BIBECHANA Vol. 22, No. 3, December 2025, 266-279 ISSN 2091-0762 (Print), 2382-5340 (Online) Journal homepage: http://nepjol.info/index.php/BIBECHANA Publisher:Dept. of Phys., Mahendra Morang A. M. Campus (Tribhuvan University)Biratnagar Assessing the antioxidant antibacterial and toxic potential of quercus floribunda lindl bark Govinda Bhattarai, Dipak Raj Jaishi, and Khaga Raj Sharma∗ Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract Since ancient times, herbal medical practitioners have been treating a wide range of condi- tions, from basic to life-threatening, with traditional or folk remedies. Phytochemicals are incredibly helpful in defending against numerous diseases in human beings. Due to their af- fordability and lack of side effects, herbal medicines have replaced synthetic drugs in many countries. The goal of the present study is to analyze the total phenolic content (TPC) and total tannin content (TTC) by the Folin-Ciocalteu method, total flavonoids content by the Aluminum chloride method, DPPH radical scavenging activity, evaluation of antimicrobial activity by agar-well diffusion method and evaluation of cytotoxic activity of crude extract. The dried bark of the Quercus floribunda Lindl was powdered, and then the extract was pre- pared in different solvents chosen based on their polarity using the cold maceration technique. Among different crude extracts, ethyl acetate extract has a high TPC (127.23 ± 1.65 mg GAE/g), and Dichloromethane (DCM) has the lowest TPC (9.25 ± 0.69 mg GAE/g). Also, hexane extract shows high TFC (61.72 ± 4.01 mg QE/g), and ethanolic extract shows the least TFC content (3.97 ± 1.03 mg QE/g). Furthermore, ethanolic extract had the highest TTC which is 49.91 ± 1.06 mg TAE/g, and least TTC value in hexane extract which is 5.51 ± 1.3 mg TAE/g. Along with this, methanolic extract showed good antioxidant ability with IC50 of 12.70 ± 0.37 µg/ mL. In the case of the antimicrobial susceptibility test, the ethanolic and ethyl acetate extract of bark was found to be effective against Escherichia coli (ATCC 25912) with a zone of inhibition of 15 mm, equal to positive control, and neomycin. Ethyl acetate extract is also found effective against Shigella sonnei (ATCC 25931) with a zone of inhibition of 22 mm. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were also estimated in two different bacterial strains. For methanolic extract, the MIC value for Gram-positive Staphylococcus aureus (ATCC 43300) was 3.125 mg/mL, and Gram- negative Shigella sonnei was 3.125 mg/mL, and the MBC was 25 mg/mL for both strains. The methanolic extract shows good cytotoxicity against brine shrimp nauplii with LC50 of 40.55 µg/mL. Thus, from the overall study, the bark of Quercus floribunda Lindl could be used as a natural source to isolate antibiotics and antioxidants. Keywords Quercus floribunda Lindl, Antimicrobial, Antioxidant, Cytotoxicity, Phytochemistry. Article information Manuscript received: April 25, 2025; Revised September 8, 2025; Accepted: September 11, 2025 DOI https://doi.org/10.3126/bibechana.v22i3.77948 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 266 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v22i3.77948 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 267 1 Introduction Nature is a remarkable indicator of the numer- ous phenomena of coexistence. The foundation for treating human illnesses is natural items derived from plants, animals, and minerals. Right now, there is a growing demand for and acceptance of medicinal herbs. Plants undoubtedly contribute significantly to ecosystems by offering vital services [1]. Almost every region in the world has a remark- able history of using medicinal plants to benefit hu- mankind. This amazing knowledge has been passed down from generation to generation by traditional healers. The deep understanding of this natural medicine paradigm has remained unchanged despite modernization and cultural changes. A new revo- lution in the treatment of diseases might undoubt- edly be brought about by phytochemical research combined with pharmacological thinking in light of traditional uses [2]. Significant medications like atropine, codeine, digoxin, morphine, quinine, and vincristine are de- rived from plants and the secondary metabolite components of those plants. Plants have a long his- tory of usage in both modern "Western" medicine and some traditional medical systems [3]. Humans could choose which plant tissues to consume based on their observation that certain plant tissues, such as fruit, leaves, or roots of certain species, improved their state of mind. Aromatic and medicinal plants are a valuable source for creating novel medica- tions and treating physical and mental health is- sues [4]. The pharmaceutical industry developed various commercial synthetic medications, the past was referred to as the "synthetic era". Continu- ous usage of synthetic medications over time re- sulted in serious side effects and microbial resis- tance. Large populations cannot pay the high cost of synthetic pharmaceuticals to profit from them. A worldwide movement has emerged in recent decades that has centered on green medicines because of their low side effects and affordability. The dis- covery of medicinal plants was crucial to the cre- ation of modern herbal remedies because allopathy is unable to fully treat several illnesses, including cancer, liver disorders, and arthritis. When there are no adequate anti-diabetic, anti-inflammatory, anti-arthritic, or chemotherapy drugs available, the bioactive components of medicinal plants are uti- lized [5]. Plant-based products are expected to be worth $83 billion globally, and this sector is still expand- ing. Moreover, it is estimated that up to 60% of anticancer medications and about 25 % of contem- porary drugs are derived from natural sources. The WHO estimates that between 65%-80% of people in underdeveloped nations currently use medicinal plants as a form of treatment. Since just 15% of the world's 300,000 plant species have been assessed to establish their pharmacological potential. Research highlighting the value and effectiveness of thera- peutic plants is being conducted across the globe in a variety of nations at different stages of devel- opment [6]. According to the World Health Orga- nization, a plant is considered medicinal if it con- tains chemicals that have therapeutic value or serve as precursors for the semi-synthesis of chemother- apeutic medications (WHO). Plants consist of sev- eral parts, including leaves, stems, rhizomes, bark, seeds, flowers, and fruits. These plant sections con- sequently contain chemical substances that are use- ful for medicinal purposes [7]. According to Bhattarai and Ghimire, 143 species of commercial Medicinal Aromatics Plants (MAPS) were evaluated from the Himalayan gradi- ent [8]. A total of 161 species of medicinal plants have been reported to be used by the Tamang community in Makwanpur district [9]. The Meche people have been reported to use 64 plant species from the Jhapa district [10]. Quercus floribunda, also known as the Tilonj oak, green oak, Moru, or Mohru oak, is a species of oak that is endemic to Afghanistan, Pakistan, Nepal, and the western Himalayas of India. It is usually found at eleva- tions between 2,000-3,000 meters (6,600- 9,800 feet) above sea level. The tree is an evergreen and an important species for fuelwood and fodder, with a dense crown that reaches up to 30 meters (98 feet) [11]. Quercus flori- bunda has a straight trunk that may grow up to 45 meters in height and 2 meters in diameter. It also has a dense crown. As the bark ages, it be- comes dark grey or dark reddish brown and exfo- liates in uneven woody scales. The bright green, 4-8 cm long, lanceolate to elliptic moru leaves can have smooth or spiky edges. Catkins in male inflo- rescences are 8 cm long. The length of the female spikes is 4 cm. The fruit is an ovoid or oblong, brown, 2 cm long acorn with a sharp tip that grows alone on the branches from the previous year [12]. Quercus species, commonly referred to as oaks, are a significant genus within the Fagaceae fam- ily. It is found extensively in tropical climates and temperate woodlands in the northern hemisphere. Numerous constituents have been employed in con- ventional medicine to address and prevent a range of human diseases, including diarrhea, ulcerative gastritis, asthma, hemorrhoids, and wound healing. Bioactive chemicals, including triterpenoids, pheno- lic acids, and flavonoids, have been linked to a vari- ety of biological activities, including anti- inflamma- tory, antibacterial, hepatoprotective, antidiabetic, anticancer, gastroprotective, antioxidant, and cyto- toxic properties [13]. The decoction or infusion include styptic, hemo- static, antibacterial, antifungal, and antiseptic ef- Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 268 fects. It is taken orally to treat conditions like severe diarrhea, dysentery, and hemorrhages. It is used topically on the outside to treat burns, wounds, and a range of skin disorders, hemorrhoids, and irritation of the anal, vaginal, and oral mucosa. It can also be used as a mouthwash to treat gum disease and toothaches. Plant extracts can be used in lotions and ointments to aid in the healing of wounds [14]. The Quercus species yields the well- known acorn, which is used in traditional medicine together with bark and leaves. These parts of the plant are used as antiseptics or to treat gastroin- testinal issues. Both people and animals eat acorns because of their nutritional value. Oak wood is valuable for its color, durability, and resistance to fungal deterioration in the wood industry as well as for wine maturation in oak barrels [15]. The analysis of phytochemicals and evaluation of bio- logical properties of bark extracts, including an- tioxidant, antimicrobial, and toxicity against brine shrimp nauplii, have not been well reported yet. So, the proposed research work plays a significant role in fulfilling the research gap. The major phy- toconstituents previously reported from the genus Quercus are shown in Figure 1 Figure 1: Structure of different phenolic compounds previously reported from Quercus species. 2 Materials and Methods 2.1 Chemicals Ethyl acetate, hexane, DCM, ethanol, and methanol solvents were purchased from Merck in Germany and Thermo Fisher Scientific in In- dia. Similarly, Gallic acid and Folin Ciocal- teu (FC reagent) and other chemicals from Loba Chemie. Dimethyl sulfoxide (DMSO) (Silico Re- search Laboratory, India), acetic acid (Control Drug House, Gujarat, India), Quercetin, 2,2- diphenyl-1-picrylhydrazyl (DPPH) (Srichem, Ma- harashtra, India), potassium acetate (Loba Chemie, PVT Ltd., Mumbai, India), sodium carbonate an- hydrous, tannic acid, ferrous nitrate, Müller Hinton broth (MHB), Muller Hinton Agar (MHA), and Nu- trient Agar (NA) Growth Nutrient Agar were pur- chased from HiMedia Lab, Mumbai. 2.2 Collection and identification of the plant Based on the indigenous knowledge from the lo- cal user and literature survey, the plant was col- lected from the Himali Gaupalika Bajura district at 2500m, latitude 29.53°N and longitude 81.67°E. After plant collection, the plant parts needed to be processed for the herbarium. The herbarium was created once the plant had fully dried and was taken to the National Herbarium and Plant Laboratories in Godavari, Lalitpur, for identification. The plant was identified as Quercus floribunda Lindl by the National Herbarium and Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 269 Plant Laboratories. The voucher code is provided as KATH163578. The photograph of the herbar- ium and collection sites of Q. floribunda is shown in Figures 2 and 3. Figure 2: Photograph of the herbarium of Q. flori- bunda. Figure 3: Maps of sample-collecting sites.. The phytochemicals flavonoids, gallotannin, and ellagitannin were found in the aqueous bark extract of Quercus acutissima. This plant could be used to block 5-reductase activity and testosterone-induced sebum synthesis in rats; it decreased androgen- related pathogenes of acne, testosterone conversion, and sebum synthesis [16]. The methanolic extract of the same species contains phenolic acids and could show anticancer activity against breast can- cer cell lines, cervical cancer cells, human T lym- phocyte cells, human colon cancer cell lines, and human embryonic kidney cells [17]. The ethano- lic leaf extract of Quercus salicina Blume depicts a vasodilatation effect on porcine coronary artery en- dothelium and good antioxidant activity [18]. The Quercus sideroxyla shows antihyperglycemic activ- ity, maintaining the glucose levels at more stable levels by inhibiting the -amylase enzyme [19]. The aqueous extract of the same species consists of gal- lic acid, catechin, epicatechin, procyanidins, and proanthocyanidins, and these phytochemicals show antioxidant activity [20]. 2.3 Preparation of extract The bark of this plant was collected, cleaned, shade dried, sliced into small pieces, and dipped in six different solvents based on polarity: Aque- ous, methanol, ethanol, ethyl acetate, DCM, and Hexane. After 72 hours, filtration was done using filter paper, and the solvents were re-suspended for 48 hours and consecutively for 24 hours one last time. Every time, the filtrates were collected and concentrated under a vacuum in a rotatory evapo- rator at 40 °C. The extracts were collected in glass vials and stored at 4 ºC for future analysis. The following formula was utilized for the calculation of the yield percentage of the crude extract. 2.4 Qualitative phytochemical analysis The presence of secondary metabolites, alkaloids, flavonoids, polyphenols, lignins, tannins, steroids, and saponins was determined by observing a color change in the chemical process, and phytochemicals found in different extracts were qualitatively iden- tified by following the standard protocol [21]. 2.5 Estimation of total phenolic content (TPC) The Folin-Ciocalteu colorimetric method was used to estimate the total phenolic content of plant ex- tracts [22–24]. In triplicate, 96-well plates were loaded with 20 µL of plant extract, 100 µL of 10% FC reagent (1:10), and 80 µL of 1M Na2CO3. Be- fore an intense blue color was noticed, the reac- tion mixture was left to incubate at room temper- ature for 30 minutes. Finally, absorbance at 765 nm was measured using a spectrophotometer. The total phenolic content (TPC) was measured in mil- ligrams of gallic acid equivalent (mg GAE/g) per gram of extract dry weight, and the standard curve was created, which is the standard curve for gallic acid (7.5- 100 µg/mL). 2.6 Estimation of total flavonoid content (TFC) The total flavonoid content was estimated by us- ing the aluminum chloride method as described by Ahmed et al. [24, 25]. In triplicate, 96-well plates Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 270 were loaded with 20 µL of plant extract, 100 µL of distilled water, and 60 µL of ethanol, followed by 10 µL of 10% AlCl3 solution and 10 µL (1M) CH3COOK solution. At room temperature, the reaction mixture was incubated for half an hour. Then, using a spectrophotometer, absorbance was measured at 415 nm. A standard calibration curve for quercetin (10-100 µg/mL) was developed and quantified in milligrams of quercetin equivalent per gram of the dry weight of the extract (mg QE/g). 2.7 Estimation of total tannin content (TTC) The Folin-Ciocalteu colorimetric method was used to estimate the total tannin content by applying the standard protocol [26]. Tannic acid concentrations ranged from 7.5 to 100 µg/mL, and 10 µL of plant extract was added to 96-well plates. Then, 50 µL of 10% FC reagent and 70 µL of distilled water were added, and a microplate reader was used to capture the initial reading at 725 nm. After that, 70 µL of 35% Na2CO3 was injected following the first mea- surement. The ultimate absorbance of the 96-well plate was measured at 725 nm after it had been in- cubated for 30 minutes. TTC was expressed as mg TAE/g. 2.8 Evaluation of antioxidant activity The antioxidant activity of crude plant extract was evaluated by following a standard protocol [27,28]. The crude plant extract was diluted serially up to the required concentrations, but the positive control quercetin was serially diluted up to 0.625 µg/mL from 20 µg/mL concentrations. In triplicate, 100 µL of plant extracts and a positive control were added to 96-well plates. The first reading was then obtained at 517 nm. After that, each well received 100 µL of DPPH reagent, which was then incubated for 30 minutes. At 517 nm, the ultimate absorbance was measured. Because methanol and 50% DMSO were employed as negative controls. The following formula was used to evaluate radical scavenging ac- tivity: Radical scavenging capacity = ( Acontrol −Asample Acontrol ) × 100 (1) where, Acontrol = absorbance of control, Asample = absorbance of sample. Inhibitory concentration (IC50) was calculated by using GraphPad Prism (version 8.0.2.263). 2.9 Evaluation of antimicrobial activity The agar well diffusion method was applied to per- form an antibacterial activity using Mueller-Hinton Agar (MHA) plates [29, 30]. The test microor- ganisms, Escherichia coli, Klebsiella pneumonae, Staphylococcus aureus, and Shigella sonnei, were cultivated in Mueller-Hinton Broth (MHB) and in- cubated for 24 hours at 37 ºC. Its turbidity was maintained at 0.5 McFarland. 50 µL of plant ex- tract, 50% DMSO as the negative control, and 50% neomycin as the positive control were added to each well created by a cork borer. The Petri plates were incubated for 18 to 24 hours at 37 °C after being left for 15 minutes to allow for diffusion. After in- cubation, the zone of clearing was measured and tracked. 2.10 of minimum inhibitory concentration (MIC) and minimum bactericidal con- centration (MBC) The minimum inhibitory concentration (MIC) and minimum bactericidal concentration were deter- mined by following the standard protocol as de- scribed [31]. A final concentration of 106 CFU/mL was obtained for the bacterial inoculum by dilut- ing the 0.5 McFarland turbidity culture in MHB 1:100. Each well of the 96-well plates received an injection of 5 µL of bacteria. The positive control was a popular drug called neomycin. A sterile lid was placed over the plate, and it was incubated at 37 °C for 20 to 24 hours. 0.003% resazurin was added to the microtiter plate wells, and the mix- ture was incubated at 37 °C for three to four hours. While the wells without infection remained blue, the wells with bacterial growth became pink. The lowest concentration at which bacterial growth is inhibited was determined to be the extract's mini- mum inhibitory concentration (MIC). The MIC and MBC of the crude plant extracts were ascertained by streaking the well contents onto nutrient agar plates and then incubating them at 37 °C for over 18 hours. 2.11 Brine shrimp lethality assay (BSLA) The toxicity of plant extracts was determined by following a standard protocol [32]. By adding 1M NaOH, the pH of the artificial sea salt water was kept between 8 and 8.5. Different quantities of the plant extract, including 1000, 800, 500, 250, 100, and 10 µg/mL, were diluted. Each test tube was then filled with 4 mL of artificial seawater. 10 nau- plii and 500 µL of sample were then added in trip- licate to each test tube. The positive control was a potassium dichromate solution, while the nega- tive control was artificial sea salt water. After 24 hours, the number of dead nauplii in a test tube was counted, and the following formula was used to calculate the % mortality of nauplii: Using the Probit value table, the linear equation can be obtained as Y = mx+c, where Y is the Pro- Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 271 bit value at 50% mortality, m is the variable, and c is the intercept. X is the lethal concentration, and finally, the lethal concentration (LC50) was calcu- lated. 2.12 Statistical Analysis The data was collected by using a Gen5 Microplate reader. Data analysis was carried out by using Mi- crosoft Excel. TPC, TFC, and TTC were reported as the mean ± standard deviation. Mean ± stan- dard error reported for antioxidants. Half maximal inhibitory concentrations (IC50) were calculated by using Graph Pad Prism (version 8.0.2.263). The comparisons were made by using a one-way ANOVA test. Values with p < 0.05 were considered statisti- cally different. 3 Results 3.1 Percentage Yield The highest yield percentage was found in the aque- ous extract, which is 15.24% followed by methanol (11.29 %), Ethanol (9.53 %), Ethyl acetate (3.35 %, DCM (0.71 %), and the lowest yield percentage of 0.4% in hexane extract. This is because the aque- ous solvent is more polar than other solvents, and the bark might contain highly polar compounds. The bar diagram of the % yield of different solvent extracts is shown in Figure 4. Figure 4: Showing the yield percentage in different solvent extracts. 3.2 Qualitative phytochemical analysis Qualitative phytochemical screening of different sol- vent extracts of Q. floribunda is shown in Table 1. Table 1: Qualitative phytochemical screening of plant extracts. Phytochemicals Test Aqueous Methanol Ethanol Ethyl acetate DCM Hexane Alkaloids Dragendorff’s test + + + + - + Carbohydrates Molish’s test + + + + + - Protein test Biuret test - - - - - - Flavonoids Alkaline reagent test + + + + - - Terpenoids Salkowski’s test + - + + - + Tannins Braymer’s test + + + + - - Phenolic test FeCl3 test + + + + - - Anthraquinones Borntrager’s test + + + + - - Note: (+) = present, (-) = absent. 3.3 Total phenolic content (TPC) The TPC of this plant ranges from 127.23 ± 1.65 mg GAE/g to 9.25 ± 0.69 mg GAE/g (Figure 5). The bark of Quercus floribunda Lindl showed the highest TPC value in ethyl acetate extract (127.23 ± 1.65 mg GAE/g) followed by aqueous (123.23 ±4.28 mg GAE/g), methanol (111.13 ± 1.55 mg GAE/g), ethanol (93.65 ± 2.23 mg GAE/g), and hexane (9.29 ± 0.26 mg GAE/g), and least value in DCM (9.25 ± 0.69 mg GAE/g). The TPC values are significantly different from each other at p < 0.05. The calibration curve is shown in Figure 6. 3.4 Total flavonoid content (TFC) The TFC of Q. floribunda ranges from 61.72 ± 4.01 mg QE/g to 3.97 ± 1.03 mg QE/g (Figure 7). The plant Quercus Floribunda Lindl possesses the highest flavonoids in hexane extract (61.72 ± 4.01 mg QE/g), followed by DCM (50.21 ± 0.93 mg QE/g), aqueous (6.98 ± 0.93 mg QE/g), ethyl acetate (6.12 ± 1.47 mg QE/g), methanolic (4.08 ± 0.67 mg QE/g), and the lowest value in ethanol extract (3.97 ± 1.03 mg QE/g). Values are signif- icantly different from each other at p < 0.05. The calibration curve of quercetin is shown in Figure 8. 3.5 Total Tannin content (TTC) The TTC of Quercus floribunda Lindl ranges from 49.91±1.06 mg TAE/g to 5.51±1.3 mg TAE/g. The maximum TTC was found in ethanolic extract (49.91±1.06 mg TAE/g), followed by methano- lic extract (49.51±3.33 mg TAE/g), ethyl acetate (47.35±1.69 mg TAE/g), aqueous (37.27±2.96 mg Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 272 TAE/g), DCM (14.78±1.39 mg TAE/g), and the least content in hexane extract (5.51±1.3 mg TAE/g) (Figure 9). Values are significantly differ- ent from each other at p < 0.05. The calibration curve of tannic acid is shown in Figure 10. Figure 5: Standard calibration curve of gallic acid.. Figure 6: Total Phenolic Content in the various sol- vent extracts. Figure 7: Standard calibration curve of quercetin. Figure 8: Total flavonoid content in various ex- tracts. 3.6 Antioxidant activity The DPPH free radical inhibition by various plant extracts was shown through a graphical represen- tation (Figures 11 and 12). Based on the re- sult obtained in the bark of Quercus floribunda Lindl possesses good antioxidant activity. Among the different extracts, the methanolic extract shows good antioxidant activity with an IC50 of 12.70 ± 0.37 µg/mL, followed by ethanolic extract ( 17.08 ± 0.48 g/mL), ethyl acetate (21.38 ± 0.66 g/mL), and Aqueous extract had the least antioxidant po- tential, with its IC50 of 29.31±1.24 µg/mL. The an- tioxidant potential of DCM and hexane extract is weak (> 500). Antioxidant values are significantly different from each other at p < 0.05. The bar di- agram of IC50 of different solvent extracts of this plant is shown in Figure 13. 3.7 Antimicrobial activity The result showed the minimum zone of inhibition for the DCM and hexane extract, which confirmed their least anti-bacterial activity. In comparison to the positive control (neomycin), the aqueous ex- tract also does not show a good zone of inhibition. The aqueous extract showed a good zone of inhi- bition for E. coli 10 mm and Staphylococcus au- reus 15 mm. Methanolic (14 mm), ethanolic (15 mm), and ethyl acetate (15mm) extracts showed a good zone of inhibition in E. coli, which is exactly the same as a positive control (15mm). Methano- lic and ethanolic extracts showed the same zone of inhibition (20mm) against Staphylococcus aureus, whereas for the same bacteria, the positive control showed 25 mm of ZOI. For Klebsiella pneumoniae, methanol, ethanol, and ethyl acetate showed com- parable ZOI of 15 mm, 16 mm, and 16 mm, re- spectively, and the positive control showed 28 mm of ZOI for the same bacteria. In the same manner for Shigella sonnei, ethyl acetate extract showed a good ZOI of 22 mm, whereas the positive control showed 25 mm. Also, methanolic and ethanolic ex- Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 273 tracts showed comparable ZOI of 19 mm and 20 mm, respectively. The antibacterial activity of dif- ferent solvent extracts in terms of ZOI is shown in Table 2. Figures 14 and 15 are the test slides against different microorganisms. Table 2: Zone of inhibition (ZOI mm) shown by various extracts of Quercus floribunda Lindl against different bacteria and neomycin as a positive control. Crude extract Shigella sonnei Klebsiella pneumoniae Staphylococcus aureus Escherichia coli Aqueous 14 9 15 10 Methanol 19 15 20 14 Ethanol 20 16 20 15 Ethyl acetate 22 16 18 15 DCM 12 12 11 8 Hexane 9 9 9 9 Positive control 25 28 25 15 Negative control - - - - Figure 9: Standard calibration curve of Tannic acid. Figure 10: Total tannin content in various extracts of Quercus floribunda Lindl. Figure 11: Standard curve of DPPH inhibition by quercetin. Figure 12: Plot of DPPH inhibition against the con- centration of (a) aqueous extract, (b) ethyl acetate extract, (c) methanol extract, and (d) ethanol ex- tract. Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 274 Figure 13: Bar diagram showing the antioxidant po- tential of bark crude extracts in different solvents at various concentrations. Figure 14: The ZOI shown by the aqueous extract against the bacterial strains. SA = Staphylococcus aureus, KP = Klebsiella pneumoniae, E. coli = Escherichia coli, SS = Shigella sonnei Figure 15: ZOI of methanol, ethanol, ethyl acetate, DCM, and hexane extract against different bacte- rial strains. MeOH = Methanol extract, SS = Shigella son- nei, EtOH = Ethanol extract, SA = Staphylococcus aureus EA = Ethyl acetate extract, KP = Kleb- siella pneumoniae, DCM = Dichloromethane ex- tract, E.coli = Escherichia coli, PC = Positive con- trol, NC = Negative control 3.8 Minimum inhibitory concentration (MIC) and minimum bactericidal con- centration (MBC) Methanolic extract showed the MIC and MBC value for both bacterial strains (3.12 mg/mL and 25 mg/mL, respectively, where the positive control (neomycin) shows MIC 0.78 mg/mL for both bac- terial strains. The MBC value was 6.25 mg/mL for Shigella sonnei and 3.12 mg/mL for Staphylococ- cus aureus for neomycin. Ethanolic extract showed MIC against Staphylococcus aureus and Shigella sonnei was 1.65 mg/mL, but MBC was different for Staphylococcus aureus, 25 mg/mL, and more than 25 mg/mL for Shigella sonnei. The MBC was more than 25 mg/mL for Shigella sonnei by ethanolic ex- tract because the bacterial growth in the petri dish was not completely inhibited or killed, so more than 25 mg/mL will be the required concentration to kill the bacteria completely. Similarly, for ethyl acetate extract, it was found that 3.125 mg/mL. MIC value for Shigella sonnei and 1.652 mg/mL for Staphylo- coccus aureus, and for both bacterial strains, the MBC value was 25 mg/mL. Table 3 depicts the MIC and MBC of different solvents against two bac- teria. The results of MIC and MBC are shown in Figure 16. Table 3: MIC and MBC values of different extracts for gram-positive (Staphylococcus aureus) and gram- negative (Shigella sonnei). Extract type MIC (mg/mL) MBC (mg/mL) Shigella sonnei Staphylococcus aureus Shigella sonnei Staphylococcus aureus Methanol 3.125 3.125 25 25 Ethanol 1.562 1.562 >25 25 Ethyl acetate 3.125 1.652 25 25 Positive control 0.781 0.781 6.25 3.125 Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 275 3.9 Brine shrimp lethality activity (BSLA) The percentage mortality along with the LC50 of the methanolic extract is shown in Table 4. The methanolic extract had good lethal potential against brine shrimp nauplii. From BSLA, the LC50 value for the methanolic extract was found to be 40.55 µg/mL. A plot of probit against Log C is shown in Figure 17. Table 4: Brine shrimp activity for the methanolic extract of the plant sample. Extract type Concentration (µg/mL) Surviving nauplii after 24 h Mortality % LC50 value (µg/mL) Methanol 1000 24 80.00 40.55 800 17 56.66 500 22 73.33 250 20 66.66 100 16 53.33 10 12 40.00 Figure 16: Showing MIC and MBC values of three different extracts (methanol, ethanol, and ethyl ac- etate) along with the positive control (Neomycin). Figure 17: A plot of probit against Log C. 4 Discussion All the species of Quercus floribunda Lindl are use- ful medicinal plants. The different parts of Quer- cus species are used in various treatments, where the bark of this species is used as an astringent, antiarthritic, and for other disorders [13]. Since there is no more work in pharmacognosy of this species, my research mainly focuses on the bark of the Quercus floribunda Lindl plant, which is widely distributed in the range of the Himalayas (2500- 3000 m), Nepal. Different extraction solvent shows different yield percentages. The aqueous yield is maximum, show- ing maximum polar constituents in the bark of this plant, and minimum in Hexane, showing minimum presence of non-polar compounds in the bark. This study found the same result, where the yield was maximum in the polar solvent. According to this study, to improve extraction efficiency, a combi- nation of polar and nonpolar solvents should be used [33]. In this species, there is no specific re- search related to this work, so the data are corre- lated with other species of the same genus. In phytochemical screening of the bark of Quer- cus floribunda Lindl, it was found that the bark of the plant is highly rich in different classes of bioac- tive compounds, alkaloids, flavonoids, steroids, tan- nins, carbohydrates, and saponins. This result can be correlated to the study carried out in different species of the Quercus genus by different researchers [34]. The bark of Quercus gluca contains tannins, carbohydrates, saponins, phenols, and quinones, which make it a medicinally active plant. Also, ac- cording to [13], the Quercus genus contains various classes of compounds such as glycosides, terpenoids, flavonoids, phenolic acids, fatty acids, sterols, and tannins. The bark of Quercus floribunda Lindl had the highest TPC value in ethyl acetate extract (127.23 ± 1.65 mg GAE/g), with the lowest value in DCM (9.25 ± 0.69 mg GAE/g). There is no more sig- nificant difference in TPC value between the DCM and hexane extracts. Also, the study finds ethanol extract had the lowest TFC value (3.97 ± 1.03 mg QE/g), and hexane had the maximum value (61.72 ± 4.01 mg QE/g). The bark of Quercus floribunda Lindl shows the maximum TTC in aqueous extract (37.27±2.96 mg TAE/g) and the least in hexane extract (5.51±1.3 mg TAE/g). There is no spe- cific research conducted on this species within this solvent and bark, so it is difficult to correlate the data. Thus, the data were correlated with differ- ent species of the same genus and another part of the bark in the same plant. A study conducted by Ahmad et al. 2023 [35] on aerial part leaves and galls found the maximum TPC value and TFC value in acetonic extract, 66.9 ± 0.05 g GAE/mgE Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 276 and 38.4 ± 0.72 g QE/mgE, respectively. Thus, the bark of Q. floribunda Lindl has more phenolic and flavonoid content than the aerial part. A study con- ducted in the bark of Q.faginea by Ferreira et al. 2018 [36] found maximum TPC (630.3 mg GAE/g), TFC(207.7 mg catechin equivalents (CE)/g), and Tannin (220.7 mgCE/g) in ethanol-water extract. This shows that the bark of Q. faginea contains more phenolic compounds, flavonoids, and tannins. Among six solvent extracts, the methanolic ex- tract shows good antioxidant activity with an IC50 of 12.70 ± 0.372 µg/mL, followed by ethanol, ethyl acetate, and aqueous. The IC50 value for the crude extracts of DCM and hexane was greater than 500 µg/mL, indicating a low level of antioxidant activity in these extracts. According to the research carried out by Ferreira et al. 2018 [36] in the bark of Q. faginea IC50 was 2.6 µg/mL in polar ethanol-water extract, showing more antioxidant activity in com- parison to Q. floribunda Lindl. This variation in results is due to climatic conditions, altitude varia- tion between the collected samples, and correlated species from the literature. The value reported by Sánchez-Burgos et al., 2013 [37] for anti-oxidant ac- tivity in leaves of different species (Q. resinosa, Q. grisea, Q. laeta, and Q.obtusata) ranges from 150 – 450 µg/mL, which is more than compared to aque- ous extract (29.31±1.24 µg/mL) of Quercus flori- bunda Lindl. This data shows the bark of Quer- cus floribunda Lindl is more antioxidant than the above-mentioned plants. From the antibacterial study in the bark of Quercus floribunda Lindl, it was found that the gram-negative E. coli was inhibited equally by ethanol and ethyl acetate, with a ZOI of 15 mm, exactly as a positive control. This shows the bark of this plant is a potent source of antibiotics against E. coli. For gram-positive Staphylococcus aureus, methanolic and ethanolic extracts show a ZOI of 20 mm, a minimal zone of inhibition for hexane ex- tract and DCM, confirming their low levels of an- tibacterial activity. A poor zone of inhibition is also seen in the aqueous extract when compared to the positive control (neomycin). The bark of Quercus floribunda Lindl shows antibacterial activity. There is no exact paper to correlate the anti-microbial re- sult. Of this species, but it was found by Sarwar et al., 2015 [38] that the leaves of Q. incana have signif- icant antibacterial activity for both Gram-positive and negative. The n-butanol extract of leaves shows 32 mm and 28 mm ZOI against A.niger and A. flavus, respectively also 19 mm for Micrococcus leu- teus. This data shows that the Quercus genus has good antibacterial properties. This supports the bark of Quercus floribunda Lindl also acts as a good source of antibiotics. Similarly, according to Iqbal et al., 2023 [33], the bark of Q. glauca shows good antibacterial properties, whereas the methanolic ex- tract shows a maximum ZOI of 13.33mm against Klebsiella pneumoniae, which is similar to the methanolic extract of the bark of Quercus flori- bunda Lindl. (15 mm). MIC and MBC values were determined for gram-positive (Staphylococcus aureus) and gram-negative (Shigella sonnei) bacte- ria. For both bacterial strains, methanolic extract demonstrated the same MIC values and MBC val- ues for both strains (3.125 mg/mL and 25 mg/mL, respectively). Ahmad et al., 2023 [35] found that nuts of Quercus floribunda Lindl are moderately cytotoxic against brine shrimp nauplii. This inves- tigation revealed that methanolic extract from the bark of Quercus floribunda Lindl exhibits good tox- icity against brine shrimp nauplii with an LC50 of 40.55 µg/mL. 5 Conclusion Phytochemical screening in the bark of Quercus floribunda Lindl revealed that the bark extract in- cluded phenol, flavonoids, tannins, terpenoids, car- bohydrates, and alkaloids. It was based on the re- sult of yield percentage and phytochemical screen- ing for the extraction process; methanol, ethanol, and ethyl acetate are good solvents. The results of the comparison of TPC, TFC, and TTC of six different crude extracts found the highest TPC in ethyl acetate and the lowest in DCM. The TFC was highest in hexane and lowest in ethanolic ex- tract. Likewise, the TTC value was highest in the aqueous extract and the lowest in the hexane ex- tract. On the evaluation of the antioxidant ac- tivity of various crude extracts of Quercus flori- bunda Lindl’s bark, the methanolic extract shows good antioxidant properties, followed by ethanol, ethyl acetate, and aqueous extract. As a conse- quence, this finding has demonstrated that the pres- ence of phenols' antioxidant activity is directly re- lated. DCM and hexane extract were found to be less potent for scavenging free radicals. So, from the above-mentioned results of methanolic, ethano- lic, and ethyl acetate extracts, it was concluded that these extract shows good antimicrobial activity and toxicity assay. Ethanol and ethyl acetate showed a good zone of inhibition for E. coli, which is equal to the positive control, neomycin. Methanolic and ethanolic extracts showed a maximum zone of inhi- bition for Staphylococcus aureus, about the positive control, neomycin. For Klebsiella pneumoniae, all three extracts showed moderate ZOI in comparison to the positive control, neomycin. Ethyl acetate extract showed maximum ZOI for Shigella sonnei. These extracts showed comparable zones of inhi- bition for different bacteria. From the calculation of MIC and MBC of these three extracts against Shigella sonnei and Staphylococcus aureus, it was Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 277 concluded that for the methanolic extract, the min- imum required concentration to inhibit was 3.125 mg/mL for both bacteria, and to kill the bacte- ria completely, the required concentration bacterial growth. The MIC and MBC values are also com- parable for these three extracts. From all these re- sults, it was concluded that the methanolic extract is more potent for the toxicity assay. Therefore, further research is necessary to completely compre- hend this plant's medical potential. This plant has a wide range of biological properties, so this plant has great biological importance and could be used as a source of natural antimicrobial and antioxidant agents. Abbreviations DMSO: Dimethyl sulfoxide GAE/g: Gallic acid equivalent per gram QE/g: Quercetin equivalent per gram TAE/g: Tannic acid equivalent per gram TPC: Total phenolic content TFC: Total flavonoid content TTC: To- tal tannin content IC50: Half-maximum inhibitory con- centration DPPH: 2,2-diphenyl-1- picrylhydrazyl ZOI: Zone of inhibition MIC: Minimum inhibitory concentra- tion MBC: Minimum bactericidal con- centration LC50: Lethal concentration 50% Funding Statement This research did not receive any financial support and was conducted through the independent efforts of the authors. Conflicts of Interest The authors declare that there are no conflicts of interest associated with the publication of this re- search paper. Author’s Contribution • Govinda Bhattarai: Performed laboratory work, writing, review, editing, and original draft. • Dipak Raj Jaishi: Writing, reviewing, for- mal analysis, and editing • Khaga Raj Sharma: Writing, reviewing, editing, supervision, and conceptualization Acknowledgments The authors express their gratitude to the Na- tional Herbarium and Plant Laboratories, Go- dawari, Nepal, for their assistance in the identifi- cation of the plant. References [1] F. Jamshidi-Kia, Z. Lorigooini, and H. Amini- Khoei. Medicinal plants: Past history and fu- ture perspective. Journal of Herbmed Pharma- cology, 7(1):1–7, 2017. [2] H. Khan. Medicinal plants in light of history: Recognized therapeutic modality. Journal of Evidence-Based Complementary & Alternative Medicine, 19(3):216–219, 2014. [3] IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some Tra- ditional Herbal Medicines, Some Mycotox- ins, Naphthalene and Styrene. International Agency for Research on Cancer, Lyon, France, 2002. [4] M. Inoue, S. Hayashi, and L.E. Craker. Role of medicinal and aromatic plants: Past, present, and future. In Pharmacognosy - Medicinal Plants. IntechOpen, 2019. [5] S. S. Nair, V. Kavrekar, and A. Mishra. Eval- uation of in vitro anti diabetic activity of se- lected plant extracts, 2016. Unpublished work. [6] R.M. Palhares, M. Gonçalves Drummond, B. dos Santos Alves Figueiredo Brasil, G. Pereira Cosenza, M. das Graças Lins Brandão, and G. Oliveira. Medicinal plants recommended by the world health organiza- tion: Dna barcode identification associated with chemical analyses guarantees their qual- ity. PLoS ONE, 10(5):e0127866, 2015. [7] J. Hamuel. Phytochemicals: Extraction meth- ods, basic structures and mode of action as po- tential chemotherapeutic agents. In V. Rao, editor, Phytochemicals - A Global Perspective of Their Role in Nutrition and Health. InTech, 2012. [8] K.R. Bhattarai and M. Ghimire. Commercially important medicinal and aromatic plants of nepal and their distribution pattern and con- servation measure along the elevation gradient of the himalayas. Banko Janakari, 16(1):3–13, 2016. Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 278 [9] D.R. Luitel, M.B. Rokaya, B. Timsina, and Z. Münzbergová. Medicinal plants used by the tamang community in the makawanpur district of central nepal. Journal of Ethnobiology and Ethnomedicine, 10(1):5, 2014. [10] S. Thapa, S. Rawal, A. Prasai, J. Adhikari, S. Bist, and A. Ghimire. A case study of medic- inal plants and their usage by the local com- munity of dilasaini gaunpalika, baitadi district, nepal. Archives of Agriculture and Environ- mental Science, 5(1):50–54, 2020. [11] Quercus floribunda. Wikipedia, 2023. Accessed 2024-04-15. [12] D. Inam-ur Rahim, H. Maselli, Rueff, and U. Wiesmann. Indigenous fodder trees can increase grazing accessibility for landless and mobile pastoralists in northern pakistan. Pas- toralism: Research, Policy and Practice, 1(1):2, 2011. [13] M. Taib, Y. Rezzak, L. Bouyazza, and B. Ly- oussi. Medicinal uses, phytochemistry, and pharmacological activities of quercus species. Evidence-Based Complementary and Alterna- tive Medicine, pages 1–20, 2020. [14] Antibacterial and antifungal activities of some quercus species growing in turkey. ProQuest. Accessed 2024-04-15. [15] E. Burlacu, A. Nisca, and C. Tanase. A com- prehensive review of phytochemistry and bio- logical activities of quercus species. Forests, 11(9):904, 2020. [16] J. Koseki, T. Matsumoto, Y. Matsubara, K. Tsuchiya, Y. Mizuhara, K. Sekiguchi, H. Nishimura, J. Watanabe, A. Kaneko, T. Hattori, K. Maemura, and Y. Kase. Inhibition of rat 5-reductase activity and testosterone-induced sebum synthesis in ham- ster sebocytes by an extract of quercus acutis- sima cortex. Evidence-Based Complementary and Alternative Medicine, page e853846, 2015. [17] H.O. Elansary, A. Szopa, P. Kubica, H. Ekiert, M.A. Mattar, M.A. Al-Yafrasi, D.O. El-Ansary, T.K. Zin El-Abedin, and K. Yessoufou. Polyphenol profile and phar- maceutical potential of quercus spp. bark extracts. Plants, 8(11):486, 2019. [18] S.H. Park, H.J. Kim, J.S. Yoon, H.W. Lee, C.G. Park, E. Yi, G. Yoon, V.B. Schini-Kerth, and M.H. Oak. The effect of quercus salicina leaf extracts on vascular endothelial function: Role of nitric oxide. Journal of Nanoscience and Nanotechnology, 16(2):2069–2071, 2016. [19] M. Soto-García, M. Rosales-Castro, G.N. Escalona-Cardoso, and N. Paniagua-Castro. Evaluation of hypoglycemic and genotoxic ef- fect of polyphenolic bark extract from quer- cus sideroxyla. Evidence-Based Complemen- tary and Alternative Medicine, page e4032618, 2016. [20] R. Sarwar, U. Farooq, A. Khan, S. Naz, S. Khan, A. Khan, A. Rauf, H. Bahadar, and R. Uddin. Evaluation of antioxidant, free rad- ical scavenging, and antimicrobial activity of quercus incana roxb. Frontiers in Pharmacol- ogy, 6, 2015. [21] J. Shaikh and M. Patil. Qualitative tests for preliminary phytochemical screening: An overview. Chemical Science Review and Let- ters, 8:603–608, 2020. [22] X. Lu, C.F. Ross, J.R. Powers, D.E. As- ton, and B.A. Rasco. Determination of to- tal phenolic content and antioxidant activity of garlic (allium sativum) and elephant gar- lic (allium ampeloprasum) by attenuated total reflectance–fourier transformed infrared spec- troscopy. Journal of Agricultural and Food Chemistry, 59(10):5215–5221, 2015. [23] B. Sapkota, K. Khadayat, B. Adhikari, D. Poudel, P. Niraula, P. Budhathoki, B. Aryal, K. Basnet, M. Ghimire, R. Mara- hatha, and N. Parajuli. Phytochemical analy- sis, antidiabetic potential and in-silico evalua- tion of some medicinal plants. Pharmacognosy Research, 13(3):140–148, 2021. [24] C.C. Chang, M.H. Yang, H.M. Wen, and J.C. Chern. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10(3), 2010. [25] T. Sabudak, O. Demirkiran, M. Ozturk, and G. Topcu. Phenolic compounds from trifolium echinatum bieb. and investigation of their ty- rosinase inhibitory and antioxidant activities. Phytochemistry, 96:305–311, 2013. [26] N. Tamilselvi, P. Krishnamoorthy, R. Dhamotharan, P. Arumugam, and E. Sagadevan. Analysis of total phenols, total tannins and screening of phytocompo- nents in indigofera aspalathoides (shivanar vembu) vahl ex dc. Journal of Chemical and Pharmaceutical Research, 4(6):3259–3262, 2012. [27] T.H.A. Alabri, A.H.S. Al Musalami, M.A. Hos- sain, A.M. Weli, and Q. Al-Riyami. Compara- tive study of phytochemical screening, antiox- idant and antimicrobial capacities of fresh and Govinda Bhattarai et al./ BIBECHANA 22 (2025) 266-279 279 dry leaves crude plant extracts of datura metel l. Journal of King Saud University - Science, 26(3):237–243, 2014. [28] L.F. Shyur, J.H. Tsung, J.H. Chen, C.Y. Chiu, and C.P. Lo. Antioxidant properties of extracts from medicinal plants popularly used in tai- wan. International Journal of Applied Science and Engineering, 3(3):195–202, 2005. [29] M. Balouiri, M. Sadiki, and S.K. Ibnsouda. Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2):71–79, 2016. [30] T.C. Abbey and E. Deak. What’s new from the clsi subcommittee on antimicrobial susceptibil- ity testing m100, 29th edition. Clinical Micro- biology Newsletter, 41(23):203–209, 2019. [31] S.D. Sarker, L. Nahar, and Y. Kumarasamy. Microtitre plate-based antibacterial assay in- corporating resazurin as an indicator of cell growth, and its application in the in vitro an- tibacterial screening of phytochemicals. Meth- ods, 42(4):321–324, 2007. [32] Y. Baravalia, Y. Vaghasiya, and S. Chanda. Brine shrimp cytotoxicity, anti-inflammatory and analgesic properties of woodfordia fruti- cosa kurz flowers. Iranian Journal of Pharma- ceutical Research, 11(3):851–861, 2012. [33] H. Nawaz, M.A. Shad, N. Rehman, H. An- daleeb, and N. Ullah. Effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals from bean (phaseolus vul- garis) seeds. Brazilian Journal of Pharmaceu- tical Sciences, 56:e17129, 2020. [34] R. Banc, M.E. Rusu, L. Filip, and D.S. Popa. Phytochemical profiling and biological activi- ties of quercus sp. galls (oak galls): A system- atic review of studies published in the last 5 years. Plants, 12(22):3873, 2023. [35] F.M. Ahmad, A. Zafar, M. Ahmed, N. Akhtar, M.M.U. Hasan, M.A. Abdel-Maksoude, and M. Aufy. Quercus floribunda lindl. ex a. ca- mus; a tremendous remedy against inflamma- tion and associated symptoms. Fitoterapia, 170:105628, 2023. [36] J.P.A. Ferreira, I. Miranda, V.B. Sousa, and H. Pereira. Chemical composition of barks from quercus faginea trees and characteriza- tion of their lipophilic and polar extracts. PLoS ONE, 13(5):e0197135, 2018. [37] J.A. Sánchez-Burgos, M.V. Ramírez-Mares, M.M. Larrosa, J.A. Gallegos-Infante, R.F. González-Laredo, L. Medina-Torres, and N.E. Rocha-Guzmán. Antioxidant, antimicrobial, antitopoisomerase and gastroprotective effect of herbal infusions from four quercus species. Industrial Crops and Products, 42:57–62, 2013. [38] R. Sarwar, U. Farooq, A. Khan, S. Naz, S. Khan, A. Khan, A. Rauf, H. Bahadar, and R. Uddin. Evaluation of antioxidant, free rad- ical scavenging, and antimicrobial activity of quercus incana roxb. Frontiers in Pharmacol- ogy, 6:277, 2015. Introduction Materials and Methods Chemicals Collection and identification of the plant Preparation of extract Qualitative phytochemical analysis Estimation of total phenolic content (TPC) Estimation of total flavonoid content (TFC) Estimation of total tannin content (TTC) Evaluation of antioxidant activity Evaluation of antimicrobial activity of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Brine shrimp lethality assay (BSLA) Statistical Analysis Results Percentage Yield Qualitative phytochemical analysis Total phenolic content (TPC) Total flavonoid content (TFC) Total Tannin content (TTC) Antioxidant activity Antimicrobial activity Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Brine shrimp lethality activity (BSLA) Discussion Conclusion