Free Project- Determination of phytochemicals composition and antibacterial activity of Ziziphu mauritiana methanolic stem bark extraction

Free Project- Determination of phytochemicals composition and antibacterial activity of Ziziphu mauritiana methanolic stem bark extraction             



Introduction and Literature review 

1.1 Introduction

 Medicinal plants are plants that have a recognized medical use. They range from those used in the production of mainstream pharmaceutical products to plants used in herbal medicinal preparations (Chopra et al., 1956). Herbal medicine is one of the oldest forms of medical treatment in human history and could be considered one of the forerunners of the modern pharmaceutical trade. High  population  growth  rate  (2.8%  per annum)  and  poverty  coupled with  dwindling  economic  reserves  in  the  country  make Nigerians  resort  to  more  affordable  sources  for  their  immediate  health  needs.  As the population increases, demand for traditional medicine will increase (Samy and Ignacimuthu, 2000)  Plants that have medical uses can be found growing in many settings all over the world. Many plants contain pharmacologically active compounds that can be accessed by making teas, tisanes and other preparations. Plants can also be blended with each other to achieve a desired outcome or processed to make homeopathic medicines, along with medicines designed for topical application, such as oils and creams (Gupta et al., 2005).  Bacteria causes a significant amount of illnesses in our society and these illnesses if not treated can be life threatening. Moreover, emergence of multiple drug resistant strains of microorganisms due to in discriminate use of antibiotics to treat infectious diseases has generated a renewed interest in herbal medicine (Chopra et al., 1997). Antibacterial potential of different medicinal plants is being extensively studied all over the world (Kaur and Arora 2009). 

Bioactive  components  are  naturally  found  everywhere  in  most  dietary  higher  plants available  to  humans  and  livestock.  The  natural  products   such   as   plant   extracts   provide   unlimited   opportunities for  new  drug  discoveries,  mostly  because  of  plethora  of  varieties of  phytochemicals  (Cos et al., 2006; Sasidharan et al., 2011)   Literally  Phytochemicals  (from  the  Greek  word phyto, meaning plant) are   biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans  and  livestock  further  than  those  attributed  to  macronutrients  and  micronutrients (Saxena et al.,  2013) Plant  extracts  have  therapeutic  effect  with  or without chemical modification for various infectious diseases cause by bacterial  activities  and other  disease-causing  organisms  (Najafi, 2013; Saxena et al., 2018) Plants  used  for  the  treatment  of  diseases  is  as  old as  mankind,  medicinal plants  are  an  important  source  of  potentially  useful  structures  for the  development  of  new  chemotherapeutic  agents.  The first step towards achieving this goal is the in vitro (Samy and Ignacimuthu, 2000)  antibacterial  activity  studies  (Najafi,  2013) which  reported  that  the  methanol  leaf  extracts  of Ziziphus  mauritiana showed  significant  activity  against  some  certain  bacteria  and  shows potentials  to  have  powerful  antibacterial  effect.  Also  reports  (Palombo and Morton, 1987; Semple 2001)  on the antiviral,  antibacterial,  antifungal,  anti-helmintic,  antimolluscal  and anti-inflammatory  properties  of  plants,  some  of  these  observations have  helped  in  identifying  the  active phytochemicals  responsible  for such  therapeutic  activities  and  in  developing  synthetic drugs  for  the treatment and management of a number of ailments in human beings.

1.1.1 Statement of research problems

The issue of diseases and disease treatment in Nigeria has increasingly becoming alarming. The use of synthetic drugs is usually expensive and often accompanied with various side effect and complications. The production of a given synthetic drug can take a couple years involving multidiscipline as well as expensive. However, the economic status of Nigeria and it concomitant increased in population necessitate the exploitation of the traditional medicinal plants of which they were endowed with.

1.1.2 Justification for the study 

Nigeria is endowed with a multiple varieties of medicinal plants use of the traditional treatment of various diseases. However, only few of these plants ware being exploited and utilized whereas many were left unexploited. The present research work is therefore design hoping to provide scientific proofs on the medicinal potential of Ziziphu mauritiana stem bark extract.

1.1.3 Aim

The aim of this study is to determine the phytochemicals composition and antibacterial activity of Ziziphu mauritiana methanolic stem bark extract.

1.1.4 Objectives 

The objective of the present research is based on the following

Methanolic exctraction of Ziziphu mauritiana stem back.

To determine some Phytochemical compounds present in Ziziphu mauritiana methanolic stem bark extract.

To test for the Antibacterial activity/sensitivity of Ziziphu mauritiana methanolic stem bark extract.

To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ziziphu mauritiana stem bark extract.

1.2  Literature review

1.2.1 Plant under study

Ziziphu mauriatiana is a straggling herb of about 50cm high, of waste moist sites occurring throughout the region of the tropics and part of the subtropics. Zizphu maurtiana is known for its medicinal values in unconventional system of herbal medicine. Ziziphu mauritiana is reported to possess hepatoprotective, antibacterial, anti-inflammatory, analgesic, immuno-modulatory, antiviral, leprosy, asthma and bronchitis activities (Arunachalam et al., 2009; Leal et al., 2000)

Ziziphus mauritiana is a spiny, evergreen shrub or small tree up to 15 m high, with trunk 40 cm or more in diameter; spreading crown; stipular spines and many drooping branches. The fruit is of variable shape and size (It is oval, obovate, oblong or round, and that can be 2.5- 6.25 cm long, depending on the variety). The flesh is white and crisp. When slightly underipe, this fruit is a bit juicy and has a pleasant aroma. The fruit's skin is smooth, glossy, thin but tight. It is the most commonly found in the tropical and sub-tropical regions. Originally native to India it is now widely naturalized in tropical region from Africa to Afghanistan and China, and also through Malaysia and into Australia and in some Pacific regions. It can form dense stands and become invasive in some areas, including Fiji and Australia and has become a serious environmental weed in Northern Australia. It is a fast growing tree with a medium life span, that can quickly reach up to 10–40 ft tall. Ziziphus mauritiana can be propagated from seeds, or vegetatively, through in situ grafting or budding on to rootstocks. A spacing of 5-6 m or 8-9 m is often used (Latiff, 1991). The trees begins to fruit within 2 years but do not yield sizeable crop before the fourth year (Latiff, 1991). Once the fruits have been harvested, the trees must be pruned so that new fruits are produced on the next year shoots. In India, the best cultivars yielded 77 kg fruit/year but in Mali only 2.5 kg fruit/year were reached (Morton, 1987; Houérou, 1980). The amount of fodder (including leaves and fruits) from Ziziphus mauritiana available to livestock in Sahelian sandy pastures was only 0.5 kg/ha and 14.8 kg/ha in low lying pastures (Sanon et al., 2005).  Plants of the Ziziphus genus can withstand extreme stress caused by drought, salinity, and in some cases water logging. Cultivated Ziziphus mauritiana are thus ideal for planting in sites unfit for other crops, such as marginal or degraded areas, provided that the right genotypes are selected for alkali or sodic soils (Hebbara et al., 2002; Dagar et al., 2001). Ziziphus mauritiana can be used for coastal dune sand fixation (Orwa et al., 2009; Azam et al., 2006). Ziziphus mauritiana grafted on Ziziphus nummularia can be grown on saline soils (Ecocrop et al .,2013). In the Sahelian climate, Ziziphus mauritiana provides permanent cover to the soil, with the abundant and deep root system helping to maintain soil structure (Arndt et al., 2001;Depommier, 1988).

      The different parts of the plant are used as cuts and ulcers healer, pulmonary ailments, fevers, laxative, sedative, antinausea, anti-rheumatic areas, anti-diarrhoeal, wounds and abscesses healer, swelling, gonorrhoea curer (Michel, 2002) and also used as anthelmintic in ethnoveterinary medicinal system in Pakistan (Hussain et al., 2008). 

              The plant is also used in liver diseases, asthma and fever, gingivitis, febrifugee and epilepsy (Msonthi and Magombo, 1983; Morton, 1987). Ziziphu mauritiana is a plants that holds a reputable position in both Ayurvedic and Unani Systems of medicine. (Michel, 2002) Different parts of the plant are useful as cardiovascular tonic, alexteric, styptic and anthelmintic. The plant is also used in diuresis, ulcers, asthma, heart disease, biliousness, fractures, tumours, leucoderma, anemia, excessive perspiration, internal and external problems of urinary discharge, endocarditis, mitral regurgitation, pericarditis, angina and heart tonic (Bharani et al., 1995; Bharani et al., 2002; Karthikeyan et al., 2003; Sarwat et al., 2006). Plant Description

Ziziphu mauritiana is also called Ziziphus mauritianais a shrub or small thorny tree that can grow to a height of 3-15 m. Deciduous or almost evergreen, Ziziphus mauritianahas an erect or spreading habit (US Forest Service, 2013; Ecocrop et al., 2013; Orwa et al., 2009). It has a deep taproot (Morton, 1987). The trunk is around 40 cm in diameter, covered with a dark grey or dull black, irregularly fissured bark (Orwa et al., 2009). The branches are numerous and drooping. The twigs are tomentose. The spines are solitary or borne in pairs at the base of the leaves, 5to 7 mm long (Latiff, 1991). In cultivated Indian Ziziphus mauritiana, the spines may be absent (Azam et al., 2006). The leaves are simple, alternate, ovate, and 2-9 cm long with 3 conspicuous longitudinal veins. The upper surface of the leaves is dark glossy green and the lower one is densely hairy (Latiff, 1991; Morton, 1987).This dense silky underside helps distinguish Ziziphus mauritianafrom Chinese Ziziphus mauritiana (Ziziphus jujuba Mill.) (Morton, 1987). The flowers are pentamerous, greenish yellow in colour, hairy outside and about 5 mm wide. They are borne on 1-2 cm long axillary cymes (US Forest Service, 2013; Latiff, 1991). The fruit is an ovoid drupe, 6 cm x 4 cm in size. The fruit skin may be smooth or rough, glossy, yellowish to reddish. The flesh is white, juicy, slightly acid to sweet, turning mealy when fully ripe (Latiff, 1991). The stone is single, central, hard, oval or oblate, with a rough surface. It contains 2 elliptic, brown seeds, 6 mm long (Morton, 1987).

Ziziphu mauritiana  is a multi-purpose tree mainly grown for its fruits. It starts bearing fruits 6-8 years after planting, and yield increases until the tree is 15-20 years old (Ecocrop et al., 2013). The fruit is edible and can be eaten fresh, dried like dates, candyied, salted or pickled (Ecocrop et al., 2013; Latiff, 1991). It can be processed into flour meal, paste, juice, syrup or an alcoholic beverage (Latiff, 1991). It is a good source of carotene, vitamins A and C, and fatty oils (Orwa et al., 2009). Young leaves are edible and are cooked as a vegetable in Indonesia. Fruits and bark are used to make dye and medicinal preparations (Ecocrop et al., 2013; Orwa et al., 2009). Ziziphus mauritiana wood is reddish, fine-textured, hard and durable. It can be used in rural house construction, posts and tool manufacturing. It makes excellent firewood. The Ziziphus mauritianatree hosts lac insects, and is also fodder for the tasar silkworm that makes high-prized silk in India (Orwa et al., 2009). It is a minor source of pollen for bees (Orwa et al., 2009). A potential agroforestry species, this thorny tree can grown to provide windbreaks and living fences. It is browsed by livestock and its leaves are nutritious fodder for sheep and goats (Ecocrop et al., 2013; Orwa et al., 2009; Nair, 1993). Taxonomical classification

The taxonomical classification of Ziziphu mauritiana ranged from kingdom to the specie level.

Kingdom                        Plantae 

Subkingdom                 Viridiplantae 

Infrakingdom              Streptophyta 

Superdivision               Embryophyta

Division                        Tracheophyta 

Subdivision                  Spermatophytina 

Class                             Magnoliopsida

Superorder                  Rosanae

Order                            Rosales

Family                           Rhamnaceae 

Genus                           Ziziphus Mill.

Species                        Ziziphus mauritiana Lam. Origin of the plants

Ziziphu mauritiana originated from Central Asia and then spread to North Africa and India through Afghanistan, South China, Malaysia and Australia. It was in use around 1000 BCE and is now widely naturalized in the tropics (Africa, Central and South America, and the West Indies), in the Pacific Islands and in the Mediterranean region (Orwa et al., 2009; Morton, 1987). It is only commercially important in India and China (Orwa et al., 2009). In South-East Asia, it is mostly found in Thailand (Latiff, 1991). It is becoming increasingly important for its wide adaptation, easy management, early fruit bearing, value as a food and feed, and multiple uses (Liu et al., 2014). Ziziphus mauritiana can be grown in semi-arid and arid regions as it thrives under very dry conditions. In the Sahelian zone, Ziziphus mauritiana is one of the most persistent trees, like Acacia raddiana and Balanitesaegyptiaca, (Sanon et al., 2012). Ziziphus mauritiana is found from sea level up to an altitude of 1600 m in India, though it is mostly cultivated under 1000 m. It grows where annual rainfall ranges from 150 mm to 4000 mm. Fruits need hot, sunny and dry condition. 

Ziziphus mauritiana can be propagated from seeds, or vegetatively, through in situ grafting or budding on to rootstocks. A spacing of 5-6 m or 8-9 m is often used (Latiff, 1991). The trees begins to fruit within 2 years but do not yield sizeable crop before the fourth year (Latiff, 1991). Once the fruits have been harvested, the trees must be pruned so that new fruits are produced on the next year shoots. In India, the best cultivars yielded 77 kg fruit/year but in Mali only 2.5 kg fruit/year were reached (Morton, 1987; Houérou, 1980). The amount of fodder (including leaves and fruits) from Ziziphus mauritiana available to livestock in Sahelian sandy pastures was only 0.5 kg/ha and 14.8 kg/ha in low lying pastures (Sanon et al., 2005). but moisture is necessary to support growth and flowering. During hot summers, with temperatures up to 49-50°C, the tree may shed its leaves and stop growing. New shoots can grow with onset of the rains (Azam et al., 2006). In Burkina Faso, Ziziphus mauritiana remains green later in the season than acacia species (Sanon et al., 2012). Ziziphus mauritiana does well on a wide range of soils but prefers well-drained, deep sandy, neutral or even slightly acidic or alkaline loams. It can withstand some salinity (Latiff, 1991). Plant Distribution 

Ziziphus mauritianais widely distributed in warm regions of the world but India is its main site of cultivation. Currently, it is grown over approximately 49,000 hectares with 481,000  production per year in India (Anon, 2017). It is an important fruit crop of the hot arid regions in India as it forms an integral part of the life of the locals as a source of nutrition, fodder (leaves), fuel (pruned wood) and has several ethnobotanical uses (Krishna and Parashar, 2013; Morton, 1987). It is a dominant component of the natural vegetation in the desert of India and thrives under a maximum annual temperature of 35–42°C and minimum temperature of 4–12°C (Azam et al., 2006; Meghwsal and Tewari (2007), asserted that ber trees should not be grown in areas experiencing temperatures lower than 4°C for prolonged periods. The plant can tolerate temperatures as high as 49–50°C and as low as −2°C (Awasthi and More, 2009). It can produce fruits even in areas which receive average annual rainfall of only 150–200 mm and thus is a suitable crop for the arid and semi‐arid regions of the world (TelZur and Schneider, 2009). Ziziphus mauritiana is a spiny shrub or a small tree with ends of branches decurved or drooping. It has a characteristic shoot growth pattern which may be an adaptation to arid and semi‐arid conditions. It produces three types of proleptic shoots: vigorous, normal and spur‐type by reiteration or following pruning of the previous season's growth. It also produces a dense canopy by sylleptic branching when conditions are favorable for growth, and shows rapid morphogenetic adjustments by the way of shoot tip abscission or dormancy of apical buds at the onset of drought (Kurian and  Reddy, 1999). The plant is vigorous and has a rapidly developing tap root. The bark is dark grey or nearly black in colour, with deep vertical cracks and a reddish, fibrous interior. Leaves are simple, alternate, distichous, ovate or oblong elliptic in shape, while stipular spines are borne solitary or in pairs, both recurved or with one of them straight. Leaves are distinguished from those of the Chinese Ziziphus mauritiana (Ziziphus Mauritiana) by the presence of dense, silky, whitish or brownish hairs on their underside (Morton, 1987). The leaf surface is glabrous above and dark‐green in colour with three conspicuous, depressed, longitudinal veins originating from the base, which later converge at the tip (Krishna et al., 2014). The plant sheds leaves and enters into dormancy for several weeks in hot summers (May–June in North Indian States) which is an adaptive mechanism to escape damage through desiccation during hot weather (Awasthi and More, 2009). This strategy is different from that of the Chinese Ziziphus mauritiana, which goes leafless during cold winters. The flowers are bisexual, greenish yellow in colour and are borne in 15–20 flowered, dense cymose axillary fascicles. The ovary has two chambers, each with a single ovule, and is broadly attached at the base. Each fruit can bear two viable embryos (TelZur and Schneider, 2009). The fruit varies in size depending upon the genotype and management practices. The shape may be oval, obovate, round or oblong; the skin smooth or rough, glossy, thin but tough. They turn from light green to yellow, later becoming partially or wholly burnt orange or red brown or red. When slightly under‐ripe, the flesh is white, crisp, juicy, acid or sub acid to sweet and somewhat astringent. Fully ripe fruits are less crisp and somewhat mealy; overripe fruits are wrinkled, the flesh buff‐coloured, soft, spongy and musky. At first, the aroma is apple‐like and pleasant but it becomes peculiarly musky as the fruit ripens (Pareek, et al., 2009). Flowering begins in July and continues until September, while fruiting occurs from October to January. However, time of pruning may influence onset of flowering (TelZur and Schneider, 2009). Growth pattern

Ziziphus mauritiana has been recognized as an underutilized plant worthy of further research and development by the Indian National Genetic Resources Programme and the International Centre for Underutilised Crops, UK. It is an important fruit crop of the hot arid regions in India as it forms an integral part of the life of the locals as a source of nutrition, fodder (leaves), fuel (pruned wood) and it has several ethnobotanical uses. (Hernández, et al., 2015). The plant form dense stands and is invasive in some regions like Fiji and Australia. These days it an environmental weed in Northern Australia causing problems. It is grows very fastly with a medium life span, that can quickly reach up to 10–40 ft. tall. (Hernández, et al., 2015).

1.2.2 Ethno-botanical uses 

Ziziphus mauritiana commonly  called,  Red  Date  or  Chinese  date  or  Bera (Pushto),  belonging  to  family Rhamnaceae,  is  used  primarily  for  its  fruits.  Ziziphus mauritiana,  a  delicious  fruit,  is  an effective  herbal remedy improving stamina and muscular strength and aids weight gain ((Hernández, et al., 2015). It strengthens liver function  and  increases  immune  system  resistance (Hernández, et al., 2015).  It  functions  as antidote,  diuretic, emollient and expectorant (Chen et al., 2015). The leaves are febrifuge, astringent and said to promote the hair growth (Sanon et al., 2005) In the treatment of strangury they are used to form a plaster (Berger et al., 2010)  The dried fruits are anticancer, anodyne, refrigerant, sedative, styptic, pectoral, tonic and stomachic (Costa et al., 1999). They  help  in  digestion  and  blood  purification. (Sanon et al., 2005). They  are  used  internally  to  treat  loss  of appetite,  chronic  fatigue,  hysteria,  diarrhea,  irritability  and  anemia (AlHavi 2001 and Razi)  The  seed  is sedative,  stomachic,  hypnotic,  tonic  and  narcotic (Sina,  2005 ). It  is  used  internally  to  treat insomnia, nervous exhaustion, palpitations, excessive perspiration and night sweats (Costa et al., 1999). For the treatment of dyspepsia and fevers, root is used (Morton, 1987). The powdered root is applied to old ulcers and wounds (Latiff, 1991).  The plant is a folk remedy in China as a treatment for burns, anemia,  nephritis,  hypertonia  and  nervous  diseases  (Ecocrop et al., 2013 The seeds  have  been  used  as tranquilizer, analgesic, convulsant in oriental countries like Korea and China  Ziziphin, a compound in the leaves of the Ziziphus mauritiana, suppresses the ability to perceive sweet taste in humans (Pareek, et al., 2009)  The mucilaginous nature of the fruit of Ziziphus mauritiana makes them a candidate in pharmacy to treat  sore  throats. Ziziphus mauritiana extracts  exhibited  a  protection  against hydroquinone  induced cytogenesis (Pareek, et al., 2009)  Extracts of Ziziphus mauritiana fruits  and  seeds  exhibited moderate  activity  against Lycoriella ingenua and Coboldia fuscipes, which are important mushroom pests (Costa et al., 1999).

1.2.3 Medicinal use 

Ziziphus mauritiana is used for improving muscular strength and weight, for preventing liver and bladder diseases and stress ulcers, and as a sedative (Hernandez et al., 2016). It is also used to reduce constipation and to reduce symptoms of some medications. Zizyphus mauritiana is also used for various skin conditions including dry and itchy skin, purpura, wounds, and ulcers; digestive problems including lack of appetite and diarrhea; and circulatory problems including high blood pressure, high cholesterol, and anemia. Other uses are for diabetes, fatigue, hysteria, anxiety, insomnia, seizures, fever, obesity, cancer, inflammation, asthma and other lung disorders, and eye diseases. In newborns, it is used for jaundice (yellowing of the skin). The use of the Ziziphus mauritiana fruit in traditional medicine has a long history due to some of its pharmacological properties and benefits for blood purification and digestion. It also slows down the process of aging in women (Chen et al., 2015). It is also important in the treatment of rectal and intestinal ulcers/diseases as well as liver diseases. Ripe Ziziphus mauritiana fruit has laxative properties, but unripe Ziziphus mauritiana heals diarrhea.  This plant is difficult to digest, and thus it might inhibit digestion in people suffering from digestive system problems. Flatulence is also a side effect of the excessive intake of Ziziphus mauritiana. Depending on the patient’s physical condition, honey and libido-enhancing medicines are prescribed to avoid the decrease in libido following the consumption of Ziziphus mauritiana (AlHavi 200;  Razi and Sina  2005 ).

1.2.4 Phytochemical properties

Phytochemicals are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those attributed to macronutrients and micronutrients (Blumberg and Harsler, 1999). They protect plants from disease and damage and contribute to the plant’s color, aroma and flavor. In general, the plant chemicals that protect plant cells from environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack are called as phytochemicals (Gibson et al., 1998). Recently, it is clearly known that they have roles in the protection of human health, when their dietary intake is significant. More than 4,000 phytochemicals have been cataloged and are classified by protective function. A wide-ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices (Mathai, 2000). Broccoli, cabbage, carrots, onions, garlic, whole wheat bread, tomatoes, grapes, cherries, strawberries, raspberries, beans, legumes, and Soy foods are common sources (Moorachian, 2000).

  Phytochemicals accumulate in different parts of the plants, such as in the roots, stems, leaves, flowers, fruits or seeds (Costa et al., 1999). Many phytochemicals, particularly the pigment molecules, are often concentrated in the outer layers of the various plant tissues. Levels vary from plant to plant depending upon the variety, processing, cooking and growing conditions (King, 1999). Phytochemicals are also available in supplementary forms, but evidence is lacking that they provide the same health benefits as dietary phytochemicals (American cancer society, 2000). Flavanoids

Flavonoids are polyphenolic compounds that are ubiquitous in nature. More than 4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridahm, 1960). Flavonoids have been reported to exert multiple biological property including antimicrobial, cytotoxicity, anti-inflammatory as well as antitumor activities but the best-described property of almost every group of flavonoids is their capacity to act as powerful antioxidants which can protect the human body from free radicals and reactive oxygen species (Pridahm, 1960).

Figure 1: Basic structures of some derived flavonoids (Pridahm, 1960) Tannins

It might be said that the tannins are a heterogeneous group of high molecular weight polyphenolic compounds with the capacity to form reversible and irreversible complexes with proteins (mainly), polysaccharides (cellulose, hemicellulose, pectin, etc.), alkaloids, nucleic acids and minerals, etc. (Schofield et al., 2001). In medicine, especially in Asian (Japanese and Chinese) natural healing, the tannin-containing plant extracts are used as astringents, against diarrhea, as diuretics, against stomach and duodenal tumors (Debruyne et al., 1991) and as anti-inflammatory, antiseptic, antioxidant and haemostatic pharmaceuticals (Dolara et al., 2001).

                     Daidzein                                                                                                   Gallic acids



Figure 2: Basic structures of some derived tannins (Debruyne et al., 1991) Saponins

Saponins are a group of secondary metabolites found widely distributed in the plant kingdom. They form a stable foam in aqueous solutions such as soap, hence the name “saponin” Chemically, saponins as a group include compounds that are glycosylated steroids, triterpenoids, and steroid alkaloids (Dolara et al,  2001). Alkaloids 

Alkaloids are natural product that contains heterocyclic nitrogen atoms, are basic in character. The name of alkaloids derives from the “alkaline” and it was used to describe any nitrogen-containing base (Mueller and McAllan, 1992). Alkaloids are significant for the protecting and survival of plant because they ensure their survival against micro-organisms (antibacterial and antifungal activities), insects and herbivores (feeding deterrents) and also against other plants by means of allelopathically active chemicals (Molyneux et al., 1993).

  The use of alkaloids containing plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization. Alkaloids have many pharmacological activities including antihypertensive effects (many indole alkaloids), ant arrhythmic effect (quinidine, spareien), anti malarial activity (quinine), and anticancer actions (dimericindoles, vincristine, vinblastine). These are just a few example illustrating the great economic importance of this group of plant constituents (Wink et al., 1998). Some alkaloids have stimulant property as caffeine and nicotine, morphine are used as the analgesic and quinine as the anti malarial drug  (Wink et al., 1998)

Figure 3: Basic structures of some derived alkaloids Glycoside

Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. These can be activated by enzyme hydrolysis, which causes the sugar part to be broken off, making the chemical available for use (Arias and Brito, 2007). Steroids

 A steroid is an organic compound with four rings arranged in a specific configuration. Examples include the dietary lipid cholesterol, the sex hormones estradiol and testosterone and the anti-inflammatory drug dexamethasone. Steroids have two principal biological functions: certain steroids (such as cholesterol) are important components of cell membranes which alter membrane fluidity, and many steroids are signaling molecules which activate steroid hormone receptors. (Mueller and McAllan, 1992).

Figure 4: Basic structures of some derived steroids (Mueller and McAllan, 1992).

1.2.6 Test organisms

Some organisms that were tested for the antibacterial activity of this plant stem back extract include the following: Escherichia coli (E. coli)

Escherichia coli are part of the natural flora of many animals. Human infections occur through consumption of contaminated food products (undercooked meat, or contaminated fresh produce such as salad leaves), drinking water contaminated with animal or human waste, or through direct person-to-person spread from poor hygiene (Berger et al., 2010). Accurate figures of the incidence of enteric E. coli infections. As the causative agents of diarrhogenic infections are often not identified. And appear to be major causes of infantile diarrhea with potentially fatal consequences when untreated. These infections are mild and self-limiting and more recently are the main E. coli pathotypes associated with food poisoning out breaks in the developed Country and is the most commonly isolated bacterial entero-pathogen in children under 5 yrs of age in developing countries, accounting for approximately 20% of cases, equivalent to several hundred million cases of diarrhea and several tens of thousands of deaths each year (Qadri et al.,2005), and  is also the most common cause of travelers’ diarrhea accounting for 10–60% of infections depending on the region visited (Black, 1990; Gascón et al., 1998). Infectious Disease of E. coli

Escherichia coli is one of the most frequent causes of many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI) and traveler's diarrhea and other clinical infections such as neonatal meningitis and pneumonia. The genus Escherichia is named after the author Escherich, who isolated the type species of the genus. (Postgate, 1998) Escherichia organisms are gram-negative bacilli that exist singly or in pairs. E coli is facultatively anaerobic with a type of metabolism that is both fermentative and respiratory (Black ,1990 ) They are either non motile or motile by peritrichous flagella. E coli is a major facultative inhabitant of the large intestine (Black ,1990 ). Klebsiella pneumoniae

Klebsiella pneumoniae is a Gram-negative, non-motile, encapsulated, lactose-fermenting, sfacultative anaerobic, rod-shaped bacterium. It appears as a mucoid lactose fermenter on MacConkey agar. Although found in the normal flora of the mouth, skin, and intestines, (Ryan et al, 2004). It can cause destructive changes to human and animal lungs if aspirated, specifically to the alveoli resulting in bloody sputum. In the clinical setting, it is the most significant member of the genus Klebsiella of the Entero-bacteriaceae. K. oxytoca and K. rhinoscleromatis have also been demonstrated in human clinical specimens. In recent years, Klebsiella species have become important pathogens in nosocomial infections. It naturally occurs in the soil, and about 30% of strains can fix nitrogen in anaerobic conditions (Postgate, 1998). Infectious Disease of klebsiella pneumoniae

Klebsiella pneumoniae is second to Escherichia coli the most common gram-negative pathogen associated with a wide spectrum of infections, such as urinary tract infection (UTI), pneumonia, intra-abdominal infection, bloodstream infection (BSI), meningitis and pyogenic liver abscess (PLA) (Ullmann and  Podschun, 1998) during the last decades the rates of extended spectrum cephalosporin-resistant K. pneumoniae producing extendedspectrum β-lactamases (ESBL) have dramatically increased worldwide and in most parts of the world K. pneumoniae is the pathogen mostly associated with dissemination of ESBLs and other horizontally transmissible resistance genes  (Bush, 2007; Iredell, 2016; Queenan,  2007). Invasive infections caused by K. pneumoniae have been associated with such as cancer, diabetes, and previous organ transplantation (Meatherall et al., 2009; Wang et al., 1999) Staphylococcus aureus

Staphylococcus aureus is a Gram-positive, round-shaped bacterium that is a member of the Firm cutes, and it is a usual member of the micro biota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive for catalase and nitrate reduction and is a facultative anaerobe that can grow without the need for oxygen (Masalha et al, 2001).

Although S. aureus usually acts as a commensal of the human microbiota it can also become an opportunistic pathogen, being a common cause of skin infections including abscesses, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. The emergence of antibiotic-resistant strains of S. aureus such as methicillin-resistant S.aureus (MRSA) is a worldwide problem in clinical medicine.                    Infectious Disease of staphylococcus aureus

Staphylococcus aureus is the most dangerous of all of the many common staphylococcal bacteria. These gram-positive, sphere-shaped (coccal) bacteria  often cause skin infections but can cause pneumonia, heart valve infections and bone infections (Masalha et al, 2001)

The most common staphylococcal infections are Skin infections: often causing abscesses. However, the bacteria can move through the bloodstream (called bacteremia) and infect almost any site in the body, particularly heart valves (endocarditis) and bones (osteomyelitis). The bacteria also tend to accumulate on medical devices in the body, such as artificial heart valves or joints, heart pacemakers, and catheters inserted through the skin into blood vessels.

Certain staphylococcal infections are more likely in certain situations (Masalha et al, 2001) Bloodstream infections: When a catheter that is inserted in a vein has remained in place for a long time Endocarditis: When people inject illegal drugs or have an artificial heart valve or when a catheter inserted in a vein is infected Osteomyelitis: When Staphylococcus aureus spreads to the bone from an infection in the bloodstream or from an infection in nearby soft tissue, as may occur in people with deep pressure sores or foot sores due to diabetes Lung infection (pneumonia): When people are attacked with influenza (particularly) or a bloodstream infection, when people are taking corticosteroids or drugs that suppress the immune system (immune suppressant), or when they are hospitalized because they need tracheal intubation and mechanical ventilation (called hospital-acquired pneumonia) (Postgate, 1998) Infectious Disease of K. oxytoca

Klebsiella oxytoca  is one of several Klebsiella bacteria. These bacteria are naturally found in the intestinal tract, mouth and nose. They are considered healthy gut bacteria inside intestines. (Ryan et al, 2004).  outside the gut, however, these bacteria can cause serious infections (K. oxytoca) is commonly spread in healthcare environments. These environments include nursing homes and intensive care units. K. Oxtoca can cause a serious infection.  K. Oxytoca can also lead to urinary tract infections (UTIs), wound infections (Berger et al., 2010 ). The type of bacteria and where it infects the body determines the symptoms of the disease.  Chemical constituent

The major chemical constituent found in plant is a follow

  Plant contains crude protein, fat, fiber, ash, calcium, phosphorus, magnesium, potassium, sodium, chlorine, Sulphur (Morton, 1987; Palombo and Semple 2001) They also contain ceryl alcohol and the alkaloids, protopine and berberine, quercetin, kaempferol, sitosterol, stigmasterol, lanosterol, diosgenin. The leaves contain flavonoids, tannins, oses and holosides, mucilages, sterol, triterpenoids, cardiotonic glucosides, and leucoanthocyanes. Plant also contain Protein, Fat, Fiber, Carbohydrates  (Palombo and Semple 2001; Morton, 1987).

                                                  CHAPTER TWO

2.0 Chemicals/reagent

Table 1: List of apparatus and equipment used

S/no      Name        Model          Manufacturer


  1.   Beaker                                     Duran                                 Duran glass, U.S.A

  2.   Pippette                                   Duran                                 Duran glass,U.S.A

  3.   Water Bath           -                                                             Japan Ernainc

  4.   Conical flask                            Pyrex                                  Pyrex, England

  5.   Test tube                                  Pyrex                                  Pyrex, England

  6.   Measuring cylinder                  Pyrex                                  Pyrex, England

  7. Weighing balance                      Mettler PC440                    Mettler, Switzerland

Table 2: List of Chemical Reagents used

S/no   Name          Formula                Grade                 Purity%            Manufacturer   

Sulphuric acid     H2SO4         G.R                    98                  Loba chemie, India.

Sodium Hydroxide  NaOH     LC                      97                 P.I SCI. Park, U.K

Ferric chloride     Fecl2            LC     -                                     P.I SCI. Park, U.K

Hydro chloric Acid  HCl         G.R                    35.                 Loba chemie, India 

Acetic anhydride                     L.R    -                                     Loba chemie, India

Ethyl acetate          -                 L.R    -                                     Loba chemie, India

Chloroform        CCL4             LC    -                                      P.I SCI. Park, U.K

Fehling’s solution     -               L.R    -                                    Loba chemie, India

Balget’s reagent       -                L.C    -                                    P.I SCI. Park, U.K

Lead Acetate    PbCH2CHCH3    G.R    -                                    Loba chemie, India 

Ammonium       NH4                 L.R    -                                    Loba chemie, India

Potassium ferrocynide               L.C   -                                     P.I Park, U.K 

Fehling’s solution                      G.R                            Loba chemie, india 

Picric acid                                  L.R                                         P.I Park, U.K

2.1 Test organisms

The test microorganisms which include Escherichia coli, Staphylococcus aureus, Klebsiella Pneumoniae and Klebsiella Oxytoca  were obtained from the Department of Microbiology, Kebbi state university of science and technology, Aliero, and were maintained in nutrient agar slant at 37°C before used as described by Jumare et al., (2015). The plate of the test organisms was obtained  from isolate culture of agar slants. The isolates were sub-cultured on selective and differential media and re-identified using colony morphology, gram reaction mortality test and haemolytic activity and biochemical test  Wire loop colonies of culture of bacterial culture were picked and suspended in 5 ml nutrient broth in 10 ml bottles and were incubated at 37°C for 24 hrs.

2.2 Collection of plant material

The leaves of Z. mauritiana were collected from Kebbi State University of Science and Technology Aliero, Aliero Local Government Kebbi State, Nigeria.  identified and authenticated at the herbarium of the department of biological science Kebbi State University of Science and Technology Aliero. With the voucher number of 295.

2.3 Preparation of plant extracts

The fresh stem bark of the tree was air-dried under shed for four weeks, and it was pulverized into powdered form using a mortar and pestle and sieved. About (100g) of the coarse  powdered sample was extracted with 500ml of 90% Methanol for 72 hours and the mixture was filtered using Muslim Cloths The filtrate was stored in hot air Ovum before the experiment at 37oC Distilled water was used to reconstitute the solid extract to obtain a desired concentration for the studies.


2.4 Preparation of stock solution of extract: 

The stock solution was prepared by dissolving 1.0 g of the extract in 10 ml of distilled water to obtained a Final concentration of 100 mg/ml. The stock solution was reconstituted to a graded concentration of 50 mg/ml, 25 mg/ml and 12.5 mg/ml by two fold dilution this was stored at 37°C.

2.5 Preliminary phytochemical screening.

Preliminary phytochemical screening for the presence of phenols, tannins, flavonoids, alkaloids, steroid and saponins was carried out using standard test protocols (Msonthi and Magombo, 1983). These phytochemicals were identified by characteristics colour change using standard procedures (Trease and Evans, 1983).

2.5.1 Test for alkaloids: To the 1ml of the extract, 2 ml of picric acid was added. Orange colouration indicates the presence of alkaloids.

2.5.2 Test for saponins: The plant extract (1ml) was shaken with water in a test tube and was heated to boil. Frothing was observed which was taken as a preliminary evidence for the presence of saponin.

2.5.3 Test for tannins: Extract (1ml) was added to 10 ml of water in a test tube and filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or blue-black coloration. as an indication for the presence of tannins.

2.5.4 Test for steroids: Acetic anhydride (2 ml) was added to 1ml of methanol extract of each sample with 2 ml sulphuric acid. A colour change from violet to blue or green in some samples was an indication of the presence of steroids.

2.5.5 Test for flavonoids: Ethylacetate (5 ml) was added to small portion of the powdered plant material and it was held over steam bathe for 3 minutes. The mixture was filtered and 1 ml of dilute ammonia solution was added to 4 ml of the filterate and then shaken. A yellow colouration indicates the presence of flavonoids (Sofowora, 1993).

2.5.6 Test for Phenols: The powdered (1 ml) plant material was boiled with 5 ml of sulphuric acid and filtered while hot. 5 ml of chloroform was added and shaken. The chloroform layer was then pipetted into another test tube and 1 ml of dilute ammonia was added to it. The resulting solution was observed for colour changes to green-blue or violent.

2.5.7 Test for cynogenic glycosides: To 1ml of the extract, 5 ml of 50% H2SO4 was added and the mixture heated in boiling water for about 15 min. Fehling’s solution (5 ml) was then added and the mixture boiled. A brick-red precipitate was confirmatory for the presence of glycosides.

2.6 Preparation of nutrient agar

Nutrient agar was prepared for this study by weighing 28 g of the powdered agar and dissolved in 1000 ml of distilled water in a conical flask. It was then autoclaved at 121°C for 15 minutes. After sterilization, it was allowed to cool at room temperature and the content was poured aseptically into a sterile petri-dish and allowed to solidify.

2.7 Antimicrobial activity study of the extract.

Agar well diffusion and broth dilution technique was used to test for antimicrobial activity in accordance with the method described by Irobi et al., (1994). The antibacterial  activity of the extract  was tested on  E.coli, S. aureus, K. pneumoniae and K. oxytoca. Isolates were first grown in a nutrient broth for 18 h before use and standardized to 0.5 McFarland standards (106 cfu/ml). Two hundred micro (200µL) liters of the standardized cell suspensions was spread on a Mueller-Hinton agar. Wells were then bored into the agar using a sterile syringe. A few drops of different concentration of the crude extract at 100 mg/ml,   50 mg/ml and 25 mg/ml  was then introduced into the wells, and was allowed to stand at room temperature for about 2 h and then incubated at 37°C. The plates were observed for zones of inhibition after 24 h (Michel, 2002).

2.8 Minimum Inhibitory Concentration (MIC)

Minimum Inhibitory Concentration (MIC) of the extract was carried out on the microorganisms that were sensitive to the extract and was done using broth dilution method (Vollekova et al., 2001). Different concentrations of the extract that exhibited antimicrobial activity against the test organisms were prepared in the test tube containing Mueller Hinton Broth (MHB). The organisms were inoculated into each tube containing the diluted extracts. The plates were incubated at 370C for 24 hours. The lowest concentrations of the extract which shows no turbidity was recorded as the minimum inhibitory concentrations. (MIC)

2.9 Minimum Bactericidal Concentration (MBC)

Minimum Bactericidal Concentrations (MBC) of the extracts were carried out to check whether the test microbes were killed or only their growth was inhibited. Mueller Hilton agars were prepared according to the manufacturer’s instruction, boiled to dissolve and were sterilized at 1210C for 15 minutes, the media were cooled to 450C and the medium (20 ml) was poured in to sterile Petri dishes, the plates were covered and allowed to cool and solidify. The contents of the MIC in the serial dilution was inoculated on to the media, the plates were incubated at 370C for 24 hrs, after which the plate were observed for colonies growth. The MBC was the plate with lowest concentrations of the extract without colony growth (Mann et al., 2008).


3.0 Results.

3.1 Phytochemicals screening

The result of the phytochemicals screening on the methalonic Extract of Ziziphu mauritiana is Present in table 1 

The results indicate  the presence of phytochemicals compounds such as saponins, tannins, alkaloids, phenolics, steroids, and Cyanogenicglycosides. however  Flavonoids. not detected in the Stem bark Extract.

Table 3.0: Result on phytochemical screaning of Methanolic extract of Z. mauritiana

         S/N                   Phytochemicals           Effectiveness of Stem bark extract

1.      Alkaloids                                                    ++

2.                    Tannins                                                     ++

3.                   Steroids                                                      ++

4.                   Saponins                                                     ++

5.                   Flavonoids                                                 __

6.                  Phenolics                                                     ++

            7                Cyanogenicglycosides                                   ++

Keys (+) detect, (-) Not detect 

3.2  Antibacterial activity of crude Methalonic extract of Ziziphu mauritiana

For antibacterial test, the test organisms were Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca  and  Staphylococcus aureus, which the activity of the methanol Stem bark  extract of Z.mauritiana base on different concentration were tested against the listed organisms. It was found that at 100mg/ml concentration the extract shows zone of inhibition 25mm against K.oxytoca, while it  shows 20mm against K.pneumonae. while it shows 24mm against E.coli  and expressed 20mm zone of inhibition against Staph. aureus,  At 50mg/ml  The extract Shows 22mm zone of inhibition against K.oxytoca, it also  shows 20mm against  E.coli, while the extract shows  18mm against Staph. aureus, and shows 16mm against K.pneumonae.  while the extract at 25mg/ml the shows 17mm against E.coli, it also shows 15mm zone of inhibition against Staph. aureus, it  shows 18mm, against K.oxytoca and expressed low inhibition of 9mm against K.pneumonae as shown in Table 2

Table 4.0  Antibacterial on activity of Z. mauritiana Stem bark extract on test organisms

   Test organisms Zone of growth inhibition at varying concentrations


                                            100mg/ml         50mg/ml    25mg/ml            ciprofloxacin


E. coli                                 24                   20                17                      25

Klebseilla pneumonae              20                   16                 10                     30

Klebseilla oxytoca                     25                 22                  18                     27

Stph. aureus                               20                 18                  15                    30            

3.3 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) 

The result for the minimum inhibitory concentration (MIC) showed that  Klebseilla oxytoca  has the highest MIC of 25mm. E.coli has the 24mm while the Staphylococcus aureus and  Klebseilla pneeumonae has the least MIC of 20mm. the result shows that Klebseilla oxytoca  were observed to have the highest MBC 25mm. the MBC/MIC ratio was ranged from 2 to 4. 

Table 5: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) 


Test organism

                                                   MIC (mg/ml)               MBC (mg/ml)            MBC/MIC

          E. coli 25 50    2           

        Klebseilla pneumonae     25    50    2

       Klebseilla oxytoca                    12.5         25                                   2

          Stph. aureus                           12.5                             50                                 4


                                                               CHAPTER FOUR

4.0 Discussion

The preliminary phytochemical screening showed various bioactive ingredients such as saponins, steroids, Tannins, alkaloids, phenolic compounds and Cyanogenicglycosides. and This Indicates that the plants may have medicinal importance as a herbal remedy for several diseases. The presence of these components is an indication that this plant has some medicinal properties. The findings of current study agreed with previous studies, Najafi (2013) reported that stem bark  of Ziziphus mauritiana revealed the presence of saponins, phenolic compounds, steriods and glycosides.

 Parmar et al. (2012) stated that Stem bark  of Ziziphus mauritiana contain glycosides, saponins, phenols, lignins, Steriods and Tannins. Plants rich in saponins have anti-inflamatory activity and strengthen the immune system, Tannins are antibacterial compounds which damages the bacterial cell wall (Mainasara et al., 2012). Phenolic compounds, alkaloids, flavonoids, tannins, saponins and glycosides are good antioxidant compounds  and controls the oxidative stress related disorders and as an agents of other mechanisms that contribute in their anti-carcinogenic or cardio protective actions. (Biapa et al., 2007).

The methanol Stem bark extract of Ziziphus mauritiana resulted in variable zone of inhibitions. However, E.coli and Klebsiella oxytoca shows the highest sensitivity. However, Staphylococcus aureus shows the least inhibition under different concentration used. Similar studies published that Stem bark of Ziziphus mauritiana have antibacterial activity against different bacterial strains; Najafi (2013) cited that the methanol stem bark extracts of Ziziphus mauritiana exhibited significant activity against Staphylococcus aureus and Escherichia coli. Ashraf et al., (2015) claimed that the methanol Stem bark extract of Ziziphus mauritiana has potent antibacterial effects against Escherichia coli, Klebsiella oxytoca and Staphylococcus aureus.

It is considered that, to classify any plant extract as bactericidal the ratio of MBC/ MIC should be ≤ 4, but if this ratio is > 4, so the plant extract classified as bacteriostatic (Djeussi et al., 2013). Accordindly, the methanolic stm bark extract of Zizipus mauritiana shows a bacteriostatic effect on the tested organism. The ratio of MIC/MBC obtained for this plant leaves extract was ranged from 2 for E.coli, klebseillia pneumonae, and Klebseillia oxytoca, to 4 for Staphylococcus areus.

4.1 Conclusions

Based on the result presented above, it can be concluded that the methanolic stem bark extract of Zizipus mauritiana stem bark extract  has bactericidal activity due to the presence of some pharmacologically bioactive compounds in the plant such as tannins, steriods, phenolics, alkanoids, cyanogenic glycosides. that has a direct bactericidal activity. It can also be concluded  that the stem bark extract of Zizipus mauritiana can be used traditional in the treatment of various ailment. due to the presence of phytochemicals compounds. However, when these active compounds from this plant are extracted and isolated, it can be used to cure a wide range of diseases.

4.2 Recommendations

Based on the above findings, the following recommendations can be deduced from the present research.

1) It is recommended to design research on the phytochemical screaning of Zizipus mauritiana stem bark under different solvent.

2) A research should be conducted on the chromatographic profile of Zizipus mauritiana stem bark extract.

3) Test for anti-oxidant activity of Zizipus mauritiana stem bark Extract on albino rats.

4) It is also recommended to design a research on the in vivo pharmacological activity of Zizipus mauritiana stem bark  in the treatment of various ailments. 



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