ABSTRACT Background: Infectious diseases are becoming major health concern. One of the main causes of this problem is the widespread emergence of acquired bacterial resistance to antibiotics.
The development of microbial resistance towards antibiotics has heightened the accumulation of different antibiotic resistance mechanisms within the same strains has led to the appearance of multi-drug resistant bacteria.This needs asearch of alternative source of antimicrobials. Objective: The overall objective of the study was to screen the preliminary phytochemical constituent and evaluate the antibacterial activity of crude hydroalcolic root extract of Cucumis ficifolius A.rich. Methods: The roots of Cucumis ficifolius A. rich was collected and dried under the shade.
The dried plant material was pulverized and extracted with 80% methanol. The crude extract was evaluated for the antibacterial activity against six standard bacteria by agar well diffusion methods. Preliminary photochemical analysis was conducted using standard procedures. Result: reddish powder crude extract (25% on dry weigh base) was obtained.
Preliminary phytochemical screening revealed the presence of secondary plant metabolites such as; alkaloids, terpinoid, flavonoids, phenols, antraqunolones and steroids. Despite ethnobotanical claim for antimicrobial activity, the extract did not show any significant inhibitory activity against the tested bacteria’s in this study. Conclusion: From the results of this study it can be concluded that phytochemical screening of the root extract of Cucumis ficifolius A. rich indicated the possible presence of alkaloids, steroid, terpinoid, flavonoids, phenols and antraqunolones.
The presence of these phytochemicals in this plant enhances their pharmaceutical and therapeutic potentials. However the antimicrobial results reported in this study doesn’t have a correlation between the traditional uses which might be due to some methodological error. Keywords:antibacterialactivity,Infectiousdisease,Hydroalcoholicextract, CucumisficifoliusA.rich ACKNOWLEDGMENT First of all, we would like to thank almightily “GOD” the most gracious and merciful for his kindness, love and help for our success and achievement. Our heart full great gratitude goes to our advisors MrYohannes Kelifa and Mr.
Abyot Endale for their invaluable advice, support and unrelenting encouragement .We wish God to give them the fruits of life. We also want to extend our acknowledgment to Mr Gashaw Sisay and Mr Edeshaw the laboratory technicians and all other people who support us techniqueally and morally. Last but not least our finial gratitude goes to Mr. Getenet, lecturer of Microbiology, and Mr. Azanew,microbiology laboratory technitian for their invaluable support and guidance during antimicrobial activity test.
? TABLE OF CONTENTS Contents pageno ABSTRACT………………………………………………………………….….I ACKNOWLEDGMENT…………………………………………………………………………..II TABLE OF CONTENTS…………………………………………………………………………III LIST OF TABLES IV LIST OF FIGURES……………………………………………………………………..V LIST OFABBERIVATIONS AND ACRONOMYS…………………………. VI 1 Introduction 1 1.1 Background 1 1.2 Statement of the problem 3 1.3 Significance of the study………………………………………………………………….4 1.4 The role of phytochemicals in management of bacterial infections 5 1.5 The family Curcurbitaceae 5 1.6 The Genus Cucumis 6 1.6.1 Pharmacological activity of genus Cucumis 7 1.6.2 Isolated compounds 9 1.7 Cucumis ficifolius A. Rich 12 1.7.1 Description 12 1.7.2 Ethnobotanical uses 12 1.7.3 Pharmacological activity 13 2 OBJECTIVES 14 2.1 General objectives 14 2.2 Specific objectives 14 3 .MATERIALS AND METHOD 14 3.1 Chemicals 15 3.2 Plant materials 15 3.3 Bacterial strains 15 3.4 Preparation of plant extract 15 3.5 Determination of anti bacterial activity……………………………………………………………16 3.6 preliminary phytochemical screening……………………………………………16 3.7 Ethical consideration………………………………………………………………17 4.RESULTS…………………………………………………………………………………19 4.1 .Percentage yield……………………………………………………………..…19 4.2 . Phytochemical screening………………………………………………………19 4.3 .
Antibacterial assay…………………………………………………………………20 5 .DISCUSSION………………………………………………………….……21 6. CONCLUSION……………………………………………………………..24 7. RECOMMENDATION……………………………………………………25 8 . REFERENCE: ….26 9.
ANNEXES……………………………………………………………………………….31 ? List of tables Table 1: Results of the Preliminary phytochemical screening of the hydroalcoholic root extract ……………………………………………………………………………………………………….. 19 Table 2: Results Antibacterial activity of crude extracts Cucumis ficifolius A. Rich (Curcurbitaceae )………………………………………………………………20 ? List of figures Figure1: Some secondary metabolites isolated from the cucumis melo …………10 Figure2: Some secondary metabolites isolated from the cucumis sativasL………..11 Figure3:Morphological view of cucumis ficifolius…………………………………12 ? List of abbreviations and acronyms DMSO: Dimethyl Suphoxide DPPH Diphenylpicrylhydrazyl INT Iodonitrotetrazolium chloride MHA: Mueller Hinton Agar RA: Reference antibiotic SOD: Super Oxide Dismutase UOG: University of Gondar WHO: World Health Organization ZI Zone of inhibition ? 1 Introduction 1.1 .Background Infectious disease is defined as a disease caused by a specific infectious agent or itstoxicproduct that results from transmission of that agent or its product from an infected person, animal, or reservoir to a susceptible host, either directly or indirectly through an intermediate plant or animal host, vector or inanimate environment(1). Infection is the term that defines the entrance and development of infectious agent in a human body or animal body, whether or not it develops into a disease(1).Some of pathogenic infectious agent include Staphylococcus aureus (infections of skin, septic arthritis and food intoxication) (2),Bacillus subtilis and Bacilluslicheniformis (food intoxication)(3),Esherichia coli (foodborne illness, diarrhea) (4).Salmonella enterica and Salmonella typhimirium (food intoxication) (5,6). The development of pathogenesis of infectious diseases into the host body involves different periods of time (7)between the host becoming infected and developing the disease or becoming a new transmitter of the agent. These different periods includes; Incubation period is the time interval between the effective exposure of the susceptible host to an infectious agent and the appearance of signs and clinical symptoms; latent period is the time period from infection to onset of the ability to infect; Prodromal period is the time between the insight of illness by the host and the appearance of signs and symptoms based on a clinical diagnosis of the disease; communicable period is the time interval during which the infected host eliminates an agent to the environment and new susceptible individuals can become infected.
The epidemiology of infections has significantly changed in the last 30 years. As a result, bacterial illnesses are currently considered as emerging diseases. World Health Organization (WHO) reports that infectious diseases are responsible for over 50% deaths worldwide, occurring mainly in tropical and developing countries(8). Infectious diseases constituted the most serious health issue in the world until the beginning of the 20th century when chronic degenerative diseases began to dominate this scenario in developed countries.(1).Presently, they are on the rise because of antimicrobial resistance (9),and are more frequent in developing countries where one out of two people is dying prematurely from infections, when compared to developed countries(10). ? 1.2. Statement of the problem To date even though a wide range of synthetic and semi-synthetic antibacterial agents is available for the control of infectious diseases (11);resistance of bacteria to the available antibiotic agent is a growing and worrisome problem that continues to challenge both developing and developed countries (12).
Although health care providers treat infected patients with narrow and broad spectrum antibacterial drugs, the use of these synthetic drugs may subject the patient to a higher risk, due to the possibility of the occurrences of more harmful adverse effects(12). Consequently, infectious disease complications remain an important cause of mortality and morbidity among hospitalized patients. Antimicrobial resistance is harmful to mankind, because most of the infectious microorganisms understand the mechanism of drug action and develop tolerance to it(13). Due to these negative effects, and the constant development of bacterial resistance, there is a continuous need to develop newer antimicrobial agents effective against microorganisms and less harmful to the host (11).Moreover, the current cost of most of the chemotherapeutic agents is unaffordable to the public especially in developing countries(14).Numerous authors have therefore explored the activity of plant extracts, fractions and pure compounds contained in the extracts relevant bacterial infections in Asia and in Africa (15). Due to restricted access to proper medicine, in many developing countries, people are still using plants to treat the most prevalent infections(16).Hence, plant derived antimicrobials have received considerable attention in recent years. Therefore, discovery of new generation drugs against these infections from natural products is highly desired for development of effective, affordable and safe antibacterial agents that would be used as complimentary or alternative with convectional medicines.
To the best of our knowledge, there was no previous studies have been conducted so far on the antibacterial activities of the extracts of Cucumis ficifolius A. Rich. The findings will be obtained from the present study may provide the foundation for clinical research to pave the way for new therapeutic option against various microorganisms. ? 1.3. Significance of the study Treating bacterial infections with antibiotics is beneficial, but their indiscriminate use has led to an alarming rate of resistance among microorganisms.
Multiple drug resistance in microbial pathogens is an ongoing global problem, these results in loss of effective antibiotics and loss of budget for infectious disease treatment. Thus, there is an urgent and constant need for exploration and development of cheaper, effective, new plant-based drugs with better bioactive potential(17). Development of new antimicrobials is among the proposed solutions to curb this problem. In this regard, plants could provide a good alternative in search for new chemical agents with a wide-ranging antimicrobial activity(18).Therefore, the search for new anti-microbial agents is a high priority, especially in the tropical countries and other developing nations where infectious diseases are out of control. In light to the ethno botanical claim, it can increase the therapeutic alternative for the currently challenging infections disease. Screening of medicinal plants for their bioactivity and phytoconstituents is extremely important to identify promising candidates that are sources of potential therapeutic agents, which can be used as a guide for the selection this medicinal plant as a potential species in order to establish further structural elucidation and structure activity relationship, which lead the way to discover a novel antimicrobial agent for therapeutic use.
The emergence of multidrug resistant strain of many pathogens such as S.aureus, E.coli, E.fecalis, P.aurginosa, K. Pneumonia andS.pyogens is a serious threat and makes chemotherapy more difficult. Moreover, the current cost of most of the chemotherapeutic agents is unaffordable to the public especially in developing countries. Therefore attempts must be made towards the development of effective natural, non-toxic drug for treatment. The antibacterial activity cucumisficifolius have not been evaluated for antimicrobial activity exhaustively although it is used traditionally for treatment of infectious disease. Therefore, this paper reports the preliminary phytochemical constituents and antibacterial activities of the hydro alcoholic leaf extract of cucumisficifolius.
1.4 .The role of phytochemicals in management of bacterial infections The medicinal and pharmaceutical properties of plants are due to the type of chemical substance they produce and store. These include compounds that are utilized as food by man and other animals and also other compounds that exert physiological effects on them. This second group of chemical substances often referred to as secondary metabolites, give plants their therapeutic properties. The usual term used to refer to these various chemical substances present in plant is “constituents(19) Phytochemicals (from the Greek word phyto, meaning plant) are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those attributed to macronutrients and micronutrients(20).
Phytochemicals obtained from natural products have been used worldwide in traditional medicine since antiquity and area source of potential and powerful drugs.(11). For thousands of years, natural products have been used in traditional medicine all over the world and predate the introduction of antibiotics and other modern drugs (21). Since, medicinal plants constitute a wide variety and diversity of secondary metabolites, medicinal plants could be used as a good source of antibacterial agents(22).Natural products, either pure compounds or standardized plant extracts, provide unlimited opportunities for novel and suitable additives and drug treatments because of their constituent a wide range of chemical diversity(23).Usage of plant-derived antimicrobial agents might be effective in reducing the dependence on antibiotics and minimizing the chances of antibiotic resistance in foodborne pathogenic microorganisms (24). Phytochemicals found in plants such as tannins and alkaloids, which have been found in vitro to have antimicrobial properties(25).In addition, polyphenolic biomolecules have been expected to play important roles in creating new and better therapeutic agents (26).Indeed, the flavonoids can be inhibited the nucleic acid synthesis, cytoplasmic membrane function, and can act on energy metabolism(27).Flavonoids include flavonols, flavones, isoflavones, flavanones, anthocyanidins most commonly known for their antioxidant and antimicrobial biological activities(28,29).
1.5.The family Curcurbitaceae The family cucurbitaceae includes a large group of crops like cucumbers, and melon which are medicinally essential. It is a distinct family without any close relatives (30). Plants of this family have many medicinal and nutritional benefits(30) The major phytocompounds present are the phytochemicals like Glycosides, Terpenoids, Saponins, Tannins, Steroids, Carotenoids, and Resins etc. and most commonly the terpenoid substance called Cucurbitacins.(30) Cucurbitaceae family is also called vine family which consists of 825 species under 125 genera (31). Cucurbitacins constitute a group of diverse triterpenoid substances which are well known for their bitterness and toxicity (32).They are highly oxygenated, tetracyclic triterpenes containing a cucurbitane skeleton characterized as 19-(10?9?)-abeo-10?-lanost-5ene(32).The plants of this family are mainly grown around the tropics and to some extent in temperate areas(31). Some of the important plants that have been extensively studied in this family are Mukia maderaspatana, Solena amplexicaulis, Citrullus colocynthis, Citrullus lanatus, Coccinia indica, Cucumis sativus, Cucurbita pepo, Lagenaria siceraria, Luffa acutangula, Trichosanthes cucumerina, Corallocarpus epigaeus, Luffa cylindrica, Momordica charantia, Trichosanthes dioica and Kedrostis foetidissima(31).
The various cucurbitacins differ with respect to oxygen functionalities at various positions(32).Many pharmacological and clinical investigations have verified that cucurbitacin B (CuB) and cucurbitacin E (CuE) possess various pharmacological activities, such as, antitumor, anti-hepatitis and immunopotentiating effects(30). The cucurbitacin preparation used clinically contains mostly CuB and CuE, which are obtained from the calyx of Cucumismelo L., a Chinese medicinal plant that is effective against chronic hepatitis and primary liver carcinoma(31). Seeds or fruit parts of some cucurbits are reported to possess purgative, emetic and antihelmintic properties due to the secondary metabolite cucurbitacin contents(33). Previous literature have found that Cucurbitaceae Family have numereus medicinal properties such as antiHIV, anxiolytic,anti-pyretic, anti-diarrheal, carminative, antioxidant, anti-diabetic, antibacterial, laxative, anti-helminthic, anti-tuberculosis, and purgative(32). It is also employed as an abortifacient, diuretic, andcardiotonic agent.
They also show strong anti-inflammatory, antitussive, cytotoxic, and expectorant properties(32). 1.6 .The Genus Cucumis The genus name Cucumis is the Latin name for the cucumber which was already cultivated in Ancient Egypt(34). The genus Cucumis belongs to the family Cucurbi-taceae, order Cucurbitales is represented by 32 species(35). According to specific morphological features of tendrils pollen grains and ovules, there are clear relation of this taxon with the order Passiflorales(35). The plants are annual herbs, exceptionally typically having a climbing growth habit or trailing and semi shrubs, although few cucumber and C.
melo cultivars have a root systems, rarely woody (C. trigonus) are extensive, bush habit, but not often tuberous (C. kalahariensis), usually shallow . Stems are sulcate, angled, not aculeate or rarely aculeate (C. ficifolius), rarely glabrous or variously pubertal, rarely breakaway hairs or with nonbreak away hairs (C. sacleuxii)(36).
The genus Cucumis indigenous mainly to Africa, also Asia, Australia and some islands in the Pacific. It includes two major commercial vegetable crops: C. sativus (cucumbers, from Asia) and C. melo (melons, from Africa and Australia, asia), and two minor ones: the West Indian gherkin (C.
anguria) and the kiwano (C. metuliferus). These last two species became cultivated crops outside their native Africa(34). The South Africa is main hub of the center of multiplicity for the genus Cucumis.Pharmacological experiment conducted on C. melo and some Cucumis spp., point to its immense prospective in the management of conditions such as inflammation, pain, cancer, cough, liver diseases, and cardiovascular disorders(36).
Cucumismelo whole fruit is useful in chronic eczema. The fruit is tonic, laxative, and galactagogic, diuretic and diaphoretic. The flowers used as expectorant and induce vomiting. The seeds are used as cough suppressant, fever reducer, and a digestive aid.
A seed powder is mixed with water and used as anti-helminthic(32). Cucumis sativus fruit help in removing constipation and ingestion the fruits are used during summer as cooling food, seeds as anthelmintic(32). 1.6.1 .Pharmacological activity of genus Cucumis Antimicrobial activity In the previous study the crude extract of the whole plant of Cucumis melo.L (Cucurbitaceae) was screened for activity against bacterial strains like E. coli, K. pneumoniae, S.paratyphi, S.
aureus and a fungal strain C. barbicans using aqueous, heptane, petroleum ether and acetone as a solvent by using disk diffusion method. Highest zone of inhibition was shown by whole plant and fruit extract of Cucumis melo L. with aqueous and acetone with C. albicans and E. coli 08 and 12 mm respectively(37).The previous study on the Cucumis sativus using disk diffusion shows higher antibacterial and antifungal activity against following microorganisms like S.typhi, E.coli, E.faecalis,B.cereus and C.lunata, C.albicans(38).
Also it justifies. Antimicrobial activities are aggravated by increasing the quantity of this compound, which can be used as an alternative for antibiotics(38). Antibacterial and antifungal activity was studied by Agar well diffusion method and was concluded that the fruit ethanolic extract of Cucumis anguria shows stronger antimicrobial activity than its methanolic,Chloroform,ethylacetate extracts(39). Antidiabetic activity On the basis of previous evidences obtained in laboratory animal study, the researchers concluded that the Cucumis sativus extract possesses hypoglycemic properties which suggests the presence of biologically active components such as terpenoids, alkaloids, flavonoids, and phenolics have shown antidiabetic potential through the insulino mimetic activity of the plant extract which may be worth further investigation and elucidation(40). Data show that 1mL of the extract almost normalize the blood glucose level of rats. Hence, it might help in preventing diabetic complications and may serve as a good alternative of antidiabetic drugs(40).
C. melo leaf methanolic extract was investigated for its antihyperglycemiceffects against streptozotocin induced hyperglycemia in rats and concluded that methanolic extract of C. melo leaf have greater anti-hyperglycemic activity than aqueous extract in streptozotocin induced hyperglycemia model and when compared with Glibenclamid treated group(41). Antioxidant Activity Qualitative DPPH spray method was used for checking free radical scavenging activity of cucumis anguria extract and free radical in it gives a strong absorption maximum at 517 nm and purple colour were changed to yellow which can be concluded as extract has anti-oxidant properties(42).The methanolic extract of Cucumis trigonus roxb seeds gave concentration-dependent DPPH radical scavenging activity.
The highest free radical scavenging activity was found to be 83.66% at concentration of 200 ?g/ml which was comparable to the free radical scavenging activity of standard (0.05 mM) ascorbic acid(43). In DPPH assay – the percentage of antioxidant activity of Cucumis melo Seed extracts values recorded at 30 minutes were analyzed. Cucumis melo seed extracts were analysed the highest radical scavenging activity (73.2 %) and followed by others(44). Another previous study showed that Response of Cucumis dipsaceus fractional extracts towards various antioxidant assays was appreciable especially in ABTS+ , metal chelating, nitric oxide and DPPH assays. Methanol extract of Cucumis dipsaceus fruit showed the highest activity (4907.22 µg TE/g) to stabilize ABTS radical(45). 1.6.2 .Isolated compound Preliminary phytochemical screening of the ethanol and crude chloroform extract of leaves and steam of cucumis sativus possessed phytoconstituent such as alkaloid, glycoside, and steroid, saponin and tannins where as cucumissativus fruits revealed the presence of glycosides, steroids, flavonoids, carbohydrates and tannins(30).Flavone glycosides such as isovitexin, saponarin and various acylated flavone C-glycosides are present in the leaves.
Antiulcer 9-beta-methyl-19-norlanosta-5-ene type Glycosides have been isolated from Cucumis sativus Seeds(33). Cucumis melofruit has a high Superoxide dismutase activity (SOD) which is responsible for the in vitro and in vivo antioxidant and anti-inflammatory properties of the extract.Furthermore, number of phenolic glycosides have been isolated from the seed(32). Cucumis anguria showed to possess Antioxidant activity and various constituentsSuch as Flavonoids, Alkaloids, Triterpenoids, Carbohydrates, Tannins, Sterols, Anthraquinoneglycosides and Saponins are present in it(42). Phytochemical screening of Cucumis metuliferus revealed the presence of alkaloids, flavonoids, triterpenoid, saponins, steroids, volatile oils, total glycosides, cardiac glycosides and saponin glycosides(46). ? The following figure shows some isolated structure from some species of genus cucumis (47,48). where 1 Cucumins, 4 R=H, luteolin ,5R=OH, quercetin, 2 R1=H,R2=R3=OH, 5,7-dihydroxy-2- 2-(4-hydroxyphenyl)ethylchromone 3 R1=R2=R3=OH,5,7-dihydroxy-2-2-(3,4- dihydroxyphenyl)ethylchromone 6 R1=H,R2=OH,R3=O-O-Glu,7- glucosyloxy-5-hydroxy-2-2-(4-hydroxyphenyl)ethylchromone Figure 1: Some secondary metabolites isolated from the cucumismelo 1(2S,3S,4R,10E)-2-(2’R)-2-hydroxytetracosanoylamino-1,3,4-octadecanetriol-10-ene 2,1-O-?-D-glucopyranosyl-(2S,3S,4R,10E)-2-(2’R)-2-hydroxytetracosanoylamino-1,3,4-octadecanetriol-10-ene 3, soya-cerebroside Figure2:Sphingolipids Isolated from the Stems of Cucumber (Cucumis sativas .L) 1.7.
Cucumis ficifolius Figure3: Morphological view of Cucumis ficifolius A. Rich 1.7.1 . Botanical Description C. ficifolius is a perennial herb climbing to 2m long from a perennial wood root stock up to1mm long. The stems are hairy with short fine hairs and also with small aculeate hairs of varying degrees or stoutness; Leaves are blade ovate to broadly rotundate in outline; Seeds are Elliptic (49). In Ethiopia, the plant is locally known as Yemidier embuay (Amharic language)(49).
The plant distributed in different part of Ethiopia mainly Tigray, Go jam, Shewa, Welega, Gamo Gofa and Harare(49). This plant mainly growing at the altitude ranging from 1300-2400 m (49). 1.7.2 .Ethnobotanical uses In Ethiopia, the fruit part reported to treat rabies(49). And also, fresh fruit used as addressing for inflamed fingers in Nigeria and Ethiopia(49). In Some places it is an ingredient of medicine for syphilis and as an emetic and in small doses with honey to relieve stomach ache for children, in Ethiopia it also used for the treatment of various ailments such as “Kuruba”, “Chiffea”, “Mageriat geter” (meningitis), “nessir” (epistaxis),”Wefbeshita” (50).
And also the roots is used for complete treatment of malaria. The seeds are oil giving. The Root extract of C. ficifolius is recorded to be used in local Honey-wine or “Tej” to make beverage more intoxicating (49).
Previous study in Dek island by by Tilahun Teklehymanot confirmed that different parts of cucumis ficifolius used for different ailments such as Stomach-ache, nail injury, wart, dysentery with blood, ‘mich’, meningitis, evil eye(50) 1.7.3. Pharmacological activity In the previous investigation, the antimicrobial activity of the chloroform, ethyl acetate,acetone, ethanol and methanol extracts of the leaves of C. ficifolius were evaluated using disk diffusion method .Tetracycline and erythromycin were used as positive control where as Tween 20 served as a negative control and extracts showed varying degree of inhibitory activity against S. aureus, E.
coli, P. aeruginosa and S.boydii at concentration of 400mg/ml (49). 2 .Objectives 2.1. General objectives • The aim of this study was evaluation of antibacterial activity of the root extracts of Cucumis ficifolius A.
Rich against selected test microorganisms. 2.2. Specific objectives • To undertake preliminary phytochemical screening of the crude hydro alcoholic root extract of C. ficifolius; • To evaluate the antimicrobial activity of the crude root extract of C. ficifolius using well diffusion method; and • To determine the MIC and MBC of root extract of C. ficifolius ? 3 .Material and Methods 3.1.
Chemicals Standardized ciprofloxacin 50 ?g/mL was diluted in Dimethyl sulphoxide (DMSO) used as positive controls for the antibacterial susceptibility test; where as DMSO serves as a negative control), p-Iodonitrotetrazolium chloride (INT), Muller-Hinton agar was used as a microbial growth indicator. 3.2. Plant materials The root of C. ficifolius was collected from Estie town which is located South Gondar, Amhara region, in January 2018 Ethiopia, 165 km far from Gondar.
Then the Botanical identification and authentication of the plant materials were performed and voucher specimens (No……….) has been deposited in Herbarium Biology Department, Faculty of Natural and Computational Science, University of Gondar. 3.3.Bacterial strains The test was car¬ried out against three Gram positive bacterial strains: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis ATCC 1912/R and Streptococcus pyogenes (ATCC 19615) and three Gram negative bacterial strains: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 2706) and Klebsiella pneumonia (ATCC 700603). All the standard bacterial strains were obtained from Department of Microbiology, University of Gondar, and Amhara Regional Laboratory, Bahr Dar. 3.4.
Preparation of plant extract The roots were washed thoroughly under tap water and shad dried at room temperature for three weeks. There after, the dried roots were grinded into a coarse powder and 600 g of powder was macerated in 80% methanol for the period of 72 h with occasional shaking and stirring. This was repeated for three times and the extracts thus obtained were filtered through filter paper (Whatman Fitter Paper No. 1).
Then, the filtrate was allowed to evaporate using oven at temperature 40 °C. Finally, a highly concentrated methanol extracts were obtained and kept in refrigerator for further experiment. 3.5. Determination of antibacterial activity Bacterial broth culture was prepared to a density of 108 cells ml-1 of 0.5 Mc Farland standards and on each plate; equidistant wells were prepared with a 6 mm diameter sterilized cork borer then randomly labelled. The aliquot was spread evenly onto Muller Hinton agar using sterilized cotton swab. Next, 100 ?L of each extract at concentrations of 100, 200 and 400 ug/mL, by using dimethyl sulphoxide (DMSO) as solvent, were applied aseptically into a respective agar wells.
Ciprofloxacin 50 µg/mL, dissolved by s dimethyl sulphoxide (DMSO), was used as positive control and DMSO (100 ?L) was included as negative control. The agar plates were allowed to stand on bench for 30 minutes at room temperature for pre-diffusion and then incubated at 37oC for 24 hours. Antibacterial activity was determined by measuring the Zone of inhibition (ZI) around each well (excluding the diameter of the well by subtracting 6 mm from ZI results). For each concentration, three replicate trials were conducted against six organisms(51). 3.6 .Preliminary Phytochemical screening The crude extract of cucumis ficifolius was screened for the presence or absence of secondary metabolites such as alkaloids, steroidal compounds, terpenoids, phenolic compounds, flavonoids, tannins, phlobatanins, and saponins by using the methods as described by standard procedures.edeoga (52)and jones and kinghorn(53) Terpenoids (Salkowski test) One hundred milligram of the extract was mixed with 2 ml chloroform and 3 ml of sulfuric acid was added to form a layer. A reddish-brown coloration of the interface was an indication of terpenoids.
Tannins A half gram of the dried powdered plant sample was boiled in 20 ml of water in a test tube and filtered. Few drops of 0.1% ferric chloride were then added to the filtrate and the formation of brownish green or a blue-black coloration was regarded as indicator of the presence of tannins. Detection of alkaloids Mayer’s test: To a few ml of filtrate, 2-3drops of Mayer’s reagent (Potassium mercuric iodide solution) was added along the slides of the test tube. Formation of white or creamy precipitate indicates presence of alkaloids.
(ii)Wagner’s test: To a few ml of filtrate, 2-3drops of Wagner’s reagent (Potassium iodide solution) was added along the sides of the test tube. Formation of reddish brown precipitate indicates presence of alkaloids. Detection of flavonoids NaOH test: 50mg of extract was dissolved in 2ml of alcohol and to the extract, increasing amount of NaOH was added. It shows yellow coloration which decolorizes after addition of an acid. Detection of saponins Foam test: About 50mg of extract was dissolved in 2ml of alcohol, diluted with20ml of distilled water and shaken for 15min in a graduated cylinder. A layer of stable foam indicates presence of saponin glycosides.
Detection of phenolics FeCl3 test: About 50mg of extract was dissolved in 2ml of distilled water, add 2drops of neutral 5% FeCl3 solution and observed for colouration. Formation of blue, green and violet colour indicates the presence of phenolic compounds Detection of steroids: Salkowski test: To about 50mg of extract, 2ml of chloroform and 2mlof concentratedH2SO4were added and shaken well. Then observed for coloration of CHCl3 and acid layer .appearance of Chloroform layer in red color and acid layer as greenish yellow fluorescence indicates the presence of steroids. Detection of anthraquinone Boil 200 mg of extract with 6 ml of 1% HCl and filtered. Shake the filtrate with 5 ml of benzene, filtered and add 2 ml of 10% ammonia solution to the filtrate.
Then shake the mixture and the presence of a pink, violet or red colour in the ammoniacal phase indicated the presence of free hydroxyl anthraquinones 3.7. Ethical consideration This experiment was conducted as per the internationally accepted guideline and protocol. Furthermore, letter of approval was obtained from School of Pharmacy, College of Medicine and Health Sciences, University of Gondar ? 4. Results 4.1. Percentage yield of crude extract The 80% methanolic crude extracts of the dried root of cucums.
Ficifolius (600 gm) gave a shiny reddish powder of 150 gm. Thus the percentage yield becomes 25% on dry weight basis. Percentage yield (%yield) =Actual yield/Theoretical yieldx100=150gm/600gmx100=25%. 4.2.
Preliminary phytochemical screening As shown in Table1 preliminary phytochemical screening of the hydroalcoholic root extract of cucumis ficifolius revealed the presence of bioactive alkaloids, steroids, flavonoids, phenols terpinoids and antraqunolones while Alkaloid(mayer’stest) ,tannin and saponin are relativelyabsent. Results of the Preliminary phytochemical screening of the hydro alcoholic root extract of Cucumis ficifolius A. Rich (Curcurbitaceae). Table1: Preliminary phytochemical screening result Photochemical group Specific tests Standard color changes expected The actual result obtained Presence/ Absence Alkaloids Mayer’s Test White/creamy precipitate Dark white colored precipitate weakly (+)ve Wagner’s Test Brown or reddish precipitate Brown precipitate Strongly (+)ve Tannins FeCl3 Brownish, blue black Green precipitate purple precipitate weakly (+)ve Flavonoids NaoH test Yellow colour discoloration yellow colour discoloration Strongly (+)ve phenol Only 1% FeCl3 Violet, green and blue colour Violet like colour Strongly (+)ve Anthraquinone Born Trager’s test Pink, red or violet colour Dark red, pink strongly (+)ve Steroids Salkowski test) Greenish/reddish color Greenish color strongly (+)ve Terpinoid Salkowski test) Reddish brown color Reddish brown color strongly (+)ve Saponins Froth Test Frothing(foam) persist for 15 minute Frothing(foam) which disappear immediately weakly(+)ve 4.3.
Anti-bacterial activity The antibacterial assay was done for six bacterial strains, namely S.aureus, S.pyogens and E.fecalis which are gram positive and E.coli, P.aurginosa and K. Pneumonia which are gram negative. Triplicate was done for all six bacterial strains. .Unfortunately all six bacterial strains did not grow at all so that it was impossible to detect ZI for the entire triplicate. Ciprofloxcilln show a potent antibacterial effect against all bacterial strain except S.pyogen bacterial strain which has zero ZI.
DMSO has better ZI than three concentration of the crude extract.Hence crude extract has almost zero ZI and doent show anti bacterial activity. Antibacterial activity of crude Root Extracts of Cucumis ficifolius A. Rich (Curcurbitaceae) at different concentration on different bacterial strains is shown below. Table 2: anti bacterial activity result Tested bacteria Etract dose(µg/ml) Hydroalcoholic root extract ZI (mm) Ciprofloxacin (50?g/ml) DMSO (100?l) ZI(mm) S.
aureus ATCC 25923 100µg /ml 0 33.333 0 200µg /ml 0 400µg /ml 0 S.pyogenes ATCC 19615 100µg /ml – – – 200µg /ml – 400µg /ml – E.Faecalis ATCC 1912R 100µg /ml 0 35.666 11.333 200µg /ml 0 400µg/ml 0 P.aeruginosa ATCC 2706 100µg /ml 0 39 0 200µg/ ml 0 400µg/ ml 0 K. pneumoniae ATCC 700603 100µg/ml 0 23.666 0 200µg/ ml 0 400µg /ml 0 E.coli ATCC 25922 100 µg /ml 0 35.666 10 200µg/ml 0 400µg/ml 0 5.DISCUSSION The phytochemical analysis of the plants is very important commercially and has great interest in pharmaceutical companies for the production of the new drugs for curing of various diseases(54).Medicinal plants need to be tested for their claimed activity and safety and efficacy profiles. The assessment is important for the anticipated dosage form of the extract before it is made available for use(55).This is because not all medicinal plants possess the expected activity. Studies need to be done using scientific and experimental protocols to approve the presence of the claimed traditional use of in the plant. The result of such investigations could promote the traditional practice or it also can be used to evade ineffective practices.
Ethino-medicinally different part of Cucumis ficifolius A. Rich is used for the treatment of various ailments in traditional medicine .Root is dried and crushed then the powder is directly applied on the affected body part, which is used for skin disease, swellings in the body, and wound dressings on Dek island (50). On other local claim of south gondar showed that concentrated fresh leaves and fruit is widely used for sever stomath ache. However, claims of treatment success have been made without scientific basis.
This research was conducted to validate or invalidate the folkloric uses of the plant. The investigation covers crude extraction, preliminary phytochemical constituent screening and antimicrobial evaluation. All stages of extractions, from the pre-extraction and extraction are equally important in the study of medicinal plants. The sample preparation such as grinding and drying affected the efficiency and phytochemical constituents of the final extractions; that eventually have an effect on the final extracts.Both fresh and dried sample is used in medicinal plants studies.
In most cases, dried sample is preferred as fresh samples are fragile and tend to deteriorate faster than dried samples. Grinding resulted in coarse smaller samples and leading to better surface contact with extraction solvents.However very fine powder is not recommended as it affects the process of filtration(56). Maceration technique was used because it is reliable and effective for heat susceptible phytochemicals. The effect of of botanical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure.
Traditional healers use primarily water as the solvent but in our studies we found that plant extracts in organic solvent (methanol) provided more consistent antimicrobial activity compared to those extracted in water. Despite of water is a universal solvent,it is exposed to microbial growthand and canot be used as asolvent alone .These observations can be rationalized in terms of the polarity of the compounds being extracted by each solvent and, in addition to their intrinsic bioactivity, by their ability to dissolve or diffuse in the different media used in the assay(57).The use of simple maceration which made the extraction method less efficient give smaller yield (25%) than electrical rotatory shaker. Previous study on anti microbial activity of the leaf of Cucumis ficifolius A. Rich recommend that further investigation for the presence of secondary metabolites while it has good antibacterial and anti fungal activty . The finding of this work is encouraging and indicates that the herb should be studied more extensively for its therapeutic benefits(49).The present preliminary phytochemical screenings on root extract revealed that the possible presence of Alkaloids, flavonoids, steroid, phenolic, anthraquinone and terpinoids.These secondary plant metabolites have confirmed and versatile medicinal properties which may be a reason for the use of the plant material for different health problems traditionally.
These secondary metabolites are known to be biologically active and play significant roles in bioactivity of medicinal plants, because the medicinal values lies in these phytochemical compounds which produce a definite and specific action on the human body. Phytochemicals exert antimicrobial activity through different mechanisms. For example, flavonoids exhibit a wide range of biological activities which include antimicrobial, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, cytostatic and antioxidant properties. Flavonoids’ ability to scavenge hydroxyl radicals, superoxide anion radicals and lipid peroxyl radicals highlights many of the health-promoting functions of flavonoids in organisms which are important for prevention of diseases associated with oxidative damage of membranes, proteins and DNA. Flavonoids in the human diet may reduce the risk of various cancers, as well as preventing menopausal symptoms. The antibacterial activity of flavonoids had been reported to be a result of their ability to form complexes with bacterial cell walls, extracellular and soluble proteins(58).
Alkaloids have been associated with medicinal uses for centuries and other possible roles have not been examined. One of the most common biological properties of alkaloids is their toxicity against cells of foreign organisms. These activities have been widely studied for their potential use in the elimination and reduction of human cancer cell. In addition, alkaloids possess anti-inflammatory, anti-asthmatic and anti-anaphylactic activities with consequences of altered immunological status in vivo(58).Many synthetic and semisynthetic drugs are structural modifications of the alkaloids, which were designed to enhance or change the primary effect of the drug and reduce unwanted side-effects(60). Alkaloids and terpinoids have biological function of neuropharmacological agents, anti- oxidants, cancer chemoprevention.
Terpenoids, alkaloids and phenolics have apotenicial to Inhibit of micro-organisms, reduce the risk of fungal infection. Since these compounds were found to be present in the extracts, it might be a great potential responsible for Anticancer,Antioxidants and Antibacterial ; Antifungal capacity(61). A large body previvious stuidies has demonstrated that the naturally occurring anthraquinones possess a broad spectrum of bioactivities, such as cathartic, anticancer, antiinflammatory, antimicrobial, diuretic, vasorelaxing, and phytoestrogen activities, suggesting their possible clinical application in many diseases(62).Since this compound was found to be present in the extracts, it might be a great potential responsible in such disease. In contrast to the current study, previous study on crude extract and solvent fractions of Cucumis ficifolius A. Rich exhibited significant broad spectrum inhibitory effect against all the test bacteria(49).This difference may be due to various reasons.
The content of active constituents in plants varies due to factors such as geographical location of the plant material, season of plant material collections, soils, rainfall, altitudes. (63). Previous study suggest the existence of a correlation between environmental factors such as average annual temperature, climate, vegetation, geomorphology, latitude and altitude and tannin production.Alkaloid and tannin and saponin content increases with increase in altitud (59). This clarifies that our plant was collected from low land area of South gondar,komafasiledes and may a possible reason for the possible absence of tannins and saponin. Thus, the variation may be due to those case or some other methodological errors such as inappropriate dying.
Here in this study the negative control (DMSO vehicle) has higher zone of inhibition than the fraction of concentrations of the crude extract of cucumis ficifolius A.rich on some tested common pathogenic bacteria. This might clarified lack of potential antibacterial activity of the plant materials. This result does not need further confirmation by using another solvent (i.e. sterile water) to confirm whether negative control or extract has anti bacterial activity as the initial extract does not show any activity. This is done because the negative control DMSO has some anti bacterial activity. Despite of plant material has important secondary plant metabolites,Lack of effective antibacterial activity of might be due to degradation of the chemical constituents due to heating in oven for long time, procedural and methodological limitation , actual lack of antibacterial activity or error in concetration of the of extract.
The recommended and standard method of antibacterial assay is serial dilution techniques but here in this study since there was no satisfactory result in the disk diffusion test it is not necessary to conduct serial dilution test. 6.Conclusion In the current study, preliminary phytochemical screening and antimicrobial activity evaluations of the 80% methanol extract of Cucumis ficifolius A. Rich were carried out. Phytochemical screening of the total root extract indicated the possible presence of alkaloids, flavonoids, phenols, steroids, terpinoids and antraqunionens.
However the extract didn’t show any significant antimicrobial activity at all tested dose against all tested bacteria. The results reported in this study illustrate that no correlations exist between the traditional uses of root extracts of cucumis ficifolius A.Rich which may be due to different factors. ? 7.Recommendation Based on the present study the following recommendations are proposed: ? Additional study using solvent other than methanol for further proofing/disproving ? The use of fresh root instated of dried root may preserve volatile oils which might have a therapeutic value. ? The use of lyophilizer instead of oven which may prevent degradation of thermolabile constituents.
? Other parts of the plant like the leaf, fruit and seed might require investigations for their phytochemicals and antimicrobial activity. ? Use of plantfrom differentclimaticcondition,altitude,latitude,temprature,rainfall,seasos
