PHYTOCHEMICAL SCREENING and ANTIMICROBIAL ACTIVITIES OF STINGING NETTLE (Urtica dioica L.) LEAF, FRUIT AND ROOT EXTACTS
MSc. THESIS
MEGERSA IDRIS AHMED
FEBURARY 2021
HARAMAYA UNIVERSITY, HARAMAYA Phytochemical Screening and Antimicrobial Activities of stinging Nettle (Urtica dioica L.) Leaf, Fruit and Root Extracts
A Thesis Submitted to the School of Biological Sciences and Biotechnology Postgraduate Programs Directorate Haramaya University
In Partial Fulfillment of the Requirements for the Degree of Master of Science in Biotechnology
Megersa Idris Ahmed
January, 2021
Haramaya University, Haramaya APPROVAL SHEET HARAMAYA UNIVERSITY POSTGRADUATE PROGRAM DIRECTORATE
As thesis Research advisors, we hereby certify that we have read and evaluated this Thesis, prepared, under our guidance by Megersa Idris entitled Phytochemical Screening and Antimicrobial Activities of stinging Nettle (Urtica dioica L.) Leaf, Fruit and Root Extracts.We recommend that it be submitted as fulfilling the thesis requirement. Zekeria Yusuf (PhD) ______
Major Advisor Signature Date
Ashebr Abraha (DVM, MSc, Assoc.Prof.) ______
Co- Advisor Signature Date
As member of the Board of Examiners of the M.Sc. Thesis Open Defense examination, we certify that we have read and evaluated the Thesis prepared by Megersa Idris and examined him. We recommend that the thesis be accepted as fulfilling the thesis requirements for the degree of Master of Science in field of Biological Sciences (Biotechnology).
______
Chairperson Signature Date
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Internal examiner Signature Date
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External examiner Signature Date
Final approval and acceptance of the Thesis is contingent upon the submission of its final copy to the council of Graduate Studies (CGS) through the candidate's department or school graduate committee (DGC or SGC).
II DEDICATION
This thesis work is dedicated to my mother Fatuma Ahmed and my wife Merkani Mohamed for their encouragement and support in different aspects.
III STATEMENT OF THE AUTHOR
By my signature below, I declare and affirm that this M.Sc Thesis is my own work. I have followed all ethical and technical principles of scholarship in conducting studies, data collection, data analysis, and compilation of this Thesis. Any scholarly matter that is included in the Thesis has been given recognition through citation.
This Thesis has been submitted in partial fulfillment of the requirements for the Degree of Master of Science in Biological Science at Haramaya University. The Thesis is deposited in the Haramaya University Library and is made available to borrowers under rules of the Library. I solemnly declare that this Thesis has not been submitted to any other institution anywhere for the award of any academic degree, diploma, or certificate.
Brief quotations from this Thesis may be made without special permission provided that accurate and complete acknowledgment of sources is made. Requests for permission for extended quotations from or reproduction of this Thesis in whole or in part may be granted by the Head of the School or Department when in his or her judgment the proposed use of the material is in the interest of scholarship. In all other instances; however, permission must be obtained from the author of the Thesis.
Name: Megersa Idris Signature ------Place: Haramaya University Date of Submission: Feburary 2021.
IV BIOGRAPHICAL SKETCH
The Author Megersa Idris Ahmed was born on April 7, 1990 G.c in Haramaya District, East Hararghe, Oromia Regional State, Ethiopia.from his father Idris Ahmed and his mother Fatuma Ahmed in December, 1992. He attended his elementary school at Bate Elementary School from 1998 to 2004.Gc. He pursued Secondary Education at Haramaya Secondary School from 2005 to 2006. After successfully passing the Ethiopian General Secondary Education Certificate (EGSEC) examination, he joined Haramaya Preparatory School from 2007 to 2008, for higher education then, joined Ambo University on 2009 and graduated with BSc degree in Veternary Laboratory Technology on June 28, 2012.G.c.
After graduation, he was employed by the Haramaya University College of Veternary Medicine, where he worked for three years. He then joined Haramaya University on October 2018 to pursue a study leading to the Degree of Master of Sciences in Biotechnology.
V ACKNOWLEDGEMENTS
Above all, I would like to praise and glorify the Almighty Allah who provided me with all the required resources, knowledge, strength and wisdom for the fulfillment of the task.
First and foremost, it is my great pleasure to wholeheartedly extend my most profound and sincere gratitude to my major advisor Dr Zekeria Yusuf and co-advisor Dr. Ashebr Abraha (DVM) for their valuable comments, suggestions, assistance and critical guidance in the preparation of the proposal and thesis write up. Their ever readiness to provide assistance, guidance and advice greatly helped me to carry out the study.
VI ACRONYMS AND ABBREVIATIONS
ALP Alkaline Phosphatase ALT Alanine Transaminase AST Aspartate Aminotransferase BHA Butylated Hydroxyanisole BHT Butylated Hydroxytoluene CLSI Clinical and Laboratory Standard Institute COX-1 Cyclo Oxygenases DPPH Diphenyl-2-Picrylhydrazyl radical EGF Epidermal Groth Factor FIRI Fasting Insulin Resistance Index LD50 Median Lethal Dose LDL Low Density Lipoprotein MBC Minimum Bactericidal Concentration MFC Minimum Fungicidal Concentration MHA Mueller-Hinton Agar MIC Minimum Inhibitory Concentration MRSA Methicillin-Resistant Staphylococcus Aureus NA Nutrient Agar OGTT Oral Glucose Tolerance Test PDA Potato Dextrose Agar PSA Prostate-Specific Antigen PSA Prostate-Specific Antigen RSV Respiratory Syncytial Virus SHBG Sex Hormone Binding Globulin SHBG Human Sex Hormone Binding Globulin TNF-α Tumor Necrosis Factor UDA Urtica Dioica Agglutinin VLDL Very Low Density Lipoprotein
VII TABLE OF CONTENTS
APPROVAL SHEET II
DEDICATION III
STATEMENT OF THE AUTHOR IV
BIOGRAPHICAL SKETCH V
ACKNOWLEDGEMENTS VI
ACRONYMS AND ABBREVIATIONS VII
TABLE OF CONTENTS VIII
LIST OF TABLE XI
LIST OF TABLE IN THE APPENDIX XII
1. INTRODUCTION 1
2. LITERATURE REVIEW 4
2.1. Botanical Description of Nettle 4
2.2. Traditional Medicinal Properties 4
2.3. Phytochemical Composition 5
2.4. Antimicrobial Properties 6
2.4.1. Root aqueous extract 6
2.4.2. Root non-aqueous extract 7
2.4.3. Root hydroalcoholic extract 7
2.4.4. Leave aqueous extract 8
2.4.5. Leave non-aqueous extract 8
2.4.6. Leave hydroalcoholic extract 9
2.4.7. Seed non-aqueous extract 11
2.4.8. Seed hydroalcoholic extract 11
VIII 2.4.9. Aerial aqueous extract 11
2.4.10. Aerial non-aqueous extract 12
2.4.11. Aerial hydroalcoholic extract 12
2.5. Pharmacological Properties 12
2.5.1. Antiproliferative activity 13
2.5.2. Anti-inflammatory activity 13
2.5.3. Antioxidant activity 14
2.5.4. Analgesic, antinociceptive and antiulcer properties 14
2.5.5. Antidiabetic and antihypertensive action 15
2.5.6. Effect on platelet aggregation, hyperlipidemia, atherosclerosis, and anti allergic activity 16
2.6. Toxicity 16
2.7. Modes of use and Precautions 17
2.8. Test methods used to Investigate the Antimicrobial Effect of Nettle 17
3. MATERIALS AND METHODS 20
3.1. Description of Study Area 20
3.2. Collection of Plant Material and Extract Preparation 20
3.2.1 Crude extraction 20
3.2.2. Preparation of different concentrations of the crude extracts 21
3.4. Phytochemical Screening of the Plant Materials 21
3.5. Media Preparation and Standardization of Inoculum 23
3.6. Disk diffusion method 23
3.6.1. Inoculation of mueller hinton agar plates 24
3.6.2. Measuring zones of inhibition 24
3.6.3. Determination of minimum inhibitory concentration 24
IX 3.6.4. Determination of minimum bactericidal concentration and minimum fungicidal concentrations 25
3.7. Data Analysis 26
4. RESULTS AND DISCUSSION 27
4.1. Qualitative Analysis of Phytochemical Composition of U. dioica L.Leaf, Fruit and Root Crude Methanolic Extracts 27
4.2. Antimicrobial Activities of Stinging Nettle Leaf, Fruit and Root Methanolic Extracts 27
4.3. Minimum Inhibitory Concentration , Minimum Bactericidal Concentration and Minimum Fungicidal Concentration of Stinging nettle Leaf, Fruit and Root Methanolic Extracts 31
5. SUMMARY, CONCLUSION AND RECOMMENDATION 34
5.1. Summary 34
5.2. Conclusion 35
5.3. Recommendation 36
6. REFERENCES 37
7. APPENDICIES 50
X LIST OF TABLE Table page 1. Preliminary phytochemical screening of crude extracts from stinging nettle (U. dioicaL.) leaf, fruit and root 27 2. Antibacterial Activity of the methanolic extracts of stinging nettle leaf, fruit and root as mean of inhibition diameter zone against Gram-Positive and gram-Negative Pathogenic Bacteria . 29 3. Antifungal Activity of the methanolic extracts of stinging nettle (Urtica dioica L.) leaf, fruit and root as mean of inhibition diameter zone against fungal spp. 30 4. Minimum inhibitory concentration and minimum bactericidal concentration of methanolic leaf, fruit and root extracts of Urtica dioica L.) 32 5. Minimum inhibitory concentration and minimum fungicidal concentration of methanolic leaf, fruit and root extracts of Urtica dioica (L.) 33
XI LIST OF TABLE IN THE APPENDIX Table page
1. Antibacteria activity based on diameter of zone of inhiontion 51
2. Data for antifungal activity 52
XII Phytochemical Screening and Antimicrobial Activities of stinging Nettle (Urtica dioica L.) Leaf, Fruit and Root Extracts
ABSTRACT Medicinal plants have been used for centuries to treat diseases. Differences in antimicrobial activities may affected by geographical area and other conditions.This study was carried out to screen the phytochemical composition and antimicrobial activities of leaf,friut and root extracts of U.dioca against pathogenic bacteria and fungus. U.dioca is a medicinal plant which belongs to the family Urticaceae.The Leaf,friut and root of this plant were collected. The phytochemical screening was conducted using methanol as an extraction solvent. The antibacterial and antifungul activities of these extracts against Four bacterial pathogen (E. coli, S. typhi, S. aureus and S. pyogenes and two fungi pathogens (A. versicolor and A. niger) were evaluated using the disc diffusion method at three different concentrations (100, 150 and 200 mg/mL) in the presence of positive and negative controls.The MIC,MBCand the MFC of these crude extracts against Four bacterial pathogen and two fungial pathogens were assessed using the broth dilution method. In this study, qualitative analysis of the composition of the leaf extract has revealed the presence of tannins, saponins, flavonoids, terpenoids, and alkaloids. However, the fruit extract revealed the presence of steroids in addition to saponins, flavonoids, and terpenoids. As tannins, phlobatannins, saponins, flavonoids, terpenoids and alkaloids were detected as the active phytoconstituents of root extract. From the antibacterial activity, S. aureus were the most susceptible bacterial species to 200 mg/mL concentration with maximum zone of inhibition (16.83mm) form root extract. While the least susceptible was E.coli at 200 mg/mL concentration with minimum inhibition zone (13.57mm) for fruit extract. Contrastingly, the 200 mg/mL concentration of the extract revealed maximum antifungal activity with the highest zone of inhibition (16.00mm) for leaf extract against A. versicolor showing its susceptibility whereas the weakest antifungal activity with minimum zone of inhibition (13.17mm) at 200 mg/mL concentration of the extract was recorded for fruit extract against A. niger. A. versiclor was more susceptible than A. niger by all of the crude extracts at a concentration of 200 mg/ml. The extract from U. dioica (L.) root demonstrated strongest bactericidal activity with MIC of 1.5mg/ml and corresponding MBC of 3.125mg/ml against S.aureus. The antifungal activity of the extracts demonstrated the strongest antifungal activity with MIC of 6.25mg/ml and corresponding MFC of 12.5mg/ml in leaf extract against A. versicolor. It can be concluded from the result antimicrobial activity that the root extract of U. dioica (L) had exhibited the strongest antibacterial potential while the fruit extract had the least antibacterial. The antifungal activity, the leaf extract presented the highest antifungal potential while the fruit extract had the least antifungal. The findings of this current study suggested that the crude extracts of U.dioca have the potential to be used as a source of alternative antimicrobial agents. However, further extensive studies have to be undertaken for developing concrete recommendations for antimicrobial agents to reduce the effects of bacterial and fungal pathogenic activities.
Keywords: Disc difusion method, Gram negative, pathogenic microbes, MIC, MBC, methanolic extract, MFC, Zone of inhibition.
XIII 1. INTRODUCTION
Medicinal plants have been used for centuries to treat diseases and It play a key role in the development and advancement of modern studies by serving as a starting point for the development of novelties in drug (Wright, 2005).
The use of plant extracts and phytochemicals, both with known antimicrobial properties, are of great significance to therapeutic treatments (Kalimuthu et al., 2010).Plants generally produce many secondary metabolites which constitute an important source of microbicides, pesticides and many pharmaceutical drugs (Bobbarala et al., 2009).These natural products provide clues to synthesize new structural types of antimicrobial and antifungal chemicals that are relatively safe to man (Kalimuthu et al., 2010). According to World Health Organization (2001),medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy (Nascimento et al., 2000).In traditional medicine, plants and herbs are widely used in treatment of the disease for their benefits such as having low side effects, being natural sources with low cost.
One among such plants possessing a number of biologically active compounds is Urtica dioica L. is a widespread wild plant that is also cultivated for specific uses. It is a member of Urticaceae family, herbaceous perennial plants which have many little hairs and contain histamine, formic acid, acetylcholine, acetic acid, etc… on the leaves and stalks that cause skin irritation after contact (Moses and Nyarango, 2013) . Its genus name is derived from the Latin urere, which means to sting, and more precisely from uro, meaning to burn by friction. This plant has been used for centuries in folk medicine to cure a wide range of diseases or disorders such as arthritis, rheumatism and eczema(Kukric et al. 2012; Zekovic et al., 2017). Besides application in medicine, this plant has also been used in human nutrition for a long time. It has been harvested commercially for high content of chlorophylls, which has been used as coloring agent in food and medical products (Brown, 1995).It is widely used as a medicine due to its many pharmacological and clinical effects.
1 Urtica dioica contains wide diversity of biologically active compounds. There are essential oils and terpenoids as the main components (Durovic et al., 2017; Gul et al., 2012), carotenoids (Kukric et al., 2012), fatty acids (Durovic et al., 2017), different phenolic and polyphenolic compounds (Orcic et al. 2014), Alkaloids, saponins, tannins, flavonoids, steroids and terpenes, polyphenols and cardiac glycosides are present in the leaves (Rosłon and Węglarz, 2003). The investigation of polyphenolic acids in male and female forms of stinging nettle showed that the male has higher polyphenolic acids content than female form and these compounds increase at the stage of full blooming in both forms (Hryb et al., 1995).
The medicinal use of nettle as an antimicrobial became the focus of research due to infectious diseases rapidly spreading in modern societies. Antimicrobials are medicinal substances which kill living microorganisms (bacteria, viruses, fungi and parasites) or inhibit their growth or reproduction. In order to examine antimicrobial properties and compounds, it is necessary to prepare various extracts. Several methods were used to examine the antimicrobial activity of extracts. However, it would be important to select the same technology and circumstances in all cases to be able to make adequate comparisons. Differences in antimicrobial activities may affected by geographical area, collection season, part of the plant has been examined for its antimicrobial activity, extracting agent is used, for how long and at what temperature is the drug being extracted, the concentration of the prepared extract, the type of microbes tested, and other environmental conditions and test method used (Koszegi et al., 2017).
Nowadays increasing attention is being paid to herbs, to avoid the undesirable side effects of synthetic drugs. This is the reason why the analysis of the antimicrobial activities of medicinal plants are increasingly in the focus of scientific experiments as well .Some ethnobotanical studies indicate that in Ethiopia people have been widely using traditional medicinal plants for upholding their primary health care system. Traditionally different medicinal plants have been in use for the treatment of infectious diseases like wounds, toothache, skin itching, abdominal pain, headache, kidney problems, malaria, etc . One of the best-known medicinal plants is nettle including stinging nettle (U. dioica L.), the most commonly utilised for medical purposes,. Nettle tea consumption is widespread in folk medicine for treating diabetes, allergies, abdominal pain, benign prostatic hyperplasia, rheumatoid arthritis and treatment of
2 infections (Koszegi et al., 2017). In Eastern Ethiopia, stinging nettle is found wide spread. It is commonly called 'doobbi' in Oromo. However, little is known about its medicinal properties. On account of such justifications the present study was designed to investigate antimicrobial activities of leaf, root and fruit extracts of stinging nettle using methanol as a solvent.
Genera Objective
To investigate qualitative analysis of phytochemicals and antimicrobial activity of leaf, fruit and root extracts of stinging nettle.
Specific Objectives
To extract and screen the photochemical constituents of methanolic extracts from leaf, fruit and root of stinging nettle; To determine the antibacterial and antifungal activities of the extracts; To determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and fungicidal concentration (MFC) of the crude extracts.
3 2. LITERATURE REVIEW
2.1. Botanical Description of Nettle
Urtica dioica L. native to Eurasia, nettle was widely distributed throughout all the temperate regions of the world. It is now found in Europe (more in northern than in southern Europe), in northern Africa, in Asia and in northern and southern America where it’s also largely widespread (Ghedira et al., 2009). Nettle is a herbaceous plant, 1 to 2m tall and perennial with rhizomes. It belongs to the Urticaceae family in the Rosales order and the genus Urtica . The erect stems are strong, hairy, mostly unbranched and quadrangular. They are green in young plants and purple/reddish in older ones. The leaves are opposite, egg-shaped, elongated, with a strongly serrated margin and a pointed tip. The leaves and stems are very hairy and bear many stinging hairs whose tips come off when touched, transforming the hair into a needle that injects a stinging liquid.
It is dioecious with separate male and female plants that flower from June to September. The flowers are unisexual, small, and are arranged in clusters on slender, branched spikes formed in the leaf axils. Female flowers are greenish and have a unilocular ovary with a solitary ovule bearing one style with a brush-like stigma. Male flowers are yellowish and composed of 4 stamens, with long elastic filaments, which are bent inwards in the bud. Stinging nettle produces oval-shaped achenes (one-seeded fruits) containing tiny dark brown or almost black seeds. The root system is composed of a taproot which branches into fine rootlets allowing the tuft nettle to expand (Ghedira et al., 2009; Bhuwan et al., 2014).
2.2. Traditional Medicinal Properties
All parts of the plant are used in traditional medicine. The whole plant is used as a diuretic, anti-hypertensive, anti-diabetic, hemostatic, anti-asthenia, antianemic, antispasmodic, antirheumatic and as a remedy for headaches and chills (Hmamouchi, 1999; Bnouham et al., 2002). Nettle is also used to treat spleen, renal and dermal disorders (Daoudi et al., 2008). The seeds are administered orally for their aphrodisiac and galactagogue effects and other traditional uses against tuberculosis and kidney stones have been described (Bellakhdar,
4 1997). External uses include the treatment of aphthae, hemorrhoids, scabies and pruritus (Hmamouchi, 1999).
2.3. Phytochemical Composition
The leaves of nettle are rich in flavonoids, as well as phenolic compounds, organic acids, vitamins and minerals. The root contains lectins, polysaccharides, sterols and lignans. The stinging action is due to the liquid contained in nettle’s hairs. This liquid contains at least three compounds that could be the cause of its allergic reactions: acetylcholine, histamine and serotonin (Bhuwan et al., 2014).
Nettles secondary metabolites have marked pharmacological properties. The main flavonoids are quercetin, kaempferol and rutin These flavonoids have antioxidant and anti-inflammatory properties that may limit oxidative damage responsible for some chronic diseases such as cancer, cardiovascular diseases and degenerative diseases. They have many other effects, such as the inhibition of lipid peroxidation of liver mitochondria and blood cells and have also been shown to have hypoglycemic, antibacterial and antiviral properties (Cushnie and Lamb 2005; Kataki et al., 2012; Kumar and Pande, 2013). The most active flavonoid is quercetin. It has strong antioxidant and anti-inflammatory actions (Nair et al., 2006). It is not only capable of reducing the incidence of mammary tumors in rats (Verma et al., 1988; Carli et al., 2009) but it also has anti-tumor activity against prostate cancer (Nair et al., 2004). Its anti- ulcerogenic activity has also been demonstrated (Shin et al., 2005). The antioxidant activity of rutin is similar to that of quercetin (La Casa et al., 2000; Torres et al., 2006; Yang et al., 2008). In addition, it has anti-inflammatory, anti-cancer properties and reduces the cytotoxicity of oxidized bad cholesterol (LDL) (Selloum et al., 2003; Tian et al., 2008). Tannins, caffeic acid, ferulic acid and coumarins also have antioxidant activity and may protect cells against damage caused by free radicals (Gülçin et al., 2010; Sorensen et al., 2014).
Nettle root contains a lectin called Urtica dioica Agglutinin (UDA). This lectin is somewhat unique. It has a low molecular weight (8 to 9 kDa) and consists of a single polypeptide chain of less than 100 amino acids (Saul et al., 2000). The UDA has immunomodulatory activity and appears to limit the autoimmune manifestations (Saul et al., 2000).
5 2.4. Antimicrobial Properties
The antibacterial properties of various extracts of U. dioica against different bacterial strains were identified by several studies. In a study conducted on nine bacteria: Citrobacter koseri, Enterobacter aerogenes, Escherichia coli, Micrococcus luteus, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae, the aqueous extract of aerial parts inhibited the growth of all these bacteria except some strains of Pseudomonas aeruginosa (Gulcin et al., 2004). Another study on 38 microorganisms brought evidence of the bactericidal effect of organic extracts of the aerial parts. These extracts inhibited the growth of Acinetobacter calcoaceticus, Bacillus cereus, Bacillus spizizenii, Bacillus subtilis, Citrobacter freundii, Entrobacter aerogenes, Erwinia sp., Escherichia coli, Klebsiella pneumoniae, Micrococcus sp., Saccharomyces cerevisiae, Salmonella paratyphi B, Serratia marcescens, Methicillin-resistant Staphylococcus aureus (MRSA) and Vibrio parahaemolyticus. Phenolic compounds in the nettle were responsible for this antibacterial effect (Modarresi-Chahardehi et al., 2012).
The antiviral activity of the nettle was evaluated in vitro. The selective and powerful inhibitory action of UDA on the intracellular replication of HIV (HIV-l and HIV-2), respiratory syncytial virus (RSV), and cytomegalovirus (CMV), was well elucidated (Balzarini et al., 1992). The antimycotic activity on some pathogenic fungi (Alternaria alternata, Aspergillus flavus, Candida albicans, Ceratcystis ulmi, Fusarium oxysporum, Fusarium solani, Phoma exigua, Phytophthora carotovora, Porphyromonas gingivalis, Microsporum cookei, Microsporum gypseum, Saccharomyces cerevisiae, Trichoderma viride, Trichophyton mentagophytes and Rizoctonia solani) was also confirmed (Balzarini et al., 1992; Hadizadeh et al., 2009).
2.4.1. Root aqueous extract
An anti prostate cancer effect of the aqueous root extract which is accomplished by inhibition of the binding of 125I-SHBG (human sex hormone binding globulin) to its receptor was reported (Schottner et al., 1997). In fact, pinoresinol, dehydrodiconiferyl alcohol, (-)- secoisolariciresinol, (+)-neoolivil, isolariciresinol, lignans, and 3, 4- ivanillyltetrahydrofuran, from the aqueous root extract of Urtica dioica bind to SHBG (Belaiche and Lievoux, 1999).
6 Moreover, this aqueous extract diminishes nocturia in men suffering from prostatic adenoma. Wagner et al. (1994) showed that aqueous root extract contains a polysaccharide mixture that can be used by some stimulated T lymphocytes and others for influencing the complement system or triggering the secretion of tumor necrosis factor-α (TNF-α) in vitro. They also indicated an extended anti-inflammatory activity while performing the rat paw edema assay (Testai et al., 2002). This extract can induce hypotensive responses due to negative inotropic effect, potassium channels opening and the production of endothelial nitric oxide (Safarinejad, 2005). Patients receiving Urtica dioica improved their international prostate symptom score, lower urinary tract symptoms, and the maximum rate of urinary flow with modest decrease in prostate size. Moreover, Urtica dioica decreased postvoid residual urine volume (PVR) but did not change serum prostate-specific antigen (PSA) and testosterone levels (Wagner et al., 1995).
2.4.2. Root non-aqueous extract
Antiprostatic effect of GlcN-Ac-( N-acetylg-lucosamine), a specific lectin from the rhizomes of stinging nettle , also called urtica dioica agglutinin (UDA), exerted by inhibiting the attachment of 125I-EGF (epidermal groth factor) to its receptor (EGF-R) in prostate tissues was demonstrated (Balzarini et al. 1992). UDA inhibits the activity of respiratory syncytial virus, cytomegalovirus (CMV), influenza A and human immunodeficiency virus types 1 and 2 (Musette et al., 1996.). It also inhibits the development of the systemic lupus erythematosus- like pathology in Murphy Roths Large (MRL) mice homozygous for the lpr (lymphoproliferation) mutation (Krzeski et al., 1993 ). The combination of Urtica dioica root and Pygeum africanum bark extracts reduce urine flow as well as residual urine and nocturia in men showing benign prostatic hyperplasia (Hirano et al., 1994).
2.4.3. Root hydroalcoholic extract
The hydroalcoholic extract of stinging nettle root has a cytotoxic activity on human prostatic epithelial cells (Gansser and Spiteller, 1995). Aromatase inhibition by the methanolic extract of Urtica dioica root was also observed (Farzami et al., 2003).
7 2.4.4. Leave aqueous extract
In vivo studies showed that aqueous leaf extract of Urtica dioica is helpful in different aspects of diabetes treatment in rats. This extract affects Langerhans islets in diabetic rats and subsequently leads to an increase of insulin secretion and decrease of blood sugar (Qujeq et al., 2011). Similarly, simultaneous increase of insulin and decrease of blood glucose after treatment of diabetic rats with Urtica dioica accompanied by an increase of the activity of coenzyme acetyl A carboxylase and nucleoside diphosphate kinase in the alloxan induced diabetic rats was reported (Das et al., 2011). Antihyperglycemic effect of leaf aqueous extract of Urtica dioica in the streptozotocin treated hyperglycemic rats as well as significant decrease of the level of lipids, cholesterol but not triglyceride and low density lipoprotein (LDL) was demonstrated (El Haouari et al., 2007). Inhibition of, protein tyrosine phosphorylation, Ca2+ mobilization and oxidant production which cause platelet hyperaggregability in type 2 diabetes mellitus is caused by Urtica dioica extracts (El Haouari et al., 2006). The aqueous leaf extract has also an antiplatelet activity in vitro (Durak et al., 2004). Investigation of the effect of aqueous extract in the prostate cancer showed a significant decrease of adenosine deaminase, an important enzyme in nucleotide synthesis (Fatthi et al., 2013). In vitro studies showed apoptosis induction in MCF-7 breast cancer cell line after exposure to aqueous leave extract of Urtica dioica (Alp and Aksu, 2010). Finally, an antibacterial effect of aqueous extract against pseudomonas and psychrotrophic bacteria present in the ground beef was reported (Riehemann et al., 1999).
2.4.5. Leave non-aqueous extract
The non aqueous leave extract of Urtica dioica have various clinical effects. The ethanolic leave extract have an antiinflammatory effect on rheumatoid arthritis via inhibition of the proinflammatory transcription factor NF-ĸB (Mittman, 1990). This extract was also shown to be efficient in allergic rhinitis treatment (Modarresi-Chahardehi et al., 2012). An antimicrobial effect of extracts on fish and human pathogenic bacteria was demonstrated by disc diffusion method. Considerable antibacterial activity against both Gram negative and positive bacteria was reported (Hadizadeh et al., 2009; Singh et al., 2012; Dar et al., 2012) with hexane extracts showing better antimicrobial activity on Gram negative bacteria (Dar et
8 al., 2012) and ethyl acetate and hexane extracts exhibiting better antimicrobial activity on Gram-positive bacteria (Singh et al., 2012). Ethyl acetate extracts were demonstrated to be more efficient than methanol extracts in preventing in vitro rat platelet aggregation induction by thrombin (Durak et al., 2004). The investigation of ethanolic extract efficiency against four main plant pathogenic fungi, demonstrated that it exert an important antifungal activity. Therefore the ethanolic extract of Urtica dioica could be substituted to chemical products routinely used for preventing fungal infections in plants (Kataki et al., 2012).
The study of antioxidant, hepatoprotective and anti helminthic activity of methanol extract of leaves of Urtica dioica in vitro and in vivo showed a significant antioxidant activity comparable to traditional antioxidant compounds such as α -tocopherol, ascorbic acid and butylated hydroxyanisole (BHA). Pretreatment of animals with this extract had a significant increase in superoxide dismutase level and inhibited lipid peroxidation (Cetinus et al., 2005). Urtica dioica leaf homogenized in 1.15% KCl decreased malonyldialdehyde level therefore preventing oxidative stress induced by tourniquet in rats (Turel et al. 2008). The methanolic extract also decreased the levels of alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin of serum which indicates its hepatoprotective effect. Antihelminthic activity of the methanolic extract was also reported in Pheretima posthuma and mice naturally infected with Aspiculuris etraptera (Roschek et al., 2009).
2.4.6. Leave hydroalcoholic extract
The hydroalcoholic extract of Urtica dioica has an anti allergic rhinitis effect exerted by preventing prostaglandin formation through inhibiting hematopoietic prostaglandin D2 synthase cyclooxygenase-1 and cyclooxygenase-2 which all play essential roles in pro- inflammatory pathways. It also inhibits the activity of mast cell tryptase and histamine-1 receptor (Guder and Korkmaz, 2012). The examination of antioxidant activity of hydroalcoholic extract by means of different antioxidant evaluation methods demonstrated an antioxidant effect comparable with traditional antioxidants such as α-tocopherol, butylated hydroxytoluene (BHT) and BHA (Hajhashemi and Klooshani, 2013). Using carrageenan- induced paw edema, formalin test and acetic acid-induced writhing, an anti-inflammatory and
9 antinociceptive effect of the hydroalcholic leaf extract was demonstrated in Swiss mice and Wistar rats. Therefore, the hydroalcoholic leaf extract may reduce pain and inflammation by suppressing histamine release from mast cells and also suppressing arachidonic acid metabolism (Ozen and Korkmaz, 2003).
In vivo evaluation of extract's effect on lactate dehydrogenase, lipid peroxidation and antioxidant enzymes showed a significant increase of superoxide dismutase, glutathione S- transferase, glutathione reductase, NADH-cytochrome b5 reductase, cytochrome b5, DT- diaphorase, glutathione peroxidase, catalase activities in the liver and a decrease of NADPH- cytochrome P450 reductase activities, cytochrome P450, total sulfhydryl groups, as well as a decrease of lactate dehydrogenase, protein bound sulfhydryl groups and nonprotein sulfhydryl groups (Golalipour et al., 2010). Injection of this extract before inducing diabetes by streptozotocin in rats, has a hypoglycemic and protective effect on the β-cells of Langerhans islets as well as morphometric features of hepatocytes and seminiferous tubules (Golalipour and Khori, 2007; Golalipour et al., 2011).
This extract also decreased the number of astrocytes in the dentate gyrus of hyperglycemic rats (Ahangarpour et al., 2012). In fructose-induced insulin resistance rats, treatment with this extract caused a decrease of insulin, LDL, leptin, fasting insulin resistance index (FIRI), serum glucose, LDL/HDL ratio and increase of very low density lipoprotein (VLDL), AST and triglyceride (TG) but no ALP of serum (Nassiri-Asl et al. 2009). In hypercholesterolemic rats a decrease of the level of total cholesterol, LDL, ALT, AST and weight was shown (Ozen and Korkmaz, 2009) while a decrease of blood glucose and increase of insulin, acetyl coenzyme A carboxylase and nucleoside diphosphate kinase activities was reported in alloxan induced diabetic rats. This extract increased aniline 4-hydroxylase activity, cofactor requirement (NADH and NADPH) and metal ions (Mg2+ and Ca2+) in mice (Said et al., 2008). The combination of Urtica dioica with Atriplex halimus, Olea europea and Juglans regia decreased glucose levels and improved sugar uptake during glucose tolerance test (Kandis et al., 2010).
10 2.4.7. Seed non-aqueous extract
The diethyl ether extract of Urtica dioica seed decreased serum aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, cerulop-lasmin and lipid hydroperoxides levels and increased serum arylesterase, paraoxonase, and catalase levels in rats (Kanter et al., 2005). The diethyl ether seed extract alone or in combination with Nigella sativa decreased liver enzyme levels and maloned-ialdehyde in rats and increased their weight as well as the levels of the reduced antioxidants during 60 days treatment (Tekin et al., 2009). The extract showed moderate anti inflammatory effects in tissue inflammation model induced by carrageenan (Korpe et al. 2013). The methanol extract of seeds of Urtica diocia was highly effective against Xanthomonas vesicatoria, a plant-borne pathogen (Bnouham et al., 2003 ).
2.4.8. Seed hydroalcoholic extract
The hydroalcoholic extract of Urtica dioica seeds had a good antioxidant effect compared to traditional antioxidants α-tocopherol, BHT and BHA as demonstrated by different antioxidant evaluation methods such as reducing power, total antioxidant activity, hydrogen peroxide scavenging, superoxide anion radical scavenging, metal chelating activity and free radical scavenging (Hajhashemi and Klooshani, 2013 ).
2.4.9. Aerial aqueous extract
Oral pretreatment of rats with the aqueous extract of aerial part of stinging nettle enabled a decrease of glucose level during oral glucose tolerance test (OGTT) (Legssyer et al., 2002). This extract was shown to produce a vasoconstriction of the aorta by activating α1-adrenergic receptors and caused a strong bradycardia through non-cholinergic and non-adrenergic pathways (Daher et al., 2006). Total LDL, cholesterol, LDL/HDL ratio and plasma total AST, lactate dehydrogenase (LDH), ALT and apo B decreased after treatment of rats with aqueous extract of aerial part of stinging nettle (Tahri et al. 2000). This extract affects also arterial blood pressure in rats by increasing diuresis and natriuresis (Gulcin et al., 2004).
Antimicrobial, antiulcer, antioxidant and analgesic activities of Urtica dioica were investigated by Gülçin et al. (2004) who showed that aqueous extract of aerial part of this plant has a remarkable antioxidant activity comparable to standard antioxidants and has
11 antibacterial effect on both Gram-negative and positive bacteria. Nevertheless, pre-treatment of this extract with metamizol and famotidine inhibits the acetic acid-induced writhing and ethanol-induced gastric mucosal injury in rats, respectively (Harput et al., 2005). The extract increased T lymphocytes proliferation with a moderate increase of CD4+ T cells proportion and due to their scavenging activity inhibited peritoneal macrophages, NO2 production without affecting cell viability (Guler, 2013). Cytotoxicity and antioxidant effect of this extract was reported on MCF-7 cell line (Namazi et al., 2011).
2.4.10. Aerial non-aqueous extract
Non-aqueous extract exhibited good antibacterial activity on both Gram negative and positive bacteria. Ethyl acetate and hexane extract demonstrated better antimicrobial activity against the Gram-positive bacteria (Singh et al., 2012). Total LDL, LDL/HDL, cholesterol ratio and plasma total apo B decreased after treating rats with petroleum ether extract of aerial parts of the plant (Tahri et al., 2000).
2.4.11. Aerial hydroalcoholic extract
The hydroalcoholic extract of aerial part of Urtica dioica increased HDL, total antioxidant capacity and superoxidant dismutase and decreased FBS, HBA1C, TG, Log (TG/HDL-c) and systolic blood pressure without any changes in malondi-aldehyde and glutathione peroxides in type 2 diabetes patients after eight weeks treatement (Namazi et al., 2012; Mobaseri et al., 2012). This extract unabled glucose utilization enhance either directly or by increasing the insulin sensitivity in vitro (Fazeli et al., 2010). Treatment with Urtica dioica reduced densities of CA3 hippocampal pyramidal cells in diabetic rats (Galelli and Truffa-Bachi, 1993).
2.5. Pharmacological properties
Many research works show that nettle root's components can interfere with several mechanisms involved in the pathogenesis of benign prostatic hyperplasia. The antiproliferative effect on prostate cancer cells of UDA and the methanolic alcoholic root extracts has been demonstrated in vivo and in vitro (Konrad et al., 2000; Chrubasik et al., 2007).
12 2.5.1. Antiproliferative activity
Lignans from root extract not only inhibit the binding of androgens to their transporter proteins SHBG (Sex Hormone Binding Globulin), but also the binding of these proteins to the membrane receptors of the prostate, thereby inhibiting their proliferative activity on prostate tissues (Hryb et al., 1995; Schöttner et al., 1997 ). The root extract reduces the production of estrogen by aromatase inhibition, thereby decreasing the conversion of androgens to estrogens. Also, it was mentioned that root extracts inhibit the enzymatic activity of the membrane of prostate cells, which would stop its growth (Chrubasik et al., 2007). Clinical studies on a root extracts showed a significant improvement of the symptoms of benign prostatic hypertrophy (Schneider and Rübben, 2004; Safarinejad, 2005).
2.5.2. Anti-inflammatory activity
Scientific research has highlighted the nettle's ability to decrease the inflammatory response, through multiple mechanisms whose consequences are the reduction of synthesis of lipid mediators and proinflammatory cytokines. Indeed, leaf extracts inhibit the biosynthesis of arachidonic acid cascade enzymes, in particular the cyclo oxygenases COX-1 and COX-2, thereby blocking the biosynthesis of prostaglandins and thromboxanes (Roschek et al., 2009). In addition, an inhibitory effect was demonstrated on the NF-kappa B (nuclear factor kappa- light-chain-enhancer of activated B cells) system involved in immune, inflammatory and antiapoptotic responses (Farahpour and Khoshgozaran, 2015) and the PAF (Platelet Activating Factor) (Roschek et al., 2009). Furthermore, several studies have shown that the extract of the leaves reduces the release of Interleukins IL-2 and IL-1β, Interferon γ (IFN γ) and Tumour Necrosis Factors TNF-α and TNF-κ (Konrad et al., 2005; Yilmaz et al., 2014). Therefore, the anti-inflammatory effect of nettle leaves suggest that it may be useful in acute inflammatory diseases but also in chronic diseases, like rheumatoid arthritis. The aqueous extract of nettle roots also has anti-inflammatory activity. Wagner had shown that a polysaccharide fraction of this extract has an inhibitory effect on the induced rat paw oedema, comparable to that of indomethacin. The anti-inflammatory effect is related to the inhibition of cyclo oxygenases and lipoxygenases, and to cytokines production (Yilmaz et al., 2014).
13 2.5.3. Antioxidant activity
Extracts of nettle have a neutralizing role of reactive oxygen species (ROS). Numerous studies have shown that the methanolic and ethanolic extracts of leaves have a remarkable antioxidant effect on the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) (Kataki et al., 2012; Khare et al., 2012). Chelation of ferrous iron was evaluated using ferrozine, which forms a red chromophore with the residual iron (FeII-Ferrozine) having an absorption maximum at 562 nm. The absorbance obtained shows that nettle has a significant chelating activity of the ferrous ions Gulcin et al., 2004). Another study conducted on rats treated with carbon tetrachloride (CCl4 Immunomodulatory activity), showed that nettle decreased lipid peroxidation and increased the activity of the antioxidant defense system playing thus a protective role against hepatotoxicity. This antioxidant activity is essentially correlated to the phenolic compounds content (Kataki et al., 2012; Kanter et al., 2005).
Many studies indicate that flavonoids are able to modulate the immune system. This modulatory effect of the aerial parts of nettle was studied on mice, using an ethanolic extract at two different doses (50 and 100 mg/kg), taken orally for 14 day. The activities of enzymes such as cytochrome , lactate dehydrogenase (LDH) and NADPH-cytochrome P450 reductase showed a significant decrease while the antioxidant enzymes showed a significant increase. In addition, the plant has also shown a modulatory effect on enzymes of the kidney, lung and stomach, such as glutathione-S-transferase, superoxide dismutase and catalase (Ozen and Korkmaz, 2003). Quercetin-3-O-rutinoside, kaempherol-3-O-rutinoside and isorhamnetin-3- O-glucoside present in the aerial parts of the nettle contributes to the immunomodulatory activity (Akbay et al., 2003; Bhuwan et al., 2014). Furthermore, the immunomodulatory effect of the UDA isolated from the roots, has been demonstrated in several studies that elucidate their action on T cells, macrophages, thymocytes and on the release of TNFα (Wagner et al., 1994).
2.5.4. Analgesic, antinociceptive and antiulcer properties
In addition to its anti-inflammatory action, the nettle has an analgesic effect, proved in vivo in rats and mice. The aqueous extract of the leaves at the dose of 1200 mg/kg is capable of reducing the thermal stimulation in the hot plate test at 55 °C and causes a greater resistance
14 to pain. The antinociceptive effect of the hydroalcoholic extract of nettle leaves was evaluated through the acetic-acid writhing test and formalin-induced paw licking test. The results obtained show that the hydroalcoholic extract significantly reduces in a dose-dependent manner the nociceptive response in mice and rats. Flavonoids, the caffeoyl malic acid and the caffeic acid could be responsible for these analgesic properties (Farahpour and Khoshgozaran, 2015). The protective effect of the nettle against gastric ulcers is dose dependent. The aqueous extract of aerial parts, at doses of 50 and 200 mg/kg protected rats against gastric ulcer, with significant protection rates ranging from 67.7 to 77.8%. Moreover, this extract showed analgesic activity against gastric dilatation caused by acetic acid (Gulcin et al., 2004).
2.5.5. Antidiabetic and antihypertensive action
A study conducted to evluate the anti-diabetic activity in vivo showed the hypoglycemic effect of aqueous extracts of leaves of nettle on diabetic rats. These results are explained by the inhibition of the intestinal absorption of glucose (Bnouham et al., 2003). Furthermore, studies performed on the islets of Langerhans have demonstrated the stimulatory action of nettle on insulin secretion, accompanied by a decrease in blood sugar. Tests performed on normal and diabetic rats after intra peritoneal injection of aqueous extracts confirmed this result (Farzami et al., 2003).
Intravenous injections of an aqueous extract of the aerial parts of the nettle, using two concentrations: 4 and 24 mg/kg/h resulted in a blood pressure drop of 15% and 38% proportionally to the administered dose. This decrease was correlated with an increase in diuresis and natriuresis. However, the hypotensive effect was reversible after one hour if a low concentration (4 mg/kg/h) had been used, while it persisted when using a high concentration (24 mg/kg/h) (Tahri et al., 2000). Moreover, root extracts tested on isolated pieces of vaso constricted aorta showed a relaxant activity. This vasodilator effect is due to the release of the endothelial nitrogen oxide, potassium channel opening and a negative inotropic action (Testai et al., 2002).
15 2.5.6. Effect on platelet aggregation, hyperlipidemia, atherosclerosis, and anti allergic activity
Several studies indicate that extracts of nettle strongly inhibit platelet aggregation. The inhibitory effect of the aqueous extract of the leaves on platelet aggregation induced by thrombin was clearly demonstrated. Flavonoids are the main compounds involved in this activity (El Houari et al., 2006; Daher et al., 2006). Daily administration of aqueous extract of Urtica dioica at 150 mg/kg for 30 d, either as part of a normal or high fat diet, caused a reduction in serum lipids and lipoproteins. Significant decreases in cholesterol and LDL/HDL ratio (Low Density/High Density Lipoproteins) were observed (Daher et al., 2006). Similarly, administration of an ethanolic extract to hypercholesterolemic rats, using doses of 100 mg/kg and 300 mg/kg, was responsible for the decreased of cholesterol and LDL levels (Avci et al., 2006; Nassiri-Asl et al., 2009 ).
The anti-allergenic activity of the nettle is mainly due to two mechanisms. In addition to its inhibition of histamine H1 receptors, nettle inhibits tryptase, consequently reducing mast cell degranulation and the release of proinflammatory cytokines (Roschek et al. 2009). In a randomized double-blind study with allergic patients having allergic rhinitis, an improvement in symptoms was observed after one week of treatment (Mittman, 1990).
2.6. Toxicity
Toxicological studies have shown that the LD50 (median lethal dose) of the aqueous extract of the leaves administered intraperitoneally in mice is 3.5g/Kg (Bnouham et al., 2003). While the LD50 of the hydro-alcoholic extract of the leaves administered orally is 5.77 g/Kg (Farahpour and Khoshgozaran, 2015). Toxicity studies carried out on the roots have shown that the LD50 values obtained after intravenous injection of an aqueous extract and an infusion of the roots to rats are respectively 1.721 g/kg and 1.929 g/kg (Pourahmadi et al., 2014). Whereas the LD50 of hydro-alcoholic extracts administered intraperitoneally is 600 mg/Kg. The toxic dose of the fixed oil of nettle seeds is greater than 12.8 ml/kg (Tekin et al., 2009).
16 2.7. Modes of use and Precautions
Nettle is used by oral and local routes. The most frequently used preparations in herbal medicine are the total dry powder, dry extracts, infusions, decoctions and the fresh nettle juice. Orally, aerial parts are used as diuretics and also in the treatment of arthritis, rheumatism and gout. Nettle teas are also used in the treatment of rhinitis and seasonal allergies (ESCOP, 2003). Thanks to their high content of iron and trace elements, nettle leaves infusions, tinctures or fresh juices are prescribed to treat anemia and also for asthenia, convalescence and demineralization states. In association with the marigold (Calendula officinalis) and curled dock (Rumex crispus), nettle leaves are used for the treatment of chronic skin conditions such as eczema, psoriasis and hives. Nettle fresh juice has a hemostatic effect on the skin and nasal bleeding. It also overcomes the heavy periods or menorrhagias by reducing their flow (Chevallier, 2013).
Used in mouthwash, nettle is also effective against oral infections such as aphtha, gingivitis and tonsillitis [86]. External preparations like fresh nettle poultices are used in cases of acne and to alleviate arthritic and rheumatic pain (Bellakhdar, 2006). Nettle preparations are also applied externally in hair care against dandruff and oily hair. Furthermore, the nettle roots, alone or associated with saw palmetto (Serenoa repens), are used as teas or extracts in mictional disorders due to benign prostatic hyperplasia (Chevallier, 2013).
The adherence to dosage recommendations is essential. The recommended adult dosage of the dried aerial parts is 1.2 to 18g per day. For fresh juice, the recommended dose is 15 to 45 ml per day. Dosages for the dried root preparations are 0.3 to 24g per day. Recommended dosages and frequency of administration for each type of preparations are shown in table 6. Despite having anti allergic properties, nettle may cause allergies in sensitive people. Some rare hypersensitivity reactions like hives, itching, edema, oliguria and gastralgia have been reported (Vontobel, 1985). Furthermore, the use of nettle orally is contraindicated in pregnant women because of the risk of abortion (Aswal et al., 1984) and in children under 12 because of a lack of clinical studies in this area (Chrubasik et al., 2007).
17 2.8. Test methods used to Investigate the Antimicrobial Effect of Nettle
The effect of antimicrobials on microorganisms is generally described by the following values: MIC (minimum inhibitory concentration), MBC (minimum bactericidal concentration and post-antibiotic effect) (Brassai et al., 2012): MIC (minimum inhibitory concentration) is the lowest concentration of the antimicrobial which sufficiently inhibits the growth of the examined microorganism. MBC (minimum bactericidal concentration) is the concentration of an antibacterial agent required to kill nearly 100% of microorganisms. These can be determined by the serial dilution method. In the case of the serial dilution method, the test is performed in a liquid growth medium. A stock solution of the desired concentration is prepared from the compound to be tested by dissolving it in a growth medium of appropriate composition. Test tubes are incubated at 37 °C temperature for 24-48 hours (control test tubes not containing inhibitor are always used to control the growth of microbes). After the incubation, test tubes are examined with spectrophotometer. If the cloudiness of the growth medium is detected, this means that microbes were able to grow in that test tube and the analysed substance was not able to inhibit their growth in that concentration (Koszegi et al., 2017).
The MIC value is determined by the test tube with the greatest degree of dilution (with the lowest concentration of the inhibitor) where we find clear, transparent growth medium. For the determination of MBC, material has to be transferred from the test tubes not showing any growth into the inhibitor-free growth medium. MBC is the concentration which does not show cloudiness even after having been transferred into the inhibitor-free growth medium. Within the serial dilution methods, agar dilution and broth dilution methods can be distinguished. The name of agar dilution method derives from the fact that the tested compounds can diffuse in the agar plate and they form the growth inhibition zone of tested microorganisms depending on the rate of their efficiency. Essentially, the procedure is feeding the active substance into the prepared growth medium by diffusion. The two most commonly used methods are disk and well diffusion methods (Koszegi et al., 2017).
In the disc diffusion method paper disks impregnated with the compound of known concentration are placed on the surface of the agar plate inoculated with the tested microbes.
18 This is then incubated for a set period of time (usually for 24-48 hours). The inhibitor diffuses into the growth medium and creates a zone of inhibition if the microorganism is susceptible to that specific agent. One can infer the efficiency of the tested substance from measuring the diameter of the zone of inhibition (Koszegi et al., 2017). The principle of the well diffusion method is spreading the tested microorganism into the agar plate or on its surface and cutting holes of equal diameter in the plate with cork borer tube in sterile conditions. The dilution series prepared from the solution of the tested substance is placed in the holes in equal quantities. Petri dishes are then placed in a refrigerator at 10 °C and kept there for 10 hours. They are then placed in a thermostat at 28 °C, and after 1-2 days of incubation, the diameter of the zones of inhibition or stimulation formed around the wells is measured, from which the toxicity of the active substance can be inferred (Oricic et al., 2014).
The broth dilution method is similar to what was described in agar dilution with the difference that this is carried out on a microplate (microdilution) or in a test tube (macrodilution) in liquid growth medium and the lowest MIC is where no cloudiness can be seen in the liquid growth medium (Coyle, 2005). A time - bactericidal effect diagram is plotted in several cases which is obtained by showing the change in germ number in the function of time. This is primarily used to assess the joint application of more than one antibiotic as with this method it can be determined how the diagrams plotted for the separately tested antibiotics relates to that of the collective test, and whether the interaction is synergistic or additive. A modern test method is the E-test which contains antibiotic on one side in a concentration gradient which diffuses into the growth medium and the MIC value is where the inhibition intersects the test strip (Jorgensen, and Ferraro, 2009).
19 3. MATERIALS AND METHODS
3.1. Description of Study Area
The study was conducted at Kombolcha districts which is located about 542 kms southeast of Addis Ababa and 16 kms northwest of Harar town, the capital of East Hararghe Zone of Oromia Region. The Woreda is strategically located between the two main cities Harar and Dire Dewa.The Kombolcha is located at latitude 42° 07' 0'' E and longitude of 9° 25' 60'' N and at an altitude of 1200 to 2460 meters above sealevel.The Woreda receives mean annual rainfall of 600-900 mm, which is bimodal and erratic in distribution. The mean annual minimum and maximum temperatures are 13.8 and 24.4 0C, respectively (CSA, 2012).
3.2. Collection of Plant Material and Extract Preparation
Urtica dioica L. was collected from Kombolcha district, East Hararghe zone, Ethiopia.The authenticity of the plant material was confirmed in Herbarium at Haramaya University.The fresh samples were manually washed with distilled water and residual moisture evaporated at room temperature. The leaf, fruit and root sample were cut into pieces then The leaf, fruit and root sample was cut into pieces then air dried under shade at room temperature ( 25 0C) for a period of 3 weeks. Thereafter, each samples were ground to a fine powder in a grinder for 2 min, sample using the standard methods of the Association of the Analytical Chemists (AOAC, 2000). The solvent used was methanol (boiling range 40-60ºC).
3.2.1 Crude Extraction Sixty grams of the powdered plant material was soaked in 400 ml of 70% methanol in a conical flask sealed with aluminum foil and allowed to stand for 72 hrs with shaking. Then it was filtered by Whatman number 1 filter paper to obtain a solution of the crude extract. The resulting alcoholic filtrate was concentrated using under the reduced pressure using a rotary evaporator at about 35-40 0C. After solvent evaporation, the remaining crude extract was kept in air tight bottle in a refrigerator until use at 4oC..(Harborne, 1991).
20 3.2.2. Preparation of different concentrations of the crude extracts
The stock solution (200 mg/ml) was prepared by reconstituting 4g of each dried extracts in 20 mL of methanol. Different concentrations (100 mg/ml, 150mg/ml and 200mg/ml) of the extracts were prepared from their respective stocks. For preparing 100 mg/mL and 150mg/ml concentrations, 1g and 1.5g of the different stock solutions of the extracts were transferred, respectively, to separate volumetric flasks and the flasks were filled up to 10 ml mark with methanol as per the method described by Abdel Wahab and Gismalla (2017).
3.3. Phytochemical Screening of the Plant Materials
Qualitative analysis of major secondary metabolites including alkaloids, flavonoids, saponins, steroids, tannins and terpenoids of the stinging nettle (Urtica dioica L.) leaf, fruit and root samples were carried out on dried and powdered plant specimens using standard methods as described by Brain and Turner (1975) and Evans (1996).
Detection of Alkaloids: 0.5g of the extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were used to test the presence of alkaloids using Mayer’s test. Filtrates were treated with Mayer s reagent. Formation of a yellow cream precipitate indicated the presence of alkaloids.
Detection of Saponins: about 0.5mg of the extract was shaken with 5ml of distilled water. Formation of frozing (appearance of creamy miss of small bubbles) was shown the presence of saponins. Tests for Flavonoids using Lead Acetate Test: 1ml of the extract was treated with few drops of lead acetate solution. Formation of yellow precipitate indicates the presence of flavonoids.
Detection of Tannins: 0.25 gm of extract was mixed with water and heated on a water bath. The mixture was then filtered and ferric chloride was added to the filtrate. Formation of a dark green color indicated the presence of tannins. Tests for terpenoids using Salkowski’s test: One ml of the extract was treated with 1ml of chloroform and filtered. The filtrate was mixed with few drops of concentrated sulphuric acid,
21 shaken and allowed to stand. If the lower layer turns red, a steroid is present. Presence of golden yellow layer at the bottom indicated the presence of triterpenoids. Test for phlobatannins: 1 ml of each solid extract was placed into separate test tubes and mixed with 10 ml of distilled water. The mixture was boiled in a water bath for 10 min. Thereafter, 1ml aqueous hydrochloric acid was added to each mixture and shaken to develop red precipitate that indicates the presence of phlobatannins.
Test for steroids (Lieberman-Burchard’s Test): 1 ml of chloroform and 10 drops of acetic acid was placed in test tube. The concentrated extract (1 ml) was added to the test tube and mixed with the solvents. Then, 2 ml of concentrated sulphuric acid was added along the side of test the tube. The change of red color through blue to green serves as an indicator for the presence of steroids.
3.4. Test of Antimicrobial Activity
Four bacteria (E. coli, Salmonella typhi, S. aureus and Strep. pyogenes) and two fungi pathogens (A. versicolor and A. niger) were used for the study. All the test pathogens were obtained from Ethiopian Institute of Food and Health. The bacterial and fungal pathogens were subcultured and maintained on nutrient agar and potato dextrose agar (PDA), respectively. The incubation temperatures used were; 27 ºC (for 72 h) for fungi and 37 ºC (18- 24 h) for bacteria. 3.5. Media Preparation and Standardization of Inoculum Nutrient Agar (NA), potato dextrose agar (PDA), and Muller Hinton agar (MHA) were used for sub-culturing of bacterial test organisms, fungal test organisms, and determination of antimicrobial activities, respectively. These media were prepared and sterilized using an autoclave according to the manufacturers’ instructions. Two to three bacterial colonies on the plate were picked up with a sterile inoculating loop and transferred into a test tube containing sterile normal saline and vortexed thoroughly. The Spores of the test fungi were harvested by washing the surface of the fungal colony using 5mL of sterile phosphate buffered saline solution. This procedure was repeated until the turbidity of each bacterial and fungal spore suspension matched the turbidity of 0.5 McFarland barium sulfate standards as described by the Clinical Laboratory Standards Institute (CLSI, 2015). The resulting suspension was used
22 as inoculum for the test pathogen in the antimicrobial susceptibility test. The cultures of bacteria were subcultured on nutrient agar and stored at 4°C until required for the experiment.
3.6. Disk Diffusion Method
The disk diffusion method was performed using Mueller-Hinton Agar (MHA), which is the best medium for routine susceptibility tests because it has good reproducibility (Matuschek et al., 2018). This method is based on the principle that antibiotic-impregnated disk, placed on agar previously inoculated with the test bacterium, pick-up moisture and the antibiotic diffuse radially outward through the agar medium producing an antibiotic concentration gradient. The concentration of the antibiotic at the edge of the disk is high and gradually diminishes as the distance from the disk increases to a point where it is no longer inhibitory for the organism, which then grows freely. A clear zone or ring is formed around an antibiotic disk after incubation if the agent inhibits bacterial growth. In this study, discs of 6 mm diameter were prepared from sterile filter paper cut into small, circular pieces of equal size by a perforator and then impregnated with a volume up to 200 μlof the antimicrobial agent or extract solution at desired concentration (100mg/ml, 150mg/ml and 200mg/ml), then after agar plates were incubated under suitable conditions depending upon the tested microorganism.The stock solution of each plant extract was prepared at different concentrations in methanol (extract solvent). The extract impregnated discs were then placed onto MHA plates evenly inoculated with test pathogens.
3.6.1. Inoculation of mueller hinton agar (MHA) plates
Within 15 minutes after adjusting the turbidity of the suspension of inoculum, a sterile cotton swab was dipped into adjusted suspension and rotated several times by pressing firmly on the inside wall of the tube above the fluid level. This removes excess fluid from the cotton swab. Then, the dried surface of Mueller Hinton Agar plate was inoculated by streaking using the swab three times over the entire surface and rotating the MHA plates approximately 60° each time to ensure an even distribution of the inoculum. Then, the MHA plates were left open for three to five minutes to allow for any excess surface moisture to be absorbed (CLSI, 2015).
Following this step, the impregnated discs were dispensed onto the surface of the inoculated agar plates using sterile forceps. Each disc was pressed down to ensure complete contact with
23 the agar surface. The discs were distributed evenly so they were closer than 24 mm from center to center. ciprofloxacin (100mg/disc) and ketokonazole (100mg/disc) were used as positive controls for bacterial and fungal pathogens, respectively and the pure solvent (methanol) impregnated discs were used as negative controls. Then the MHA plates were sealed with parafilm and incubated at 37°C for 24 hrs and 27°C for 72 hrs for bacterial and fungal pathogens, respectively.
3.6.2. Measuring zones of inhibition
After 24 hours of incubation at 37 °C, the diameters of the zone of inhibition around each disc were measured to the nearest millimeter along two axes (i.e. 90° to each other) using a transparent calibrated ruler (mm) and the means of the two readings were recorded based on the method described by Hudzicki (2009). The antimicrobial activity was expressed as the diameter of the zones of inhibition produced by the extract around the wells. All tests were carried out in triplicate and the mean of zones of inhibition and Standard deviation of Mean (STD) were calculated for each microbe. The presence of inhibition zone was regarded as the presence of antimicrobial action.
3.6.3. Determination of minimum inhibitory concentration
Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of the extract at which the microorganism does not demonstrate any visible growth, as the microorganism growth was indicated by turbidity (Mousavi et al., 2015). The methanol extracts that showed significant antimicrobial activity in the antimicrobial activity tests were selected for determination of MIC. The MIC of the crude extracts of selected plant parts were determined by broth dilution method as per the method described by Andrews (2001). In the broth dilution method, the extract solution at 100mg/ml was serially diluted in a two-fold dilution to get 50mg/ml, 25mg/ml, and 12.50mg/ml, and 6.25mg/ml and 3.125 concentrations. Two mL of nutrient broth and potato dextrose broth for bacteria and fungi were added into all test tubes and 0. 1 ml of the prepared concentration of each extract was mixed with the nutrient broth and potato dextrose. Thereafter, standardized inoculums of 0.1 ml of the respective test pathogens were dispensed into the test tubes containing the suspensions of the broth and the extract. Then, all test tubes were properly corked and incubated at 37°C for 24 hrs for bacteria
24 and 27°C for 72 hr for fungi. After which, they were observed for absence or presence of visible growth. Afterwards, the color change was assessed visually. The lowest concentration at which the color change occurred was taken as the MIC value. The average of the three values was calculated providing the MIC values for the tested extract. The MIC values were taken as the lowest concentration of the extracts that show no turbidity after incubation. The presence of turbidity was interpreted as the visible growth of the microorganism. The experiment will be carried out for each organism in triplicates (Taura et al., 2012).
3.6.4. Determination of minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) Minimum bactericidal concentration and Minimum bactericidal concentration were determined by serial double dilution of their respective Minimum Inhibitory Concentration. Antibacterial agents are usually regarded as bactericidal if the MBC is no more than four times the MIC (CLSI, 2015). Accordingly, the tubes without growth after 24 h of incubation were subcultured on Muller Hinton agar in Petri dishes for 24 h. The least concentration of extract showing no visible growth on subculturing was taken as MBC or MFC. MBC was defined as the lowest concentration of the extracts at which the incubated microorganism was completely killed The minimum fungicidal concentration (MFC) was also done accordingly. The tests were per.formed in triplicate.
3.7. Data Analysis
All data were entered into Microsoft excel. Mean comparison and Analysis of variance (ANOVA) were carried out using SAS version 9.2 software package. Statistically significant differences were indicated by p<0.05 and p<0.01.
25 4. RESULTS AND DISCUSSION
4.1. Qualitative Analysis of Phytochemical Composition of Stinging Nettle (Urtica dioica L.) Leaf, Fruit and Root Crude Methanolic Extracts
The phytochemical composition of stinging nettle (U. dioica L.) leaf, fruit and root methanolic extracts is in Table 1. Accordingly the methanolic leaf extract has revealed the presence of tannins, saponins, flavonoids, terpenoids, and alkaloids.However, the methanolic fruit extract revealed the presence of steroids in addition to saponins, flavonoids, and terpenoids. This finding was in accordance with Joshi et al. (2014) who reported chemical analyses of U. dioica and detected revealed the presence of many valuable chemical compounds like phytosterols, saponins, flavonoids and tannins. Ghaima et al (2013) reported the presence of flavonoid, glycosides and phenols, alkaloid, tannins and terpenoids were present in nettle.
Table 1. Preliminary phytochemical screening of crude extracts from stinging nettle (Urtica dioica L.) leaf, fruit and root
Crude extract
Phytoconstituents Leaf Fruit Root
Tannins + - +
Phlobatannins - - +
Saponins + + +
Flavonoids + + +
Terpenoids + + +
Steroids - + -
Alkaloids + - +
(+): detected; (- ): not detectable.
26 Alkaloids are naturally occurring chemical compounds containing basic nitrogen atoms. They often have pharmacological effects and are used as medications and recreational drugs (Rhoades, 1979). Tannins have shown potential Antiviral, Antibacterial and Antiparasitic effects. Flavonoids enhance the effects of Vitamin C and also known to be biologically active against, tumors, and other microbes (Korkina et al., 1997). Plant terpenoids are used extensively for their aromatic qualities. They play a role in traditional herbal sonedies and are under investigation for antibacterial, and other pharmaceutical functions (Yamunadevi et al., 2011).
4.2. Antimicrobial Activities of Stinging Nettle (U. dioica L.) Leaf, Fruit and Root Methanolic Extracts
The disc diffusion method for measuring diameter of inhibition zone of methanolic extracts of U. dioica (L.) leaf, fruit and root is shown in Table 2. Significant antimicrobial activities based on diameter of inhibition zone were observed for all extracts. The antibacterial activity of methanolic extracts of U. dioica (L.) at the highest concentration of the extracts has recorded mean zone of inhibition ranging from 13.57±0.60 to 16.83±0.76mm. While the antifungal activity (Table 3) of the methanolic extract with colony growth inhibitory effect at the highest dose recorded a mean zone of inhibition ranged from 13.17 to 16.00mm.
As compared to most of the extracts, ciprofloxacin (used as positive control) showed a significant superiority (p<0.05) in the zone of inhibition (Table 2&3). The superiority of antibiotics might be due to the method of extraction and the type of solvent used for the extraction. For most of the test extracts, the highest concentration (200mg/mL) exhibited a significantly higher (P<0.05) zone of inhibition as compared to the respective lower concentrations (100mg/ml & 150mg/ml).
27 Table 2. Antimicrobial Activity of the methanolic extracts of stinging nettle (U. dioica L.) leaf, fruit and root as mean of inhibition diameter against Gram-Positive and negative pathogen.
Crude Mean of inhibition diameter at the specified Test pathogens extract concentration of methanolic crude extracts Ciprofloxacin
100mg/ml 150mg/ml 200mg/ml (100mg/ml) E. coli Leaf 9.83±0.67deC 11.83±0.70bcC 14.83±0.76defB 18.17±0.28aA (Gram negative) Fruit 9.00±0.95eC 10.50±0.50eC 13.57±0.60efB 18.87±0.64aA
Root 11.00±0.51abcC 12.50±0.50abC 15.10±0.36c-fB 18.33±0.28aA
S. aureus Leaf 11.00±0.50abcD 13.17±0.76aC 15.53±0.50bcdB 18.17±0.28aA
(Gram positive) Fruit 9.97±0.45cdeC 11.50±0.50cdC 15.17±0.76fB 18.87±0.63aA
Root 11.50±0.29aC 13.17±0.50aB 16.83±0.76aA 18.33±0.29aA
S. pyogenes Leaf 10.00±0.23cdeD 12.67±0.50abC 15.43±0.57cdeB 18.17±0.28aA
(Gram positive) Fruit 9.50±0.29deC 10.83±0.40deC 14.57±0.36efB 18.87±0.64aA
Root 10.50±0.76a-dD 12.50±0.50abC 15.73±0.53bcdB 18.33±0.25aA
S. typhi Leaf 10.33±0.87bcdD 13.00±0.76aC 15.00±0.40abcB 18.17±0.28aA
(gram negative) Fruit 9.83±0.50deC 10.90±0.35deC 13.90±0.40defB 18.87±0.63aA
Root 11.33±0.45abD 13.00±0.60aC 15.40±0.64abB 18.33±0.23aA
Means followed by same letter within a column were not significantly different at 0.05 probability level based on LSD (Least Significance difference) test. Small letters: significance within column; capital letters: significance across row. E. coli: Escherichia coli; S.aureus: staphylococcus aureus; S. typhi; Salmonella typhi; S.pyogenes: Streptococcus pyogenes.
The highest concentration of the methanolic extract (200mg/ml) presented the strongest antibacterial activity with maximum zone of inhibition (16.83mm) for root extract against S. aureus, indicating that S. aureus is the most susceptible to the crude extracts. On the other hand, the weakest antibacterial activity with minimum inhibition zone (13.57mm) was
28 observed with inhibitory effect at the highest dose for methanolic fruit extract against E. coli, suggesting that E. coli was the least susceptible to antimicrobial extract. Contrastingly, the highest concentration of the extract revealed maximum antifungal activity with the highest zone of inhibition (16.00mm) for methanolic leaf extract against A. versicolor showing that its susceptibility, whereas the weakest antifungal activity with minimum zone of inhibition (13.17mm) (at the highest concentration of the extract) was recorded for methanolic fruit extract against A. niger. It can be observed from the result of zone of inhibition diameter (Tables 2) that the methanolic root extract of U. dioica (L) had exhibited the strongest antibacterial potential while the methanolic fruit extract had the least antimicrobial (both antibacterial and antifungal) potentials. For antifungal activity, the methanolic leaf extract presented the highest antifungal potential (Table 3).
Table 3. Antifungal Activity of the methanolic extracts of stinging nettle (Urtica dioica L.) leaf, fruit and root as mean of inhibition diameter zone against fungal spp.
Test pathogens Crude Concentration of methanolic crude extracts Ketokonazole extract 100mg/ml 150mg/ml 200mg/ml (100mg/ml)
A. versicolor Leaf 10.80±0.72aD 12.50±0.40aC 16.00±0.45aB 18.50±0.50aA
A. versicolor Fruit 9.00±0.76bD 10.83±0.36bcC 13.50±0.76cdB 18.50±0.50aA
A. versicolor Root 9.83±0.95abD 11.60±0.76abC 14.83±0.50abB 18.50±0.50aA
A. niger Leaf 9.83±0.76abD 11.52±0.50bC 15.00±0.50abB 18.00±0.20aA
A. niger Fruit 8.83±0.65bD 10.40±0.52cC 13.17±1.0dB 18.00±0.50aA
A. niger Root 9.67±0.76abD 11.27±0.25bcC 14.50±0.40cbB 17.33±0.50aA
29 4.3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), Minimum Fungicidal Concentration (MFC) of Stinging Nettle (U. dioica L.) Leaf, Fruit and Root Methanolic Extracts
The plant extracts with high activity against a particular organism usually give low MIC value while the extracts with low activity give high MIC value Fabry et al (1998). In the present study, the effectiveness of the methanolic leaf, fruit and root extracts of U. dioica (L) against test pathogens were evaluated using MIC, MBC and MFC (Table 4). The methanolic extract from U. dioica (L.) root demonstrated strongest bactericidal activity with MIC value 1.5mg/ml and corresponding MBC of 3.125mg/ml against S.aureus while the weakest bactericidal activity with MIC of 100mg/ml and corresponding MBC 200mg/ml was observed for methanolic fruit extract against E. coli.
30 Table 4. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of methanolic leaf, fruit and root extracts of Urtica dioica L.)
Test pathogenes crude extract MIC (mg/ml) MBC(mg/ml)
E. coli Leaf 50 75
Fruit 100 200
Root 25 50
S. aureus Leaf 12.5 25
Fruit 6.25 12.5
Root 1.56 3.125
S. pyogenes Leaf 25 37.5
Fruit 25 50
Root 12.5 25
S. typhi Leaf 25 50
Fruit 50 100
Root 12.5 37.5 MIC:Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC).
It can be observed from this result that methanolic extract of U. dioica (L.) root extract possessed strongest antibacterial potential while methanolic fruit extract of the plant revealed the least antibacterial potenial. Furthermore, S. aureus was the most susceptible to the U. dioica (L.) methanolic extracts and E. coli was the most resistant among test bacterial spp. All bacterial pathogens showed different MIC and MBC with gram posetive bacteria was more susseptable than garam negetive bacteria at all concentration levels. It is also the Gram negative bacterium, in which naturally resistance to many antibiotics due to the permeability barriers afforded by its outer membrane composed of lipopolysaccharide.
31 The antifungal activity of the extracts (Table 5) demonstrated the strongest antifungal activity with MIC of 6.25mg/ml and corresponding MFC of 12.5mg/ml in methanolic leaf extract of Urtica dioica (L.) against A. versicolor whereas the weakest antifungal activity with largest MIC of100mg/ml and corresponding MFC of 200mg/ml in methanolic fruit extract against A. niger suggesting that A. versiclor was more susceptible to the methanolic extract than A. niger. Extracts that MICs below 25 mg/mL were indcated potent antimicrobial activity According to Fabry et al. (1998). In the present study, the MIC value of the extracts agreed with their corresponding antibacterial activities. The methanolic extracts of leaf, fruit and root extracts of U.dioica (L.) demonstrated significant antibacterial and antifungal activity.
Table 5. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of methanolic leaf, fruit and root extracts of Urtica dioica (L.)
Test pathogens Crude extract MIC (mg/ml) MFC(mg/ml)
A. versicolor Leaf 6.25 12.5
Fruit 50 100
Root 25 50
A. niger Leaf 25 37.5
Fruit 100 200
Root 25 50
The antimicrobial activity of the plant may also be attributed to the presence of some biologically active constituents.The widespread use of commercially available antimicrobials led to the consequence of emergence of antimicrobial resistant pathogens.
32 5. SUMMARY, CONCLUSION AND RECOMMENDATION
5.1. Summary
U. dioica L. is perennial, wild-growing plant from Urticaceae botanical family. Differences in antimicrobial activities may affected by geographical area and other environmental conditions and test method used. On account of such justifications the present study was designed to investigate antimicrobial activities of leaf, root and fruit extracts of stinging nettle (U. dioica L.) using methanol as a solvent.
The phytochemical screening was conducted using methanol as an extraction solvent. The experiment was arranged as 3 x 1 x 3 x 6 (3 source extracts: leaf, fruit and root extracts of stinging nettle at three concentration levels; 1 solvent system i.e. methanol and; 6 test pathogenic microbes (4 bacteria: Escherichia coli, Salmonella typhi , Staphylococcus aureus (gram positive), and Streptococcus pyogenes; two fungi pathogens: Aspergillus versicolor and A. niger) in three replications. Antimicrobial activities were determined by using disc diffusion and broth dilution methods. The least concentration of extract that show antimicrobial activity was selected for further determining the MIC: MBCand MFC.The leaf extract has revealed the presence of tannins, saponins, flavonoids, terpenoids, and alkaloids. The fruit extract revealed the presence of saponins, flavonoids, terpenoids and steroids. As tannins, phlobatannins, saponins, flavonoids, terpenoids and alkaloids were detected as the active phytoconstituents of in root extract.
The highest concentration of the extract (200mg/ml) presented the strongest antibacterial activity with maximum zone of inhibition (16.83mm) for root extract against S. aureus, indicating that S. aureus is the most susceptible to the crude extracts. On the other hand, the weakest antibacterial activity with minimum inhibition zone (13.57mm) was observed with inhibitory effect at the highest dose for fruit extract against E. coli, suggesting that E. coli was the least susceptible to antimicrobial extract.
Contrastingly, the highest concentration of the extract revealed maximum antifungal activity with the highest zone of inhibition (16.00mm) for leaf extract against A. versicolor showing that A. versicolor was more susceptible among tested fungal pathogens whereas the weakest
33 antifungal activity with minimum zone of inhibition (13.17mm) (at the highest concentration of the extract) was recorded for fruit extract against A. niger.
The effectiveness of the leaf, fruit and root extracts of Urtica dioica (L) against test pathogens were evaluated by MIC, MBC and MFC. The methanolic extract from Urtica dioica (L.) root demonstrated strongest bactericidal activity with MIC value (1.5mg/ml) and corresponding MBC (3.125mg/ml) against S.aureus while the weakest bactericidal activity with MIC (100mg/ml) and corresponding MBC (200mg/ml) was observed for methanolic fruit extract against E. coli.
The antifungal activity of the extracts demonstrated the strongest antifungal activity with MIC of 6.25mg/ml and corresponding MFC of 12.5mg/ml in methanolic leaf extract of U. dioica (L.) against A. versicolor whereas the weakest antifungal activity with largest MIC of 100mg/ml and corresponding MFC of 200mg/ml in methanolic fruit extract against A. niger suggesting that A. versiclor was more susceptible to the methanolic extract than A. niger.
5.2. Conclusion
Based on results of qualitative phytochemical screening of methanol crude extracts of the leaves ,root and fruit of U. dioica, various bioactive secondary compounds like alkaloid, tannins, flavonoids, terpenoids, steroids, saponnins and were presented. This indicated the leaf,root and friut of this plant is rich with various bioactive secondary chemical compounds and could be taken as a potential source of various pharmacologically active phyto- constituents which contribute medicinal role. It can also be observed from the result of zone of inhibition diameter that the methanolic root extract of U. dioica (L) had exhibited the strongest antibacterial potential while the methanolic fruit extract had the least antibacterial potentials. For antifungal activity, the methanolic leaf extract presented the highest antifungal potential while methanolic fruit extract had the least antifungal potential. Furthermore, S. aureus was the most susceptible to the Urtica dioica (L.) methanolic extracts and E. coli was the most resistant among test bacterial spp. All bacterial pathogens showed different MIC and MBC with gram posetive bacteria was more susseptable than garm negetive bacteria at all concentration levels. It is also the Gram negative bacterium, in which naturally resistance to
34 many antibiotics due to the permeability barriers afforded by its outer membrane composed of lipopolysaccharide.
5.3. Recommendation
Based on the findings of this study the following can be recommended : The present study used methanol as a solvent as it is known for one of the most effective solvent to test antimicrobial activities. However, different extraction methods and extraction solvents could have been used. Bacterial death rate can be cheeked by taking broth samples at different time intervals (1hr,3hrs, 6hrs, 12 hrs, and 24hrs post-treatments). The findings of the study reflect potential antibacterial and antifungal capabilities of this plant. Further researches are highly recommended to investigate the bioactive molecules of this medicinal plant and to investigate their prospective clinical outcomes in the treatment of microbial infections. Further investigation is required for this plant exhibited the highest bactericidal activity with a view to identifying and isolating their active principles. The Extract can be investigated in other bacterial strains regarding their ethnopharmacological studies. More specific methods can be used for detection of MIC e.g. Micro-dilution method and agar well diffusion methods.
35 6. REFERENCES
Abdel Wahab, M., Gismalla, M., 2017. Antimicrobial Activity of Aqueous Methanol Extracted Rigla (Portulaca oleracea Linn).M.Sc Thesis. University of Gezira. Ahangarpour A., Mohammadian M., Dianat M. 2012. Antidiabetic Effect of Hydroalcholic Urtica dioica Leaf Extract in Male Rats with Fructose-Induced Insulin Resistance. Iran J Med Sci. 37:181-6. Akbay P, Basaran AA, Undeger U, Basaran N. 2003. In vitro immunomodulatory activity of flavonoid glycosides from Urtica dioica. Phytother Res 2003;17:34-7. Alp E., Aksu M. I. 2010. Effects of water extract of Urtica dioica L. and modified atmosphere packaging on the shelf life of ground beef. Meat Sci. 2010;86:468-73. Amini K., Hemmatpour B., Moradi P., Nazari Z., & Mahvar T. 2014. Antibacterial activity of the ethanol and water extracts and investigating the chemical composition of different parts of Urtica dioica L. Science Road Journal, 2 (2): 57-65. Andrews, J. M. 2001. JAC Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy.48, Suppl. S1, 5–16. AOAC. 2000. Association of Official Analytical Chemists. Official Methods of Analysis of AOAC International, 17th ed., AOAC, Washington D.C, USA. Aswal B. S., Bhakuni D. S., Goel A. K., Kar K. 1984. Screening of indian plants for biological activity: Part X. Indian J Exp Biol 22:312-32. Avci G, Kupeli E, Eryavuz A, Yesilada E, Kucukkurt I. 2006. Antihypercholesterolaemic and antioxidant activity assessment of some plants used as a remedy in Turkish folk medicine. J. Ethnopharmacol. 107:418-23. Balzarini Jan., Neyts Johan., Schols Dominique. 1992. The mannose-specific plant lectins from Cymbidium hybrid and Epipactis helleborine and the (N- acetylglucosamine)n- specific plant lectin from Urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro. Antiviral Res. 18:191-207. Belaiche P, Lievoux O. 1991. Clinical studies on the palliative treatment of prostatic adenoma with extract of Urtica root. Phytother Res. 5:267-9. Bellakhdar J. 1997. La pharmacopée marocaine traditionnelle: Médecine arabe ancienne et savoirs populaires. France: Ibis Press; 1997.
36 Bellakhdar J. 2006. Plantes medicinales du Maghreb et soins de base. Maroc: Le Fennec. Bhuwan C.J, Minky M, Ajudhia NK. 2014. Pharmacognostical review of Urtica dioica L. Int J. Green Pharm. 8:201-9. Bnouham M, Mekhfi H, Legssyer A, Ziyyat A. 2002. Medicinal plants used in the treatment of diabetes in morocco. Int J Diabetes Metab 10:33-50. Bnouham M, Merhfour F Z, Ziyyat A. 2003. Antihyperglycemic activity of the aqueous extract of Urtica dioica. Fitoterapia. 74:677-81. Brassai, A., Dóczi, K. and Bán, E. G. 2012. Az élő kórokozókra ható szerek Gyógyszertan. In p. 7. Brown D. Encyclopedia of Herbs and their Uses. London: Dorling Kindersley; 1995. Bucar F, Britzmann B, Streit B, Weigend M. 2006. LC-PDA-MS-profiles of phenolic compounds in extracts aerial parts of Urtica species. Planta Med 72:152. Carli C. B., de Matos DC, Lopes F. C. 2009. Isolated flavonoids against mammary tumour cells LM2. Z Naturforsch C. 64:32-6. Cetinus E, Kilinc M, Inanc F. 2005. The role of Urtica dioica (urticaceae) in the prevention of oxidative stress caused by tourniquet application in rats. Tohoku J Exp Med. 205:215-21. Chaurasia N., Wichtl M. 1986. Phenylpropane und lignane aus der wurzel von Urtica dioica L. Dtsch Apothek Zeitung. 126:1559-63. Chevallier A. 2013. Plantes médicinales. France: Grund; 2013. Chrubasik, J. E., Boutogalis, B. O., Hagner, H. and chrubasik, S. A. 2007. Comprehensive review on the stinging nettle, effect and efficacy profile. Phytomedicine, 14(7):568- 579. Clinical and Laboratory Standards Institute (CLSI) (2015). Document M45. Methods for Antimicrobial Dilution and Disk Susceptibility of Infrequently Isolated or Fastidious Bacteria; Approved Guideline. Third Edition. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Clinical and Laboratory Standards Institute (CLSI, 2012). Performance standards for antimicrobial disk susceptibility tests; approved standards – Eleventh Edition. CLSI document M02-A11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, PA, 19087 USA, 2012.
37 Coyle, M. B. 2005. Manual of antimicrobial susceptibility testing. American Society for Microbiology 2005 . CSA (Central Statistical Authority), 2012. Agricultural sample survey report on area and production of crops (private peasant holdings, meher season). Volume I. CSA, Ababa, Ethiopia. 128 pp. Cushnie T. P. T., Lamb A. J.2005. Antimicrobial activity of flavonoids. Int J Antimicrobial Agents 26:343–56. 2012;6:5714-9. Daher, C. F., Baroody K. G., Baroody G. M. 2006. Effect of Urtica dioica extract intake upon blood lipid profile in the rats. Fitoterapia. 77:183-8. Daoudi A, Benboubker H, Bousta D, Aarab L.2008. Screening of fourteen moroccan medicinal plants for immunomodulating activities. Moroccan J Biol 4-5:24-30. Dar, S. A., Ganai, F. A., Yousuf, A. 2012. Bioactive potential of leaf extracts from Urtica dioica L. against fish and human pathogenic bacteria. Afr Microbiol J Res. 6:6893-9. Das D, Sarma B P, Rokeya B . 2011. Antihyperglycemic and antihyperlipidemic activity of Urtica dioica on type 2 diabetic model rats. J of Diabetol. 2:2:1-6. Durak I, Biri H, Devrim E. 2004. Aqueous extract of Urtica dioica makes significant inhibition on adenosine deaminase activity in prostate tissue from patients with prostate cancer. Cancer Biol Ther. 3:855-7. Durovic S., Pavlić B, Šorgić S, Popov S, Savić S, Pertonijević M. 2017. Chemical composition of stinging nettle leaves obtained by different analytical approaches. J. of Functional Foods. 32:18-26. El Haouari M, Jardin I, Mekhfi H. 2007. Urtica dioica extract reduces platelet hyperaggregability in type 2 diabetes mellitus by inhibition of oxidant production, Ca2+ mobilization, and protein tyrosine phosphorylation. J Appl Biomed. 5:105-13. El Houari M., Bnouham M., Bendahou M., Aziz M, Ziyyat A., Legssyer A. 2006. Inhibition of Rat Platelet Aggeration by Urtica dioica leaves extracts. Phytother Res, 20:568-72. ESCOP 2003. Monographs of the Scientific Foundation for Herbal Medicinal Products. 2nd ed. Exeter (UK): European Scientific Cooperative on Phytotherapy and Thieme; 2003. Farahpour MR, Khoshgozaran L. 2015. Antinociceptive and anti-inflammatory activities of hydroethanolic extract of Urtica dioica. Int J Biol Pharm Allied Sci., 1:160-70.
38 Farzami B, Ahmadvand D, Vardasbi S, Majin FJ, Khaghani Sh. 2003. Induction of insulin secretion by a component of Urtica dioica leave extract in perfused islets of langerhans and its in vivo effects in normal and streptozotocin diabetic rats. J Ethnopharmacol 89:47-53. Fatthi S, Motevalizadeh Ardakani A, Zabihi E. 2013. Antioxidant and apoptotic effects of aqueous extract of Urtica dioica on MCF-7 human breast cancer cell line. asian pacific of cancer prevention. APJCP. 2013;14:5317-23 Fazeli S A, Gharravi A M, Ghafari S. 2010. Effects of Urtica dioica extract on CA3 hippocampal pyramidal cell loss in young diabetic rats. Neural Regen Res. 5:901-5. Fu HY, Chen SJ, Chen RF, Ding WH, Kuo-Huang LL, Huang RN. 2006. Identification of oxalic acid and tartaric acid as major persistent pain‑ inducing toxins in the stinging hairs of the nettle, Urtica thunbergiana. Ann Bot 98:57-65. Galelli A, Truffa-Bachi P. 1993. Urtica dioica agglutinin. A superantigenic lectin from stinging nettle rhizome. J. Immunol. 151:1821-31. Gansser D, Spiteller G. 1995. Aromatase inhibitors from Urtica dioica roots. Planta Med. 61:138-40. Ghaima K. K., Hashim N. M., Ali S. A.2013. Antibacterial and antioxidant activities of ethyl acetate extract of nettle (Urtica dioica) and dandelion (Taraxacum officinale). Journal of Applied Pharmaceutical Science Vol. 3 (05), pp. 096-099.. Ghedira K, Goetz P, Jeune L. 2009. Urtica dioica L., Urtica urens et ou hybrides (Urticaceae). Phytothérapie 7:279-85. Golalipour M J, Ghafari S, Afshar M. 2010. Protective role of Urtica dioica L. (Urticaceae) extract on hepatocytes morphometric changes in STZ diabetic Wistar rats. Turk J Gastroenterol. 21:262-9. Golalipour M J, Kabiri Balajadeh B, Ghafari S. 2011. Protective Effect of Urtica dioica L.(Urticaceae) on Morphometric and Morphologic Alterations of Seminiferous Tubules in STZ Diabetic Rats. Iran J Basic Med Sci. 14:472-7. Grevsen K, Frette XC, Christensen LP. 2008. Concentration and composition of flavonol glycosides and phenolic acids in aerial plants of stinging nettles (Urtica dioica L.) are affected by nitrogen fertilization and by harvest time. Eur J Hortic Sci., 73:20-7.
39 Güder A, Korkmaz H. 2012. Evaluation of in-vitro Antioxidant Properties of Hydroalcoholic Solution Extracts Urtica dioica L., Malva neglecta Wallr and Their Mixture. Iran J Pharm Res.11:913-23. Guil-Guerrero J. L., Rebolloso-Fuentes MM, Isasa MET. 2003. Fatty acids and carotenoids from stinging nettle (Urtica dioica L.). Journal of Food Composition and Analysis. 16:111-119. Gul S., Demirci B, Başer KHC, Akpulat HA, Aksu P. 2012. Chemical composition and in vitro cytotoxic, genotoxic effects of essential oil from Urtica dioica L. Bulletin of Environmental Contamination and Toxicology. 88:666-671. Gülçin İ, Huyut Z, Elmastas M, Aboul-Enein HY. 2010. Radical scavenging and antioxidant activity of tannic acid. Arabian J Chem 3:43-53. Gulcin I, Kufrevioglu O I, Oktay M. 2004. Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.). J Ethnopharmacol. 90:205-15. Guler E R. 2013. Investigation of Chemopreventif Properties of Urtica Dioica L., in MCF-7 and MDA 231 Breast Cancer Cell Lines. The new journal of medicine. 30:50-3. Hadizadeh I, Peivastegan B, Kolahi M. 2009. Antifungal activity of nettle (Urtica dioica L.), colocynth (Citrullus colocynthis L. Schrad), oleander (Nerium oleander L.) and konar (Ziziphus spina-christi L.) extracts on plants pathogenic fungi. Pak J Biol Sci. 12:58- 63. Hajhashemi V, Klooshani V. 2013. Antinociceptive and anti-inflammatory effects of Urtica dioica leaf extract in animal models. Avicenna J Phytomed. 3:193-200. Harborne, J. B. 1991. Phytochemical methods. Second Edition. Chapman and Hall Ltd. London,288.
Harput U S, Saracoglu I, Ogihara Y. 2005. Stimulation of lymphocyte proliferation and inhibition of nitric oxide production by aqueous Urtica dioica extract. Phytother Res.19:346-8. Harput US, Saracoglu I, Ogihara Y. 2005. Stimulation of lymphocyte proliferation and inhibition of nitric oxide production by aqueous Urtica dioica extract. Phytother Res 19:346-8.
40 Hirano T, Homma M, Oka K 1994. Effects of stinging nettle root extracts and their steroidal components on the Na+,K(+)-ATPase of the benign prostatic hyperplasia. Planta Med. 60:30-3. Hmamouchi M. 1999. Les plantes medicinales et aromatiques marocaines. Morocco: Imprimerie de Fédala. Hryb D J, Khan M S, Romas N A. 1995. The effect of extracts of the roots of the stinging nettle (Urtica dioica) on the interaction of SHBG with its receptor on human prostatic membranes. Planta Med. 61:31-2. Hudzicki J. 2009. Kirby-Bauer Disk Diffusion Susceptibility Test Protocol. American Society for Microbiology. pp 1.23. Jorgensen, J. and Ferraro, M. 2009. Antimicrobial susceptibility testing: A Review of General Principles and Contemporary Practices, Medical Microbiology, pp. 1749- 1755. Joshi BC, Mukhija M, Kalia AN. 2014. Pharmacognostical review of Urtica dioica L. Int J Green Pharm. 8:201-209. Kandis H, Karapolat S, Yildirim U. 2010. Effects of Urtica dioica on hepatic ischemia- reperfusion injury in rats. Clinics (Sao Paulo). 65:1357-61. Kanter M, Coskun O, Budancamanak M. 2005. Hepatoprotective effects of Nigella sativa L and Urtica dioica L on lipid peroxidation, antioxidant enzyme systems and liver enzymes in carbon tetrachloride-treated rats. World J Gastroenterol 11:6684-8. Kara D.2009. Evaluation of trace metal concentrations in some herbs and herbal teas by principal component analysis. Food Chemistry. 2009;114:347-354. Kataki M. S., Murugamani V., Rajkumari A., Mehra P. S., Awasthi D., Yadav R. S. 2012. Antioxidant, Hepatoprotective, and anthelmintic activities of methanol extract of Urtica dioica L. Leaves. Pharm Crops 3:38-46. Kavalali G. 2003. The chemical and pharmacological aspects of Urtica. In: Kavalali GM, (Ed.). Urtica. Therapeutic and Nutritional Aspects of Stinging Nettles. London, New York: Taylor and Francis, p. 47-55. Kavtaradze NS, Alaniya MD. 2003. Anthocyanins glycosides from urtica dioica. Chem Nat Comp 39:315.
41 Khare V., Kushwaha P., Verma S., Gupta A., Srivastava S., Rawat A. K. S. 2012. Pharmacognostic Evaluation and Antioxidant Activity of Urtica dioica L. Chin Med. 3:128-35. Koch E. 2001. Extracts from fruits of saw palmetto (Sabal serrulata) and roots of stinging nettle (Urtica dioica): viable alternatives in the medical treatment of benign prostatic hyperplasia and associated lower urinary tracts symptoms. Planta Med. 2001;67:489- 500. Konrad A, Mahler M, Arni S, Flogerzi B, Klingelhöfer S, Seibold F. 2005. Ameliorative effect of IDS 30, a stinging nettle leaf extract, on chronic colitis. Int J Colorectal Dis 20:9- 17. Konrad L., Müller H. H., Lenz C., Laubinger H., Aumüller G, Lichius J. J. 2000. Antiproliferative effect on human prostate cancer cells by stinging nettle root (Urtica dioica) extract. Planta Med 66:44-7. Korkina, L.G., and Afanas'ev, I.B. 1997. Antioxidant and chelating properties of flavonoids. Adv Pharmacol ;38:15163. Korpe D. A., Iseri O. D., Sahin F. I. 2013. High-antibacterial activity of Urtica spp. seed extracts on food and plant pathogenic bacteria. Int J Food Sci Nutr. 64:355-62. Koszegi, K., Kocsis, J. M., Vatai,G., Bekassy-Molnar, E. 2017. Antimicrobial effects of the stinging nettle (Urtica dioica L.). Review. Analecta Technica Szegedinensia. 2:2064- 7964. Krystofova O, Adam V, Babula P, Zehnalek J, Beklova M, Havel L. 2010. Effects of various doses of selenite on stinging nettle (Urtica dioica L.). Int J Environ Health Res Public Health, 7:3804-15. Krzeski T, Kazon M, Borkowski A. 1993. Combined extracts of Urtica dioica and Pygeum africanum in the treatment of benign prostatic hyperplasia: double-blind comparison of two doses. Clin Ther. 15:1011-20. Kukric Z, Topalić-Trivunović L, Kukavica B, Matoš S, Pavičić S, Boroja M. 2012. Characterization of antioxidant and antimicrobial activities of nettle leaves (Urtica dioica L.). Acta Periodica Technologica. pp. 257-272. Kumar S, Pande AK.2013. Chemistry and biological activities of flavonoids: an overview. World J. Sci, 2013:1-16.
42 La Casa C, Villegas I, Alarcan de la Lastra C, Motilva V, Martan Calero MJ. 2000. Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. J Ethnopharmacol 2000;71:45-53. Legssyer A, Ziyyat A, Mekhfi H. 2002. Cardiovascular effects of Urtica dioica L. in isolated rat heart and aorta. Phytother Res. 16:503-7. Mihaljev E, Eivkov-Baloa M, Cupic E, Jakaic S. 2014. Levels of some microelements and essential heavy metals. Acta Pol Pharm 71:385-91. Mittman P. 1990. Randomized, double-blind study of freeze-dried Urtica dioica in the treatment of allergic rhinitis. Planta Med. 56:44-7. Mobaseri M, Aliasgarzadeh A, Bahrami A. 2012. Efficacy of the Total Extract of Urtica dioica on the glucose utilization by the Human Muscle Cells. J. of Clinical and Diagnostic Research. 6:437-40. Modarresi-Chahardehi A, Ibrahim D, Fariza Sulaiman S. 2012. Screening antimicrobial activity of various extracts of Urtica dioica. Rev Biol Trop. 60:1567-76. Morshed, H., Islam, M. S., Parvin, S., Ahmed, M. U., Islam, M. S., Mostofa, A G. M. and Bin Sayeed, M. S. 2012. Antimicrobial and Cytotoxic Activity of the Methanol Extract of Paederia foetida Linn. (Rubiaceae). Journal of Applied Pharmaceutical Science 02 (01); 77-80. Moses A. G., M, Nyarango, M. 2013. Fourier Transformer Infra-Red Spectrophotometer Analysis of Urtica dioica Medicinal Herb Used for the Treatment of Diabetes, Malaria and Pneumonia in Kisii Region, Southwest Kenya. World App Sc J. 21:1128-35. Mousavi S.M., Bagheri G., Saeidi S. 2015 Antibacterial Activities of the Hydroalcoholic Extract of Portulacaoleracea Leaves and Seeds in Sistan Region, Southeastern Iran. Int J Infect. 2(2):e23214. Musette P, Galelli A, Chabre H. 1996. Urtica dioica agglutinin, a V beta 8.3-specific superantigen, prevents the development of the systemic lupus erythematosus-like pathology of MRL lpr/lpr mice. Eur J Immunol. 26:1707-11. Nahata A, Dixit V K. 2012. Ameliorative effects of stinging nettle (Urtica dioica) on testosterone-induced prostatic hyperplasia in rats. Andrologia. 2012;44:396-409.
43 Nair H. K, Rao K. V. K., Aalinkeel R, Mahajan S, Chawda R, Schwartz S. A. 2004. Inhibition of prostate cancer cell colony formation by the flavonoid quercetin correlates with modulation of specific regulatory genes. Clin Diagn Lab Immunol 11:63-9. Nair M. P., Mahajan S., Reynolds J. L. 2006. The Flavonoid quercetin inhibits proinflammatory cytokine (Tumor Necrosis Factor Alpha) gene expression in normal peripheral blood mononuclear cells via modulation of the NF-κβ system. Clin Vaccine Immunol 13:319-28. Namazi N, Esfanjani A T, Asghari M. 2011. effect of hydroalcholic nettle (urtica dioica) extract on some cardiovascular risk factor in patients with type 2 diabetes. J Med Sci. 11:138-44. Namazi N, Tarighat A, Bahrami A. 2012. The effect of Hydro Alcholic Nettle ( Urtica Dioica) extract on oxidative stress in patients with type 2 diabetes: A randomized double-blind clinical trial. Pak J Biol Sci. 15:98-102. Nassiri-Asl M, Zamansoltani F, Abbasi E, Daneshi MM, Zangivand A. A. 2009. Effects of Urtica dioica extract on lipid profile in hypercholesterolemic rats. Zhongxiyi Jiehe Xuebao 7:428-33. Orcic D., Francišković M., Bekvalac K, Svirčev E, Beara I, Lesjak M. 2014. Quantitative determination of plant phenolics in Urtica dioica extracts by high- performance liquid chromatography coupled with tandem mass spectrometric detection. Food Chemistry. 143:48-53. Otles S, Yalcin B. 2012. Phenolic compounds analysis of root, stalk, and leaves of nettle. The Scientific World Journal. :1-12. Ozen T, Korkmaz H. 2003. Modulatory effect of Urtica dioica L. (Urticaceae) leaf extract on biotransformation enzyme systems, antioxidant enzymes, lactate dehydrogenase and lipid peroxidation in mice. Phytomedicine, 10:405-15. Ozen T, Korkmaz H. 2009. The effects of Urtica dioica L. leaf extract on aniline 4- hydroxylase in mice. Acta Pol Pharm. 66:305-9. Pourahmadi M, Jashni HK, Bagheri M, Jahromi AS. 2014. The effect of hydro-alcoholic extract of Urtica dioica root on testes in adult rats. Life Sci. J. 11:420-4.
44 Pourmorad F., Hosseinimehr S. J., Shahabimajd N. 2006. Antioxidant activity, phenol and flavonoid contents of some selected Iranian medicinal plants. Afr J. Biotechnol 5:1142-5. Pradhan S., Manivannan S., Tamang J. P. 2015. Proximate, mineral composition and antioxidant properties of some wild leafy vegetables. J Sci Ind Res 74:155-9. Qujeq D, Davary S, Moazzi Z. 2011. Effect of Urtica dioica leaf extract on activities of nucleoside diphosphate kinase and acetyl coenzyme, a carboxylase, in normal and hyperglycemic rats. Afr J Pharm Pharmacol. 5:792-6. Rafajlovska V, Kavrakovski Z, Siminovska J, Srbinoska M. 2013. Determination of protein and mineralcontents in stinging nettle. Quality Life 2013;4:26-30. Rafajlovska V, Najdenova V, Cvetkov L. 2001. Influence of some factors at chlorophyll extraction from stinging nettle (Urtica Dioica L.). Herba Pol 2001;47:304-14. Ramtin M., Massiha A., Khoshkholgh-Pahlaviani M. R., Issazadeh K., Assmar M., & Zarrabi S. 2012. In vitro antimicrobial activity of Iris pseudacorus and Urtica dioica, Zahedan Journal of Research in Medical Sciences, 16(4): 35-39. Rhoades, David F., 1979. Evolution of Plant Chemical Defense against Herbivores. In Rosenthal, Gerald A., and Janzen, Daniel H. Herbivores: Their Interaction with Secondary Plant Metabolites. New York: Academic Press. p. 41. Riehemann K, Behnke B, Schulze-Osthoff K. 1999. Plant extracts from stinging nettle (Urtica dioica), an antirheumatic remedy, inhibit the proinflammatory transcription factor NF- kappa B. FEBS Lett. 442:89-94. Roschek BJ, Fink RC, McMichael M, Alberte RS, Roschek BJ, Fink Ryan C. 2009. Nettle extract (Urtica dioica) affects key receptors and enzymes associated with allergic rhinitis. Phytoter Res 23:920-6. Rosłon W, Węglarz Z. 2003. Polyphenolic Acids of Female and Male Forms of Urtica dioica. Acta Hortic. 597. 597:101-4. Rutto LK, Xu Y, Ramirez E, Brandt M. 2013. Mineral properties and dietary value of raw and processed stinging nettle (Urtica dioica L.). Int J Food Sci. 2013:1-9. Safarinejad M. R. 2005. Urtica dioica for treatment of benign prostatic hyperplasia: a prospective, randomized, double-blind, placebo-controlled, crossover study. J Herb Pharmacother 5:1-11.
45 Said A. A. H., El Otmani, I. S., Derfoufi, S., Benmoussa, A. 2015. Highlights on nutritional and therapeutic value of stinging nettle (Urtica dioica). Int. J. of Pharmacy and Pharmaceutical Sciences. 7 (10): 0975-1491. Said O, Fulder S, Khalil K, et al 2008.. Maintaining a physiological blood glucose level with 'glucolevel', a combination of four anti-diabetes plants used in the traditional arab herbal medicine. Evid Based Complement Alternat Med. 5:421-8. Saul F. A., Rovira P., Boulot G., Damme E. J., Peumans W. J. 2000. Crystal structure of Urtica dioica agglutinin, a superantgen presented by MHC molecules of class I and class II. Structure, 8:593-603. Schneider T, Rübben H. 2004. Stinging nettle root extract in long term treatment of benign prostatic syndrome. Results of a randomized, souble-blind, placebo controlled multicenter study after 12 mo. Urologe A 43:302-6. Schöttner M., Gansser D., Spiteller G. 1997. Lignans from roots of Urtica dioica and their metabolites bind to human sex hormone binding globulin (SHBG). Planta Med 63:529-32. Sekeroglu N, Ozkutlu F, Deveci M, Dede O, Yilmaz N. 2006. Evaluation of some wild plants aspect of their nutritinal values used as vegetable in eastern black sea region of Turkey. Asian J Plant Sci. 5:185-9. Seliya M, Kothiyal P. 2014. Urtica dioica (stinging nettle): a review of its chemical, pharmacological, Toxicological and ethnomedical properties. Int J Pharm. 4:270-7. Selloum L, Bouriche H, Tigrine C, Boudoukha C. 2003. Anti-inflammatory effect of rutin on rat paw oedema, and on neutrophils chemotaxis and degranulation. Exp Toxicol Pathol 54:313-8. Semih, O., Buket, Y.2012. Phenolic Compounds Analysis of Root, Stalk and Leaves of Nettle. The Scientific World Journal . Article ID564367, 1-12. Shibuya N, Goldstein IJ, Shafer JA, Peumans WJ, Broekaert WF. 1986. Carbohydrates binding properties of the stinging nettle (Urtica dioica) rhizome lectin. Arch Biochem Biophys 249:215-24. Shin J. E., Kim J. M., Bae E. A., Hyun Y. J., Kim D. H. 2005. In vitro inhibitory effect of flavonoids on growth infection and vacuolation of Helicobacter pylori. Planta Med 71:197-201.
46 Singh R., Dar S. A., Sharma P. 2012. Antibacterial activity and toxicological evaluation of semi purified hexane extract of Urtica dioica leaves. Res J Med plant. 6:123-35. Sokeland J. 2000. Combined sabal and urtica extract compared with finasteride in men with benign prostatic hyperplasia: analysis of prostate volume and therapeutic outcome. BJU Int.86:439-42. Sorensen A. D., Durand E., Laguerre M. 2014. Antioxidant properties and efficacies of synthesized alkyl caffeates, ferulates, and coumarates. J Agric Food Chem 62:1253-6. Sultan J. I., Rahim I. U., Yaqoob M., Mustafa M. I., Nawaz H., Akhtar P. 2009. Nutritional evaluation of herbs as fodder source of ruminants. Pak J Bot. 41:2765-76. Tahri A, Yamani S, Legssyer A. 2000. Acute diuretic, natriuretic and hypotensive effects of a continuous perfusion of aqueous extract of Urtica dioica in the rat. J Ethnopharmacol 73:95-100. Tekin M, Özbek H, Him A.2009. Investigation of Acute Toxicity, Anti-inflammatory, and Analgesic Effect of Urtica dioica L. Pharmacologyonline. 1:1210-5. Testai L, Chericoni S, Calderone V. 2002. Cardiovascular effects of Urtica dioica L. (Urticaceae) roots extracts: in vitro and in vivo pharmacological studies. J Ethnopharmacol. 81:105-9. Tian X, Li F, Zhu L, Ye B. 2008. Study on the electrochemical behavior of anticancer herbal drug rutin and its interaction with DNA. J Electroanal Chem 621:1-6. Torres R, Faini F, Modak B, Urbina F, Labba C, Guerrero J. 2006. Antioxidant activity of coumarins and flavonols from the resinous exudate of Haplopappus multifolius. Phytochemistry 2006;67:984-7. Turel D, Oto G, Ayaz E. 2008. anthelmintic activity of urtica dioica l. in mice naturally infected with aspiculuris tetaptera. J anim vet adv 8:1628-30. Uncini Manganelli R. E., Zaccaro L., Tomei P. E. 2005. Antiviral activity in vitro of Urtica dioica L., Parietaria diffusa M. K. and Sambucus nigra L. J Ethnopharmacol 98:323- 7. Upton R. 2013. Stinging nettles leaf (Urtica dioica L.): Extraordinary vegetable medicine. J. of Herbal Medicine. 20133;:9-38.
47 Verma A. K., Johnson J. A., Gould M. N., Tanner M. A. 1988. Inhibition of 7,12- Dimethylbenz(a)anthracene-and N-Nitrosomethylurea-induced rat mammary cancer by dietary flavonol quercetin. Cancer Res 48:5754-8. Vontobel H. P. 1985. Ergebnisse einer doppelblindstudie über die wirksamkeit von ERU- Kapseln in der konservativen behandlung der benignen Prostatahyperplasie. Urologie 24:49-51. Wagner H, Geiger W N, Boos G. 1995. Studies on the binding of Urtica dioica agglutinin (UDA) and other lectins in an in vitro epidermal growth factor receptor test. Phytomedicine. Apr;1:287-90. Wagner H, Willer F, Samtleben R. 1994. Search for the antiprostatic principle of stinging nettle (Urtica dioica) roots. Phytomedicine. 1:213-24. Wagner H., Willer F., Kreher B. 1989. Biologically active compounds from the aqueous extract of Urtica dioica. Planta Med 1989;55:452-4. Wetherilt H. 1992. Evaluation of Urtica species as potential sources of important nutrients. Dev Food Sci 29:15-25. Yamunadevi, M., E.G.Wesely, Johnson M. 2011. Phytochemical studies on the terpenoids of medicinally important plant AervalanataL. using HPTLC. A.Pacific J. of Trop. Biomedicine. S220-S225. Yang J, Guo J, Yuan J. 2008. In vitro antioxidant properties of rutin. Food Sci Technol 41:1060-6. Yilmaz B, Basar Ö, Aktas B, Altinbas A. 2014. Effects of Urtica dioica extract on experimental acute pancreatitis model in rats. Int J Clin Exp Med 7:1313-8. Zekovic Z., Cvetanović A, Švarc-Gajić J, Gorjanović S, Sužnjević D, Mašković P. 2017. Chemical and biological screening of stinging nettle leaves extracts obtained by modern extraction techniques. Industrial Crops and Products. 108:423-430.
48 7. APPENDICIES
49 Appendix Table 1. Antibacteria Activity Based on Diameter of Zone of Inhiontion Test pathogens crude rep Concentration of the methanolic extract Ciprofloxacin extract 100mg/ml 150mg/ml 200mg/ml (100mg/ml) E.coli Leaf 1 10 12 14 18 E.coli Leaf 2 9.2 11.1 15 18.5 E.coli Leaf 3 10.5 12.5 15.5 18 E.coli Fruit 1 9 10 14 18.5 E.coli Fruit 2 8.1 11 14.5 19.6 E.coli Fruit 3 10 10.5 15.2 18.5 E.coli Root 1 11.2 12 14.8 18.5 E.coli Root 2 10.5 12.5 13 18 E.coli Root 3 11.5 13 15.5 18.5 S. aureus Leaf 1 11 13 15 18 S. aureus Leaf 2 10.5 12.5 15.6 18.5 S. aureus Leaf 3 11.5 14 16 18 S. aureus Fruit 1 9.5 11 14 18.5 S. aureus Fruit 2 10 11.5 13.5 19.6 S. aureus Fruit 3 10.4 12 15 18.5 S. aureus Root 1 12 13 16 18.5 S. aureus Root 2 11 13.5 17.5 18 S. aureus Root 3 11.5 13 17 18.5 S. typhi Leaf 1 10.5 13.5 16.2 18 S. typhi Leaf 2 10.1 13 16.5 18.5 S. typhi Leaf 3 10.5 12.5 15.4 18 S. typhi Fruit 1 10 11.2 15 18.5 S. typhi Fruit 2 9.5 11.2 14.5 19.6 S. typhi Fruit 3 10 10.5 15.2 18.5 S. typhi Root 1 12 13.5 16.2 18.5 S. typhi Root 2 10.5 12.5 16 18 S. typhi Root 3 11.5 13 17 18.5 S. pyogenes Leaf 1 10.5 13.5 15.8 18 S. pyogenes Leaf 2 9 12 15 18.5 S. pyogenes Leaf 3 10.5 12.5 15.5 18 S. pyogenes Fruit 1 10 11.2 15 18.5 S. pyogenes Fruit 2 9.5 10.8 14.5 19.6 S. pyogenes Fruit 3 9 10.5 14.2 18.5 S. pyogenes Root 1 11 12 16.2 18.5 S. pyogenes Root 2 10.5 12.5 16 18.1 S. pyogenes Root 3 10.1 13.2 15 18.5
50 Appendix Table 2. Data for Antifungal Activity Test pathogenes Ctude extract Rep Concentration of the methanolic extract Ketokonazole 100mg/ml 150mg/ml 200mg/ml (100mg/ml) A. versicolor Leaf 1 10 12 16 18.5 A. versicolor Leaf 2 11 13 16.5 17.5 A. versicolor Leaf 3 11.4 12.5 15.5 18 A. versicolor Fruit 1 9 10 13 18 A. versicolor Fruit 2 8 11 13.5 18.5 A. versicolor Fruit 3 10 11.5 14 19 A. versicolor Root 1 10 11.5 14 17.5 A. versicolor Root 2 10.5 12 15 18 A. versicolor Root 3 9 11.3 15.5 18.5 A. niger Leaf 1 10.5 11 15 15 A. niger Leaf 2 9 12 15.5 18.5 A. niger Leaf 3 10 11.55 14.5 18.5 A. niger Fruit 1 9 10 12 19 A. niger Fruit 2 8 11 13.5 18.5 A. niger Fruit 3 9.5 10.2 14 18 A. niger Root 1 9 11.5 14 18 A. niger Root 2 10.5 11 15 18.5 A. niger Root 3 9.5 11.3 14.5 19
51