BioMed Research International

BioMed Research International / 2021 / Article

Research Article | Open Access

Volume 2021 |Article ID 3123476 | https://doi.org/10.1155/2021/3123476

Shaymaa Fadhel Abbas Albaayit, Rukesh Maharjan, Rasedee Abdullah, Mohd Hezmee Mohd Noor, "Anti-Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer Studies on Clausena excavata Burum. f. (Rutaceae) Leaves", BioMed Research International, vol. 2021, Article ID 3123476, 9 pages, 2021. https://doi.org/10.1155/2021/3123476

Anti-Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer Studies on Clausena excavata Burum. f. (Rutaceae) Leaves

Academic Editor: Gail Mahady
Received08 Jun 2020
Revised15 Jan 2021
Accepted27 Jan 2021
Published23 Feb 2021

Abstract

Background. Clausena excavata Burum. f. has long been applied in ethnomedicine for the treatment of various disorders like rhinitis, headache, cough, wound healing, fever, and detoxification. This study is aimed at investigating the antibacterial activity against Enterococcus faecalis ATCC 49532 using AlamarBlue assay and atomic force microscopy (AFM) as well as the cytotoxicity, anticancer, and phytotoxicity of C. excavata. Method. Bacterial cell viability was performed by using microplate AlamarBlue assay. Atomic force microscopy was used to determine morphological changes in the surface of bacterial cells. Cytotoxicity and phytotoxicity were determined by brine shrimp lethality and Lemna minor bioassay. Caco-2 (colorectal adenocarcinoma) cell line was used for the evaluation of the anticancer effects. Result. Among the fractions tested, ethyl acetate (EA) fraction was found to be active with minimum inhibitory concentration (MIC) of 750 μg/mL against E. faecalis, but other fractions were found to be insensitive to bacterial growth. Microscopically, the EA fraction-treated bacteria showed highly damaged cells with their cytoplasmic content scattered all over. The value of the EA fraction against brine shrimp was more than 1000 μg/mL showing the nontoxic nature of this fraction. Chloroform (CH), EA, and methanol (MOH) fractions of C. excavata were highly herbicidal at the concentration of 1000 μg/mL. EA inhibited Caco-2 cell line with an of 20 μg/mL. Conclusions. This study is the first to reveal anti-E. faecalis property of EA fraction of C. excavata leaves, natural herbicidal, and anticancer agents thus highlight the potential compound present in its leaf which needs to be isolated and tested against multidrug-resistant E. faecalis.

1. Introduction

Since few decades, the emergence of antibiotic-resistant microbial infections has created alarming condition in global health care system due to significant increase in mortality in intensive care unit (ICU) [1, 2]. Development of resistant bacteria is governed by the indiscriminate use of the antibiotics. Other factors governing towards resistance include the usage of antibiotics as growth enhancers in livestock feed [3]. Hence, it is necessary to develop new antibacterial drugs to fight against multidrug-resistant pathogens. Medicinal plants have been subjected for treatment of many diseases from ancient times around the world. These products highly triggered the researchers to obtain natural and safe antibacterial agents due to lack of adverse side effects in comparison to the synthetic antibiotics, such as hypersensitivity, allergic reaction, and immunosuppression [410]. Atomic force microscopy (AFM) has been employed to scan the surface structures in nanoscales, and this property has been used to scan the morphology of bacterial cells [1113].

Clausena excavata Burm. f. is a widespread plant in tropical and subtropical Asian regions. In popular medicine, leaves are used to treat wound, stomachache, headache, fever, malaria, snakebite, and poisoning [7, 11, 14]. C. excavata leaves contain high pharmacologically active coumarins such as furanocoumarins, xanthoxyletin, and nordentatin. These compounds are responsible for the antibacterial activities [11, 1519]. In addition, our previous studies had reported that C. excavata leaves are rich in phenolic content quercetin, myricetin, and kaempferol, which help to suppress bacterial infections [20, 21].

Cytotoxicity testing provides important preliminary data for the selection of natural compounds with potential antimicrobial properties for future work [22]. Brine shrimp lethality bioassay plays an important role for getting information about the safety of compounds from crude extracts which exhibited antimicrobial activity. Due to the sensitivity of brine shrimp to a variety of chemical substances, many researchers had considered this assay for searching potent noncytotoxic natural compounds having biological and pharmacological properties [2325]. Although previous studies showed antimicrobial activity of the bark, leaf, and stem of this plant [26, 27], there is no recorded data for anti-Enterococcus faecalis.

Use of herbicides has enhanced economic growth by increasing the production of food but its excessive use has resulted serious health implications to environment especially in soil and water ecosystem, thus caused health hazards to man and animals [28]. In comparison to synthetic compounds, natural compounds were found to be less toxic due to the absence of halogenated molecules, hydrophilic property, and a lower half-life, providing suppressive or inhibitory activity at lower concentrations [29]. The phytotoxicity (Lemna minor) assay is a general useful assay for screening plants having weedicidal property. Some natural antitumor compounds showed positive correlation with Lemna growth inhibition [30].

Cancer is one of the life threatening diseases responsible for high mortality all over the world. Colon cancer is the third leading cause of cancer-related deaths across the globe [31]. Recent studies done in Iraq showed an escalating rise in the incidence of colorectal cancer [32]. In some cases, even surgical or radio therapy could not cure or prevent the recurrence and metastasis of tumor. Hence, many investigators are focusing on natural products to have safe and effective anticancer drugs from natural source [33].

The antibacterial activity of C. excavata and other species of this genus Clausena had already been reported for their antibacterial property against some gram-positive and gram-negative bacteria [3437], but no any records against E. faecalis have been reported during our literature survey, which are naturally present in gastrointestinal tract; therefore, it is recommended to traditional medicine practitioners to use this plant against gastrointestinal infections caused by this pathogen. Thus, this study was undertaken to evaluate the anti-E. faecalis as well as cytotoxic, phytotoxic, and antiproliferation activity towards Caco-2 cell line of C. excavata leaf solvent fractions.

2. Materials and Methods

2.1. Plant Material

The leaves of C. excavata plant were obtained from Pendang, Kedah, Malaysia (5°59N, 100°28E) on December 2010 and were identified by Dr. Shamsul Khamis (Resident Botanist) at the Biodiversity Unit, Institute of Bioscience, Universiti Putra Malaysia, voucher specimen (TI-013201-CE). Naturally grown plants in fields were collected. As it was not listed in endangered species, therefore, no specific permission was required to collect plant. Fresh leaves were dried in room temperature, powdered, and macerated in 1 : 5 dried leaf weights to solvent (petroleum ether (PET)) volume ratio for 3 days. The filtrate was collected, and the residues were subjected to further macerate with chloroform (CH), ethyl acetate (EA), and methanol (MOH) sequentially. The suspensions were collected, and solvents were removed under reduced pressure using rotary evaporator at 45-50°C to obtain crude extracts [19]. EA was sent for LCMS/MS analysis to screen for the presence of phytochemicals (Advance Chemistry Solution, GHOD Sdn Bhd ACD/Labs Inc., Malaysia).

2.2. Antibacterial Activity

The minimum inhibitory concentration (MIC) for the E. faecalis ATCC 49532 was determined by colorimetric indices in 96-well plates. The fractions were prepared using Mueller Hinton Broth (MHB) media to make concentration range (46.18-3000 μg/mL) such that their total volume became 100 μL in each well. Fully grown E. faecalis was diluted 1000 times in MHB media, and 100 μL of this suspension was aliquoted to all wells containing 100 μL of fraction such that bacteria will be approximately of CFU/mL. Untreated control was with only cells and media. All samples were in triplicates. The plate was sealed and incubated at 37°C for 18-20 h. The next day, all wells were visually checked to confirm the clear and turbid wells, and, then, 20 μL of AlamarBlue dye was added, and the plate was incubated in the dark for 2 h in a shaking incubator at 37°C. Absorbance were recorded at 570 and 600 nm using a spectrophotometer (Thermo Scientific, USA), and the percent of inhibition of bacteria due to fractions was calculated by putting the absorbance values in the formula mentioned by Lancaster and Fields [38]. where is molar coefficient of AlamarBlue dye at different wavelengths (570 nm) and (600 nm) and and are absorbance of test and control wells, respectively.

2.3. Atomic Force Microscopy (AFM)

The EA fraction which was active against E. faecalis was subjected to the AFM study. Bacterial samples were prepared using inoculum of CFU/mL and treated with the MIC concentration of EA fraction while, untreated control contained only media and bacteria. After incubation for 3 h, cells were centrifuged at 5000 rpm for 5 min and washed twice with double distilled water (D.W). These cells were resuspended in 50 μL of sterile distilled water, and 10 μL of this suspension from treated and nontreated samples was spread in poly-l-lysine (0.01%) precoated silicon wafer slides and left it overnight for drying [11, 39, 40]. Sample scanning was carried out with AFM (Agilent Technologies-5500, AZ, USA) in tapping mode. All 2D and 3D topographical and pseudocolor images were analyzed using the PicoView 1.2 imaging analysis software.

2.4. Brine Shrimp (Artemia salina) Lethality Bioassay

A stock solution of artificial seawater was made by dissolving 38 g of sea salt in 1 L of distilled water; thus, filtered solution would be pH 7.4. This solution was kept in the hatching tray ( cm) with perforated partition. On one half of this tray, 50 mg of brine shrimp eggs (San Francisco Bay Brand) was sprinkled and covered by aluminium foil on its top so that light could not penetrate inside, whereas lamp was kept on the other half so that larvae after growth will come out towards the light portion through partition pores. Stock solutions of fractions (10 mg/mL) were made in methanol, and 5, 50, and 500 μL from this stock solution were dispensed in new vial in triplicates such that final concentration would be 10, 100, and 1000 μg/mL, respectively. These vials were allowed for drying overnight. After 24 h of hatching, 10 active, motile nauplii were transferred in vial in triplicates by using a Pasteur pipette, and 5 mL of sea water was added in each vial and incubated for 24 h at 25-27°C under illumination. Vial with Etoposide as standard positive control at value of 7.5 μg/mL was used. Only solvent was used as untreated control. Next day, the numbers of live and dead larvae was counted and find the percentage inhibition of larvae due to test fractions [7, 41, 42].

2.5. Phytotoxicity

A one-liter stock solution of inorganic E-medium was made in distilled water by mixing appropriate inorganic constituents [43], and pH 7 was maintained by adding potassium hydroxide pellets. Subsequently, a working solution was made by diluting the stock solution 10 times with distilled water. A stock solution (20 mg/mL) of different fractions of C. excavata leaves was made in methanol and dispensed 10, 100, and 1000 μL of these solutions to the flasks in triplicates so that final concentration would become 10, 100, and 1000 μg/mL, respectively, and left overnight for solvent evaporation. Next day, all flasks were filled with 20 mL of working E-medium solution and then 20 fronds of green, healthy L. minor were added to each flask and sealed the flask by parafilm and pricked the parafilm to make hole so that air passes inside the flask. In untreated control, there were no any compounds and served as the positive growth control, whereas standard plant growth inhibitor drug (paraquat at 0.015 μg/mL) served as negative growth control. All these flasks were placed in growth cabinet for 7 days with maintained temperature at 27°C, relative humidity %, and light intensity of 9000 lux. On the 7th day, the numbers of dead and live fronds in each flask was counted and calculated the percentage inhibition of each compound by using below mentioned formula [44].

2.6. 3-(4,5-Dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium Bromide (MTT) Assay of Fractions against Caco-2 Cell Line

The cytotoxicity of samples against human colon cancer Caco-2 (ATCC® HTB37™) cell line was determined by using the MTT assay [45, 46]. This cell line was obtained from the cell culture bank, Panjwani Centre for Molecular Medicine and Drug Research, ICCBS, Karachi. These cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% nonessential amino acid and incubated at 37°C in 5% CO2 incubator. Fetal bovine serum is required for proper growth, and it is also required to neutralize trypsin during trypsinization process. DMEM was replaced every 48-72 h until 80% confluence was achieved.

Cells at passage number 5 were seeded into 96-well plates at density 5000 cells/well in 100 μL culture medium. After 24 h incubation, the medium was replaced with fresh medium containing different fractions (EA, CH, and MOH) in various concentrations (100, 50, 25, and 12.5 μg/mL) and incubated for 24 h. Untreated well served as positive growth control. After incubation, old medium was replaced by fresh medium containing 0.5 mg/mL of MTT dye and incubated further for 4 h. The medium containing MTT dye was removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to all wells to solubilize the formed formazan crystals. After 10 min, plate was shaken for 30 sec and absorbance was recorded at 570 nm in spectrophotometer (Multiskan GO, Thermo Scientific). In this assay, doxorubicin at 0.501 μg/mL was used as a standard drug. The percentage of cytotoxicity of compounds was determined by comparing with the untreated positive growth control. All experiments were performed in triplicate [45].

3. Statistical Analysis

Data of each fraction were presented in () values and compared with value of positive drug control. One-way analysis of variance (ANOVA) was used in the GraphPad Prism software version 5.0 and analyzed with level of significance ().

3.1. Results
3.2. Antibacterial Study

The change in color of the dye from blue to pink indicated the reduction of dye due to reducing environment of viable cells, whereas blue color indicated nonviable cells. The decrease in AlamarBlue dye reduction suggested that the fractions have antibacterial properties. The antienterococcal activities of different fractions of C. excavata leaves are presented in Table 1. The study showed that the EA fraction was found to be active against E. faecalis at MIC of 750 μg/mL, whereas other fractions were found to be inactive. Thus, EA fraction was carried out for the AFM study.


Fraction namesEnterococcus faecalis (ATCC 49532)
MIC (μg/mL)% inhibition ()

Chloroform (CH) fractionNot active
Ethyl acetate (EA) fraction750
Methanol (MOH) fractionNot active
Petroleum ether (PET) fractionNot active

3.3. Atomic Force Microscopy

AFM technique was used to visualize the morphological changes on bacterial strain due to antibacterial effect of ethyl acetate fraction at its MIC value. The images of untreated E. faecalis cells showed cocci arranged in clusters, with some short chains. These cells were typically oval shaped, smooth surfaces with a mean diameter of 0.5-1.0 μm (Figure 1(a)). No visible pores or rupture were seen on surface indicating the well-preserved structural integrity. Upon treatment with EA fraction, significant morphological damages were seen with loss of cluster formation (Figure 1(b)). Large amount of cytoplasmic content was spilled all over, thus confirming the antibacterial effect of EA fraction.

3.4. Brine Shrimp Lethality Assay

Among different fractions, only ethyl acetate showed slight toxicity (36.6% lethality) against A. salina larvae at 1000 μg/mL whereas lethality of other fractions even at 1000 μg/mL was less than 25% showing the nontoxic nature of these fractions (Figure 2). This result showed that these fractions are noncytotoxic even at higher concentrations so these could be used in ethnomedicine with minimal side effects.

3.5. Phytotoxic Activity

EA, CH, and MOH fractions were found to be highly active at 1000 μg/mL, but inactive in lower concentrations of 100 and 10 μg/mL, while PET showed moderate phytotoxic activities at same dose (Table 2). The of paraquat was 0.02 μg/mL which was much lower as compared to EA fraction. There was significant difference () between fraction and drug control value.


Concentration (μg/mL)% inhibition at different concentrations
Methanol (MOH)Ethyl acetate (EA)Chloroform (CH)Petroleum ether (PET)Paraquat

100010010010048
100025.78.5714.28
100005.7
value (μg/mL)313.1213.3283.1>10000.02

3.6. Effect of C. excavata Leaf Fractions on Caco-2 Colorectal Cancer Cell Lines

The two fractions (CH and EA) of C. excavata showed in vitro growth inhibition effects on the Caco-2 cell line. At 100 and 50 μg/mL, EA fraction showed more than 90% inhibition, whereas CH fraction showed 70-80% inhibition at 50 μg/mL. of EA and CH fraction were found to be 20 and 40 μg/mL, respectively, which were significantly higher () as compared to doxorubicin (μg/mL). Therefore, EA fraction found to be more suitable for further study on colorectal cancer activity. The MOH showed less effect throughout the range of tested concentrations in Caco-2 cell line (Table 3).


Concentration (μg/mL)Ethyl acetate (EA)Chloroform (CH)Methanol (MOH)Doxorubicin

10097.797.003<20
5096.274.1<20
2588.3924.2<20
12.537.4121.1<20
value (μg/mL)2040Not active0.501

4. Discussion

With the escalation of multidrug-resistant bacterial pathogens, the advancement of ethnopharmacology became a focus area for researchers in the discovery of alternative natural drug with potent antimicrobial activities [47]. C. excavata leaves have been widely used in folklore medicine, but only a few scientific evidences proved its therapeutic properties and mechanism of action [14]. C. excavata was reported to possess antibacterial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Proteus vulgaris, and Shigella flexnari [11, 48]. In this study, the C. excavata leaves fractions were first time reported for their anti-E. faecalis properties by using AlamarBlue assay. In this assay, resazurin, a redox-sensitive dye which is originally blue in color has the ability to penetrate the viable cells. Inside the cell, due to reducing environment, resazurin is reduced and converted to resorufin giving pink coloration [49]. Thus, changing of color from blue to pink shows the viability of cells. This assay is rapid, simple, and low cost with inexpensive instrument; therefore, it is widely used in several viability/proliferation assays for bacteria, fungi, cancer lines, and other viable cells. By using this assay, we assigned the MIC value to that concentration in which there was blue coloration, and below this concentration, there was pink coloration due to growth of bacteria. Among different fractions, EA fraction showed good anti-E. faecalis activity and its further analysis by AFM technique showed scattered cytoplasmic content confirming their lost cellular integrity.

The nonpolar solvent (EA) fraction was active at 750 μg/mL; hence, it proved that the nonpolar active compounds present in nonpolar fraction are responsible for the anti-E. faecalis activity, as previously reported in other plants [50]. Coumarin and carbazole derivatives, isolated from nonpolar solvent extract of roots and leaves of C. excavata, have been reported for their antimicrobial, wound healing, and antioxidant properties [1518]. Previous studies reported that the mechanism of action of natural drug was focused to show their effects on surface morphology [51].

In brine shrimp lethality assay, the nonpolar solvent (EA and CH) fractions reduced brine shrimp survival as compared with the other fractions at 1000 μg/mL. This may be due to high coumarin content in these fractions [25]. Earlier reports have shown that substances from natural products were known as toxic if the μg/mL. Thus, EA and CH fractions were considered nontoxic on brine shrimp since the is more than 1000 μg/mL [25, 52]. The outcome is in agreement with article reported by Albaayit et al. [15], who demonstrated that EA and CH fractions did not inhibit Vero, a kidney epithelial cell line.

Since last few decades, overuse of herbicides led to the emergence of herbicide-resistant weeds, because of which conventional synthetic herbicides became less effective and caused low crop yield with massive economic loss and health and environmental-related concerns. Therefore, herbicide researchers are trying to make new herbicides by isolating potent compounds from natural sources [53]. The CH, EA, and MOH fractions showed the most significant phytotoxic (100% mortality) effect at tested concentrations of 1000 μg/mL, while PET showed moderate phytotoxic activities at same dose. The findings are similar to that of previous studies, which have reported that plants containing phenolic compounds and their derivatives as potential inhibitors of seedling growth [54, 55].

Till date, many anticancer compounds have been isolated from this plant. Excavatine A, a carbazole alkaloid, has been reported for its anticancer effect on adenocarcinomic human alveolar basal epithelial cells and cervical cancer. Clausine-TY, a carbazole alkaloid, showed anticancer activity against CEM-SS cell line [56]. Propyl-cannabinol allyldimethylsilyl ether compound showed anticancer effect on HT29, MCF-7, and HepG2 cell lines [57]. Clausine B, a carbazole alkaloid, showed antiproliferative activities against MDA-MB-231, HeLa, CAOV3, and HepG2 cell lines [58]. Three carbazole alkaloids (Clausine-E, Murrayanine, and Clauszoline J) also showed potent antiproliferative activity against NCI-H187, MCF-7, and KB cell lines [59]. The inhibition of Caco-2 cell line growth by C. excavata (EA and CH fractions) might be due to the presences of phenolic compounds like coumarin that induces apoptosis by activating the caspase-3-dependent apoptotic pathway and the mitochondrial pathway, by downregulating antiapoptotic genes (Bcl-2 and Bcl-xL), upregulating caspase-3 with the release of cytochrome c. and arrest and premature aging, and enhancing the immune system to destroy cancer cells [6062].

5. Conclusion

This study first time reports the anti-E. faecalis activity of ethyl acetate fraction of C. excavata leaves; thus, the antibacterial compounds previously isolated from this plant may be active against MDR E. faecalis and could be used in gastrointestinal infections after further investigation. The ethyl acetate fraction is noncytotoxic to brine shrimp larvae and possesses anticolorectal activity. Due to its phytotoxic property, it could be used as a herbicide. In our future work, we will test its in vivo efficacy in the treatment of gastrointestinal infections caused by E. faecalis.

Data Availability

Data used to support the findings of this study are included within the article

Conflicts of Interest

We wish to confirm that there are no known conflicts of interest associated with this publication.

Acknowledgments

The authors show utmost gratitude and appreciation to NAM-ICCBS (International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan) and Prof. Dr. M. Iqbal Choudhary (Director, ICCBS) for fellowship Award to Shaymaa Fadhel Abaas Albaayit.

References

  1. A. Haddadin, S. Fappiano, and P. Lipsett, “Methicillin resistant Staphylococcus aureus (MRSA) in the intensive care unit,” Postgraduate Medical Journal, vol. 78, no. 921, pp. 385–392, 2002. View at: Publisher Site | Google Scholar
  2. H. T. Ibraheem and H. J. F. Al-Mathkhury, “pvl-carried methicillin resistant Staphylococcus aureus isolated from hospitalized patients in Baghdad, Iraq,” Iraqi Journal of Science, vol. 59, pp. 1967–1972, 2018. View at: Publisher Site | Google Scholar
  3. C. P. Mudzengi, A. Murwira, M. Tivapasi, C. Murungweni, J. V. Burumu, and T. Halimani, “Antibacterial activity of aqueous and methanol extracts of selected species used in livestock health management,” Pharmaceutical Biology, vol. 55, no. 1, pp. 1054–1060, 2017. View at: Publisher Site | Google Scholar
  4. L. K. Al-Ani, M. I. Yonus, B. A. Mahdii et al., “First record of use Fusarium proliferatum fungi in direct treatment to control the adult of wheat flour Tribolium confusum, as well as, use the entomopathogenic fungi Beauveria bassiana,” Ecology, Environment and Conservation, vol. 24, no. 3, pp. 29–34, 2018. View at: Google Scholar
  5. R. M. Al-Bahrani, H. M. Radif, and S. F. A. Albaayit, “Evaluation of potent silver nanoparticles production from Agaricus bisporus against Helicobacter pylori,” Pakistan Journal of Agricultural Sciences, vol. 57, no. 4, pp. 1197–1201, 2020. View at: Google Scholar
  6. T. H. S. Al-naddawi, S. F. A. Albaayit, and Z. Z. Khalaf, “The synergism and antagonism behavior of aqueous extraction for black tea, green tea and coffee against the effectiveness of certain antibiotics,” The Eurasia Proceedings of Science Technology Engineering and Mathematics, vol. 6, no. 6, pp. 39–42, 2019. View at: Google Scholar
  7. S. F. A. Albaayit, R. Abdullah, N. Abdullah, and Y. Abba, “Methanolic extract of Clausena excavata promotes wound healing via antiinflammatory and anti-apoptotic activities,” Asian Pacific Journal of Tropical Biomedicine, vol. 10, no. 5, p. 232, 2020. View at: Publisher Site | Google Scholar
  8. S. F. A. Albaayit and R. Maharjan, “Immunomodulation of Zerumbone via decreasing the production of reactive oxygen species from immune cells,” Sciences, vol. 21, pp. 475–479, 2018. View at: Google Scholar
  9. K. Dhama, R. Tiwari, S. Chakraborty et al., “Evidence based antibacterial potentials of medicinal plants and herbs countering bacterial pathogens especially in the era of emerging drug resistance: an integrated update,” International Journal of Pharmacology, vol. 10, pp. 1–43, 2014. View at: Google Scholar
  10. S. F. A. Albaayit, R. Abdullah, and N. Abdullah, “Zerumbone-loaded nanostructured lipid carrier gel facilitates wound healing in rats,” Revista Brasileira de Farmacognosia, vol. 30, no. 2, pp. 272–278, 2020. View at: Publisher Site | Google Scholar
  11. S. F. A. Albaayit, “Evaluation of anti-methicillin resistant Staphylococcus aureus property of Clausena excavata leaves by using atomic force microscopy and flowcytometry techniques,” Pakistan Journal of Agricultural Sciences, vol. 58, no. 1, pp. 315–320, 2021. View at: Publisher Site | Google Scholar
  12. A. Farhan, A. A. Mehde, W. A. Mehde, R. A. Jassim, N. J. Kadhim, and N. A. Jasim, “Synthesis of silver nanoparticles from leaf extract of olive and fig with silver nitrate and effect on Ecto-5- nucleotidase (5-NT), ADA and AMPDA enzymes in sera of arthrosclerosis patients,” Journal of Chemical Sciences, vol. 14, pp. 1805–1817, 2016. View at: Google Scholar
  13. K. D. Song, K. Liu, T. Q. Liu, and X. H. Ma, “Characterization and effect of modified surface on the morphology, structure and function of rabbit bone marrow-derived mesenchymal stem cells 1,” Asia-Pacific Journal of Chemical Engineering, vol. 4, no. 5, pp. 765–770, 2009. View at: Publisher Site | Google Scholar
  14. S. F. A. Albaayit, Y. Abba, R. Abdullah, and N. Abdullah, “Prophylactic effects of Clausena excavata Burum. f. leaf extract in ethanol-induced gastric ulcers,” Drug Design, Development and Therapy, vol. 10, pp. 1973–1986, 2016. View at: Publisher Site | Google Scholar
  15. S. F. A. Albaayit, Y. Abba, R. Abdullah, and N. Abdullah, “Effect of Clausena excavata Burm. f. (Rutaceae) leaf extract on wound healing and antioxidant activity in rats,” Drug Design, Development and Therapy, vol. 9, pp. 3507–3518, 2015. View at: Publisher Site | Google Scholar
  16. A. Sunthitikawinsakul, N. Kongkathip, B. Kongkathip, S. Phonnakhu, and J. W. Daly, “Coumarins and carbazoles from Clausena excavata exhibited antimycobacterial and antifungal activities,” Planta Medica, vol. 69, no. 2, pp. 155–157, 2003. View at: Publisher Site | Google Scholar
  17. V. B. Tsassi, H. Hussain, B. Y. Meffo et al., “Antimicrobial coumarins from the stem bark of Afraegle paniculata,” Natural Product Communications, vol. 5, no. 4, pp. 559–561, 2010. View at: Google Scholar
  18. T. S. Wu and H. Furukawa, “Biological and phytochemical investigation of Clausena excavata,” Journal of Natural Products, vol. 45, no. 6, pp. 718–720, 1982. View at: Publisher Site | Google Scholar
  19. L. Huang, Z. L. Feng, Y. T. Wang, and L. G. Lin, “Anticancer carbazole alkaloids and coumarins from Clausena plants: a review,” Chinese Journal of Natural Medicines, vol. 15, no. 12, pp. 881–888, 2017. View at: Publisher Site | Google Scholar
  20. S. F. A. Albaayit, A. S. K. Al-Khafaji, and H. S. Alnaimy, “In vitro macrophage nitric oxide and interleukin-1 beta suppression by Moringa peregrina seed,” Turkish Journal of Pharmaceutical Sciences, vol. 16, no. 3, pp. 362–365, 2019. View at: Publisher Site | Google Scholar
  21. S. F. A. Albaayit, Y. Abba, R. Abdullah, and N. Abdullah, “Evaluation of antioxidant activity and acute toxicity of Clausena excavata leaves extract,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 975450, 10 pages, 2014. View at: Publisher Site | Google Scholar
  22. N. H. Harlita, N. S. M. Sagi, and P. Astuti, “Acute toxicity of cashew nut Shell extract (Anacardium occidentale L.) in albino rat (Rattus norvegicus Berkenhout 1769),” Pakistan Journal of Biological Sciences, vol. 19, no. 2, pp. 89–94, 2016. View at: Publisher Site | Google Scholar
  23. R. R. Bomfim, A. A. Araujo, S. Cuadros-Orellana, M. G. Melo, and L. J. Quintans-Junior, “Larvicidal activity of Cladonia substellata extract and usnic acid against Aedes aegypti and Artemia salina,” Latin American Journal of Pharmacy, vol. 28, pp. 580–584, 2009. View at: Google Scholar
  24. S. Hassarangsee, J. Uthaibutra, N. Nomura, and K. Whangchai, “Degradability of treated ethion insecticide by TiO2 photocatalysis,” Pakistan Journal of Biological Sciences, vol. 18, no. 1, pp. 27–31, 2015. View at: Publisher Site | Google Scholar
  25. S. Sasidharan, I. Darah, and K. Jain, “In vivo. and in vitro. toxicity study of Gracilaria changii,” Pharmaceutical Biology, vol. 46, no. 6, pp. 413–417, 2008. View at: Publisher Site | Google Scholar
  26. S. Chakthong, N. Bindulem, S. Raknai, S. Yodwaree, S. Kaewsanee, and A. Kanjana-Opas, “Carbazole-pyranocoumarin conjugate and two carbazole alkaloids from the stems of Clausena excavata,” Natural Product Research, vol. 30, no. 15, pp. 1690–1697, 2016. View at: Publisher Site | Google Scholar
  27. P. Lim, H. Ramli, N. Kassim et al., “Chemical constituents from the stem bark of Clausena excavata Burm. f,” Biochemical Systematics and Ecology, vol. 82, pp. 52–55, 2019. View at: Publisher Site | Google Scholar
  28. M. Imatomi, P. Novaes, M. A. F. M. Miranda, and S. C. J. Gualtieri, “Phytotoxic effects of aqueous leaf extracts of four Myrtaceae species on three weeds,” Acta Scientiarum Agronomy, vol. 37, no. 2, pp. 241–248, 2015. View at: Publisher Site | Google Scholar
  29. Z. Xiao, S. L. Morris-Natschke, and K. H. Lee, “Strategies for the optimization of natural leads to anticancer drugs or drug candidates,” Medicinal Research Reviews, vol. 36, no. 1, pp. 32–91, 2016. View at: Publisher Site | Google Scholar
  30. F. Ayaz, N. Küçükboyacı, B. Bani, B. Şener, and M. I. Choudhary, “Phytotoxicity, toxicity on brine shrimp and insecticidal effect of Chrysophthalmum gueneri Aytac & Anderb. Growing in Turkey,” Turkish Journal of Pharmaceutical Sciences, vol. 15, no. 3, pp. 382–385, 2018. View at: Publisher Site | Google Scholar
  31. S. H. Arani and M. A. Kerachian, “Rising rates of colorectal cancer among younger Iranians: is diet to blame?” Current Oncology, vol. 24, pp. e131–e137, 2017. View at: Publisher Site | Google Scholar
  32. K. I. Mahmood, K. F. Namiq, C. A. Muhamad, B. O. Muhamad, K. M. Majed, and H. N. Fattah, “A descriptive study of colorectal cancer in Hiwa Cancer Hospital, Sulaymaniyah, Iraq,” Science Journal of University of Zakho, vol. 5, no. 3, pp. 244–248, 2017. View at: Publisher Site | Google Scholar
  33. P. C. Jimenez, D. V. Wilke, and L. V. Costa-Lotufo, “Marine drugs for cancer: surfacing biotechnological innovations from the oceans,” Clinics, vol. 73, article e482s, Supplement 1, 2018. View at: Publisher Site | Google Scholar
  34. N. Agyepong, C. Agyare, M. Adarkwa-Yiadom, and S. Y. Gbedema, “Phytochemical investigation and anti-microbial activity of Clausena anisata (Willd), Hook,” African Journal of Traditional, Complementary and Alternative Medicines, vol. 11, no. 3, pp. 200–209, 2014. View at: Publisher Site | Google Scholar
  35. R. Begum, M. S. Rahman, M. R. Haque, A. S. Chowdhury, and M. A. Rashid, “Antimicrobial activity and cytotoxicity of Clausena suffruticosa,” Dhaka University Journal of Pharmaceutical Sciences, vol. 5, pp. 81–83, 2006. View at: Google Scholar
  36. P. T. Diep, A. M. Pawlowska, P. L. Cioni, C. V. Minh, M. le Huong, and A. Braca, “Chemical composition and antimicrobial activity of Clausena indica (Dalz) Oliv. (Rutaceae) essential oil from Vietnam,” Natural Product Communications, vol. 4, no. 6, pp. 869–872, 2009. View at: Google Scholar
  37. M. Fakruddin, K. S. Mannan, R. M. Mazumdar, and H. Afroz, “Antibacterial, antifungal and antioxidant activities of the ethanol extract of the stem bark of Clausena heptaphylla,” BMC Complementary and Alternative Medicine, vol. 12, no. 1, p. 232, 2012. View at: Publisher Site | Google Scholar
  38. M. V. Lancaster and R. D. Fields, “Antibiotic and cytotoxic drug susceptibility assays using resazurin and poising agents,” 1996, US Patent 5-501959. View at: Google Scholar
  39. Y. Aqeel, R. Siddiqui, M. Ateeq, M. Raza Shah, H. Kulsoom, and N. A. Khan, “Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts,” The Journal of Eukaryotic Microbiology, vol. 62, no. 1, pp. 85–94, 2015. View at: Publisher Site | Google Scholar
  40. N. K. Abass, Z. J. Shanan, T. H. Mohammed, and L. K. Abbas, “Fabricated of Cu doped ZnO nanoparticles for solar cell application,” Baghdad Science Journal, vol. 15, no. 2, 2018. View at: Publisher Site | Google Scholar
  41. S. F. A. Albaayit, R. Maharjan, and M. Khan, “Evaluation of hemolysis activity of Zerumbone on RBCs and brine shrimp toxicity,” Baghdad Science Journal, vol. 18, no. 1, article 0065, pp. 65–69, 2021. View at: Publisher Site | Google Scholar
  42. S. F. A. Albaayit and M. Ozaslan, “Cytotoxic and urease inhibition potential of Moringa peregrina seed ethanolic extract,” International Journal of Pharmacology, vol. 15, pp. 151–155, 2019. View at: Google Scholar
  43. A. Bashir, N. Ali, S. Bashir, and M. Choudhary, “Biological activities of aerial parts of Tylophora hirsuta Wall,” African Journal of Biotechnology, vol. 8, no. 18, 2009. View at: Google Scholar
  44. M. Khurm, B. A. Chaudhry, M. Uzair, and K. H. Janbaz, “Antimicrobial, cytotoxic, phytotoxic and antioxidant potential of Heliotropium strigosum Willd,” Medicine, vol. 3, no. 3, p. 20, 2016. View at: Publisher Site | Google Scholar
  45. S. F. A. Albaayit, “In vitro evaluation of anticancer activity of Moringa peregrina seeds on breast cancer cells,” The Eurasia Proceedings of Science Technology Engineering and Mathematics, vol. 11, pp. 163–166, 2020. View at: Google Scholar
  46. J. Kowapradit, P. Opanasopit, T. Ngawhirunpat et al., “In vitro permeability enhancement in intestinal epithelial cells (Caco-2) monolayer of water soluble quaternary ammonium chitosan derivatives,” AAPS PharmSciTech, vol. 11, no. 2, pp. 497–508, 2010. View at: Publisher Site | Google Scholar
  47. F. Benbelaïd, A. Khadir, M. A. Abdoune, M. Bendahou, A. Muselli, and J. Costa, “Antimicrobial activity of some essential oils against oral multidrug–resistant Enterococcus faecalis in both planktonic and biofilm state,” Asian Pacific Journal of Tropical Biomedicine, vol. 4, pp. 463–472, 2014. View at: Publisher Site | Google Scholar
  48. Y. Jagadeesan and E. Kuppusamy, “Antioxidant activity, mosquitocidal activity, antibacterial activity of solvent extract and synthesized nanoparticles of Clausena excavata (burm.) f.(Rutaceae) against selected mosquitoes and pathogenic bacteria,” LIFE: International Journal of Health and Life-Sciences, vol. 5, no. 1, pp. 19–39, 2019. View at: Publisher Site | Google Scholar
  49. E. Travnickova, P. Mikula, J. Oprsal et al., “Resazurin assay for assessment of antimicrobial properties of electrospun nanofiber filtration membranes,” AMB Express, vol. 9, no. 1, pp. 1–11, 2019. View at: Publisher Site | Google Scholar
  50. S. Priya, A. Noor, and P. K. Satheeshkumar, “Antioxidant and anti-proliferative activity of different solvent extracts of Casuarina equisetifolia needles,” Phytomedicine, vol. 4, pp. 99–107, 2014. View at: Google Scholar
  51. P. Skandamis, K. Koutsoumanis, K. Fasseas, and G. J. E. Nychas, “Inhibition of oregano essential oil and EDTA on Escherichia coli O157: H7,” Italian Journal of Food Science, vol. 13, pp. 65–75, 2001. View at: Google Scholar
  52. A. Riaz, A. Rasul, G. Hussain et al., “Astragalin: a bioactive phytochemical with potential therapeutic activities,” Advances in Pharmacological Sciences, vol. 2018, Article ID 9794625, 15 pages, 2018. View at: Publisher Site | Google Scholar
  53. A. Rauf, N. Muhammad, A. Khan, N. Uddin, and M. Atif, “Antibacterial and phytotoxic profile of selected Pakistani medicinal plants,” World Applied Sciences Journal, vol. 20, pp. 540–544, 2012. View at: Google Scholar
  54. G. Dastagir and F. Hussain, “Phytotoxic and insecticidal activity of plants of family Zygophyllaceae and Euphorbiaceae,” Sarhad Journal of Agriculture, vol. 29, pp. 83–91, 2013. View at: Google Scholar
  55. S. Shah, S. M. Shah, Z. Ahmad et al., “Phytochemicals, in vitro antioxidant, total phenolic contents and phytotoxic activity of Cornus macrophylla Wall bark collected from the North-West of Pakistan,” Pakistan Journal of Pharmaceutical Sciences, vol. 28, no. 1, pp. 23–28, 2015. View at: Google Scholar
  56. Y. H. Taufiq-Yap, T. H. Peh, G. C. L. Ee et al., “A new cytotoxic carbazole alkaloid from Clausena excavata,” Natural Product Research, vol. 21, no. 9, pp. 810–813, 2007. View at: Publisher Site | Google Scholar
  57. A. S. Al-Abboodi and M. Al-Farawn, “Evaluation the anticancer activity of propyl-cannabinol allyldimethylsilyl ether against different cancer cell lines,” in 2nd International Conference on Materials Engineering & Science (IConMEAS 2019), 2020. View at: Publisher Site | Google Scholar
  58. W. N. I. W. Mohd, A. R. Zain, F. Othman, and T. Y. H. Yap, “Antiproliferative properties of Clausine-B against cancer cell lines,” The Malaysian Journal of Medical Sciences, vol. 16, no. 3, p. 29, 2009. View at: Google Scholar
  59. T. Sripisut, S. Cheenpracha, T. Ritthiwigrom, U. Prawat, and S. Laphookhieo, “Chemical constituents from the roots of Clausena excavata and their cytotoxicity,” Records of Natural Products, vol. 6, no. 4, pp. 386–389, 2012. View at: Google Scholar
  60. J. Chuang, Y. Huang, H. Lu et al., “Coumarin induces cell cycle arrest and apoptosis in human cervical cancer HeLa cells through a mitochondria- and caspase-3 dependent mechanism and NF-kappaB down-regulation,” In Vivo, vol. 21, no. 6, pp. 1003–1009, 2007. View at: Google Scholar
  61. W. Y. Huang, Y. Z. Cai, and Y. Zhang, “Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention,” Nutrition and Cancer, vol. 62, no. 1, pp. 1–20, 2009. View at: Publisher Site | Google Scholar
  62. N. E. B. Saidu, S. Valente, E. Bana, G. Kirsch, D. Bagrel, and M. Montenarh, “Coumarin polysulfides inhibit cell growth and induce apoptosis in HCT116 colon cancer cells,” Bioorganic & Medicinal Chemistry, vol. 20, no. 4, pp. 1584–1593, 2012. View at: Publisher Site | Google Scholar

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