Identification of Dandelion Taraxacum Officinale Leaves Components and Study Its Extracts Effect on Different Microorganisms

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Journal of Al-Nahrain University Vol.15 (3), September, 2012, pp.7-14 Science Identification of Dandelion Taraxacum officinale Leaves Components and Study Its Extracts Effect on Different Microorganisms

Abdul Kadir M. N. Jassim*, Safanah Ahmed Farhan** and Omar Mohammed Noori*** *Department of Chemistry, College of Science, Al-Mustansiriyah University, Baghdad-Iraq. **Polymer research center, College of Science, Al-Mustansiriyah University, Baghdad-Iraq. ***Industries of Chemical and Physical Materials, Ministry of Science and Technology.

Abstract Natural plant Dandelion Taraxacum officinale have been used as a phytomedicine, in this study, the chemical components of the Dandelion Taraxacum officinale leaves in watery and alcoholic extracts were identified. The results showed that watery extract was alkaline (presence of alkaloids) while the alcoholic extract was acidic in these contained: glycosides, alkaloids, phenolic compounds, tannins, flavonoids and proteins, while the saponins and resins were not found. The result also showed that high concentrations of the following trace elements were found in the leaves (K ,Ca , Na ,Fe) with 185.1, 22, 19.5, 11.2 ppm, respectively and low concentrations of (Zn, Cd, Cu) with 6.3, 1.3, 0.2 ppm, respectively. The effect of these extracts on the different microorganisms were studied. It has been found that the concentration 0.5 mg/ml was effective on the inhibition of the growth of the intended bacteria (for both extracts) especially Gram positive, Staphylococcus aureus and the alcoholic extracts with concentration 0.5 mg/ml was more effective on the Gram negative, E.coli, than the watery extract, while less than 0.1 mg/ml failed to inhibit any microorganisms. The High Performance Liquid Chromatography (HPLC) was used to identify some flavonoids as compared to standard one; the analysis showed that both kaempferol and morin were absent.

Keyword: Dandelion Taraxacum officinale, Plant extraction, Trace elements, HPLC.

Introduction at the same time as a cleanser of the blood and Herbs have been used for a large range of liver. An active substance of dandelion purposes .They are found to be potent sources reduces serum cholesterol and triglycerides of natural components .Some have been used because it intensifies bile secretion [1,6]. for hundreds of years, and their clinical and Dandelion improves the function of liver, pharmacological effects have been extensively pancreas and stomach. It is used to treat studied from various viewpoints [1].This anemia, cirrhosis of the liver, hepatitis and study focused on a used in folk medicine, rheumatism [1,7] anti-inflammatory, anti- Taraxacum officinale, commonly called oxidative, anti-carcinogenic, analgesic, anti- Dandelion, which is a herbaceous perennial hyperglycemic, anti-coagulatory and prebiotic plant of the family Asteraceae (Compositae). effects [8-13]. It can be found growing intemperate regions of The leaves can be eaten cooked or raw in the world, in lawns, on roadsides, on disturbed various forms, for assembling salads or soup, banks and shores of water ways and other which also recommended as a natural areas with moist soils. Dandelion is considered source of vitamin C in the early spring. a weedy species [2]. It has many english Dandelion water extract has anti-tumor common names including: Blowball, Lion's- activity attributed to polysaccharide [1].The tooth, Cankerwort, Swine's snout, …etc [3], most important biologically active components Arabic names :Hindiba, Khas berri [4], The are sesquiterpenic lactones, biotin, inositol genus name Taraxacum ,might be from the and vitamins B, D, E and phosphorous (P). Arabic word "Tharakhchakon" [2], or from the The leaves possesses a higher content of Greek word "Tarraxos" .The common name β-Carotene than carrot and more Fe and Ca "Dandelion" comes from the French phrase level than spinach, along with macro- and "Dent de lion" which means "Lion's tooth", in micro- elements [1,14]. references to the jagged shaped foliage [5]. Flavonoids and coumaric acid derivatives Dandelion serves mainly as a diureticum, and were identified from dandelion flower [7].Its

7 Abdul Kadir M. N. Jassim roots have been used to make a coffee like The pH of the extract was determined using drink and the plant had used by Native pH-meter (Orion,SA720).

Americans as a food and medicine [15]. b) Alcoholic extract: However, its use has mainly been Prepared as in water extract described based on empirical finding .This contribution above, but with using ethanol 70% (v/v) provides a comprehensive review of the instead of water to give alcoholic extract pharmacologically relevant compounds of powder [16-19]. The pH of this extract was Taraxacum officinale characterized so far and also determined. of the studies supporting its use as a medicinal [C] Determination of Ash content: plant. Dried leaves 2 g were taken and heated at Accordingly, the leaves of Taraxacum 900ºC for 20 min. in muffle furnace until the officinale has drawn the attention of researches material converted to white powder, after its and consumers and an understanding of the cooling the percentage of ash content was chemical and pharmacological properties determined [20]. should be of importance from scientific points [D]Chemical detection of the plant of view, therefore, this study is aimed at components: evaluating the ash contents, pH, leaves The chemical components of the prepared components, trace elements, effect of its watery and alcoholic extract were detected as extracts on different microorganisms and shown in Table (1). They included: glycosides, enzyme,checking of some the presence of alkaloids, saponins, phenolic compounds, flavonoids in these extracts. tannins, resins, flavonoids [16-18] and proteins Materials and Methods [21].

Protocol of Method [E] Determination of trace element levels: [A] Collection and treatment of samples: Dried leaves 3 g were taken and mixed The leaves of Taraxacum officinale were with 8 ml of concentrated nitric acid and 2 ml collected from the north of Baghdad, Iraq.The of 60% prechloric acid in a conical flask, the leaves were transported to the laboratory, mixture was kept for 24hrs covered with watch washed with water, cleaned with filter paper or glass. After that it was left for 6hrs at sand soft clothes to remove all traces of dust and bath at 80ºC, until the digestive material insects, then dried in shad 25-30ºC for one converted to white powder. Deionized water week, with continuous overturn to prevent 8 ml were added to this powder, and the mould. weighed, ground in a mortar and trace elements were determined [20] by pestle, placed in airtight bottles and stored in (Shimadzu AA-670, Flame Atomic Absorption dissicator to by used for extraction. Spectrophotometer).

[B] Preparation of extracts: [F] Inhibition of hyaluronidase: a) Watery extract: Hyaluronidase inhibition activity was Air dried leaves 50 g were suspended in 1 determined turbidimetrically by the method of liter of distilled water and left for 24 hrs at Kass et al [22] by using 0.01 mg/ml enzyme 35ºC with continuous stirring in shaking mixed with 250 µg/ml from the extracts with incubator. Then the mixture was filtered by inhibition time 45 min. and the percentage of filter paper. The filtrate was centrifuged for 10 inhibition %I was calculated according to this min. at 2500 rpm, and the extract was equation [23]: evaporated to dryness at 40ºC in the incubator.

Activity of control - Activity in the presence of Extract % Inhibition  100 Activityof control [G]The biological activity: isolates were obtained from department of The biological activity against various Biology/College of Science /Al-mustansiriyah bacterial species was determined. As gram University .The concentrations used for each negative bacteria, Proteus mirabilis and E.cole extracts were 0.1, 0.5 and 1 mg/ml [18, 19]. were used, while Staphylococcus aureus was used as gram positive bacteria. These 8 Journal of Al-Nahrain University Vol.15 (3), September, 2012, pp.7-14 Science

[H] HPLC Analysis: Results and Discussion Analysis of flavonoids, Kaempferol and Our results showd that pH value for watery Morin, were carried out in the chemical and alcoholic extracts are pH =8.15 and 4.81, research center, ministry of science and respectively (This may reflect the presence technology, Baghdad, Iraq, by using of alkaloids in the watery extracts). The (Shimadzu, LC2010A, Japan) HPLC. Standard Ash content for the Taraxacum officinale solution were prepared (standard Kaempferol leaves is (12.8%). The qualitative chemical and Morin were gift supplied from analysis of the watery and alcoholic extracts Dr.Mohammed Mustafa Radi). are represented on Table 1,which show that A luna 5U C-18Column(250mm x 4.6 x leaves contents are (glycosides, alkaloids, 5µm)Was used and coupled with 20µl of phenolic compounds, tannins, flavonoids and sample at 40ºC with a linear gradient mobile proteins) in watery extract and the absence of phase containing solvent A (water), solvent B alkaloids in the alcoholic extract. Other studie (acetonitrile) and solvent C (5% formic acid show that the major phenolic fraction in in water, v/v) with flow rate set at 0.2ml/min. dandelion is anthocyanins, a type of flavonoids The linear gradient program started with [1,14], phenolic acid [25] and poly phenolic 88% A:10% B:2% C (v/v) and finished at 73% compounds [26], these phenolic compound A:25% B:2% C (v/v) with retention time are an ecologically significant class of 10min. The chromatograms were recorded at secondary metabolites expressed in all higher 280nm. Standard and leaves extracts samples plants with high chemopreventive potential were made at 25ppm then analyzed directly by [27]. The saponins and resins are not found in HPLC [24]. both extracts.

Table (1) Chemical components analysis (qualitative methods) for watery and alcoholic extracts of Taraxacum officinale leaves. Result Result Components Reagents Note Watery Alcoholic extract extract Iodine test Brown ppt. Ve+ Ve- Glycosides Molish test Violet ring Ve+ Ve+ Benedict test Orange ppt. Ve+ +Ve Mayer's reagent No white ppt. Ve- Ve- Alkaloids Wagner reagent Brown ppt. Yellow Ve- Ve- Picric acid ppt. Ve- Ve- No dense foam for Fast stirring Ve - Ve- Saponins long time Mercuric Chloride Ve - Ve- No White ppt. Phenolic Aqueous%1 Green ppt. Ve+ Ve+ compounds Ferric chloride Aqueous%1 Green ppt. Ve+ Ve+ Tannins Ferric chloride Preface yellow ppt. Ve+ Ve+ Lead acetate%1 Ethanol + Boiling + Resins No turbidity Ve- Ve- Distilled Water aqueous%1 Ferric chloride Green ppt. Ve+ Ve+ Flavonoids Ethanol hydroxide Yellow ppt. Ve+ Ve+ alcohol Proteins Folin-Ciocalteau reagent Blue color Ve+ Ve+

9 Abdul Kadir M. N. Jassim The concentration trace elements in This work shows, the two extracts were Taraxacum officinale leaves are represented in examined for their effects on hyaluronidase. Table (2) which shows, high concentrations The percentage of inhibition for each extract of (K, Ca, Na, Fe) with (185.1, 22, 19.5, 11.2) was 1.3% with respect to control assays run ppm, respectively, and low concentrations simultaneously. Kuppusamy, et al. show that of (Zn, Cd, Cu) with (6.3, 1.3, 0.2) ppm, morin and kaempferol, types of flavonoids, respectively, other reports indicated the having potent inhibitory effect on this enzyme presence of these metal in this plant by using [30] with 56%, 31%, respectively. Absence of different method [28,29], while (Cr, pb) were these two flavonoids, as shown latter by not found. The high concentration of HPLC, may cause the lower inhibitory effects potassium cause the leaf acts as a diuretic; this of these extracts on hyaluronidase. spares the human body's potassium, which The effect of these extracts on different tends to be excreted with diuretic use. The microorganisms were studied and compared. effect is more therapeutic and beneficial However, the results in Table (3), show that overall. The young leaves that come up in the the concentrations 0.5 and 1 mg/ml exhibit spring may also be add to spring salads for this effective inhibition towards the growth of benefit [14]. Regardless of its intended use, the intended bacteria for both extracts specially on high mineral content of this herb greatly Gram positive, Staphylocoous aureu, while increases the chance of drug-drug interactions less inhibition effects were seen for Gram with the conventional medicines that are negative, Proteus mirabilis and E.coli. sensitive to cations [13]. Alcoholic extract with concentrations

Table (2) 0.5 mg/ml was more effective inhibitor for the The concentration trace elements content of Gram negative, E.coli than that for the Taraxacum officinale leaves. watery extract; and the concentration 0.1 mg/ml of this extract failed to inhibit any Concentration Trace elements symbol microorganisms. (ppm) Potassium K 185.1 Calcium Ca 22 Sodium Na 19.5

Iron Fe 11.2 Zinc Zn 6.3 Cadmium Cd 1.3 Cupper Cu 0.2 Chrome Cr Nil Lead Pb Nil

Table (3) The effect of watery and alcoholic extracts of Taraxacum officinale represented by inhibition zone (mm) against different bacteria species.

Bacterial species Alcoholic extract (mg/ml) Watery extract (mg/ml)

0.1 0.5 1 0.1 0.5 1 Staphylococcus - ++ ++ - ++ ++ aureus (gram + ve) Proteus mirabilis + + + + (gram - ve) - - E.coli + + + (gram - ve) - - -

(-) No inhibition zone (+) Inhibition zone between (1-4) mm .

(++) Inhibition zone between (4-10) mm . 01 Journal of Al-Nahrain University Vol.15 (3), September, 2012, pp.7-14 Science This inhibitory effects may be duo to the The HPLC chromatogram in Fig.(1), (glycosides and/or phenolic compounds and/or shows the standard Morin (A), standard tannins and/or flavonoids and/or alkaloids Kaempferol (B) and leaves extract sample (C). and/or proteins) presence in the plant extracts. Identification of these flavonoids in leaves Such compounds had been reported to have an extract sample was checked according to active effect on the bacterial cells membrane, retention time obtained from standard run at which may caused destroy these identical conditions. microorganisms [7, 18]. The alkaloids interact The major peaks for Morin standard had with the DNA, the tannins inhibit the carrier retention time was 2.35min (Fig.(1, A)), while enzymes and proteins present in the cells for that of Kaempferol was 3.3 and 3.56 min. membrane, while the phenolic compounds (Fig.(1, B)). As it is clear from the form complex with dissolved protein out of the chromatogram. These two flavonoids were cells or with cells membrane which made to absent in the leaves of Taraxacum officinale. destroy the bacteria [18, 22].

HO 1 O OH HO 7 B A C 3 OH 5 HO O

(A) Standard Morin

OH 1 B O HO 7 A C

5 3 OH HO O (B) Standard Kaempferol

Fig.(1) HPLC chromatogram of (A) standard Morin, (B) standard Kaempferol, (C) leaves watery extract for Taraxacum officinale leaves. Using luna 5U C-18Column(250mm x 4.6 x 5µm) the mobile phase at 40 ºC containing solvent A (water) ,solvent B (acetonitrile) and solvent C (5% formic acid in water ,v/v) with flow rate set at 0.2ml/min. The absorbance at (280nm).

00 Abdul Kadir M. N. Jassim

Conclusion [8] chutz, k.; Carle, R. and Schieber, A., The present study confirm that plant leaves "Taraxacum–A review on Its phytochemical extracts (watery and alcoholic) for Dendelion, and Pharmacological Profile, Journal of Taraxacum officinale leaves posses in vitro Ethnopharmacology", 107,3, 2006, 313-323. antibacterial activity because of its content, [9] Shi, S.; Zhao, Y.; Zhang, Y.; Huang, K. glycosides, alkaloids, phenolic compounds, and Liu, S., "Phenylpropanoids From tannins, flavonoids, proteins and trace Taraxacum mongolicum", Biochemcal elements, however, if plant leaves extracts are Systematics and Ecology, 36, 2008, 716- to be used for food preservation or medical 718. purposes, issues of safety and toxicity will [10] Shi, S.; Zhao, Y.; Zhao, H.; Zhang, Y.; need to be addressed, and this results will Jiang, X. and Huang, K.,". Identification serve as a pilot experiment for further research Antioxidants From Taraxacum mongolicum and improvement strategies of this important by High-performance Liquid Chromato plant . raphy – Diode Array Detection – Radical -

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01 Journal of Al-Nahrain University Vol.15 (3), September, 2012, pp.7-14 Science

Activity of Mentha Longifolia Leaves [27] Sueriro, L.; Yousef, G.G.; Seigler, D.; Composites", Iraqi Journal of Chemistry, DeMeija, E.G.; Grace, M.H. and Lila, 27, 2001, 1. M. A., "Chemopreventive Potential of [18] Jassim, A.M.N., "Study of Some Flavonoid Extracts from Plantation-Bred Eucalptus Rostrata Leaves Components and Wild Aronia melanocarpa (Block and Effect of Its Extract on Different Chokeberry) Fruits", J. Food Sci., 71, 8, Microorganisms", Al-Mustansirya J. Sci., 2006, C 480-488. 16, 2, 2005,62-71. [28] Ertas, Ö.S.; Aktas H.F. and Hazedaroglu, [19] Mohammed, M.T.(2007). Study of Some M.Z., "Analysis of Sodium and Potassium Vinca Rosea l. (Apocynaceae) Leaves Levels in Taraxacum officinale by Flame Components and Effect of Its Extract on Emission Photometry", Acta Pharm Different Microorganisms, Al-Mustansirya Turcica, 47, 2005, 127-130. J.Sci.,18,1, 28-36 . [29] Królak, E. ,"Accumulation of Zn, Cu, Pb and Cd by Dandelion (Taraxacum ]02[ الدليمي، نصر حامد، دراسة مظيريو و فسمجيو وخموية officinale WEB.) in Environment with Various Degrees of Metallic كمؤشر عمى آلية تحمل نبات الحنطة لجفاف، أطروحة Contamination", Pol J Environ. Studies, ماجستير، 7991. 12, 2003, 713-721. [21] Plummer, D.T, "An Introduction of [30] Kuppusamy, U.R.; Khoo, H.E. and Practical Biochemistry", 2ed, McGRAw- Das, N.P., "Structure–Activity Studies HILL Book Co., England, 1978, pp.145- of Flavonoids as Inhibitors of 146. Hyaluronidase", Biochem. Pharm., 40, [22] Kass, E. and Seastone, C. ,"The Role of 1990, 397-401. the Mucoid Polysaccharide (Hyaluronic Acid) In the Virulence of Group A الخالصة ,Hemolytic Streptococci", J.Exp. Med., 79 1944, 319. شممت الد ارسة معرفة المكونات الكيميائية الف ّعالة [23] Jassim, A. M. N., "Inhibition Effects Dandelion الموجودة في أوراق نبات اليندباء ,of Flavylium Salt on (Serum Taraxacum officinale، حيث أظيرت الدراسة أن ,"Testicular, Bacterial) Hyaluronidase Al-Mustansiriya J.Sci., 15, 4, 2004,32-43. محمول المستخمص المائي ذا طبيعة قاعدية الحتوائو عمى [24] Hu, C. and Kitts, D. D., "Dandelion القمويدات بينما كان محمول المستخمص الكحولي ذا طبيعة Taraxacum Officinale) Flower Extract) Suppresses Both Reactive Oxygen Species حامضية، وتحتوي ىذه المستخمصات عمى مجموعة من and Nitric Oxide and Prevents Lipid المركبات الكاليكوسيدية، القمويدات، المركبات الفينولية، ,Oxidation In Vitro", Phytomedicine, 12 2005, 588-597. العفصيات، الفالفونويدات والبروتينات في حين ال تحتوي [25] Schütz,K. ;Kammerer, D.R.; Carle, R. and عمى الصابونيات والراتنجات. Schieber, A., "Characterization of Phenolic كما اثبت التحميل الدقيق لمعناصر المعدنية احتواء Acid and Flavonoids in Dandelion (Taraxacum officinale WEB.ex WIGG.) Fe Na Ca K االوراق عمى تراكيز عالية من ، ، ، وىي Root and Herb by High Performance ppm 99.2,91.1 ، 22، 9.1.9، عمى التوالي وكميات Liquid Chromatography/ Electrospray Ionization Mass Spectrometry", Rapid اقل من Cu , Cd ,Zn وىيppm 2.2,9.2 ، 6.6، عمى Commun Mass Spectrom., 19, 2005, 179- التوالي. كما تم دراسة تأثير المستخمصات المائية والكحولية .186 عمى أنواع مختمفة من األحياء الدقيقة حيث لوحظ أن لمتركيز Wolbis, M.; Krolikowska, M. and [26] Bednarek, P., "Polyphenolic Compounds 2,1 ممغم \مل تأثي ار فعاال تجاه تثبيط نمو البكتريا المستخدمة ّ ,.in Taraxacum officinale", Acta Pol Pharm ولكال المستخمصين خاصة تجاه البكتريا الموجبة الغرام .153-158 ,1993 ,50 Staphylococcus aureus، في حين كان لمتركيز 2,1

02 Abdul Kadir M. N. Jassim

ممغم \مل لممستخمص الكحولي فعالية تثبيطية اكبر تجاه البكتريا السالبة الغرام E.coli من المستخمص المائي، كما لوحظ أن التركيز األقل من 2,9 ممغم \مل ليس ليا تأثيرا تثبيطيا ولكال المستخمصين عمى البكتريا المدروسة. كما تم استخدام كروماتوغرافيا السائل عالي األداء HPLC لتشخيص المركبات الفالفونويدية ومقارنتيا مع المركبات القياسية، إذ اثبت التحميل عدم احتواء أوراق النبات عمى مركبي الكمبفيرول و المورين.