American-Eurasian J. Agric. & Environ. Sci., 15 (9): 1753-1759, 2015 ISSN 1818-6769 © IDOSI Publications, 2015 DOI: 10.5829/idosi.aejaes.2015.15.9.9514

Phytochemical Screening, Antioxidant, Total Phenolic Contents, Cytotoxic and Phytotoxic Potential of the Methanolic Extract of macrophylla Wall Leaves

1Syed Muhammad Mukarram Shah, 23 Syed Muhammad Hassan Shah, Muhammad Ali, 4Syed Muhammad Saifur Shah, 22 Frahat Ali khan and Naseem Ullah

1Department of Pharmacy, University of Malakand, Chakdara, Dir, 2Department of Pharmacy, Sarhad University of Science and Information Technology, KPK, Pakistan 3Department of Biotechnology, Bacha Khan University, Charsadda, KPK, Pakistan 4Hayatabad Medical Complex, Peshawar, KPK, Pakistan

Abstract: Phytochemical screening of the methanolic extract of Cornus macrophylla leaves (MECML) revealed various classes of phytochemicals. MECM demonstrated a concentration dependent antioxidant activity, in DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging assay. Maximum activity (69.08%) was observed at

100 µg/ml, with IC50 , 57.87 µg/ml. Total Phenolic contents (TPC) were determined by Folin-Ciocalteu reagent (FCR) assay and was found 97.3±2.0 mg of gallic acid equivalent per gram of dry weight of the material. MECML exhibited profound cytotoxic potential (91%, 1000 µg/ml) against brine shrimp’s nauplii and was

directly proportional to the concentration (LC50, 200 µg/ml). Similarly MECML displayed the phytotoxic activity

in a dose dependent manner (LD50 , 24 µg/ml) and has shown remarkable phytotoxic potential, 95%±0.8 against Lamina minor at 1000 µg/ml. Thus on the basis of our findings one can conclude that the methanolic extract of C. macrophylla leaves is a rich source of antioxidants and natural herbicides.

Key words: Cornus macrophylla Phytochemicals Antioxidant TPC Cytotoxic Phytotoxic

INTRODUCTION pathogen [6]. Thus it is important to characterize different types of medicinal for their antioxidant and Medicinal plants have been used in different antimicrobial potential. cultures of the world as remedies for human diseases. Phytochemicals are naturally occurring compounds Plants produce a diverse range of bioactive synthesized by medicinal plants. Flavonoids are a group molecules, making them a rich source of different of polyphenolic compounds with known properties, which types of medicines [1-2]. Higher plants, as sources of include free radical scavenging, inhibition of hydrolytic medicinal compounds, have continued to play a dominant oxidative enzymes and anti-inflammatory action [7]. role in the maintenance of human health since ancient Besides, phenolic compounds, flavonoids are also widely times [3]. distributed in plants which have been reported to exert According to the world Health care Organization multiple biological effects, including antioxidant, free (WHO) up to 80% of the population in Africa depends on radical scavenging abilities, anti- inflammatory and anti- traditional herbal medicine for primary health care [4]. carcinogenic etc [8]. Medicinal plants exhibits various pharmacological, such Cornus macrophylla, Wall is a key member of as cytotoxic, phytotoxic and insecticidal activities [5]. family. This is a deciduous tree, which Numerous studies have shown that aromatic medicinal normally grow up to 15 meter height. The fruits of C. plants are sources of different nutrient and non nutrient macrophylla are the active ingredient of some of the molecules, many of which display antioxidant and traditional medicines used for the improvement of liver antimicrobial properties which can protect the human and kidney function [9]. Furthermore it is also used for the body against both cellular oxidation reaction and treatment of infections, inflammation and cancer.

Corresponding Author: S.M. Mukarram Shah, Department of Pharmacy, University of Malakand, Chakdara Dir, KPK, Pakistan. Cell: +92333-9256603. 1753 Am-Euras. J. Agric. & Environ. Sci., 15 (9): 1753-1759, 2015

In our previous research work we explored the Anthraquinonnes: Half gram of the extracts was boiled antioxidant and phytotoxic activities in the methanolic with 2 ml HCl (10%) for few minutes in a water bath. It was extracts of the bark of C. macrophylla. Thus looking to filtered and allowed to cool. After cooling, 2 ml of CHCl3 the previous result we designed the current study to was added to the filtrate. Then 2-4 drops of NH3 (10%) determine the Phytochemicals, total phenolic contents, were added to the mixture and heat. Formation of antioxidant, cytotoxic and phytotoxic potential of the rose-pink colour shows the presence of Anthraquinone methanolic extracts of the leaves of the same plant. [10].

MATERIAL AND METHODS Glycosides: The extract was hydrolyzed by adding 5 ml HCl solution and neutralized by adding 5 ml NaOH Collection of Plant Material and Identification: The fresh solution. After this a few drops of Fehling’s solution A leaves of Cornus macrophylla were collected from the and B were added to the mixture and observed the sample Hazarnau hill of Kot Manzary Baba, District, Malakand for red precipitates [11]. agency Khyber Pakhtunkhwa (KPK) Pakistan during June 2014. The plant were identified by Professor Dr. Nasrullah Reducing Sugars: The extracts (5 g) was shaken with in the Department of Botany, University of Malakand, distilled water and filtered. The filtrate was boiled with few Khyber Pakhtunkhwa, Pakistan. Voucher specimen was drops of Fehling’s solution A and B for few minutes. deposited to the herbarium of the same Department. Then formation of orange red precipitates will confirm the occurrence of reducing sugars. Extraction: After collection, the plants leaves were washed with water, shad dried under room temperature for Saponins: About 0.2 g of the sample (Extract) was shaken 10 days. The dried samples were then grounded into a with 5 ml of distilled water and heated to boil. Then coarse powder with electric blender and stored in observe the sample for the froth formation (Appearance polythene bag at room temperature. A portion of the of creamy miss of small bubbles) [11]. samples was used for detection of different classes of chemical constituents and the remaining plant material Flavonoids: Sample (0.2 g) was dissolved in 2 ml diluted was soaked in methanol (80%) for 15 days. After 15 days, (2%) NaOH and make a solution (a) having yellow colour. the extracts were filtered using Whatman’s No; 1 filter After this 2% HCl was added to solution (a) and observe paper. The extract was concentrated to dryness under the sample for discoloration. reduced pressure at temperature of (45°C), using rotary evaporator. Phlobutanins: Half gram was dissolved in distilled water and filtered. The filtrate was boiled with 2% HCl solution. Phytochemical Screening: Chemical tests were carried After heating the sample were observed for red precipitate out on crude methanolic extract and powder material of [12]. Cornus macrophylla leaves using standard procedures to identify the constituents as described by Sofowora, Terpenoids (Salkowski test): The extract (0.2 g) was

Trease and Evans and Harborne [10-12]. mixed with 2 ml of chloroform (CHCl3 ) and 3 ml

concentrated H24 S0 was carefully added to form a layer. Alkaloids: About 0.2 g of the extracts was warmed Then observe a reddish brown colouration of in the with 2% H24 S0 for two minutes on a boiling water bath. interface to indicate positive results. The mixture was then cooled, filtered and treated with the Dragendorff’s reagent. Orange red precipitate indicates Antioxidant Activities the presence of alkaloids. DPPH Radical-scavenging Activity: The hydrogen atom or electron donation abilities of the corresponding Tannins: The extract (2 g) was mixed with 1 ml distilled methanolic extract and standards were measured from the water and heated on water bath. The mixture was filtered change of the dark purple-coloured methanol solution of and 1-3 drops of ferric chloride solution was added to the DPPH (1, 1- Diphenyl-2-picryl hydrazyl radical). sample. Then observe the sample for a dark green Experiments were performed according to the method of coloration, which indicates the presence of tannins [10]. Yildirim and Blois [13-14] with a slight modification.

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Methanolic solution (1 mM) of DPPH free radical was triplicate in separate containers. Solvent (Methanol) was prepared and 1ml of this solution was mixed with 3 ml of evaporated and 30 brine shrimps nauplii were transferred sample solutions (Containing 20-100 µg) and control into each container using a Pasteur pipette. Five milliliters (Without sample). All the samples were incubated for half of sea water (Sea salt 38 g/l of distilled water, pH 7.4) was an hour and then the absorbance was taken at 517 nm. added into each container. One container survived as The lowering of absorbance of DPPH solution indicates negative control, 5 ml sea water and 30 shrimps, while an increase of the DPPH radical-scavenging activity. etoposied served as positive control. All the containers Scavenging of free radicals by DPPH as percent radical were incubated at room temperature under ordinary light scavenging activities (%RSA) was calculated as follow. and the numbers of survived and dead brine shrimps were counted after 24 h. Percent lethality was calculated by control absorbance - applying the following formula; sample absorbance %RSA = x 100 Control absorbance

The assays were carried out in triplicate and the results were expressed as mean values±standard Phytotoxic Activity of Cornus Macrophylla Leaves: deviations. The extract concentration showing 50% Phytotoxicity assay was carried out for the crude methanolic extract and subsequent solvent dilution inhibition (EC50 ) was calculated from the graph of% RSA against extract concentration with quercetin and gallic against Lemna minor. The medium was prepared and acid used as standards. sterilized in autoclave at 121°C and 15 Psi for 15 min. Test samples (10 mg) of Cornus macrophylla leaves Determination of Total Phenolic Compounds: Antioxidant dissolved in methanol (1ml) served as stock solution. compounds generally contains phenolic group. This Nine flasks (Three for each dilution) were inoculated with assay was carried out as described by Singleton et al [15], 1000, 100 and 10 µl of the stock solution (500, 50 and with slight modification. Briefly, 1ml of each of the already 5ppm). The solvent was evaporated overnight under prepared extract solution (500 µg) was transferred to 50-ml sterilized conditions and add 20 ml of the medium to each volumetric flask then volume was adjusted to 45 ml by flask. Thereafter, 10 plants were added to each flask. One addition of distilled water. Afterward, 1ml of Folin- other flask, supplemented with solvent served as control Ciocalteu Reagent (FCR) was added into this mixture and and reference plant growth inhibitor (Paraquat), served as a standard phytotoxic drug. The flasks were plugged with after three minutes 03 ml of Na23 CO (2%) was added. th Subsequently, the mixture was shaken for 2 h at room cotton and placed in growth cabinet for 7 days. On the 7 temperature and then absorbance was measured at 760 day, the number of fronds per flask was counted. Results nm. Estimation of the Phenolic compounds was carried were analyzed as growth regulation in percentage, out in triplicate. The results obtained were expressed as calculated with reference to the negative control. mg of gallic acid equivalents µg/mg of extract (GAEs) by using the following equation; RESULTS

A =1156 C + 0189 Phytochemicals: The Phytochemical screening of crude methanolic extracts of Cornus macrophylla leaves where “A” is the absorbance and “C” is the gallic acid revealed the presence of 07 different classes of equivalent (mg/g). phytochemicals, while glycosides and phlobutanins were not identified as shown in Table 1. Brine Shrimps Lethality Bioassay: Brain shrimps toxicological assay is a standard protocol for Antioxidant Potential and Total Phenolic Contents: determination of preliminary cytotoxic effect of the test The methanolic extracts of C. macrophylla leaves have samples. Cytotoxic activity was conducted according to shown a concentration dependent antioxidant potential in the standard procedure with slight modification [16]. DPPH radical scavenging assay. Maximum (69.08%) Stock solution of MECML was prepared by dissolving 20 activity was observed at 100 µg/ml, while 58.33, 50.44, mg of each sample in 2 ml of methanol. Then, 10, 50, 100, 45.82 and 43.99% activity were observed at 80, 60, 40 and 200, 250, 500 and 1000 ppm test doses were prepared in 20 µg/ml respectively, depicted in Table 2. The activity

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80

70 69.08

60 58.33 50 50.44 45.82 43.49 Cornus macrophylla leaves 40

30

% DPPH RSA 20

10

0 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Concentration (µg/ml), IC50 : 57.87 µg/ml

Fig. 1: %RSA graph of Cornus macrophylla leaves for determination of IC50 value

Table 1: Phytochemical screening of crude methanolic extracts of Cornus macrophylla leaves S. No Phytochemicals Reagents/Chemicals Results Observations 1 Alkaloids Dragendorff’s + Orange red precipitation was found 2 Tannins Ferric chloride + Dark green colouration

3 Anthraquinonnes HCl+CHCl33 +NH + Rose pink colour 4 Glycosides Fehling's solution - Red precipitate was not observed 5 Reducing sugars Fehling's solution + Orange red precipitation 6 Saponins Distilled water + Frothing 7 Flavonoids NaOH + HCl + Discoloration 8 Phlobutanins HCL _ Red precipitate not found

9 Terpenoids CHCl324 +H SO + Dark green colouration Key + = Present - = Absent

Table 2: Comparison of%RSA of methanolic extract of Cornus macrophylla leaves (MECM) with Quercetin and Gallic acid Concentration (µg/ml) Test Solutions ------Sample/Standards 20 40 60 80 100 MECM 43.9±1.4 45.8±1.6 50.4±0.9 58.3±2.1 69.0±1.7 Quercetin 95.3±1.5 96.3±1.1 97.1±0.6 98.0±0.8 98.2±0.6 Gallic acid 85.4±1.3 92.4±1.1 95.1±0.7 96.2±0.8 98.0±0.4 Results are expressed as mean±SD, of three independent observations.

was compared with reference standard drugs, quercetin 200, 250, 500 and 1000 µg/ml respectively with LC50 200 and gallic acid. Quercetin and gallic acid both displayed µg/ml. Etoposide was used as standard drug and showed marked activity (95.35 and 85.49%) at lowest profound cytotoxic effect (LC50 , < 0.1 µg/ml), depicted in concentration (20 µg/ml). The IC50 value determined for Table 3. MECM was 57.87 µg/ml (Figure 1). Total Phenolic Contents (TPC) determined in the crude methanolic extract Phytotoxic Potential: In the phytotoxic assay of the of Cornus macrophylla leaves was 97.3±2.06 mg of gallic crude methanolic extract and subsequent dilutions of acid equivalent per gram of dry weight of the plant Cornus macrophylla leaves displayed a material. concentration dependent activity. The test sample at a concentration of 10 µg/ml has shown good activity (45%), Brine Shrimps Lethality Bioassay: In cytotoxicity assay excellent activity (65%) at 100 µg/ml, while profound MECML displayed remarkable percent lethality against activity (95%) was observed at 1000 µg/ml (Table 4). The brine shrimp’s nauplii in a concentration dependent reference standard drug Paraquat exhibited excellent manner, as shown in Table 3. The test sample exhibited 11, activity at comparatively low dose (LD50 , 0.90 ìg/ml), 22, 36, 51, 57, 66 and 91% lethality at a dose of 10, 50, 100, depicted in Table 4.

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Table 3: Concentration dependent cytotoxic effect of CMMEL against Brine shrimp’s nauplii

Concentration of extract (µg/ml) No. of Brine shrimps Tested No. of dead Brine shrimp % Lethality LC50 (µg/ml) 10 30 3.3±0.8 11.1 200 50 30 6.6±1.2 22.2 100 30 11.0±1.7 36.6 200 30 15.3±0.8 51.1 250 30 17±1.4 57.7 500 30 20±1.0 66.6 1000 30 27.3±0.8 91.1

Data is represented as mean±SEM of the mean of three replicates of each dose (n=30). Etoposide LC50 = 9.8 ìg/ml.

Table 4: Concentration dependent phytotoxic potential of Cornus macrophylla leaves No of Frounds ------

Plant name Conc. (µg/ml) Test Samples Control % Growth Inhibition LD50 (µg/ml) Lemna minor 10 11±0.8 20 45±1.1 24 100 07±1.7 65±1.9 1000 01±1.8 95±0.8

Conc; concentration. Values are expressed as the mean±SD of three independent observations. Standard drug; Paraquat, LD50 = 0.90 ìg/ml.

DISCUSSION can be quantitatively measured from the changes in absorbance. In DPPH radical scavenging assay the The medicinal value of the plants is due to crude methanolic extract of Cornus macrophylla phytochemical constituents that cause definite leaves exhibited marked antioxidant activity i:e 69.08% pharmacological actions [17-19]. Plant produces a wide (100 µg/ml) with IC50 ; 57.87 µg/ml, as shown in Table 2. variety of secondary metabolites many of which have Total Phenolic Contents (TPC) determined in the crude been reported to be of therapeutic value. Secondary methanolic extract of Cornus macrophylla leaves was metabolites comprise of alkaloids, flavonoids, saponins, 97.3±2.06 mg of gallic acid equivalent per gram of dry tannin, phenolic compounds and many more [20]. weight of the plant material. Phenolic compounds are one Literature survey shows that out of 25000 species of of the largest groups of phytochemicals and have been higher plants in the world, only 5-10% has been tagged for most of the antioxidant activity of plants or chemically investigated. While a vast majority of the plant plant products. The antioxidant activity of C. resource is waiting for discovery [21]. In the current study macrophylla might be due to their high phenolic the methanolic extract of C. macrophylla leaves (MECM) contents. was investigated for secondary metabolites. Tests for Numerous researchers are focusing medicinal plants alkaloids, tannins, terpenoids, saponins, reducing sugar to discover novel, effective and safe compounds for the and anthraquinonnes were positive in MECML, while treatment of various challenging diseases like neoplasia phlobutanins and glycosides were not detected in the [23]. A plethora of research studies are available which methanolic extract of C. macrophylla, depicted in Table 1. reflects the effectiveness and strong safety profile of Our results are in conformity to the results obtained from various classes of phytochemicals. Saponins are group of Cornus controversa, members of the cornaceae family, secondary metabolites which possesses diversified which have shown the presence of same classes of pharmacological activities, like insecticidal [24], compounds including flavonoids, phenolic compounds antioxidant [25] and anthelmintic [26]. Crude saponins and terpenoids [22]. The medicinal property of the plant extracted from Teucrium stocksianum possesses could be attributed to phytochemicals residing in it. awesome cytotoxic effect (LC50 <10 ìg/ml) [27]. The Antioxidants are vital substances, which possess the cytotoxic effect of saponins has been previously reported ability to protect the body from damages caused by free by a number of researchers [28]. Human population is radical-induced oxidative stress. DPPH antioxidant assay increasing at high rate which has a direct impact on the is based on the ability of 1, 1-diphenyl-2-picryl-hydrazyl food resources. Numerous researchers are trying to (DPPH) to decolorize in the presence of antioxidants. elevate the per acre yield of the crops. Weeds are one of When DPPH accepts an electron (Donated by an the major stumbling blocks in the crops production. antioxidant compound), the DPPH is decolorized, which Currently a number of effective synthetic weed killers are

1757 Am-Euras. J. Agric. & Environ. Sci., 15 (9): 1753-1759, 2015 available in the market but most of them are associated 6. Ahmad, B., S. Mukaram Shah, S. Bashir and with serious hazardous effects. Therefore scientists are H. Begum, 2008. Antibacterial and antifungal trying to discover plant based novel compounds for the activities of Teucrium royleanum (Labiatea). Journal eradication of weeds. A plethora of research is available of Enzyme Inhibition and Medicinal Chemistry, which shows phytotoxic potential of medicinal plants [29]. 23(1): 136-139. In the current study the crude methanolic extract and 7. Torel, J., J. Cillard and P. Cillard, 1986. Antioxidant subsequent dilutions of Cornus macrophylla leaves activity of flavonoids and reactivity with peroxy displayed profound phytotoxic activity (95% at 1000 radical. Phytochemistry, 25(2): 383-385. µg/ml) against Lemna minor (Table 3). Thus it is 8. Clavin, M., S. Gorzalczany, A. Macho, E. Munoz, concluded that the methanolic extract of C. macrophylla G. Ferraro, C. Acevedo and V. Martino, 2007. Anti- s a potential source of antioxidant and herbicidal inflammatory activity of flavonoids from constituents and need further scientific evaluation. Eupatorium arnottianum. Journal of Ethnopharmacology, 112(3): 585-589. ACKNOWLEDGEMENTS 9. Alexiou, P. and V.J. Demopoulos, 2010. Medicinal plants used for the treatment of I am thankful to Dr. Nasrullah for the identification of diabetes and its long-term complications. Plants in plant and Department of Pharmacy, University of Traditional and Modern Medicine: Chemistry and Malakand for the laboratory facilities. Activity, 69: 175. 10. Sofowora, A., 1982. Medicinal plants and traditional REFERENCES medicine in Africa. 1982: John Wiley and sons LTD. 11. Evans, W.C., 2009. Trease and Evans' 1. Shah, S.M., A. Sadiq and F. Ullah, 2014. Antioxidant, pharmacognosy. 2009: Elsevier Health Sciences. total phenolic contents and antinociceptive potential 12. Harborne, J.B., 1984. Methods of plant analysis. 1984: of Teucrium stocksianum methanolic extract in Springer. different animal models. BMC Complementary and 13. Y ld r m, A., A. Mavi and A.A. Kara, Alternative Medicine, 14(1): 181. 2003.Antioxidant and antimicrobial activities of 2. Khan, S., A.S. Shah, H. Rahim, F.M. Gillani, Polygonum cognatum Meissn extracts. Journal of the K.A. Chisti, M. Shah and A.G. Khan, 2015. Effect of Science of Food and Agriculture, 83(1): 64-69. Nigella Sativa Tablets on Various Parameters of 14. Blois, M.S., 1958. Antioxidant determinations by the Insulin Resistance Syndrome in Fatty Liver Disease use of a stable free radical. at Peshawar Cantonment. American-Eurasian Journal 15. Singleton, V., R. Orthofer and R. Lamuela-Raventos, of Toxicological Sciences, 7(4): 243-246 1999. Analysis of total phenols and other 3. Shah, S., S.M.M. Shah, Z. Ahmad, M. Yaseen, oxidation substrates and antioxidants by means of R. Shah, A. Sadiq, S. Khan and B. Khan, 2015. Folin-Ciocalteu Reagent.Method in Enzymology, Phytochemicals, in vitro antioxidant, total phenolic 299c(1): 152-178. contents and phytotoxic activity of Cornus 16. Meyer, B., N. Ferrigni, J. Putnam, L. Jacobsen, macrophylla Wall bark collected from the North- D.J. Nichols and J. McLaughlin, 1982. Brine shrimp: West of Pakistan. Pakistan Journal of Pharmaceutical a convenient general bioassay for active plant Sciences, 28(1): 23-28. constituents. Planta medica, (45): 31-4. 4. Bryce, J., C. Boschi-Pinto, K. Shibuya, R.E. Black and 17. Shah, S.M.M., F.A. Khan, S.M.H. Shah, K.A. Chishti, W.C.H.E.R. Group, 2005. WHO estimates of the S. Pirzada, M.A. Khan and A. Farid, 2011. Evaluation causes of death in children. The Lancet, of phytochemicals and antimicrobial activity of white 365(9465): 1147-1152. and blue capitulum and whole plant of Silybum 5. Shah, S.M.M. and A. Sadiq, 2014. Biological activities marianum. World Appl Sci J., 12(8): 1139-1144. of crude saponins, methanolic extract and sub 18. Zahoor, M. and S.M.M. Shah, 2012. Evaluation of fractions of Teucrium stocksianum Bioss collected inorganic profile and anti-nutritional values of from north west of pakistan. Pharmacologyonline, Cocculus hirsutus. African Journal of Pharmacy and 03: 145-152. Pharmacology, 6(3): 144-147.

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