Phytochemical analysis, anticancer screening and antimicrobial activity of Ballota undulata Fresen (Benth.) from Al- Baha region Saudi Arabia

Plants of medicinal value play vital roles in prevention of disease and their use and promotion is fitting into all current prevention strategies. Nevertheless, diligent effort is required to properly identify, recognize and locate medicinal plants in the design and implementation of these strategies.  The medicinal plant Ballota undulata Fresen (Benth.) that belonging to family Lamiaceae has been reported to exhibit antispasmodic, anti-allergic, anti-inflammatory and antimicrobial properties. In this context, this work aimed to assess the biological activity of its three different extracts (chloroform, ethyl acetate and methanol) as antitumor against different cancer cell lines as hepatocellular (HEPG-2) carcinoma, breast (MCF-7) carcinoma and colon (HCT-116) carcinoma cell lines. Further determine its activity as antimicrobial against a diverse human pathogen (five Gram negative bacteria, two Gram positive bacteria and yeast) and its chemical composition using Gas Chromatography–Mass Spectrometry (GC-MS) analysis and HPLC. Our results showed that the different extracts of B. undulata  have antitumor activity against tested cell lines. In addition, the chloroform and methanol extracts showed marked antimicrobial activity against  the tested strains. Furthermore, the percentage of the MIC activity of B. undulata methanolic extract can help as sources for treatment compounds assay. So, this results of B. undulata shed the light on the antimicrobial ability of extracts from Saudi Arabia medicinal flora, which can be used as antimicrobial natural agents in pharmaceutical drugs and food preservation strategy.

Ballota undulata, Lamiaceae, GC-mass spectrometry, HPLC, cytotoxicity, Antimicrobial activity, herbal medicine, MCF-7, HCT-116, and HepG-2 cell lines A Plants of medicinal value play vital roles in prevention of disease and their use and promotion is itting into all current prevention strategies. Nevertheless, diligent effort is required to properly identify, recognize and locate medicinal plants in the design and implementation of these strategies. The medicinal plant Ballota undulata Fresen (Benth.) that belonging to family Lamiaceae has been reported to exhibit antispasmodic, anti-allergic, anti-in lammatory and antimicrobial properties. In this context, this work aimed to assess the biological activity of its three different extracts (chloroform, ethyl acetate and methanol) as antitumor against different cancer cell lines as hepatocellular (HEPG-2) carcinoma, breast (MCF-7) carcinoma and colon (HCT-116) carcinoma cell lines. Further determine its activity as antimicrobial against a diverse human pathogen ( ive Gram negative bacteria, two Gram positive bacteria and yeast) and its chemical composition using Gas Chromatography-Mass Spectrometry (GC-MS) analysis and HPLC. Our results showed that the different extracts of B. undulata have antitumor activity against tested cell lines. In addition, the chloroform and methanol extracts showed marked antimicrobial activity against the tested strains. Furthermore, the percentage of the MIC activity of B. undulata methanolic extract can help as sources for treatment compounds assay. So, this results of B. undulata shed the light on the antimicrobial ability of extracts from Saudi Arabia medicinal lora, which can be used as antimicrobial natural agents in pharmaceutical drugs and food preservation strategy.

INTRODUCTION
Cancer, which is known to be one of the deadliest diseases in the world, is estimated at about 10 million carcinomas globally, half of which are in the developing nations (Khalil et al., 2015;Khafagi and Dewedar, 2000). The prevalence of this disease has a greater impact in underdeveloped countries. Recently, in many countries, cancer is the second leading cause of death after heart diseases (Kau et al., 2005;Adamczak et al., 2019). A combination of surgery or radiation with chemotherapy is generally used for the treatment of this disease, however, it is not excused from adverse effects or resistance of the tumor to this type of treatment, for this reason the search for new drugs is still continuous. The plants are a promising source to attain this. The use of complementary and alternative medicinal products as herbal medicinal products, expressed mainly by plants, ranges from 30 to 75% among cancer patients in many countries (Khafagi and Dewedar, 2000). This also supports the interest in searching for anti-cancer agents derived from natural products from different countries particularly from the Arab Peninsula.
As an antimicrobial medicine are commonly and sometimes indiscriminately used, many microorganisms have developed resistance to different antibiotic treatments and these strains are particularly evident in the hospital setting. This has developed a major clinical issue in infectious diseases care. Furthermore, synthetic chemical antibiotics are often associated with adverse effects on host, including hypersensitivity, degradation of healthy intestines and mucous membranes, immuno suppression and allergic reactions (Al-Bakri and A i i, 2007).
Various plants with alkaloids, polyphenols and volatile oils as active ingredients are used as traditional medicinal products, while others are more traditional as inished products known as phytomedicines. The growth of microbial antibiotic resistance in the second half of the 20th century led scientists to explore multiple antimicrobial activity in many medicinal plants as an alternative form of health care and traditional medicine (Kau et al., 2005). Plants bioactive components contain a variety of compounds (e.g. lignans, quinones, coumarins, tannins, lavones, catechins, phenolic acids, anthocyanins, proanthocyanins, and stilbenes) that may increase life expectancy and slow or inhibit the development of degenerative diseases (Al-Bakri and A i i, 2007).
The world's leading cause of death is infectious diseases. Worldwide, about 50,000 people die from infectious illnesses every day. Antimicrobials originated from plants have huge therapeutic potential. They are effective in infectious diseases treatment, while at the same time mitigating numerous side effects that are often related to synthetic antimicrobials. In modern drug production, the bioactive compounds produced from wild plants play a key role. The development of many human diseases is driven by oxidative damage. Generally, the redox enzymes of aerobic bacteria mediate oxidative phosphorylation with an oxygen molecule, they produce reactive oxygen species (ROS) which have a pathological role to play in different infections. Plants are well known for generating a large number of biologically active metabolites with highly unusual structures because of their distinctive environments, cultivation and physiological behavior (Abdallah et al., 2017). This promotes the opportunity to explore new class chemicals with the prospect of therapy. Several studies have reported that these metabolites exhibit various bioactivities, such as antimicro-bial, antiviral, anti-in lammatory, and antimicrobial activity (Abdallah et al., 2017;Hsieh et al., 2001). However, for herbal medicine, natural derivatives from wild plants are used as therapeutically agents. It is a rich source of many medicines which are strong and effective.
Phytochemical studies are of great importance commercially for development of new herbal medicines by pharmaceutical companies. The growing use of pharmaceutical extracts shows further that a systematic study of medicinal plants is essential in order to identify active compounds (Majdi et al., 2020;Bader et al., 2003). One of these medicinal plants Ballota undulata which belonging to family Lamiaceae and has been reported to exhibit anti-allergic, antispasmodic, anti-in lammatory and antimicrobial properties (Bader et al., 2003). The genus Ballota comprises around 33 species growing mostly in the Arabian Peninsula and Mediterranean regions. Ballota undulata is a woody perennial herb with an ovate-cordate and reticulate leaves, distributed in Arabian Peninsula, mainly in high elevated regions and mountains of Saudi Arabia (Majdi et al., 2020;Siciliano et al., 2005).
Bedouins use this plant for the treatment of wounds, bee, wasp, and scorpion stings. B. undulata aqueous extract has been investigated in several literatures for antimalarial and antitumor activities. In addition, plants from family lamiaceae, which are common in the world, have historically been used for the treatment of various diseases and have recently undergone tests for anti-cancer activities and have been used as a source of anticancer drugs. Essential oils also act as anti-multidrug resistant agents, tumor reduction and metastases inhibition (Mesquita et al., 2019). Methods included in the essential oils-mediated antiproliferative activity comprise apoptosis, cell cycle arrest, and DNA repair mechanisms (Zingue, 2014).
This research focuses on extracting essential oils from B. undulata as a bioactive component and evaluating its biological activity as an antitumor against various cancer cell lines. Further, determine its activity as antimicrobial against a diverse human pathogen. The current research was carried out to track the different B. undulata plant sample extracts from Saudi Arabia lora in keeping with this international pattern. Phytochemical analysis, separation and identi ication of the plant extracts must be done.

Plant collection and preparation of the extracts
The fresh aerial parts of B . undulata were collected at its growth period of spring season from Wadi Turbah Zahran, Albaha regoin, southwestern Saudi Arabia. The plant was air dried at lab-temperature till constant weight, then ground to ine powder and kept being used for different plant analysis. Two hundred grams of plant powder were successively extracted by soxhlet apparatus using different organic solvents with analytical reagent (AR) quality. These solvents were chloroform, ethyl acetate, and inally methanol for 10 h. Each extract collected separately into dry clean beakers, after that they were evaporated under reduced pressure using rotavapor apparatus at 60 • C, then were dried in desiccators for 1 h and inally all the dried residues were stored in refrigerator at 5 ºC until the use.

Mammalian cell lines
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 were determined and the percentage of viability was calculated as [(ODt/ODc)] x100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cell. The survival curve of each tumor 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 concentration.

Bacterial and fungal pathogens Five Gram negative bacteria
Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi, two Gram positive bacteria: Streptococcus mutans, Staphylococcus aureus, and yeast: Candida albicans), were isolated from microbiological lab of King Khaled Hospital, Al-Baha region, Saudi Arabia.

Culture media
Nutrient broth (NB) and agar (NA) media for both Gram-negative, Gram-positive bacteria Mueller-Hinton broth & agar medium (MHB & MHA) and potato dextrose agar (PDA) for yeast fungi. All media were purchased from (Sigma-Aldrich) California, USA. Gentamicin 32 µg as antibacterial and Fluconazole 32 µg as antifungal were purchased from (Sigma-Aldrich) California, USA. Incubation period for all bacteria was at 37°C for 18 hours and for fungi at 28°C for 48 hours.

Antibacterial Activity Assay
The antibacterial activity of all plant extracts was determined by disk diffusion method (Balouiri et al., 2016). Antibiotic Gentamicin was used as positive control and sterilized Miller Hinton broth were used as negative controls.

Antifungal Activity assay
The same for antibacterial activity assay except that the medium used is PDA and the incubation period is 48 hr. and using the luconazole as a positive antifungal antibiotic control.

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., (Balouiri et al., 2016;Murray, 1999) using modi ied Broth dilution assay with the help of Spectrophotometer at 595 nm in mg/ml. Minimum Fungicidal Concentration (MFC) were done according to Mosmaan (Mosmann, 1983) by using Candida albicans as a pathogen and Fluconazole 32 µg antifungal as positive control, on PDA medium.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The GC-MS analysis of various crude extracts were 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.

Statistical analysis
The results were analyzed using 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
Our results showed that, the chloroform extract of B. undulata achieved cytotoxic effect against HEPG2, MCF7, HCT 116 With (IC 50 =76.2, 113.9 and 93.6 µg/ ml, respectively), ethyl acetate extract was (IC 50 =91.7, 188.4 and 101.8 µg/ ml, respectively) and methanol extract was (IC 50 = 101.2, 148.1 and 120.9 µg / ml, respectively) all these data were recorded at Table 1 and illustrated at Figures 1, 2 and 3 with cisplatin as control. The results of the previous protocols of the American National Cancer Institute NCI (Boyd, 1997) expressed strong when IC 50 less than 20 µg/ml and moderate activities when IC 50 21 -50 µg/ml. So our plant showed weak cytotoxic activity against HEPG2, MCF7, HCT 116 cell lines.

Minimum inhibitory concentration and minimum bactericidal concentration assay
Minimum inhibition concentration and minimum bactericidal concentration (MIC and MBC) of various extracts of Ballota undulata (Sieb. ex Fres.), are registered in the Tables 3, 4 and 5. B. undulata, is a genus of family lamiaceae and responsible for a diverse mode of action, however the bioactivities of this plant have not yet been exploited before in Arabian Peninsula. The collected wild plants were extracted by (chloroform, ethyl acetate and methanol) exhibit powerful antimicrobial action more than the antagonistic values of chloroform and ethyl acetate. From our results and the MIC values indicated that the methanol fraction was reported as a signi icant activity toward tested microorganisms lacking for C. albicans including E. coli,Klebsiella pneumoniae, S. aureus, and S. mutans both of which are important for human health. This inding like the results documented by Majdi et. al. (Majdi et al., 2020), where the important activity against S. mutans 28 mm (inhibition zone) and lesser activity on P. mirabilis 9 mm. Chloroform extract shows moderate action on tested strains, extract gives high effect on S. aureus about 25 mm, E. coli 17 mm, and K. pneumoniae 13 mm diameter of inhibition zone respectively. On the other hands P. mirabilis, and P. aeruginosa exhibiting resistance to chloroform extract. Ethyl acetate extract giving the below activity on examined microorganisms, where the greatest action of the extract on S. aureus 20 mm of clear zone, while P. mirabilis, P aeruginosa, and S. typhi exhibit insensitivity for extract (no inhibition zone). S. mutans, S. aureus, and E. coli have not resistancefor all extracts and on another hand, P. mirabilis has no sensitivity for all extracts. All examined isolates show stability for standard antibiotics as recorded in a Table 2.
Methanol and chloroform extracts show the high signi icant antagonistic results of MIC and MBC activity, while ethyl acetate extract gives the weakest results of MIC and MBC action. S. aureus and S. mutans recorded the highest degree of MIC and MBC action each, while P. mirabilis shows the lower value of MIC and MBC in ethyl acetate extract as recorded in a Tables 3, 4 and 5. This disagree with Khalil et al. (Khalil et al., 2009) who noted that B. undulata has some medical uses, as, Antioxidant, diuretic, hemostatic, and ethanolic extract has         (Khafagi and Dewedar, 2000) recorded that various extracts (hexane, ethyl acetate, and ethanol) of B. undulata (collected from Sinai, Egypt) have not any antimicrobial activity on tested microorganisms either Grampositive bacteria: Bacillus subtilis, Staphylococcus aureus, or Gram-negative bacteria: Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, and fungi: Candida albicans, Microsporum canis, and Trichophyton mentagrophytes. This may be due to antimicrobial activity is related to the presence of different secondary metabolites; alkaloids, phenolics and terpenoids reported or identi ied in these plant species and the habitat affects the production and activity of these metabolites (Khafagi and Dewedar,    (Majdi et al., 2020).
luteolin 7-O-glucoside, apigenin 7-Oglucoside, and rutin were isolated from B. undulata by majdi et al (Majdi et al., 2020) and the composition of its essential oil has been reported recently. Further Sesquiterpenes were the main constituents, which caused the most antiproliferative essential oil against HepG2 cells with high percentage of inhibition. However, 22 compounds which represent more than 98% of the total essential oil were extracted and evaluated as strong to moderate antimicrobial agent to treat infections and as food preserver in many cases. These compounds are four phenylpropanoids, forsythoside B1, lysionotoside 2, verbascoside 3, betonyoside F4, an iridoid, verminoside 5, seven lavonoids, compounds 6-12, and two betaine derivatives 13 and 14 were isolated from the aerial parts of B. undulata (Mesquita et al., 2019). Several compounds have been reported in Ballota sps., including terpenes, lavonoids and tannins (Khalil et al., 2009). The chemical composition of the essential oil of B. nigra and B. undulata shows some similarities: monoterpenes are little represented, while sesquiterpenes are present in large amounts. In both cases germacrene D is the main compound. Previous phytochemical studies on the genus Ballota evidenced the presence of labdane diterpenoids, lavonoids, and phenylpropanoids (Rigano et al., 2017). The aqueous extract of B. undulata has different pharmacological properties with a diverse bioactivity as antitumor and antimalarial activities. Ballota species have been widely used in traditional medicine as sedative, antispasmodic, diuretic, choleretic, and ant hemorrhoidal agents (Mesquita et al., 2019;Rigano et al., 2017). Literature surveys suggest the important role played by plant-based drugs in treating infectious diseases (Khalil et al., 2009).
Due to the diversity and complexity of natural phenolic compounds, it is dif icult to characterize every compound present in the crude extract to elucidate its structure (Zou et al., 2015), qualitative estimation for some phenolic and lavonoids compounds for different successive extract of B. undulata was observed at Table 8 by HPLC. The chloroform extract contains kaempferol (Figure 7), the ethyl acetate extract contains caffeic acid, kaempferol, rutin and quercetin ( Figure 8) and the methanolic extract contain chlorogenic acid and Caffeic acid (Figure 9).

CONCLUSIONS
Our study showed that all different extracts of B. undulata possess weak cytotoxic activity against different three cell line using MTT assay in addition, the antimicrobial activity of methanol and chloroform of B. undulata shown the most important value of action on most of the tested strains. Furthermore, the percentage of the MIC activity of B. undulata methanolic extract can help as sources for treatment compounds assay.