ZnO Nanocomposites of Juniperus procera and Dodonaea viscosa Extracts as Antiproliferative and Antimicrobial Agents

Cancer and microbial infections constitute a major burden and leading cause of death globally. The development of therapeutic compounds from natural products is considered a cornerstone in drug discovery. Therefore, in the present study, the ethanolic extract and the fractions of Dodonaea viscosa and Juniperus procera were evaluated for anticancer and antimicrobial activities. It was found that two fractions, JM and DC, exhibited promising anticancer and antimicrobial activities. The JM and DC fractions were further modified into ZnO nanocomposites, which were characterized by SEM, XRD, TGA, and EDX. It was noted that the synthesized nanocomposites displayed remarkable enhancement in cytotoxicity as well as antibacterial activity. Nanocomposite DC–ZnO NRs exhibited cytotoxicity with IC50 values of 16.4 ± 4 (HepG2) and 29.07 ± 2.7 μg/mL (HCT-116) and JM–ZnO NRs with IC50 values of 12.2 ± 10.27 (HepG2) and 24.1 ± 3.0 μg/mL (HCT-116). In addition, nanocomposites of DC (i.e., DC–ZnO NRs) and JM (i.e., JM–ZnO NRs) displayed excellent antimicrobial activity against Staphylococcus aureus with MICs of 2.5 and 1.25 μg/mL, respectively. Moreover, these fractions and nanocomposites were tested for cytotoxicity against normal fibroblasts and were found to be non-toxic. GC-MS analysis of the active fractions were also carried out to discover the possible phytochemicals that are responsible for these activities.


Introduction
Despite technological and medical developments, cancer and microbial infections constitute a major burden and leading cause of death globally [1]. Due to the impaired immunity in cancer patients, microbial infection is more prevalent than in non-cancer patients [2]. Moreover, the treatment of these diseases has many downfalls in terms of toxicity, selectivity, resistance, and non-differentiation between normal and cancerous cells [3]. Therefore, it is highly desirable to develop new anticancer and antimicrobial agents with minimal side effects and excellent selectivity.
The development of therapeutic compounds from natural products is considered a cornerstone in drug discovery due to the fact of their vast structural diversity with fewer side effects [4]. They have provided important anticancer and antimicrobial leads [5]. For example, paclitaxel, podophyllotoxins, and vinblastine are obtained directly from plants, whereas etoposide, teniposide, irinotecan, and docetaxel are semisynthetic derivatives of these anticancer leads that are being used as anticancer drugs with diverse modes of action. Antimicrobial drugs, such as penicillin, cephalosporins, and fusidic acid, are obtained directly from fungi, while fusidin and tigecycline, which are antibacterial drugs, are semisynthesized from the leads [5]. Moreover, it has been reported that crude plant extracts contain combinations of many phytochemicals that cause a synergistic effect with available drugs leading to better therapeutic candidates [6].

Extraction
Dodonaea viscosa and Juniperus procera were collected from Madarah, Albaha region, Saudi Arabia, and identified by Haider Abd Algadir, Chemotaxonomist, Department of Biology, Albaha University, Saudi Arabia. Both plants were shade-dried and ground into powder. They were Soxhlet extracted with ethanol (95% v/v) and completely dried under vacuum at 45 • C in a rotary evaporator to afford Juniperus ethanolic extract (JE) and Dodonaea ethanolic extract (DE) [25]. Both JE and DE were then fractionated with solvents of increasing polarity using petroleum ether, chloroform, and methanol to give Juniperus

Gas Chromatography/Mass Spectrometry
The most promising fractions (i.e., JM and DC) from each plant were selected for GC-MS. The fractions (1 µL) were placed onto a Hewlett Packard 7673C Automatic Liquid Sampler (ALS) carousel (Hewlett Packard, Palo Alto, CA, US, where the sample was directly injected into the heated injection port of an Agilent 6890 Gas Chromatograph (GC) Agilent 5973 Mass Selective Detector (MSD) system (Agilent Technologies, Palo Alto, CA, USA). The method used for GC-MS was 50rtndi40.M, and the front inlet parameters were 225 • C and a 50:1 split (2% on-column) direct injection. A MeOH blank was analyzed prior to the sample analysis to identify any residual contaminants present in the system and/or the solvent used to reconstitute the sample.

Identification of the Components
The compounds present in the fractions were identified by comparison of their mass spectra with those from the Wiley 275 mass spectral database provided by the GC manufacturer and from the literature. The relative percent was computed by dividing the individual compound response by the total outgassing response for the sample and the resultant multiplied by one hundred (100). The Wiley database provides a match quality factor for each search. The match quality indicates how well the unknown spectra match that of a reference library. The components are listed in Tables 1 and 2. Table 1. Chemical constituents of the JM fraction seen by GC-MS.
To prepare plant-based ZnO nanocomposites, berries of J. procera and the aerial part of D. viscosa extracts were used. For the synthesis of JM-ZnO and DC-ZnO nanocomposites, formerly described methods were implemented with sufficient amendments [78,79]. Briefly, approximately 0.5 g of synthesized ZnO NRs were dissolved in methanol in a conical flask under continuous stirring for 3 h. Then, 1 g of JM solution (dissolved in a minimal amount of methanol) was added to the above solution. The ZnO NR solution containing the extract was constantly stirred in a water bath shaker at 60 • C for 48 h. The residual solvents were removed using a rotary evaporator. The DC-ZnO nanocomposites were also prepared following the aforementioned procedure using chloroform as a preparation solvent. The prepared DC-ZnO and JM-ZnO nanocomposites were in powder form and preserved in the dark till further use.

Characterization of Nanocomposites
The X-ray diffraction (XRD) spectra of the synthesized samples (i.e., ZnO NRs and the JM-ZnO and DC-ZnO nanocomposites) were recorded on a Bruker D8 Advance X-ray diffractometer (Bruker, Munich, Germany) with a power of 40 KV and current of 40 mA using CuKα radiation (wavelength: 1.5418 Å), and the X-rays were detected using the fast Lynx Eye one-dimensional detector. The scan was performed using a step size of 0.02 with 2 θ range: 10-70 degrees and a time/step: 0.2 s. A scanning electron microscope (SEM; JEOL JSM6700) (Jeol, Ltd., Tokyo, Japan) was used to observe the morphology of the samples. Energy-dispersive X-ray (EDX) spectroscopy attached to the SEM was used to analyze the composition of the ZnO NRs and the JM-ZnO and DC-ZnO nanocomposites. All the samples were coated with Pt before SEM-EDX analyses.
The thermogravimetric analysis (TGA) was carried out using a Mettler Toledo DSC/TGA instrument (Mettler-Toledo, Columbus, OH, USA). All samples were placed into Al 2 O 3 crucible and then heated from 25 to 800 • C with a heating rate of 10 • C/min under an inert nitrogen stream with a flow rate of 50 mL/min. Weight loss was recorded as a function of temperature, and organic decompositions were determined from the weight loss curve.

Biological Activities
In the present study, the extracts (i.e., JE and DE) and all the fractions (i.e., JP, JC, JM, DP, DC, and DM) were tested for their antimicrobial and anticancer activities. The most active fractions were taken for the synthesis of the nanocomposites.

Antimicrobial Evaluation
The antimicrobial activity of the extracts (i.e., JE and DE), the fractions (i.e., JP, JC, JM, DP, DC, and DM), and the nanocomposites were screened against Staphylococcus aureus (S. aureus, ATCC 29213), Escherichia coli (E. coli, ATCC 35218), and the fungus Candida albicans (C. albicans, ATCC 76615). The microorganisms were provided from Dr. Mahmoud Elfaky, (Microbiology Laboratory, King Abdulaziz University Hospital, Jeddah, Saudi Arabia). The bacterial and fungal stock cultures were maintained on Muller-Hinton agar (MHA) plates (Himedia Laboaratory, Mumbai, India), and Sabouraud dextrose agar (SDA) (Himedia Laboaratory, Mumbai, India), respectively. A loopful of overnight microbial cells from the agar plates was inoculated into 5 mL normal saline (85% NaCl), and the turbidity was adjusted to 1-5 × 10 6 CFU/mL. To check the antimicrobial activity, 100 mg of each extract was dissolved in 1 mL dimethyl sulfoxide (DMSO) (100 mg/mL), vortexed until dissolved completely, then diluted serially to obtain five different concentrations (i.e., 10, 5, 2.5, 1.25, and 0.6125 µg/mL). Similarly, different concentrations of nanocomposites, as mentioned above, were prepared and screened. Preliminary screening of the antibacterial and antifungal activities was conducted using an agar diffusion technique as described previously [80]. Briefly, Petri dishes (90 mm) were filled with 25 mL agar containing 1 mL microbial culture (1 × 10 6 CFU/mL). The strains were inoculated separately, and 40 µL of each concentration was added to a well of 4 mm in diameter. Dishes were pre-incubated for 2 h at 4 • C to allow pre-diffusion of extracts, then incubated for 24 h at 37 • C. DMSO was used as a negative control, and ciprofloxacin was used as a positive control. Inhibitory activity was defined as the absence of bacterial growth in the area surrounding the holes. The inhibition zone was measured using a caliper.

MTT Assay
The anticancer activity was performed according to a reported method [81]. To assess the cytotoxic effect of the extracts (i.e., JE and DE), fractions (i.e., JP, JC, JM, DP, DC, and DM), and nanocomposites against HCT-116 and HepG2 and cell lines, MTT viability assays were carried out by culturing the cells in a 96-well plate at a density of (3 × 10 3 cells/well). After 24 h of incubation, the cells were then treated in triplicate with the extracts and fractions at 7 serial dilutions (1000-3.9 µg/mL) for 48 h. Extracts and fractions were dissolved in DMSO with sonication. Appropriate control wells from untreated cells and the vehicle DMSO (0.5%) were prepared at the same time. Then, the medium was removed and replaced with MTT solution (2 mg/mL). The plates were covered with aluminum foil and incubated at 37 • C for 4 h. The purple formazan product was dissolved by adding 200 µL of 100% DMSO. The plates were then incubated for 5 min at 37 • C in a 5% CO 2 incubator, and the colorimetric signals were measured at 570 nm with a SpectraMax M3 plate reader.
The cytotoxic effect of the extracts and nanocomposites against fibroblast normal cells (3T3) was carried out by an MTT viability assay at 4 serial dilutions (125-15.62 µg/mL) for 48 h. The activity was performed as mentioned above. The colorimetric signals were measured at 490 nm with BioTek plate reader.

Extraction
The yields of the JE and DE were found to be 13.62% w/w and 11.58% w/w, respectively. The yields of the fractions of J. procera were 29.7% w/w (JP), 37.4% w/w (JC), and 32.9% w/w (JM), while the fraction yields from D. viscosa were 31.2% w/w (DP), 29.1% w/w (DC), and 39.7% w/w (DM).
Juniperus sp. literature revealed the presence of limonene, cedrene, camphene, caryophyllene, muurolene, humulene, calocorene, cedrol, valencene, neophytadiene, and phenoxy phenol in the essential oils of J. foetidissima leaves and berries grown in R. Macedonia [49]. Dehydroabietic acid and totarol occur widely in several Juniperus species including J. procera [52]. Figure 1 shows the XRD spectra of pure ZnO NRs and nanocomposites of ZnO. As implied in Figure 1a, in the spectrum of ZnO NRs, all diffraction peaks were similar to standard hexagonal wurtzite ZnO [77,85] and showed strong and sharp peaks indicating a superior crystallized morphology of the prepared nanorod samples. No obvious additional peaks of impurities were observed in the obtained spectrum of ZnO NRs. After modification of ZnO nanoparticles with J. procera methanol (JM) and D. viscosa chloroform (DC) fractions, several other phases apart from ZnO were observed. These peaks were found to be comparatively less intense, and also two small additional peaks appeared at 2θ = 20.5 • and 21.8 • , respectively, which may be attributed to the presence of unknown biomolecules in the plant extracts that capped ZnO NRs (Figure 1b,c). Therefore, these results clearly indicate that the ZnO and plant extract nanocomposites were synthesized. The diffractograms of the DC-ZnO NRs [86] as well as the JM-ZnO NRs [87] confirmed the presence of both ZnO and plant extracts; however, no sharp peaks originating from respective plant materials were present due to the amorphous nature of organic extracts. Figure 2 shows SEM morphological images of the prepared ZnO and the DC-ZnO and JM-ZnO nanocomposites. Uniformly distributed nanorods can be observed for the pure ZnO sample (Figure 2a). The average size of the nanorods was calculated to be approximately 80 nm in width and 200 nm in length. Whereas in the case of the DC-ZnO and JM-ZnO composites, the organic fractions of J. procera and D. viscosa showed smooth surfaces with ZnO NRs embedded in the surface (Figure 2b,c). The SEM, again, confirmed the formation of the DC-ZnO and JM-ZnO nanocomposites. The EDX spectrum of the prepared ZnO NRs is shown in Figure 3, displaying that the composition was Zn and O only, without any impurities. The EDX spectrum of ZnO NRs revealed the absolute elimination of unwanted organics and the formation of metal oxide species (Figure 3a). The EDX of JM-ZnO composites revealed the presence of principally C and O in addition to Zn (Figure 3b). Whereas, in the case of the DC-ZnO nanocomposite, the presence of C, Zn, O, and a small percentage of S was detected due to the presence of minerals and trace elements in the DC extract [88] or the presence of the organic biomolecule, 5-(3-buten-1-ynyl)-2,2'-bithienyl, which has been reported from the extract of leaves of this plant [69] (Figure 3c). The increase in the intensities of C and O molecules in both the nanocomposites further confirmed the formation of DC-ZnO and JM-ZnO NRs.  In the case of the ZnO NRs' thermogram, a total weight loss of approximately 3% was found in the temperature range from 25 to 800 • C due to the removal of the moisture and adsorbed structural water (Figure 4a). These results are in line with the results of the TG analysis of ZnO nanoparticles reported by other authors in a similar temperature range [89].

Characterization of Nanocomposites
In the thermograms of the DC-ZnO NRs and JM-ZnO NRs, a two-stage thermal degradation can be seen. In both the cases, in the first stage, a 1% and 3% weight loss was observed for the DC-ZnO and JM-ZnO nanocomposites [87], respectively, between a temperature range of 25 and 190 • C, attributed to the loss of moisture and adsorbed structural water. In the second stage, a weight loss of 64% was observed in the JM-ZnO NRs, which might be due to the decomposition of molecular organic compounds such as phenolics, 4-phenoxy phenol, sclareol, totarol, kaurenoic acid, dehydroabietic acid, totaradiol, and isopimara-7(8),15-dien-19-oic acid in the JP extract as reported in Table 2. On the other hand, a 62% weight loss was calculated in the DC-ZnO NRs between the temperature range of 180 and 800 • C, owing to degradation of organic compounds such as quercetin, velutin, eriodictyol, rhamnetin, costunilide, dodonaeaside A and B, and cleoradanes in the DV extract as reported in Table 2 (Figure 4b,c). The thermal spectra of the JM and DC extracts depicted an~87.5% degradation until 800 • C due to the disintegration of organic compounds as aforementioned in the Tables 1 and 2, respectively (Figure 4d,e). The major weight loss (~80%) was observed until 200 • C in DC extract, whereas JM demonstrated two-stage decomposition. The first stage of weight loss (~68.5%) was observed until 400 • C, and in the second stage, a weight loss of~86% was observed until 500 • C.
Consequently, a total weight loss of approximately 65% was noticed for the DC-ZnO and JM-ZnO NRs which once again established the successful formation of ZnO and plant extract (JM and DC) nanocomposites.

Antimicrobial Activity
In an in vitro antimicrobial bioassay, the eleven extracts were evaluated by agar diffusion assay using different concentrations on S. aureus (ATCC 29213), E. coli (ATCC 35218), and C. albicans (ATCC 76615). It was observed that the methanolic fraction (JM) from J. procera and the chloroform fraction (DC) from D. viscosa were the most promising fractions with an MIC of 5 µg/mL against S. aureus. The other fractions and extracts displayed moderate activity. The most promising fractions (JM, DC) were chosen for synthesis of the nanocomposites. The synthesized ZnO NRs and nanocomposites of DC (DC-ZnO NRs) and JM (JM-ZnO NRs) displayed excellent antimicrobial activity against Gram-positive S. aureus with MICs of 2.5 and 1.25 µg/mL, respectively. The results are shown in Figure 5. But moderate antibacterial action was observed with DC-ZnO and JM-ZnO nanocomposites against selected Gram-negative bacteria, E. coli. However, no prominent antifungal activity was observed with the extracts and fractions as well as the nanocomposites in the present study.
The antimicrobial activity of the extracts and fractions might be attributed to the presence of antimicrobial compounds: santin; quercetin; hautriwaic acid; 5-(3-buten-1-ynyl)-2,2'-bithienyl; 13,14 dihydroxy-15,16-dimethoxy-(-)-6α-hydroxy-5α, 8α, 9α, 10α-cleroda-3-en-18-oic acid; 1-L-O-methyl-2-acetyl-3-p-cis-coumaryl-myo-inositol in the DV extract ( Table 2). In the JM fraction, the antimicrobial compounds limonene, calacorene, umbellulone, pimarinal, and totaradiol may be imparting antimicrobial activity. The enhanced activity of the ZnO NRs and nanocomposites was attributed to the morphology and high surface area [90] as well as a synergistic effect. Although the pristine fractions (JM, DC) displayed the same activity at low concentration (5 µg), the antimicrobial effect varied in the respective nanocomposites, as both fractions contain different metabolites (Tables 1 and 2). The difference in activity was attributed due to the difference in the chemical components of the fractions and their synergism with ZnO NRs. The chemical components of DC-ZnO and JM-ZnO NRs might have interacted with the bacterial outer cell wall initially and then diffused into the inner cell wall, causing disorganization and leakage by disruption of the inner cell's content and cell deformation. In the present study, it was also noted that S. aureus was more sensitive to the tested samples compared to the other tested strains. The greater sensitivity of S. aureus has been credited to the difference in the cell organization of the strains [90,91]. Typically, the configuration of the microorganisms is essentially responsible for the differences in their sensitivity.
disruption of the inner cell's content and cell deformation. In the present study, it was also noted that S. aureus was more sensitive to the tested samples compared to the other tested strains. The greater sensitivity of S. aureus has been credited to the difference in the cell organization of the strains [90,91]. Typically, the configuration of the microorganisms is essentially responsible for the differences in their sensitivity. Figure 5. Antimicrobial susceptibility pattern of Staphylococcus aureus with fractions and nanocomposites (10-1.25 μg/mL). Ciprofloxacin was used as a reference drug (positive control). Data represent the mean values ± standard deviation of three replicates. Statistical analysis was performed by Dunnett's test. * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. the control.

Anticancer Activity
The anticancer activity of the extracts, its fractions, and the nanocomposites were performed on two cancer cell lines: HepG2 (liver cancer cells) and HCT-116 (colorectal cell lines). Tamoxifen was used as the standard anticancer agent. Firstly, the extracts (JE, DE) and their different fractions (JP, JC, JM, DP, DC, DM) were evaluated for cytotoxicity on the two cell lines. It was observed that the methanolic fraction (JM) from J. procera was the most active fraction with an IC50 of 62.98 ± 9.8 (HepG2) and 72 ± 11 μg/mL (HCT-116), whereas the chloroform fraction (DC) was potent from D. viscosa with an IC50 26.4 ± 3.3 (HepG2) and 39.8 ± 13 μg/mL (HCT-116). The remaining fractions and the ethanolic extract displayed moderate cell viability against these cell lines ( Figure 6). Therefore, we selected active fractions (JM, DC) from each plant for the preparation of their nanocomposites with zinc to see whether the synthesized nanocomposites would enhance further cytotoxicity. Interestingly, we found that the nanocomposite DC-ZnO and JM-ZnO NRs enhanced cytotoxicity in the HepG2 and HCT-116 cell lines. The nanocomposite DC-ZnO NRs exhibited cytotoxicity with an IC50 of 16.4 ± 4 (HepG2) and 29.07± 2.7 μg/mL (HCT-116), while JM-ZnO NRs had an IC50 of 12.2 ± 10.27 (HepG2) and 24.1 ± 3.0 μg/mL (HCT-116). Ciprofloxacin was used as a reference drug (positive control). Data represent the mean values ± standard deviation of three replicates. Statistical analysis was performed by Dunnett's test. * p < 0.05, ** p < 0.01, and *** p < 0.005 vs. the control.

Anticancer Activity
The anticancer activity of the extracts, its fractions, and the nanocomposites were performed on two cancer cell lines: HepG2 (liver cancer cells) and HCT-116 (colorectal cell lines). Tamoxifen was used as the standard anticancer agent. Firstly, the extracts (JE, DE) and their different fractions (JP, JC, JM, DP, DC, DM) were evaluated for cytotoxicity on the two cell lines. It was observed that the methanolic fraction (JM) from J. procera was the most active fraction with an IC 50 of 62.98 ± 9.8 (HepG2) and 72 ± 11 µg/mL (HCT-116), whereas the chloroform fraction (DC) was potent from D. viscosa with an IC 50 26.4 ± 3.3 (HepG2) and 39.8 ± 13 µg/mL (HCT-116). The remaining fractions and the ethanolic extract displayed moderate cell viability against these cell lines ( Figure 6). Therefore, we selected active fractions (JM, DC) from each plant for the preparation of their nanocomposites with zinc to see whether the synthesized nanocomposites would enhance further cytotoxicity. Interestingly, we found that the nanocomposite DC-ZnO and JM-ZnO NRs enhanced cytotoxicity in the HepG2 and HCT-116 cell lines. The nanocomposite DC-ZnO NRs exhibited cytotoxicity with an IC 50   Although the diverse applications of nanomaterials have resulted in the exponential progression of nano-textured materials over recent years. However, in spite of their wide range of applications, little information is available about their consequences on human health and the environment [92]. There are some reports in the literature that have clearly indicated that accidental exposure to nanomaterials via inhalation, skin contact, or absorption through the gastrointestinal route can pose pronounced threat to humans [93].
To assess the safety of these extracts and nanocomposites on normal cells, the cytotoxicity of these extracts and nanocomposites were performed on normal fibroblast (3T3) cells. As shown in Figure 7, the tested extracts and nanocomposites did not exhibit significant toxicity or there was no significant difference in cell viability with respect to the untreated control cells. As a result, these extracts exclusively had cytotoxic effects on malignant cells and no harmful effect on normal cells.
It has been reported that sclareol, a plant diterpene, inhibits the growth of osteosarcoma tumor cells MG63 (IC 50 of 65.2 µM); inhibits cell proliferation in leukemic, breast, and HeLa cancer cells; induces apoptosis via regulation of the caveolin-1 (Cav-1) protein P13K, STAT5, and NF-kB pathways [94]. Totarol, a diterpenoid exerts anticancer effects on gastric cancer cells, SGC-7901, by induction of apoptosis, cell cycle arrests, and suppression of cancer cell migration [51]. Phenoxy phenol downregulates the expression of α-tubulin and exerts inhibitory effects on hepatocellular carcinoma cells [41]. Isopimara-7,15-dien-19-oic acid has been reported to cause cell death in HeLa by inducing apoptosis through regulation of BCL-2 and FAS genes [50]. Kaurenoic acid, an ent-kaurene-type diterpene exhibits antiproliferative effect in breast, gastric, 293T, HeLa, PANC-1, and cervical cell lines by inducing apoptosis in tumor cells through regulation of the c-FLIP, caspase-3, caspase-8, and miR-2 pathways [95] The presence of these phytochemicals in JM fractions as observed in GC-MS are responsible for the anticancer effect of J. procera. D. viscosa is a flavonoid-rich plant. It is well known that flavonoids exert anticancer effects through many mechanisms. Naringenin has been reported to exert anticancer effects in breast (MDA-MB-231), hepatocellular (HepG2), mammary tumor (E0771), and prostate (PC3, LNCaP) cancer cells by arresting the cell cycle and inducing apoptosis. It also causes suppression of melanoma SK-MEL-28 cells by inhibiting ERK1/2 and JNK MAPKs' phosphorylation [96]. Eriodictyol exerts antiproliferation and anti-metastasis in brain tumor cells by blocking of signaling pathways (NF-kB, PI3K) and induction of apoptosis [97]. Penduletin -4'-methyl ether has been reported to be an aromatase inhibitor with an IC 50 of 1.0 µM [62]. Quercetin, a polyphenolic flavonoid, is useful in cancer prevention as it prevents the various types of cancers. It causes suppression of p38MAPK, the MAPKs pathway in CT26 cells, blocking of the NF-κB pathway, activation of caspase-3, inhibition of EGFR, and increases in the expression of miR-146a [63]. In addition to flavonoids, this plant also contains anticancer terpenoids and phenolics. Stigmasterol, a terpenoid, inhibits cell proliferation in SGC-7901and MGC-803 cells and induces apoptosis and autophagy by blocking Akt/mTOR signaling pathway [75]. Dodonaeaside A and B, triterpenoids, exerted promising anticancer activity with IC 50 values of 0.79 and 0.70 µM, respectively, towards the human ovarian (A2780) cancer cell line [76]. Costunolide, a sesquiterpene lactone, exerts anticancer activities by inducing apoptosis, inhibiting cell proliferation and metastasis as well as angiogenesis [71]. All these compounds reported in D. viscosa could be responsible for the significant anticancer activity exhibited by DC fractions.
Nanomaterials are expected to revolutionize cancer diagnosis and therapy [98]. In this regard, zinc oxide nanomaterials have proved to be one of the most effective candidates in nanomedicine. In this study, the enhanced cytotoxicity of ZnO NRs against selected cancer cells is ascribed to the release of dissolved zinc ions and the induction of the reactive oxygen species, which leads to apoptosis of cancerous cells [20,99]. Moreover, the higher in vitro anticancer activity of JM-ZnO and DC-ZnO nanocomposites over the blank nanorods is attributed to the capping of the abovementioned bioactive compounds (Tables 1 and 2) present in the extracts. The synergistic effect between nanorods and active extracts has resulted in potent anticancer activity of the JM-ZnO and DC-ZnO nanocomposites against the HCT-116 and HepG2 cancer cell lines.

Conclusions
It can be concluded that JM-ZnO and DC-ZnO nanocomposites were successfully synthesized using native Albaha resources and also characterized by various techniques. D. viscosa and J. procera and their JM-ZnO and DC-ZnO nanocomposites possessed anticancer and antimicrobial activities that might be attributed to the presence of important bioactive compounds present in these plants. Additionally, the safety of these extracts and nanocomposites was assessed on normal fibroblast cells and had non-cytotoxic effects on these cells. The GC-MS analysis of fractions directed the tentative identification of possible phytochemicals that are responsible for these activities. Future studies will be focused on the isolation of these bioactive molecules, which can be utilized in the preparation of nano-drugs with appropriate consideration of safety procedures.