Antitumor, Antimicrobial activities and Phytochemicals Constituent of different Extracts of Pulicaria undulata (Forssk.) Oliver. Grown Naturally in Saudi Arabia

Antitumor and antimicrobial resistance are a habitual global issue, which continually demands (cid:977)inding new natural compounds to encounter the resistance. Pulicaria undulata (Forssk.) Oliver. (Asteraceae family) has numerous promising medicinal properties. The recent work aimed at determination of antitumor effects of three extracts of P. undulata on three types of human carcinoma; HEPG-2 hepatocellular carcinoma, MCF-7 breast carcinoma and HCT-116 colon carcinoma cell lines. Anticancer activity was assessed through studying the viability of the cancer cells and apoptotic pathway. Also, antimicrobial potency of different extracts was assessed against studied human pathogens ((cid:977)ive Gram negative bacteria, two Gram positive bacteria and yeast). The results reveal that chloroform extract has different levels of cytotoxicity toward the three types of cancer cell lines. A considerable decline in cancer cell rates has been linked to increasing in concentration of plant extract. The half maximal inhibitory concentration IC 50 value was 3.01 (cid:22) g/ mL for the HepG-2, 16.4 (cid:22) g/mL for the MCF-7, and 7.4 (cid:22) g/ mL for HCT-116. Followed by the ethyl acetate extract which showed strong cytotoxic activity against HEPG2 with IC 50 = 12.2 (cid:22) g /ml and moderate activities against MCF7 and HCT 116 and recorded (IC 50 = 26.7 and 26.4 (cid:22) g /ml, respectively). While the crude methanol extract recorded the lowest cytotoxic effect against HEPG2, MCF7 and HCT 116 with (IC 50 = 51.4, 105.1 and 86.7 (cid:22) g / ml, respectively). Chloroform and ethyl acetate extracts have a high antimicrobial activity more than methanol extract against the pathogens being studied. HPLC and GCMs Analysis identi(cid:977)ied numerous chemical compounds of P. undulata extracts with various therapeutic bene(cid:977)its. In conclusion, P. undulata has the potential to act as an antimicrobial agent against various pathogenic microbes and is a promising wild herb for the treatment of cancer.

Pulicaria undulata, Asteraceae, GC-mass spectrometry, HPLC, Cytotoxicity, Antimicrobial activity, Herbal medicine, MCF-7, HCT-116, HepG-2cell lines ABSTRACT Antitumor and antimicrobial resistance are a habitual global issue, which continually demands inding new natural compounds to encounter the resistance. Pulicaria undulata (Forssk.) Oliver. (Asteraceae family) has numerous promising medicinal properties. The recent work aimed at determination of antitumor effects of three extracts of P. undulata on three types of human carcinoma; HEPG-2 hepatocellular carcinoma, MCF-7 breast carcinoma and HCT-116 colon carcinoma cell lines. Anticancer activity was assessed through studying the viability of the cancer cells and apoptotic pathway. Also, antimicrobial potency of different extracts was assessed against studied human pathogens ( ive Gram negative bacteria, two Gram positive bacteria and yeast). The results reveal that chloroform extract has different levels of cytotoxicity toward the three types of cancer cell lines. A considerable decline in cancer cell rates has been linked to increasing in concentration of plant extract. The half maximal inhibitory concentration IC 50 value was 3.01 µg/ mL for the HepG-2, 16.4 µg/mL for the MCF-7, and 7.4 µg/ mL for HCT-116. Followed by the ethyl acetate extract which showed strong cytotoxic activity against HEPG2 with IC 50 = 12.2 µg /ml and moderate activities against MCF7 and HCT 116 and recorded (IC 50 = 26.7 and 26.4 µg /ml,respectively). While the crude methanol extract recorded the lowest cytotoxic effect against HEPG2, MCF7 and HCT 116 with (IC 50 = 51.4, 105.1 and 86.7 µg / ml, respectively). Chloroform and ethyl acetate extracts have a high antimicrobial activity more than methanol extract against the pathogens being studied. HPLC and GCMs Analysis identi ied numerous chemical compounds of P. undulata extracts with various therapeutic bene its. In conclusion, P. undulata has the potential to act as an antimicrobial agent against various pathogenic microbes and is a promising wild herb for the treatment of cancer.

INTRODUCTION
Millions of people are diagnosed with different types of cancers worldwide every year. About 18.1 million new cases of cancer were estimated in 2018, and approximately 9.6 million deaths of cancer occurred. Cancer of lungs is the most prevalent type of cancer in both genders combined, thoroughly followed by female breast cancer, prostate cancer, colorectal cancer, stomach cancer, and liver cancer (Bray, 2018). Present medications can only, to a certain degree, inhibit the development of tumours in all forms of cancer. Therefore, to resolve the numerous pharmaceutical limitations of cancer, it is essential to ind alternative natural drugs for treating liver, colon, and breast cancers. Including immune system damage, several de iciencies have been found due to the severe side effects of chemical drugs produced in patients. Besides, the foremost causes of mortality and morbidity are the cancerous cell metastasis (Huang, 2017).
Nowadays, complementary therapies are also being used to treat and reduce the symptoms and pain of cancer. Natural products had been used in different parts of the world like the Kingdom of Saudi Arabia, India, and Egypt since the earliest eras as traditional remedies. Such natural products have diverse mechanisms of action such as cell growth inhibition, the disparity in the differentiation of cells and apoptosis initiation. These natural plant products have been used in the treatment of many infectious diseases and cancers, as they have antimicrobial and antitumor effects (Bourhia, 2019).
Recently the number of drug-resistant pathogens has increased substantially in medical investigation, although many new antibiotics were developed (Aslam, 2018). In this context, erroneous use of antibiotics has been attributed to the antibioticresistant development and the global emergence of multidrug-resistant bacteria that gradually reduce the ef icacy of existing drugs resulting in treatment failure. Infectious diseases caused by antimicrobialresistant microbes are becoming a serious problem all over the world, which leads to an increase in the morbidity and mortality of these infections (Nthulane and Patience, 2020). So, we need to explore a new active product against these multidrug-resistant microbes (MDR). In the same time, microbiologists discover a potent plant extract which can selectively antagonize with infectious microbes. These different extracts contain components of bioactive metabolites, including lavonoids, alkaloids, tannins, terpenoids, and phenolics function together in combination to compact microbial growth (Nthulane and Patience, 2020). These new classes of antimicrobial substances have been extracted from medicinal plants and strongly inhibited the growth of (MDR) organisms with novel antagonistic mechanisms. These new strategies had the potential to be used as alternative therapeutic options for the treatment of a diverse infection induced by resistant microbes (Mulani et al., 2019). Recently, the commercial importance of these secondary metabolites (SMs) has given considerable attention to its growth and to explore ways to increase its production using tissue culture technology (Aslam, 2018).
As stated by the World Health Organization (WHO), around 65% of the world's population prefers traditional herbal medicines. Nonetheless, few studies on herbal drugs in the treatment of several cancers have been carried out (Jadhav, 2008). Until now, a limited number of wild plants as herbal medicines have been investigated and analyzed chemically, given the possible anticancer effect of their unique bioactive chemicals. Wild pharmaceutical plants are a good source of highly biologically active SMs, which considered as a pivotal source of active constituents with many variations in its arrangements and structural properties (Hegazy and Emam, 2015). Family Asteraceae (Compositae) is a worldwide distributed family of about 1600 genera and comprise more than 23,000 species. The genus Pulicaria that is belonging to Asteraceae includes about 75 species distributed widely in Asia, Africa, and Europe (Kalwij, 2012). They used in traditional medication as antihyperglycemic, and antispasmodic drugs, also they show anticancer, antioxidant and antimicrobial properties (Emam et al., 2019). The chemical elaboration of Pulicaria species      revealed the presence of various SMs, such as mono-, sesqui-, and diterpenoids, lavonoids, and phenolics (Hegazy and Emam, 2015). Pulicaria undulata is one of the most common annual herb or subshrubs grown naturally in the desert with small yel- Based on the strong medicinal contextual of Asteraceae, P. undulata was selected for investigation.
The recent study has been done to assess the pharmacological properties of P. undulata. However, the vast gap of information's about the medicinal features of fresh P. undulata as fresh P. undulata is used in the utmost of traditional remedies, especially in

Plant collection and preparation of the extracts
The new aerial parts of P. undulata were collected at its growth period of spring season from its natural habitats in the Saudi Arabia Kingdom. The plant was air-dried at lab-temperature till constant weight, then ground to a ine powder and kept being used for different plant analysis. Two hundred grams of plant powder was successively extracted by soxhlet apparatus using different organic solvents with analytical reagent (AR) quality. These solvents were chloroform, Ethyl acetate, and inally, methanol for ten h.each extract collected separately into dry clean beakers, after that they were evaporated under reduced pressure using rota vapour apparatus at 60 • C, then were dried in desiccators for one hour and inally, all the dried residues were stored in a refrigerator at 5 ºC until the use.

HepG-2 cells (human hepatocellular carcinoma cell line), HCT-116 cells (human colon carcinoma cell line) and MCF-7 cells (human breast carcinoma cell line) were obtained from the American Type Culture
Collection (ATCC, Rockville, MD).

Cell line Propagation
The cells were grown on RPMI-1640 medium supplemented with 10% inactivated fetal calf serum and 50µg/ml gentamycin. The cells were maintained at 37ºC in a humidi ied atmosphere with 5% CO 2 and were sub-cultured two to three times a week.

Cytotoxicity evaluation using viability assay
The antitumor activity for different extracts and Cisplatin drug as positive control evaluated according to the method described by (Mosmann, 1983). By MTT assay the number of viable cells was determined, and the percentage of viability was calculated as [(ODt/ODc)] x100% where it is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of the untreated cell. The survival curve of each tumour cell line after treatment with the speci ied drug was plotted from the relation between surviving cells and drug concentration.By GraphPad Prism software (San Diego, CA. USA), the 50% inhibitory concentration (IC 50 ) was estimated from graphic plots of the dose-response curve for each level.

Antimicrobial activity
The antimicrobial effectiveness of the chloroform, ethyl acetate, and methanol extracts was determined using the agar well diffusion method (Murray et al., 1999). The prepared extracts were examined for its antibacterial and antifungal activities against studied pathogenic microorganisms (Gramnegative bacteria (GNB): Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi, Gram-positive bacteria (GPB): Streptococcus mutans, Staphylococcus aureus and yeast: Candida albicans).

Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and the Minimum Fungicidal Concentration (MFC)
MIC, MBC and MFC of the all studied extracts were carried out according to Murray et al. (1999) using modi ied Broth dilution assay with the help of Spectrophotometer at 595 nm in mg/ml.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The GC-MS analysis of various crude extracts was performed using Trace GC-ISQ mass spectrometer (Thermo Scienti ic, Austin, TX, USA) with a direct capillary column TG-5MS (30 m x 0.25 mm x 0.25 µm ilm thickness) with the same condition as recorded by (Hashmi et al., 2013). The components were identi ied by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 14 mass spectral database.

Qualitative Determination of Flavonoids and phenolics Using HPLC
High-performance liquid chromatography (HPLC) technique using Waters 2690 Alliance HPLC system equipped with a Waters 996 photodiode array detector., set at low 1 ml/min. Autosampler, degasser, column compartment set at 35 o C and variable wavelength detector set at 280 nm, column: Hypersil C18 Thermo 5µm, 250x4.6 mm was used and the mobile phase: Buffer (0.1 % phosphoric acid in water) and methanol. A stock solution of 8 different standards in methanol was prepared. Each of the standards was iltered using a 0.22 µm syringe ilter then 10µl were injected. The prepared concentrations were Kampferol 0.4mg/ml, Gallic acid 1.2 mg/ml, Ellagic acid 0.4mg/ml, Chlorogenic acid 0.7mg/ml, Catechin 0.7mg/ml, Quercitin 0.3mg/ml, caffeic acid 1mg/ml and rutin 1mg/ml.

Statistical analysis
The results were analyzed using a two-way analysis of variance (ANOVA). All statistical investigations were carried out using SPSS 18.0 software. The indings were reported as standard error (SE)± of three replicates, and statistical signi icance was set as p-value≤0.05.

Cytotoxic activity
The common therapies as radiation, chemotherapy, and surgery had limited ef iciency, so the mortality rate among cancer patients is high (Xu et al., 2009). Recently, the researcher has been interested in using of crude plant extracts as natural productsor a combination of different phytochemicals for cancer therapy; this course is based upon the synergistic effect of the various plant metabolites in the crude extract and its multiple points of the intervention of such extracts.
According to the previous protocols of the American National Cancer Institute NCI (Boyd, 1997), the results expressed strong when IC 50 less than 20 µg/ml and moderate activities when IC 50 =21 -50 µg/ml. It was observed from the obtained results in a Table 1 and Figure 1, Figure 2 and Figure 3 that, all extracts of P. undulata achieved a cytotoxic effect against HEPG2, MCF7, HCT 116. While chloroform extract was had strong cytotoxic activity against HEPG2, MCF7 and HCT 116 with (IC 50 =3.01, 16.4 and 7.4 µg/ ml, respectively) followed by the ethyl acetate extract which showed strong cytotoxic activity against HEPG2 with IC 50 = 12.2 µg /ml and moderate activities against MCF7 and HCT 116 and recorded (IC 50 = 26.7 and 26.4 µg /ml, respectively). While the crude methanol extract recorded the lowest cytotoxic effect against HEPG2, MCF7 and HCT 116 with (IC 50 = 51.4, 105.1 and 86.7 µg / ml, respectively). AnEgyptian study by (Emam et al., 2019) recorded that methanol crude extract of P. undulata has cytotoxic activity against HEPG2 with IC 50 = 27.7 mg /ml and Hussien et al. (2016) reported that the crude extract (CH 2 Cl 2 /MeOH) of P. undulata showed excellent cytotoxic activity against both MCF-7 cells and HEPG2 cells with IC 50 41.6 and 40.7 µg/ml respectively.
P. undulata extracts (chloroform, ethyl acetate, and methanol) show signi icant antimicrobial activity. Chloroform extract records high activity against examined microorganisms except for E. coli (no activity).
But the most signi icant activity against S. aureus and K. pneumonia (30 mm inhibition zone) and lowest activity against P. aeruginosa (9.0 mm. inhibition zone).
Ethyl acetate extract showing mild activity against tested pathogens where S. aureus shows high sensitivity for extract about 25 mm diameter of inhibition zone. At the same time, E. coli exhibits resistance for extract 10 mm (inhibition zone).
Methanol extract showing the lowest activity against tested microbes where the highest activity of the extract against S. mutans 35 mm of clear zone, while P. mirabilis shows resistance for extract 9.0 mm inhibition zone.
S. mutans and S. aureus were more sensitive microbes for all extracts, and on the other hand, E. coli was more resistance for all extracts.

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungal concentration (MFC)
(MIC), (MBC) and (MFC) of P. undulata, different extracts are recorded in the Table 3, Table 4 and  Table 5. From the results of Table 3, Table 4 and Table 5 which revealed that the MIC & MBC are going in two parallel directions. In all extracts, the best MIC will be followed by the best MBC. In the same manner, as the These results of Table 3, Table 4 and Table 5 are in agreement with the study of (Touati et al., 2018), who recorded that chloroform and methanol extracts of Pulicaria odora have potent antimicrobial activity against Gram-positive bacteria S. aureus and B. subtilis while Gram-negative P. aeruginosa and E. coli were more resistance for all studied extracts. They reported that MIC and MBC values from 1.4 to 2 mg/ml, and this result con irms our results. Another researcher con irmed that different extracts ( In the same context, the Table 3, Table 4 and Table 5 show that the MFC results of the studied extracts show high antifungal activity (MFC) against the studied Candida albicans for chloroform and ethyl acetate extracts (21 ± 0.8 and 18 ± 0.6 mm inhibition zone diameter, respectively) and low antifungal activity of methanolic extract (10 ± 0.4 mm inhibition zone diameter) comparing to the standard luconazole antibiotics (20 mm inhibition zone diameter). It agrees with (Helal, 2019) who reported that methanolic extract of P. undulata showing antifungal activity for some fungal strains, for example, C. Albicans 20 mm diameter of inhibition zone and the highest activity toward M. boulardii about 32 mm.

Phytochemical evaluation
It was performed for qualitative and quantitative detection of various chemical constituents in P. undulata which aid in tracing the presence of an active entity that elicit a signi icant biological response of the plant. The mass spectrum of the unknown component was compared with the spectrum of the known element stored in the National Institute Standard and Technology (NIST) library. The compound name, probability, molecular formula, molecular weight and peak area of the test materials were recorded. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. GC-MS analysis of the chloroform extract revealed the presence of 29 compounds ( Figure 4 & Table 6) the major components were tomentosin (48.56%), is a natural sesquiterpene lactone. Many medicinal plants from the Asteraceae family are rich by sesquiterpenes lactones which have cytotoxic and anticancer properties (Hegazy, 2015). Sesquiterpenes lactones are potentially selective toward tumour and cancer stem cells by targeting speci ic signalling pathways, which make them lead compounds in cancer clinical trials (Zhang et al., 2005). Tomentosin showed antibacterial and antifungal effects (Masoumian and Zandi, 2017) and in vitro antiproliferative activity on various human cancer cell lines (Hegazy, 2015).
It is dif icult to characterize every compound present in the crude extract to elucidate its structure, due to the diversity and complexity of natural phenolic compounds (Surveswaran et al., 2007), qualitative estimation for some phenolic and lavonoids compounds for a different successive extract of P. undulata was observed at the Table 9. Flavonoids and phenolic components have been reported as antioxidants, anticancer, antibacterial, cardioprotective agents, anti-in lammation, immune system promoting, and skin protection from UV radiation, and interesting candidate for pharmaceutical and medical applications (Tungmunnithum et al., 2018). Many studies have suggested that lavonoids like rutin, kaempferol, quercetin, apigenin etc. are well-known for its anti-in lammatory, anti-allergic, anti-thrombotic, hepatoprotective, anti-spasmodic and anticancer properties (Tungmunnithum et al., 2018).

CONCLUSION
Our study showed that all different extracts of P. undulata possess marked and moderate cytotoxic activity against different three cell line using MTT assay, besides Antimicrobial test of P. undulata proved that chloroform and ethyl acetate extracts exhibited a high value of lethal activity against most of the examined human pathogens. Also, the value of MIC, MBC and MFC activities of P. undulata extracts can be used as natural therapeutic compounds against a wide range of pathogenic microorganisms, instead of the traditional commonly used antibiotics. These activities may be due to its abundance of many biologically active phytochemical compounds which provide a useful document for further study on our plant to detect its impact on another cancer type in vivo study.

ACKNOWLEDGEMENT
I would like to express my sincere gratitude to Dr Ahmed Hashem, Assistant Professor. Ain shams University, Department of Botany, for the constant support during this research. I also wish to express my thanks for Dr MarwaAbdel-salam Ibrahim, Researcher, Desert research centre, Medicinal and Aromatic Plants Department, for her patience, motivation, and profuse knowledge.

Funding Support
Nil.

Con lict of Interest
The author declares that there is no con lict of interest.