Bull. Pharm. Sci., Assiut University, Vol. 43, Issue 2, 2020, pp. 141-147. Bulletin of Pharmaceutical Sciences ISO ISO Assiut University

Accreditated Faculty Administration Council of The National Authority for Website: http://bpsa.journals.ekb.eg/ Quality Assurance of Education and Accreditation No. (102) in 27/9/2011 9001 : 2015 and renewed in 19/7/2017 No. 168 9001 : 2008 e-mail: [email protected]

PHYTOCHEMICAL AND BIOLOGICAL STUDY OF CINERARIA L. CULTIVATED IN SYRIA

R. Joujeh*

Department of Biology, Damascus University, Damascus, Syria

In the current study, C. cineraria plant was subjected to chemical and biological study. The plant extract was screened for its phytochemical constituents, its quantitative content of phenolics, and its activity in scavenging free radical (DPPH). The plant was subjected to a biological study, which includes investigating the potential cytotoxic effect of the plant towards human erythrocytes membranes, and the ability of the plant to protect erythrocytes against oxidative hemolysis. The results indicated that the extract did not contain saponins. Our study also showed that the extract contains a good content of phenolics (41.12±0.6) mg GAE/g, and has good DPPH radical scavenging activity (79.96%±1.14). The results showed that the extract does not have a toxic effect on erythrocytes membranes, and it is highly effective in protecting the membranes from oxidative damage (IC50 value was 408.86 μg/ml). We hope that future studies will focus on the pharmaceutical aspects of this plant, and will not be considered as ornamental plant only.

INTRODUCTION - Taxonomical Position of Centaurea species7 have been a great resource of . Kingdom: Plantae medicinal products which are used for the . Phylum: Magnoliophyta (Flowering treatment of several ailments1. The Plants) is one of the largest families of flowering . Class: Magnoliopsida (Dicotyledons) plants2, with 1600–1700 genera and 24,000– . Subclass: Asteridae 30,000 species3. Plants of this family are . Superorder: Asteranae widely spread in the tropics and warm . Order: temperate regions of South, South-East and . Family: Asteraceae East-Asia, Africa and central South America4. . Subfamily: Carduoideae Centaurea L.is a large genus in the Asteraceae . Tribe: family, with over 500 species widespread . Subtribe: Centaureinae across the world. Some species are cultivated . Genus: Centaurea for spice, as ornamental plants, and in gardens. . Species: C. cineraria Centaurea species produce a vast array of secondary metabolites belonging to different In general, C. cineraria is known as classes of bioactive compounds5. The plant ornamental plant, and only very few studies Centaurea cineraria is perennial herb. Stems have gone into investigating its chemical up to 80 cm, erect, rarely procumbent, with few content and biological activities. The current branches above. Leaves more or less study includes a chemical investigation of C. tomentose, rarely glabrescent; lower lyrate to 2 cineraria plant to screen its phytochemical pinnatisect. Capitula solitary. Bracts broadly compounds, determine its quantitative content ovate; appendages usually dark brown, the of phenolic compounds, and test its activity in apex acuminate, not spinose; fimbriae 0.5-2 scavenging free radical (DPPH), in addition to mm. Pappus as long as achene, rarely absent 6. a biological study, which includes investigating ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received in 5/8/2020 & Accepted in 27/8/2020

*Corresponding author: Ruba Joujeh, E-mail: [email protected] R. Joujeh the potential cytotoxic effect of the plant in our previous study14). The total phenolic towards human erythrocytes membranes, and content was reported as mg of gallic acid the ability of the plant to protect healthy human equivalent (GAE) per gram of dry extract15. erythrocytes against H2O2 induced hemolysis. Free radical scavenging activity (DPPH METHODOLOGY method) The free radical scavenging activity of the Plant material AqME extract was determined according to C. cineraria seeds were purchased, and Rashed16, with modifications. First, DPPH then cultivated within strictly defined solution was prepared in ethanol with final environmental conditions. For this study, the concentration of 45 µg/ml. Then, 1 ml of each aerial parts of the cultivated plant were plant extract (0.005 g/ml) was added to Freshly collected, shade dried and powdered using prepared 3 ml of DPPH solution. After 30 min, mechanical grinder. the absorbance was measured at 517 nm. Control sample was prepared using distilled Preparation of plant extract water instead of the plant extract. The Aqueous methanolic extract (AqME) of C. percentage of inhibition was calculated by cineraria dried aerial parts was prepared comparing the absorbance values of the control according to Falleh et al. 8 with few and test samples using the equation: modifications. The powdered plant material (50 gm) was Free radical scavenging activity (%)  mixed with 250 ml of aqueous methanol 70% Ac  At  in a bottle. The extraction procedure was   100 A carried out using ultrasonic bath with  c  frequency of 15 kilohertz (kHz). The bottle was put in a sonic bath for 15 min. After that, the Ac is the absorbance of the control. extract was vacuum filtered, the filtrate was At is the absorbance of the test sample. collected and the residual was extracted three times using the same method. The total filtrate Hemolysis assay was evaporated by using rotary evaporator C. cineraria AqME extract was tested for under reduced pressure at 45°C, and stored in a its cytotoxic effects on normal human desiccator. Finally, the extraction yield was erythrocytes. Five milliliters of blood from calculated by the formula: fifteen healthy donors - after their approval - was collected in EDTA vials. Normal human Yield%  erythrocytes suspension (10%), and the concentrations of plant extract were prepared  weight of dry extract  in phosphate buffered saline (PBS) solution,  100  weight of dry powdered plant material  (pH 7.4). In sterile Eppendorf tubes, (1 ml) of each concentration (100-500-1000- 2000-4000)

µg/ml of the prepared extract was added to (1 Qualitative analysis of phytochemical ml) of erythrocytes suspension (isolated from components the blood samples of ten healthy donors). The C. cineraria aerial parts subjected to mixture was then incubated in a water bath at qualitative tests in order to characterize several 37°C for 30 min, then samples were chemical groups using standard procedures centrifuged (2500 rpm/min) for 10 mins, and (Phenols9, Flavonoids10, Tannins11, the amount of free hemoglobin was estimated Carbohydrates12, Saponins13). The symbols + by measuring the absorption of the supernatant and - denote present, and absent. using a Spectrophotometer at the wavelength

540 nm. Total phenolic content In order to compare the results, a negative Total phenolic content of the AqME control sample containing (1 ml) of PBS and (1 extract of C. cineraria was spectrophoto- ml) of erythrocytes suspension, and a positive metrically determined using Folin-Ciocalteu control sample containing (1 ml) of distilled method, with Gallic acid as standard (published

142 water and (1 ml) of erythrocytes suspension Statistical analysis were prepared17. Results were presented as average ± The percentage of hemolysis was standard deviation using Microsoft Excel 2010. calculated according to the following formula: Statistical Package for Social Science (SPSS) was used to perform the statistical tests. (OD  OD ) Hemolysis %  test con- 100 (OD  OD ) con con- RESULTS AND DISCUSSION

OD optical density of test sample. test Characteristics and yield of the plant extract OD optical density of negative control con- C. cineraria AqME extract was (PBS). characterized by a brown color, bitter taste and OD optical density of positive control con+ a pleasant smell. The extraction yield was (distilled water). 12.64%.

Assessment of anti-hemolytic activity Phytochemical screening (hemolysis induced by oxidative factor) The results of the qualitative chemical The anti-hemolytic activity of C. cineraria analysis showed that the extract contain AqME extract was determined by the bioactive compounds as carbohydrates, spectrophotometric procedure. Five milliliters phenols, tannins, flavonoids, while it was of blood from fifteen healthy donors -after their found that it does not contain saponins. approval- was collected in EDTA vials. (1 ml) The phytochemical analysis of plants is of each concentration of the extract (100-500- important and have commercial attention in 1000-2000-4000) µg/ml was mixed with (0.5 pharmaceuticals companies and research ml) of the erythrocytes suspension (10%) in institutions for the industrialization of the new sterile Eppendorf tubes. The mixture was 19 drugs . The chemical compounds that have incubated at laboratory temperature (5 min), been found in C. cineraria AqME extract have then (0.5 ml) of hydrogen peroxide solution important biological activities, which gives this (prepared using PBS) was added to induce species medical importance, in addition to its oxidative degradation of the membrane lipid use as an ornamental plant in different (haemolysis). The mixture was incubated for 4 countries of the world. No reference studies hrs at a temperature of 37°C. Then samples were found on the phytochemical analysis for were centrifuged at a speed of 2500 rpm for 10 this plant to discuss our results. Our study may min. be the first chemical and biological study of In order to compare the results, a negative this plant. control sample containing (1 ml) of a PBS solution and (0.5 ml) of the healthy erythrocyte Total phenolic content suspension, and (0.5 ml) of hydrogen peroxide Our results showed good phenolic content solution was prepared. in C. cineraria AqME extract with (41.12±0.6) Note: The hydrogen peroxide concentration mg GAE/g. was chosen to be able to cause hemolysis at It should be noted here that the phenolic approximately 95% in the negative control content in the plant species in general is sample. affected by the different environmental The percentage of hemolysis was conditions for each geographical area, in calculated by estimating the amount of addition to the different time of collecting the liberated hemoglobin using the plant samples, and the difference of the solvent spectrophotometer at the wavelength of 540 20 used in preparing the plant extracts . nm18. Based on this, the ability of plant extracts to protect erythrocyte from oxidative hemolysis Free radical scavenging activity was calculated according to the following Hydrogen donating ability of C. cineraria equations: extract was measured by using DPPH assay. OD of sample Hemolysis %  100 ...... (1) The results were expressed in percentage of OD of control inhibition of DPPH. The extract showed good ...... (2) Protection % 100  [hemolysis]

143 R. Joujeh

DPPH radical scavenging activity with extracts, have hemolytic properties21. When (79.96%±1.14). conducting a qualitative phytochemical When testing the free radical scavenging analysis of C. cineraria, it was found that the activity of C. cineraria extract. It showed good studied extract did not contain saponins, and DPPH radical scavenging activity. The ability the extract did not actually cause noticeable to scavenge free radicals is determined by a cytotoxic effects on erythrocytes’ membranes. 2.2-Diphenyl-1-picrylhydrazyl (DPPH) test, which is a free radical with a dark purple color Anti-hemolytic activity against oxidative in methanol and ethanol solutions. This test is hemolysis based on an evaluation of the ability of plant In this test, the extract's ability to reduce extracts to reduce DPPH, which is inferred by the damage caused by oxidative stress in the the conversion of purple color to yellow. erythrocyte membranes was evaluated. In general, the results indicated that the addition Hemolytic effect of the gradient concentrations of the C. Hemolytic assay experiment was designed cineraria extract resulted in the inhibition of to evaluate the effect of five different oxidative hemolysis, which was stimulated in- concentrations of C. cineraria extract on red vitro, and in a relationship proportional to the blood cells. The results showed that there were gradual increase in the concentrations used, as no toxic effects of the extract towards the shown in (table 2). A significant decrease in erythrocyte membranes, although there was an the percentages of erythrocyte hemolysis was increased direct relationship between the demonstrated at high concentrations (1000- concentrations used from the plant extracts and 4000) µg/ml, which indicates the effectiveness the measured absorbance values. The tested of the extract in inhibiting the action of the concentrations caused a slight hemolysis (less oxidizing agent (H2O2). The IC50 value was than 5% at the concentration 4000 µg/ml) and 408.86 μg/ml (Fig. 1). This result is consistent this percentage is very weak when compared with the result of the free radical scavenging with the corresponding high concentration test, which confirms the plant's activity in (table 1). scavenging DPPH. When comparing the results of the current study of the plant C. cineraria cultivated in Table 1: The percentages of hemolytic effect Syria with a previous study14, that we by C. cineraria extract. conducted on the plant C. iberica wild spread in Syria, it became clear to us the great Concentration Hemolysis % similarity in the chemical and biological )µg/ml) effectiveness of the two species, and this may 100 0.26 ± 0.4 support the classification of these two species within the same genus. 500 0.49 ± 0.4

1000 2.36* ± 0.6 * 2000 3.72 ± 1.2 Table 2: The percentages of hemolysis and 4000 4.84* ± 0.9 protection by C. cineraria extract against oxidative hemolysis. All values are represented as mean ± SD. (*) Significant differences at (p< 0.05). Concentration Hemolysis Protection IC50 (μg /ml) % % (μg/ml) 100 83.64 16.36±2.6 500 49.91 50.09±1.2 Before developing pharmaceutical agents 408.86 from natural sources, it is important to screen 1000 18.44 81.56±3.1 them for cytotoxicity, and hemolytic activity 2000 12.96 87.04±1.4 represents a good indicator of cytotoxicity 4000 5.54 94.46±1.5 toward healthy erythrocytes. Triterpene and steroid saponins, which are abundant in plant Protection values are represented as mean± SD.

144 et al., "Taxonomic studies on the family Asteraceae (Compositae) of the Rajshahi division", Res. J. Agric. & Biol. Sci., 4 (2), 134-140 (2008). 5- O. Politeo, M. Skocibusic, I. Carev, F. Burcul, I. Jerkovic, M. Sarolic and M. Milos, "Phytochemical profiles of volatile constituents from Centaurea ragusina leaves and flowers and their antimicrobial effects", Nat. Product Commun., 7 (8), 1087-1090 (2012). Fig 1: Anti-hemolytic activity of C. cineraria 6- J. Dostal, J. G. In Tutin, V. H. Heywood, extracts against oxidative hemolysis. D. M. Moore, D. H. Valentine, S. M. Walters and D. A. Webb, "Flora Europaea", Cambridge Univ. Press, 1976, Conclusion pp. 1-505. In the light of the results obtained, we 7- A. Takhtajan, "Flowering Plants", concluded that the aerial parts of C. cineraria, Springer Science & Business Media B.V, are very rich in phytochemical compounds, and 2nd Edn., 2009, p. 871. has very low toxicity against human 8- H. Falleh, R. Ksouri, M. E. Lucchessi, Ch. erythrocytes. The plant also has an antioxidant Abdelly and Ch. Magné, "Ultrasound- activity that is demonstrated by the ability of assisted extraction: Effect of extraction the extract to scavenge the free radical (DPPH), time and solvent power on the levels of and protect normal human erythrocytes from polyphenols and antioxidant activity of oxidative damage induced by H2O2. Mesembryanthemum edule L. Aizoaceae Shoots", Trop. J. Pharm. Res., 11 (2), Conflict of interest statement 243-249 (2012). No conflict of interest. 9- G. Sangeetha and R. Vidhya, "In-vitro anti-inflammatory activity of different Acknowledgements parts of Pedalium murex (L.)", Int. J. The author is grateful to Research Herb. Med., 4 (3), 31-36 (2016). Laboratories of Pharmacognosy Department- 10- P. Singh, R. Shrivastava, RC. Saxena, M. Pharmacy Faculty (University of Aleppo). The Sharma, M. S. Karchuli, et al., author thanks the researcher, Dima Joujeh for "Phytochemical screening and evaluation her assistance in revising the manuscript. of antioxidant activity of Parkinsonia aculeate L. (Family-Leguminoseae) leaves REFERENCES extract", Int. J. Pharmtech. Res., 3 (4), 1952-1957 (2011). 1- I. Suntar, "The medicinal value of 11- A. Pandey and S. Tripathi, "Concept of Asteraceae family plants in terms of standardization, extraction and pre wound healing activity", Fabad. J. phytochemical screening strategies for Pharm. Sci., 39, 21-31 (2014). herbal drug", J. Pharmacogn. 2- M. Tadesse, "How to study the Asteraceae Phytochem., 2, 115-119 (2014). (Compositae) with special reference to the 12- R. M. Prabhavathi, M. P. Prasad and M. Asteraceae of fee", Ethiop. J. Biol. Sci., Jayaramu, "Studies on qualitative and 13, 91-101 (2014). quantitative phytochemical analysis of 3- A. Moreira-Muñoz and M. Muñoz-Schick, Cissus quadrangularis". Advances in "Classification, diversity, and distribution Appl. Sci. Res., 7 (4), 11-17 (2016). of Chilean Asteraceae: Implications for 13- P. Tiwari, B. Kumar, M. Kaur, G. Kaur biogeography and conservation", Diversity and H. Kaur, "Phytochemical screening and Distribution, 13, 818-828 (2007). and extraction: A review", Int. Pharm. 4- A. H. M. Rahman, M. S. Alam, S. K. Sci., 1 (1), 98-106 (2011). Khan, F. Ahmed, A. K. M. Rafiul Islam,

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146 Bull. Pharm. Sci., Assiut University, Vol. 43, Issue 2, 2020, pp. 141-147.

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كلية معتمدة مجلس إدارة الهيئة القىمية لضمبن جىدة التعليم واإلعتمبد رقم (102) بتبريخ 27/9/2011 اإلعتمبد األول وتجديد شهبدة اإلعتمبد ببلمجلس رقم (168) بتبريخ 2017/7/19 جامعة أسيوط 2008 : 9001 2015 : 9001

Centaurea cineraria L.

قسم بيولوجيا النبات ، جامعة دمشق ، دمشق ، سوريا

Centaurea cineraria

DPPH

DPPH

147