin G (benzylpenicillin) is a form of penicillin administered by injection and penicillin V (phenoxymethylpenicillin) is an orally administered form of penicillin (McFerran, 2008). In addition to penicillin, amoxcillin and cephalosporins were fully active against S. pyogenes (Gueye-Ndiaye, A. et al., 2009). Azithromycin and erythromycin were very active according to the result of the study of (Camara et al., 2013), with susceptibility rates greater than 95% and could be used as final alternative choice (p.197). In addition, clindamycin and pristinamycin, less used in therapeutic settings, have shown high degree of efficacy on the ß-haemolytic Streptococcus. Clindamycin is an antibiotic administered by mouth or injection to treat serious bacterial infections and its trade name is Dalacin C (McFerran, 2008). Clindamycin may be used in cases of necrotizing fasciitis and surgical debridement of the affected area is necessary (Bessen, 2009; Collins & Kennedy, 1983). Amoxicillin, which is according to McFerran (2008), a semisynthetic penicillin used to treat infections caused by a wide range of bacteria and other microorganisms which is administered by mouth or injection (p.26). With 97% susceptibility, these two molecules could be used as an alternative or second line antibiotic. Interestingly, chloramphenicol, teicoplanin, vancomycine, and levofloxacin were also very active and could be potential alternative choices of treatment against infection with S. pyogenes (Camata et al., 2013). This bacteria is susceptible to 1% sodium hyphochlorite, 4% formaldehyde, 2% glutaraldehyde, 70% ethanol, 70% propanol, 2% peracetic acid, 3-6% hydrogen peroxide and 0-16% iodine and also susceptible to moist heat (121°C for at least 15 minutes) and dry heat (170°C for at least 1 hour) (Collns & Kennedy, 1983). This bacterium belongs to Risk group 2 (Public Health Agency of Canada, 2010). According to Limsuwan and Supayang (2012), they found out that the extracts of Boesenbergia pandurata, Eleutherine Americana and Rhodomyrtus tomentosa not only demonstrated good antibacterial activity against S. pyogenes isolated from upper respiratory tract infections but also produced similar activities against different S. pyogenes clinical isolates.
The study made by Hussain et al. (2014) showed results wherein E. hirta extract (methanolic and ethanolic) possess the potent antibacterial activity against Gram positive bacteria in which he used Bacillus pumilus, Staphylococcus aureus, and Streptococcus pneumoniae (p. 552). Methanol or methyl alcohol which is a wood alcohol, is an alcohol that is oxidized in the body much more slowly than ethyl alcohol and forms poisonous products, and as little as 10mL of pure methyl alcohol can produce permanent blindness, and 100mL is likely to be a fatal (Mcferran, 2008). In this study made by Ibrahim et al. (2012), it was observed that ethanolic extracts had a significantly higher inhibition zone than the aqueous extract (p.117). This difference is attributed to the solubility of the active component of the different solvent (Ekpo and Etim, 2009). This is an agreement with the report of Deboer et al. (2005) that stated that active components of plants are more soluble in organic solvent (p.467). Results obtained from the study of Titilope et al (2012) showed that dry ethanolic extracts proved that ethanol was better for extraction than water (p.172). According to Ibrahim et al (2012), the ethanolic extracts were more potent against the test organisms than the aqueous extracts and this could be because the active compounds are more soluble in polar solvent which gave it an edge over the sterile distilled water extract which was also noticed by Karou et al. (2013) (p.116). According to Titilope et al. (2012), the alcoholic extracts of E. hirta is highly effective against gram positive bacteria (p. 169). Another study which is made by Jyothirmayi & Saripalli (2012) reported that the methanol extract was found to inhibit the growth of Escherihia coli, Bacillus subtilis and Bacillus thuringiensis (p. 6010), which is also the same with other previous studies. According to the study made by Abu-bakar et al. (2009), the results showed that water seems be the best solvent for the E.hirta plant material, thus supporting the use of water as solvent of choice in traditional practice (p.501). This is contrary to the study made by Hussain et al. (2014), the aqueous extracts of E,hirta showed the weaker antibacterial responses against studied microbes (Bacillus pumilus, Staphylococcus aureus, Streptococcus pneumoniae, E.coli, Citrobacter freundii, Klebsiella pneumoniae, Candida albicans and Aspergillus niger) as compared to ethanolic and methanolic extracts. In proportion to Kuta et al. (2013), the inability of the aqueous extract to have significant activity on the texted organisms could explained by the fact that when ground plant tissues are in aqueous medium, phenolases and hydrolases are released by the tissues and these enzymes have effect on the active components of the plant extracts. Therefore, this may have been the reason why the aqueous crude extract was not active against the test organisms (p.68). With these conditions, Streptococcus pyogenes, which is also a Gram positive bacterium, could be susceptible to E. hirta extracts with the use of methanol mixed with the extract.
The bread spectrum antimicrobial action displayed by some of these extracts could be attributed to the presence of pronounced antimicrobial phytoconstituents such as terpenoids, tannins, flavonoids and alkanoids (Perumal et al., 2012). Same with the study of Cowan (1999) and Draughon (2004), they stated that the observed antibacterial effects on the isolates is believed to be due to the presence of alkaloids, tannins and flavonoids which have been shown to possess antimicrobial properties. Same workers have also attributed their observed antimicrobial effects of plant extracts to the presence of the secondary metabolites ( Nweze et al., 2004) and also identified tannins, flavomoids and alkanoids in the extracts of some medicinal plant (Upadhyay et al., 2010). Previous reports by Junaid et al (2006) revealed that tannins and polyphenols are more soluble in alcohol and are active against cell wall synthesis; it could be the reason for the inhibitory or cidal effects of the alcoholic crude extracts. Another reported phytoconstituents of the herb included triterpenods, while some of the reported scientific uses include its use as an anti spasmatic, antiasthmatic, expextorant, anticatarrhal and antisyphilitic (Burkill, 1994; Adepapo et al., 2005; Falodun et al., 2006). Most of the activities of the plant are believed to be due to the presence of some bioactive components and found out that tannins, phenolics, cardiac glycosides, anthraquinones, saponins, flavonoids and alkaloids (501). These compounds have potentially significant application against human pathogens, including those that cause enteric infections (Abubakar et al., (2008). Several authors have linked the presence of the bioactive compounds to the antimicrobial properties and crude plant extracts ( Adesokan et al., 2007; Ogbolie et al., 2007; Oroolabi et al., 2007; Oyelike et al., 2008). Rahila et al. (1994), Hayashi et al. (1993) and Gills (1992) also reported th presence of various phytochemicals in the leaves extract of E.hirta which has been implicated in the treatment of cough, asthma and hay fever. The presence of tannin has been found to form irreversible complexes with praline-rich protein (Trease & Evans, 1989(resulting in the inhibition of the all protein synthesis. Thus, possessing both antimicrobial and antioxidant activities (Ibrahim et al., 2012). This agreed with the study of Titilope et al. (2012), apart from antimicrobial activity exhibited by tannins, they also react with proteins to provide the typical tanning effect and this is important for the treatment of inflamed ulcerated tissues (p.173). Tannins have important roles such as stable and potent antioxidants (Trease et al., 1983). The presence of alkaloids is interesting, as significant quantities are used as antimalarial, analgesics and stimulants (Duke & Ayensi, 1985). Alkaloids are hetero cyclic nitrogenous compound and have been found to have antimicrobial effects (Trease, 1989). Herbs that have tannins as their components are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery thus exhibiting antibacterial activity (Akinpelu & Onakoya, 2006). Tannins are widely used in traditional medicine in treating wounds and to arrest bleeding (Nguyi, 1988). The presence of glycosides moieties like saponine, anthraquinones, cardiac glycosides and flavonoids which are known to inhibit tumor growth and serve also to protect against gastrointestinal infections are of pharmacognostic importance and give credence to the use of the plant in ethromedicine (Abubakar, 2009). The presence of phenolic compounds have been extensively used in disinfection and remain the standard with which other agents are compared (Okwu, 2001). In the plants were flavonoids which protectplant against allergies, inflammation, free radicals, microbial attack and tumor (Okwu, 2005), the presence of saponin in the plants could implicate them in having antihyper-cholesterol; hypotengine and cardiac depressant properties (Olayinka et al., 1992). Some of these bioactive compounds which are synthesized are secondary metabolites as the plant grows, also serve to protectthe plant against microbial attacks and predation by animals (Abubakar et al., 2008).
Most of the previous studies made, that are related to the present study conducted their experiment in vitro. Attah et al. (2013) made their experiment of E. hirta against Onchocerca volvulus Microfilariae in vitro same with the other studies, also, by isolating microorganisms. In extraction, most of the plants were air dried and grounded into fine powder (Perumal et al., 2012; Upadhyay et al., 2010), but according to Iyabo (1991), fresh whole plant grounded into particles will enhance the permeability of the extracting solvent into the cells, thus facilitating the release of active ingredients. To determine the antimicrobial activity of the plants, previous studies used standard disc diffusion method same with the process made by Hussain et al. (2014). The disc diffusion method for antibiotic susceptibility testing is the Kirby-Bauer method (Disc Diffusion Susceptibility Methods, n.d.). The Kirby-Bauer test for antibiotic susceptibility called the disc diffusion test is a standard that has been used for years (Reynolds, 2011). According to Reynolds (2011), first developed in the 1950’s, it was refined and by W. Kirby and A. Bauer, the standardized by the World Health Organization in 1961 and it has been superseded in clinical labs by automated tests., but, the K-B is still used in some labs, or used with certain bacteria that automation does not work well with (p.1). This test is used to determine the resistance or sensitivity of aerobes or facultative anaerobes to specific chemicals, which can then be used by the clinician for treatment of patients with bacterial infections and the presence or absence of an inhibitory area around the disc identifies the bacterial sensitivity to the drug (Reynolds, 2011). Well diffusion method was used by some of the studies in determination of antimicrobial activity which were used by Titilope et al. (2012) in In-vitro antimicrobial activities of Euphorbia hirta against some clinical agents. For easier disc diffusion method, paper disc diffusion method could also be done, using 6mmor 13mm paper discs or Whatman #1 filter paper (Quinto & Santos, 2005). In some previous studies to make their extracts evaporate in rapid time, they used Rotary Evaporator. According to Sepos (2012), some chemical procedures require a quick and effective separation of substances through evaporation (p. 1). The Rotary Evaporator is a tool which puts the separable substance under vacuum and heats evenly through a spinning motion, causing one component to evaporate and leaving the first component behind. The rotavap works by increasing the rate of evaporation of the solvent by (1) reducing the pressure to lower the solvent boiling point, (2) rotating the sample to increase the effective surface area and (3)heating the solution (Organic Laboratory Techniques 8, n.d.).