The cytotoxic activity of plant extracts was determined using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyltetrazolium bromide] assay (Applichem, USA). This assay detects the reduction of MTT by mitochondrial dehydrogenase to a blue formazan product, which reflects the function of mitochondria and cell viability. 19 Exponentially growing cells at 2x10 4 cells/mL were plated in triplicate into 96-well plates (Greiner, Germany) in 200 µL of growth medium and incubated for 24 hr before the addition of extracts. Plant extracts were dissolved in 10% DMSO and added to the cell culture at a final concentration of 4 mg/mL to be tested against six cell lines. Cells were incubated for 72 hr at 37°C in a 5% CO 2 incubator. For the dose and time responsiveness of the cells, plant extracts were added at 0.1 mg/mL, 0.2 mg/mL, 0.5 mg/mL, 1 mg/mL, and 2 mg/mL final concentrations, and incubated for 24, 48, and 72 hr. After that, 10 µL of PBS containing 5 mg/mL MTT was added into each well. After 4 hr incubation, the medium was discarded and the formazan blue crystals that formed in the cells were dissolved in 100 µL DMSO. Reduced MTT was quantified by reading the absorbance at 540 nm on a microplate reader (Thermo Scientific Multiscan Spectrum). The cytotoxic effects of the tested extracts were determined by comparing the optical density of the treated cells against the optical density of the untreated cells. Cytotoxicity relative to controls was calculated using the following formula: Firstly, nutrition broth media was prepared and kept for sterilization. Then, about 5 mL of nutrient broth was poured into four different sterilized test tubes, which were labeled with the name of the individual bacterial strain. From previously sub-cultured media, each bacterium was transferred into well-label respective tubes with the help of an inoculating loop. Finally, all the broth media containing bacteria were kept for incubation at 37°C for 1 day to achieve the suspensions of K. pneumoniae, E. coli, S. aureus, and S. epidermidis. The turbidity of the inoculum suspension was compared and adjusted up to the turbidity of 0.5 McFarland solutions. 2.11.4. Screening and Measurement of Zone of Inhibition (ZOI) Lobato, A.; Neto, M.; Meirelles, A.; Silva, L.; Marochio, C.; Monteiro, E.; Zeni Neto, H.; Maleia, M.; Moiana, L.; Bronzato, A. Relationship between leaf relative water content and total soluble proteins in soybean exposed to short water deficit. Res. J. Biol. Sci. 2009, 4, 1061–1067. [ Google Scholar]

Dixon, R. A. et al. The phenylpropanoid pathway and plant defence – A genomics perspective. Mol. Plant Pathol. 3, 371–390 (2002). Overall, our present findings suggested that ethyl acetate and methanolic leaf extract of P. utilis manifested significant antibacterial and antioxidant effects. Also, hexane seed extract of P. utilis displayed moderate antioxidant activity with an appraisable amount of total flavonoid and phenol content to justify the ethnomedicinal use of P. utlilis seed oil. The presence of polyphenol and flavonoid components might be responsible for these biological activities. Therefore, this study concluded that P. utilis leaf extract can be recommended for the inhibition of gram-positive bacteria and seed extract can be recommended for the inhibition of gram-negative bacteria. Their notable antibacterial potency against four different pathogenic bacteria reflects that these extracts may serve as a useful source to combat various life-threatening pathogenic bacteria to mitigate various diseases like urinary tract infection, dental problems, dysentery, diarrhea, etc. Nevertheless, further advanced research activities with great priority on an animal model, along with an understanding of the mechanism of action of antibacterial activity, are mandatory to validate the traditional utilization of this medicinally significant plant and go after the long research-based expedition of medicinal plant-derived antibacterial medicine discovery, for ensuring human safety and protection of their health. Data Availability Phan, T.; Ishibashi, Y.; Miyazaki, M.; Tran, H.; Okamura, K.; Tanaka, S.; Nakamura, J.; Yuasa, T.; Iwaya-Inoue, M. High Temperature-Induced Repression of the Rice Sucrose Transporter ( OsSUT1) and Starch Synthesis-Related Genes in Sink and Source Organs at Milky Ripening Stage Causes Chalky Grains. J. Agron. Crop Sci. 2013, 199, 178–188. [ Google Scholar] [ CrossRef] Karban, R. et al. Herbivore damage to sagebrush induces wild tobacco: evidence for eavesdropping between plants. Oikos 100, 325–333 (2003).

Author Contributions

Once your children are familiar with the types of trees, set them the challenge to match up each set of three cards. Can they work with a friend to match up each leaf with its seed and its name? The extractive yield of P. utilis seed and leaf in different solvent was calculated with the help of the following equation: 2.6. Phytochemical Screening Samarah, N.; Alqudah, A.; Amayreh, J.; McAndrews, G. The effect of late-terminal drought stress on yield components of four barley cultivars. J. Agron. Crop Sci. 2009, 195, 427–441. [ Google Scholar] [ CrossRef] Kuai, J.; Liu, Z.; Wang, Y.; Meng, Y.; Chen, B.; Zhao, W.; Zhou, Z.; Oosterhuis, D.M. Waterlogging during flowering and boll forming stages affects sucrose metabolism in the leaves subtending the cotton boll and its relationship with boll weight. Plant Sci. 2014, 223, 79–98. [ Google Scholar] [ CrossRef] [ PubMed]

Healthy and mature fruits of the P. utilis Royle and leaves (Figure 1) were collected with the help of local people of Narakot, Jumla (a temperate region, 3128 m above sea level) in November 2020. The collected plant materials were identified and authenticated by the National Herbarium and Plant Laboratory in Godabhari, Nepal (Ref no.078/079). The preparation and preservation of the herbarium were carried out in the Pharmacognosy Laboratory of the Crimson College of Technology, Nepal (Herberium number: CCT/HRB/2021-012). Collected and authenticated leaves and seeds were washed, dried, and ground to a fine powder. The fine powder mass was shifted through the (40 mesh size). 2.4. Preparation of the Plant Extracts De Schepper, V.; De Swaef, T.; Bauweraerts, I.; Steppe, K. Phloem transport: A review of mechanisms and controls. J. Exp. Bot. 2013, 64, 4839–4850. [ Google Scholar] [ CrossRef][ Green Version] The most effective extracts on cytotoxicity of K-562 cell lines were A. absinthium leaf (86%) and A. retrosa leaf (28%). In addition, the highest cytotoxicity (78%) for A-549 cells was obtained from P. major seed extract ( Figure 1h). Among the other plant extracts, P. major seed was the best extract for selective cytotoxicity on A-549 cancer cell lines because it did not cause cytotoxicity on the normal Beas-2B cell line.Eom, J.S.; Choi, S.B.; Ward, J.M.; Jeon, J.S. The mechanism of phloem loading in rice ( Oryza sativa). Mol. Cells 2012, 33, 431–438. [ Google Scholar] [ CrossRef][ Green Version] Fo