Research Paper Principal Component and Heat Map Analysis of The

Research Paper

Principal component and heat map analysis of the biological activities for some selected medicinal plants

Accepted 8th June, 2018

ABSTRACT

African and Asian countries have a gorgeous and prestigious heritage of herbal medicines. Ten well-known medicinal plants cultivated and grown under African environmental conditions (temperature, relative humidity, irradiance, photoperiod, wind and soil properties, etc) were extracted with methanol. Themethanolic extracts of these plants were screened for their antileishmanial, cytotoxic and free radical scavenging activities. The methanol extract of Colchicum autumnale and Alpinia officinarum showed high antileishmanial activity (IC5061.85±2.09 and 66.42±1.60 µg/ml, respectively). A. officinarum exhibited the most potent cytotoxicity (IC50 8.66±2.30 and 3.66±1.52 µg/ml against Hep G2 and A549, respectively). Rosa damascena, A. officinarum, and Humulus lupulus showed potent DPPH radical scavenging activity with IC50 values (2.0±0.23, 11.50±2.17, 14.0±2.0 µg/ml, respectively). Principal component analysis (PCA) was used as a Ahmed G. G. Darwish1,2*, Mamdouh N. statistical tool for experimental sets of obtained values for antileishmanial, Samy2,3, Sachiko Sugimoto2 Katsuyoshi antioxidant and anticancer activities performed statistics with person correlation Matsunami2 and Hideaki Otsuka2,4 matrix on plant samples. The results showed that there was a high correlation between anticancer activity against A549 and Hep G2 cells (0.743); moderate 1Department of Biochemistry, Faculty of Agriculture, Minia University, Minia correlation between anticancer activity against A549 cells and antileishmanial 61519, Egypt. activity (0.643); moderate correlation between anticancer activity against Hep G2 2Department of Pharmacognosy, cells and antileishmanial activity (0.555), and low correlation between DPPH Graduate School of Biomedical and radical scavenging activity and anticancer against A549 cell (0.440). The heat map Health Sciences, Hiroshima University, 1- 2-3 Kasumi, Minami-Ku, Hiroshima 734- data set was based on values of the biological activities for the plants extract 8553, Japan. percentage. The results showed that C. autumnale and A. officinarum showed high 3Department of Pharmacognosy, Faculty antileishmanial activity, while A. officinarum exhibited the most potent of Pharmacy, Minia University, Minia cytotoxicity against Hep G2 and A549 cell lines. R. damascena, A. officinarum and H. 61519, Egypt. 4Department of Natural Products lupulus showed potent DPPH radical scavenging activities. Hierarchical clustering Chemistry, Faculty of Pharmacy, Yasuda analysis (HCA) data showed that some plant extracts have similar clustering Women’s University, 6-13-1 Yasuhigashi, results associated with their biological activities. Asaminami-Ku, Hiroshima 731-0153, Japan. Keywords: Plant extract, antileishmanial, cytotoxicity, cancer cell line, DPPH *Corresponding author. E-mail: radical scavenging activity, principal component analysis, heatmap, hierarchical [email protected]. clustering analysis, Rosa damascene, Alpinia officinarum, Humulus lupulus.

INTRODUCTION

Leishmania is a genus of trypanosomatid protozoa and is Canidae, rodents and humans, and currently affects more the parasite responsible for the disease leishmaniasis. It is than 12 million people. Leishmaniasis is a disease with a spread through sandflies. Their primary hosts are prolonged worldwide distribution in 98 countries where vertebrates; Leishmania commonly infects hyraxes, about third of the cases in the Americas, the Mediterranean Academia Journal of Medicinal Plants; Benzeid et al. 102

basin and Western Asia with 70 to 75% of the cases recorded antispasmodic, astringent, carminative, diaphoretic, digestive in Afghanistan, Algeria, Colombia, Brazil, Iran, Syria Ethiopia, emmenagogue, stimulant, tonics, vasodilator, and vulnerary Costa Rica, Peru and Northern Sudan (Alvar et al., 2001). and also used against colds, cramps, fevers and kidney Chemotherapy of leishmaniasis is still inspiring due to the disorders (Stojanović et al., 2005; Yaeesh et al., 2006). C. limitation of the efficiency of the drug. N-methylglucamine, autumnale is used as anti-inflammatory, antimitotic and amphotericin B, and pentamidinethese drugs are in use for the antifibrotic and involved in the inhibition of microtubule treatment of leishmaniasis. However, many parasites are formation (Boye and Brossi, 1992; Katzung, 2004). C. resistant to these drugs. Medicinal plants are a good source of morifolium possesses antihepatotoxic and antigenotoxic effects bioactive phytochemicals used for the treatment of parasitic (Lee et al., 2011). It exhibits an allelopathic activity (Beninger diseases including leishmaniasis for people around the world and Hall, 2005) and has anti-inflammatory, (Bahmani et al., 2015). immunomodulatory humoral, cellular and mononuclear Reactive Oxygen Species (ROS),including free radicals, are phagocytic activities (Cheng et al., 2005; Su et al., 2012). H. reported to cause several diseases like liver diseases, diabetes lupulus traditionally is used to relieve insomnia, anxiety, and cancer (Ames, 1983) which are considered the most excitability, restlessness associated with tension, headache and dangerous in all countries around the world (Feher et al., gastrointestinal spasms (Newall et al., 1996; Schulz et al., 1987; Halliwell et al., 1989; Aruma, 1998; Suizane et al., 2009; 2001). M. chamomilla showed different pharmacological Alzeer et al., 2014) and the cost of chemotherapy has become activities like anti-inflammatory, anticancer, anti-allergic more expensive and the funds in many governments of some activities and is used in the treatment of stress and depression countries, particularly, the developing countries are (Shipochliev et al., 1981; Al-Hindawi et al., 1989). T. tomentosa unavailable for this treatment. Thus, it is important to have been used for diuretic, diaphoretic, antispasmodic, investigate new drugs or compounds that could be associated stomachic and sedative activities and have been taken for the with the traditional treatment. As such medicinal plants have treatment of flu, cough, migraine, nervous tension, ingestion provided some of the natural therapeutics for cancer (Alzeer et problems, various types of spasms and liver disorders al., 2014). Furthermore, the use of plant extracts provides a (Theallet, 1963; Baytop, 1984; Weiss, 1988; Toker, 1995). R. standard gauge for screening especially in its initial phase damascene has been used as cardiotonic (Hadjiakhoondi, (Ekta, 2013). The impact of environmental factors 2009), mild laxative (Zargari, 1992), anti-inflammatory (temperature, relative humidity, irradiance, photoperiod, (Loghmani- Khozani et al., 2007), cough suppressant (Shafei et wind, soil properties, fertilization and harvest time) influence al., 2003), anti HIV (Mahmood et al., 1996), antibacterial the composition of essential oils (terpenoids and aromatic (Basim and Basim, 2003) and antitussive (Shafei et al., 2003). compounds etc) (Kusumoto et al., 1995; Cordell, 1995; Baker S. marianum has hepatoprotective and antidepressant et al., 1995; Nadia, 2013). activities and used in the treatment of diabetes, varicose veins, The aim of our work was to evaluate the in vitro activity of selenic congestions, amenorrhea and uterine hemorrhage methanolic extracts of some medicinal plants such as (Talicia, 2014; Evans, 2009; Barceloux, 2008). The essential antileishmanial activity and cytotoxic activity using two oils of V. castus have antifungal and antimicrobial activities different cancer cell lines (Hep G2 and A549) and free radical (Hoffaman, 2003). scavenging activity with the DPPH assay. Regarding applied statistical analysis tool, we used principal component analysis (PCA) in order to obtain the initial MATERIALS AND METHODS solution reducing the original dataset. PCA expresses the data as a linear combination of its basic vectors. It is a non- Plant materials parametric analysis, and the correlation is called loadings. The squared loadings are easier to interpret than the loadings The plants used in this study were collected from different (Horel, 1981). Pearson’s correlation is a normalization method cities of Egypt. Plants were identified by Engineer Esraa where the standard deviation is in scaling the data between -1 Mohamed, Department of Agricultural Chemistry, Faculty of to 1 (Janzen et al., 2006). Concerning the heat map analysis, it Agriculture, Minia University, Egypt. The collected parts of is a graphical representation of data where the individual plants were separated and cleaned from dust and placed in the values contained in a matrix are signified as colors. Fractal and shade inside a well-ventilated room until were completely tree maps both often use a comparable system of color-coding dried and weight was obtained (Table 1). Dried parts of the to represent the values occupied by a variable in a hierarchy. plants were grounded to fine powder. The following plants Alpinia officinarum, Achillea millefolium, Colchicum autumnale, Chrysanthemum morifolium, Humulus lupulus, Matricaria chamomilla, Tilia tomentosa, Rosa Solvents and chemicals damascena, Silybum marianum and Vitexagnus-castus were found of multiple biological activities and broad traditional Dulbecco’s modified Eagle’s medium (DMEM), RPMI uses against infectious and non-infectious diseases. A. (R8758, SIGMA), penicillin-streptomycin (WAKO 119- officinarum is used in folk medicine as anticancer, antioxidant, 00703), fetal bovine serum (FBS) and 3-(4,5- antifungal and antimicrobial agent (Ray, 1976; Asolkar et al., Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) from 2005; An et al., 2005; Suja et al., 2008; Srividya et al., 2010). (23547-21 1g, Nacalaitesque)Trolox, 2,2-Diphenyl-1- Traditionally, A. millefolium is used as antiseptic, picrylhydrazyl (DPPH) and Doxorubicin was obtained from Academia Journal of Medicinal Plants; Benzeid et al. 103

Table 1: List of the screened Egyptian plants and their collected parts.

Scientific name Common name Family Part used Alpinia officinarum Galangal Zingiberaceae Rhizomes Achillea millefolium Yarrow Asteraceae flowers and stems Colchicum autumnale Autumn crocus Colchicaceae corms and bulbs Chrysanthemum morifolium Mums Asteraceae Flowers Humulus lupulus Hop Cannabinaceae Flowers and leaves Matricaria chamomilla Chamomile Asteraceae Flowers Tilia tomentosa Silver linden Malvaceae Flowers Rosa damascena Damask rose Roseaceae Flowers Silybum marianum Milk thistle Asteraceae Seeds Vitexagnus-castus Chaste tree Lamiaceae flowers, seeds, and leaves

(WAKO). cultured in RPMI1640 (Sigma, 500 ml) was supplemented with 10% heat-inactivation Fetal Bovine Serum (FBS) and antibiotics cocktail and incubated in 5% CO2 at 37°C. Preparation of plant extracts Human lung cancer cell, A549 (RCB004B), was obtained from (RIKEN Cell Bank) cultured in Dulbecco’s modified The air-dried fine powders of the selected plants were Eagle’s medium (DMEM; Sigma, 500 ml) supplemented with extracted by methanol thrice. The methanolic extracts were 10% heat-inactivation Fetal Bovine Serum (FBS) and concentrated under reduced pressure at 50°C using a rotary antibiotics cocktail and incubated in 5% CO2 at 37°C evaporator to yield viscous gummy materials, and then subjected to drying in vacuum desiccators (Oil pump). Finally, the weight of each plant extract was obtained. Antibiotics cocktail

Amphotericin B (Sigma A9528 100 mg) and kanamycin sulfate Determination of the antileishmanial activity 5 g (WAKO 119 to 00703) are dissolved in 8.93 and 25 ml of sterilized MilliQ water, respectively. equal volume of this The leishmanicidal activities of methanolic extracts were solution was combined with amphotericin B solution (8.93 ml) performed using the colorimetric MTT assay. Medium 199 and kanamycin sulfate solution (8.93 ml), dispensed into supplemented with 10% heat-inactivated fetal bovine serum microtube with 500 μl and stored at -20°C (FBS) and 100 µg/ml of kanamycin was used as the cell culture Heat-inactivated FBS was kept at 56°C temperature for 30 medium. The methanolic extracts were dissolved in DMSO and min and stored in a 50 ml tube at -20°C. added to each well of the 96-well micro-titration plates at 1% as the final concentration. Leishmania major cells (2×105 cells /well) were cultured in a CO2 incubator at 25°C for 72 h and Procedure for in vitro cytotoxicity assays of plants extract then MTT solution was added to each well and the plates were incubated overnight at 25°C. The absorbance was measured at This assay was performed using human lung cancer cell line 540 nm using a Molecular Device Versamex tunable microplate (A549) and human hepatocyte carcinoma cell line (Hep G2) reader. Amphotericin B was used as a positive control and the viability was estimated by the colorimetric MTT assay. (Takahashi et al., 2004). The inhibition % was calculated using Dulbecco’s modified Eagle medium (DMEM) supplemented the equation: with FBS 10% and antibiotics were used as the cell culture medium for A549 cell line and RPMI supplemented with 10% % Inhibition = [1- (A sample - A blank) / (A control - A blank)] × 100 FBS and antibiotics for Hep G2 cell line. The test plant extracts were dissolved in DMSO and added to each well of the 96-well Where Acontrol is the absorbance of the control reaction mixture micro-titration plates at 1% as the final concentration. The (containing DMSO and all reagents except for the methanolic plant extracts were tested at final concentrations of 100, 50, extracts). IC50 was determined as the concentration of sample and 25 µg/ml. A549 and Hep G2 cells (5×103 cells/well) required to inhibit the formation of MTT formazan by 50%. were cultured in a 5% CO2 incubator at 37°C for 72h and thereafter, MTT solution was added to each well and the plates incubated for a further 1.5 h. Subsequently, the Cell lines formazan precipitates were dissolved in DMSO and the HepG2 cell (RIKEN Cell Bank: RCB1886) (Courtesy of Prof. optical density value for each well was measured at 540 nm Takano). The human cell line derived from hepatocyte with a microplate reader. Doxorubicin was used as a carcinoma or hepatoblastoma of 15 years old male Caucasian positive control. The cell growth inhibition was calculated Academia Journal of Medicinal Plants; Benzeid et al. 104

Table 2: The anti-leishmanial activity of the plant extracts. Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

Antileishmanial activity (%) Plant name IC50 100 µg/ml 50 µg/ml 25 µg/ml Alpinia officinarum 97.25±1.63 26.69±4.18 8.28±4.61 66.42±1.60 Rosa damascena 9.75±4.14 6.92±3.21 5.62±4.10 ˃100 Silybum marianum 90.97±1.13 88.32±5.84 22.07±2.14 71.24±3.46 Colchicum autumnale 98.29±0.75 34.55±6.40 5.34±2.66 61.85±2.09 Humulus lupulus 28.82±6.04 2.48±2.19 4.28±2.41 ˃100 Achillea millefolium 12.63±7.71 2.26±1.15 2.21±0.83 ˃100 Chrysanthemum morifolium 7.54±1.97 3.86±1.93 3.25±0.94 ˃100 Matricaria chamomilla 11.64±6.40 6.48±3.34 4.35±3.69 ˃100 Tilia tomentosa 6.12±4.57 2.49±1.40 2.41±2.08 ˃100 Vitexagnus-castus 10.72±5.16 6.86±2.70 7.69±4.69 ˃100 Amphtericin (B) 99.13±2.08 89.33±7.63 72.20±15.52 36.11±8.03

using the equation: evaluate the dataset, reducing its dimension and conserving most of the statistical information. The analysis was Inhibition (%)= [1-(A sample- A blank)/ (A control- A performed using data analysis and statistical application blank)] available for Microsoft Excel (XLSTAT 2018.3.16, Florida, USA). Where Acontrol is the absorbance of the control reaction mixture (containing DMSO and all reagents except for the test plant extracts). IC50 was determined as the Heat maps concentration of sample required to inhibit the formation of MTT formazan by 50% (Samy et al., 2014a and b). The heat maps were created using the software (XLSTAT 2018.3.16, Florida, USA), based on their biological activities

including antileishmanial, anticancer activity against (A549 DPPH radical scavenging activity and Hep G2 cell lines) and DPPH radical scavenging activity.

The plant extracts were tested at a final concentration of The data treatment was based on several matrices 50, 25 and 12.5 µg/ml, respectively. The absorbance with containing different sets of information, depending on the various concentrations of the tested plant extracts interpretation. Hierarchical clustering analysis (HCA) was dissolved in MeOH (100 µl) in a 96-well microtiter plate carried out for columns using Euclidean distance. Color was measured at 515 nm as blank. Thereafter, 200 µM scales were adopted for each individual case based on DPPH solution (100 µl) was added to each well, followed by green (higher values), red (lower values), and black incubation at room temperature for 30 min. The (intermediate values). absorbance was measured again as a sample. The % inhibition was calculated using the equation: RESULTS AND DISCUSSION Inhibition (%) =[1-(A sample- A blank)/ (A control- A blank)] The aim of this study was to evaluate the anti-leishmanial activity, DPPH radical scavenging activity and the anti- Where Acontrol is the absorbance of the control reaction cancer activity of ten methanolic plant extracts against two mixture (containing DMSO and all reagents except for the different human cell lines (Hep G2 hepatoplastoma cell line test plant extracts). IC50 was determined as the and A549 human cancer cell line) using MTT assay. concentration of sample required to inhibit the formation of the DPPH radical by 50% (Darwish et al., 2016). Antileishmanial activity of the plant extracts

Data analysis Table 2 and Figures 1 and 2 show that the methanolic extract of C. autumnale, A. officinarum, and S. marianum had Principal component analysis (PCA) moderate antileishmania activity (IC50 61.85±2.09, 66.42±1.60 and 71.24±3.46 µg/ml, respectively) as This was used as a statistical tool; it is used with the aim to compared to the standard amphotericin B (36.11±8.03 µM), Academia Journal of Medicinal Plants; Benzeid et al. 105

Figure 1: The anti-leishmanial activity of the plant extracts. Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

Figure 2: Anti-leishmanial activity (IC50) of the plant extracts. Inhibition percentages are expressed as mean values ± S.D. of 3 experiments.

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Figure 3: The cytotoxic activity of plant extracts on human hepatocyte carcinoma cell line (HepG2). Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

Table 3: The cytotoxic activity (%) of plant extracts on human hepatocyte carcinoma cell line (HepG2) and human lung cancer cell line (A549). Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

HepG2 A549 Plant name 100 µg/ml 50 µg/ml 25 µg/ml 100 µg/ml 50 µg/ml 25 µg/ml Alpinia officinarum 98.00±0.57 94.00±0.56 85.00±5.50 93.66±6.35 70.66±3.78 58.00±13.11 Rosa damascena 93.33±3.21 57.33±9.45 17.33±4.04 82.00±6.55 51.66±11.59 29.66±15.50 Silybum marianum 86.33±7.09 21.00±7.80 21.66±4.72 85.00±19.0 18.33±3.78 3.66±1.52 Colchicum autumnale 88.00±9.50 43.67±5.50 27.66±4.93 52.00±7.0 42.66±2.68 42.00±21.07 Humulus lupulus 44.00±11.53 31.00±11.0 26.00±9.53 42.33±7.50 21.66±3.51 9.00±4.0 Achillea millefolium 25.00±11.35 15.00±5.0 5.66±1.52 44.66±2.60 6.66±2.51 2.33±0.57 Chrysanthemum morifolium 76.00±10.53 45.00±12.0 10.56±4.50 39.00±1.73 32.33±4.50 4.00±1.0 Matricaria chamomilla 45.33±3.51 26.00±7.02 3.33±1.52 20.66±1.52 5.66±2.08 3.00±1.73 Tilia tomentosa 76.66±11.23 33.00±4.0 6.00±1.0 38.00±4.0 14.66±6.42 6.66±1.52 Vitexagnus-castus 17.33±6.80 14.00±6.0 10.00±1.50 23.00±7.0 19.60±11.01 8.33±5.50 Doxorubicin 99.33±2.08 91.33±7.63 71.00±1.52 98.00±1.0 90.00±6.24 70.00±15.0

while the remaining tested extracts had no anti-leishmanial concentration 100 µg/ml against HepG2 cell line (Figure 3), activity. and A. officinarum, R. damascena and S. marianum against A549 cell line, as shown in Table 3 and Figure 4.

Growth Inhibition values in cell cultures for plant extracts IC50 values in cell cultures

The plant extracts were screened for their cytotoxic The IC50 values confirmed the highly potent cytotoxic activities in two different cancer cell lines using MTT assay. activities against HepG2 and A549 cell lines showed by A. This assay showed highly potent activities exhibited by A. officinarum (8.66±2.30 and 3.66±1.52 µg/ml, respectively) officinarum, R. damascena, S. marianum and C. autumnale at and R. damascene and (37.32±17.0 and 44.60±23.18 µg/ml, Academia Journal of Medicinal Plants; Benzeid et al. 107

Figure 4: The cytotoxic activity of plant extracts on human lung cancer cell line (A549). Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

Table 4: In vitro cytotoxicity IC50 values for the ten different plant extracts.

HepG2 A549 Plant name IC50 (µg/ml) IC50 (µg/ml) Alpinia officinarum 8.66±2.30 3.66±1.52 Rosa damascena 37.32±1.00 44.60±2.18 S. marianum 80.33±1.89 75.0±9.64 Colchicum autumnale 57.00±6.08 74.0±2.28 Humulus lupulus 113±3.0 99.0±1.50 Achillea millefolium 74.0±8.30 116.3±4.64 Chrysanthemum morifolium 80.66±4.10 114.6±4.40 Matricaria chamomilla 105.3±1.89 166.0±8.61 Tilia tomentosa 86.33±3.16 139.3±7.15 Vitexagnus-castus 391.6±1.45 234.6±7.84 Doxorubicin (µM) 7.33± 3.51 8.66±3.21

respectively) (Table 4, Figures 5 and 6). PCA Analysis

Performed statistics with Pearson Correlation matrix on DPPH radical scavenging activity plant samples based on the antileishmanial, anticancer and DPPH radical scavenging activities showed that there was a The plant extracts were examined for their 1, 1-diphenyl-2- strong correlation between anticancer activity against A549 picrylhydrazyl(DPPH) radical scavenging activities showed cells and anticancer activity against Hep G2 cells (0.743), high activity in the DPPH radical scavenging activities for R. moderate correlation between anticancer activity against damascena, A. officinarum and H. lupulus with (IC502.0±0.23, A549 cells and antileishmanial activity (0.643), moderate 11.50±2.17 and 14.0±2.0 µg/ml, respectively) as compared correlation between anticancer activity against Hep G2 cells with the standard trolox (IC50 16.0±1.0 µM) (Table 5 and and antileishmanial activity (0.555), and low correlation Figure 7). between DPPH radical scavenging activity and anticancer Academia Journal of Medicinal Plants; Benzeid et al. 108

Figure 5: IC50 of different plant extracts against A549 cell line.

Figure 6: IC50 of different plant extracts against Hep G2 cell line.

against A549 cell (0.440) as shown in Figure 8. 3.78%, respectively). Some values for factors (F1, F2, F3 Eigen values for the first and the second factor are higher and F4) are positive, while some are negative. Observation (2.404 and 0.985, respectively) (Figure 9) as compared to plot based on the activity it is shown that the highest DPPH values for the third and fourth factor (0.459 and 0.151, activity is in H. Lupulus, R. damascene and A. officinarum respectively). So, the first two factors must be used to extracts. The highest antileishmanial activity is in C. explain the obtained variabilities (60.11, 24.62, 11.48 and autumnale, A. officinarum, and S. marianum extracts. The Academia Journal of Medicinal Plants; Benzeid et al. 109

Table 5: DPPH Inhibition % are expressed as mean values± S.D. of 3 experiments and IC50for the ten different plants extract.

% of inhibition Plant name IC50 (µg/ml) 50 µg/ml 25 µg/ml 12.5 µg/ml Silybum marianum 55.03±10.14 34.66±5.54 20.46±4.17 38.66±5.77 Matricaria chamomilla 68.13±5.10 55.03±14.02 19.30±2.90 24.33±5.77 Vitexagnus-castus 42.86±1.09 23.53±1.70 17.0±3.81 75.6±17.0 Alpinia officinarum 84.56±0.83 70.60±1.57 51.40±2.68 11.50±2.17 Tilia tomentosa 12.20±2.95 6.96±4.63 5.10±2.13 151.6±4.09 Achillea millefolium 40.56±3.19 22.96±2.45 11.63±1.53 54.0±5.19 Chrysanthemum morifolium 12.30±5.28 9.10±2.53 6.46±2.11 191.3±8.50 Rosa damascena 94.33±2.02 90.0±0.05 80.0±0.92 2.0±0.23 Colchicum autumnale 27.96±3.52 13.26±1.60 8.20±2.62 123.2±4.27 Humulus lupulus 94.80±1.13 81.13±6.31 45.83±4.98 14.0±2.0 Trolox (µM) 96.0±2.51 70.0±5.0 25.0±7.63 16.0±1.0

Figure 7: DPPH radical scavenging activity of the tested plant extracts. Inhibition percentages are expressed as mean values± S.D. of 3 experiments.

highest anticancer activity against Hep G2 cells is in A. correlation between the second factor (F2) and DPPH officinarum, R. damascena, and S. marianum extract. The radical scavenging activity (r =0.900) and a moderate highest anticancer activity against A549 cells is in A. correlation between F1 and anticancer activity against Hep officinarum, S. marianum and R. damascena extracts. The G2 cells (r =0.836) and moderate correlation between the correlations between plant samples and antileishmanial, F1 and antileishmanial activity (r =0.804). The results anticancer and DPPH radical scavenging activity (Figure showed low correlation between the third factor (F3) and 10) showed that there is a strong correlation antileishmanial activity (r =0.544) and low correlation between the first factor (F1) and anticancer activity against between F1 and DPPH activity (r = 0.417). It can be A549 cells (r = 0.941), (Figures 6 and 7) and strong concluded that the highest activity among the investigated Academia Journal of Medicinal Plants; Benzeid et al. 110

Variables (Axes PC2 and PC1: 84.74%)

PC2 (24.63%)

Figure 8: Principal component score plot (PC1 and PC2) of Contribution of values %.

Figure 9: The importance of factors and values of cumulative variabilities.

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Biplot (Axes F2 and F1: 84.74%)

(60.11%)

1 1 F

F2 (24.63%)

Figure 10: Principal component score plot (F1 and F2) of the studied plant samples based on their bioactivities.

Figure 11: Heat map of the biological activities of the methanolic plants extract. Annotations on the left side of the heat map show clustering of samples. Academia Journal of Medicinal Plants; Benzeid et al. 112

samples is present in the methanolic extract of A. Aruma O (1998). Free Radicals, Oxidative stress and antioxidants in officinarum. human health and disease. J. Am. Oil Chem. Soc. 75(2): 199-212. Asolkar L, Kakkar K (2005). Glossary of Indian Medicinal plants, Part I (A- K)(2005). Council of Scientific and Industrial Research. India. 1:13. Bahmani M, Saki K, Ezatpour B, Shahsavari S, Eftekhari Z, Jelodari M Heat map analysis (2015). Leishmaniosis phytotherapy: Review of plants used in Iranian traditional medicine on leishmaniasis. Asian Pac. J. Trop. Biomed. 5(9): 695-701. The default color gradient sets the lowest value in the heat Baker J, Borries R, Carte B, Cordell G, Soejarto D, Cragg G, Gupta M, Iwu M, map to bright red, the highest value to a bright green, and Madulid D, Tyler VE (1995). Natural product drug discovery and mid-range values to black, with a corresponding transition development New perspectives on international collaboration. J. Nat. 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