BIBECHANA Vol. 22, No. 2, August 2025, 93-107 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 Evaluation of antioxidant, antimicrobial, and lethality Activities of leaf and bark extract of Alnus nepalensis D. Don Dipak Raj Jaishi, Dinesh Raj Ojha, Indra Ojha, Govinda Bhattarai, Khaga Raj Sharma Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract Herbal medicine has been used for centuries to treat both minor and life-threatening illnesses. This study aims to evaluate the total phenolic content (TPC), total flavonoid content (TFC), total tannin content (TTC), antioxidant potential, and various biological activities of Alnus nepalensis D. Don (Himalayan alder) extracts. Among the tested extracts, the ethanolic bark extract exhibited the highest TPC (300.6 ± 3.12 mg GAE/g) and TTC (120.09 ± 3.15 mg TA/g), while the methanolic leaf extract had the highest TFC (137.21 ± 4.67 mg QE/g). In terms of antioxidant activity, the ethanolic bark extract showed the lowest IC50 value (17.55 ± 1.17 µg/mL), followed by the methanolic leaf extract (IC50 = 24.03 ± 1.59 µg/mL), with quercetin as the standard (IC50 = 3.43 ± 1.61 µg/mL). For antimicrobial activity, the ethano- lic bark extract exhibited significant inhibition against Escherichia coli (20 mm) and Klebsiella pneumoniae (19 mm), comparable to the positive control neomycin (28 mm). Notably, the dichloromethane (DCM) bark extract demonstrated the highest zone of inhibition (21 mm) against E. coli. The methanolic leaf extract exhibited a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 0.78 mg/mL and 1.56 mg/mL, respectively, against K. pneumoniae, while the positive control showed MIC = 0.003 mg/mL and MBC = 0.007 mg/mL. In cytotoxicity assessment, the ethanolic bark extract had an LC50 value of 80.08 µg/mL, whereas the methanolic leaf extract had an LC50 of 468.81 µg/mL. These findings highlight the medicinal potential of Alnus nepalensis, suggesting its potential use as a source of bioactive compounds for developing novel therapeutic agents against infec- tious diseases. Keywords Alnus nepalensis D. Don, Antimicrobial activity, Cytotoxicity, Minimum Bactericidal Concentration (MBC), Minimum Inhibitory Concentration (MIC), Phytochemistry Article information Manuscript received: August 13, 2024; Revised: February 4, 2025; Accepted: February 10, 2025 DOI https://doi.org/10.3126/bibechana.v22i2.68759 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 93 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v22i2.68759 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 94 1 Introduction Natural materials, including different parts of plants, animals, and microorganisms, have been used in medicine to heal illnesses since ancient times. Fossil evidence suggests that humans have been using plants as medicine for at least 60,000 years [1]. The bioactive compounds naturally found in medicinal plants help to form various kinds of medicines, dietary supplements, and func- tional foods [2]. The broad therapeutic applica- tion range comprises, ease of accessibility, and safer side effects, natural products are extensively used as active ingredients for drug synthesis [3]. An- tioxidant, anticancer, antibacterial, antiviral, and anti-inflammatory properties have been shown by medicinal plants. Natural phytochemicals known as flavonoids and other phenolic compounds are present in the leaves, fruits, barks, stems, and roots of medicinal plants. These properties have led to the use of flavonoids and other phenolic com- pounds in medicine for a variety of conditions [4]. Oxidative stress, which is caused by oxygen-free radicals, is the primary cause of several degener- ative illnesses, such as cancer, gastric ulcers, and atherosclerosis. Medicinal herbs are rich in an- tioxidants that actively scavenge oxygen [5]. The phytochemicals contained in medicinal plants, such as phenolics, flavonoids, anthocyanins, terpenoids, and tannins, enable the prevention and treatment of disease. Many therapeutic plants are excellent providers of natural antimicrobial agents, offering viable substitutes for bacterial infection treatments of disease [6]. The usage of dietary or pharmaceuti- cal supplements was influenced by an imbalance be- tween reactive oxygen species (ROS) and the body’s natural antioxidant capacity, especially during the illness assault [7]. The chemical constituents found in medicinal plants are thought to be antibiotic, antifungal, an- tiviral, and shielded plants from infections [8]. Pri- mary metabolites are necessary to perform vari- ous biological activities such as translocation, pho- tosynthesis, and respiration. Secondary metabo- lites are those that have biological significance but are not necessary for survival. The production of primary metabolites involves several chemical pro- cesses, including methylation, glycosylation, and hydroxylation. Secondary metabolites undoubtedly have more complicated side chains and structural compositions [9, 10]. Phenolic compounds are a large family of sec- ondary metabolites that are primarily present in many higher plant parts including fruits, bark, leaves, and seeds, organs, and microor- ganisms. Phenolic substances exhibit important biological characteristics that include immune- modulating, antibacterial, anticarcinogenic, an- tithrombotic, anti-inflammatory, antiulcer, an- tiartherogenic, antiallergenic, antioxidant, and analgesic effects [11]. Phenolic compounds reduce FC reagents and produce a molybdenum-tungsten blue complex [12]. The fundamental structure of flavonoids is a subclass of phenolic compounds involved in the pro- duction of plant colour in flowers, the ability to re- sist disease, prevent ultraviolet damage, and influ- ence the formation of legume root nodules [13]. Re- active oxygen species (ROS) and reactive nitrogen species (RNS) in biological systems, such as super- oxide, hydroxyl, and nitric oxide radicals, may ox- idize lipids and proteins in cells and damage DNA [14–16]. Antioxidants can lessen oxidative stress, antioxidants have been shown in several studies cru- cial for preserving human health as well as for di- agnosing, treating, and preventing disease. Antiox- idants are substances that, in very small amounts found in either naturally occurring food or the hu- man body that delay, regulate, or stop oxidative processes. The process of preventing these antioxi- dant molecules from oxidizing involves a variety of techniques and actions [17]. The natural antioxi- dant system found in the human body may scavenge produced radicals by maintaining the equilibrium between oxidation and anti-oxidation. However, ex- posure to radiation, alcohol, tobacco smoke, or en- vironmental contaminants causes an overabundance of reactive oxygen species (ROS) and reactive nitro- gen species (RNS), which disrupts the equilibrium between oxidation and anti-oxidation and causes many degenerative and chronic illnesses [18, 19]. Alnus nepalensis D. Don is commonly known as Nepalese alder which belongs to the Betulaceae family that found in gully terrace forests or river beach wetlands throughout a wide range of eleva- tions between around 700-3600 meters [20] and is historically used to treat wounds, cuts, diarrhoea, and dysentery [21]. The analysis of phytochemicals and evaluation of biological properties of bark and leaf extracts such as antioxidant, antimicrobial, and toxicity against brine shrimp nauplii have not been well re- ported yet. So, the proposed research work plays a significant role in fulfilling the research gap. The major phytoconstituents which show richness re- ported from the genus Alnus are shown in Figure 1. Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 95 Figure 1: Chemical compounds reported from Al- nus nepalensis D. Don and Alnus nitida D. Don 2 Materials and Methods 2.1 Chemicals Merck and Scientific Fischer provided analytical grade (extra pure) solvents, such as methanol, ethanol, ethyl acetate, dichloromethane, and hex- ane. EDTA disodium salt dihydrate was purchased from SRL, and boric acid, calcium chloride fused, and sodium chloride were purchased from Merck. FC reagent and Resazurin (LOBA CHEMI Pvt. Ltd), Mueller Hinton Broth, Nutrient Agar, and Mueller Hinton Agar media were purchased from HieMedia. 2.2 Collection and Identification of the Plant The bark and leaf parts of A. nepalensis D. Don medicinal plant were collected from the Far West- ern region of Nepal (Doti). The local name, scien- tific name, parts of medicinal plants used, and their ethnomedical usage are shown in Table 1. The taxo- nomic identification of A. nepalensis D. Don medic- inal plant was conducted by National Herbarium and Plant Laboratories, Godawari, and research of- ficers of the herbarium center provided the voucher code 01KATH163159. The Photographs of the se- lected medicinal plant and the study area are shown in Figure 2 and Figure 3. Figure 2: Alnus nepalensis D. Don. Figure 3: Sample collected cite. Table 1: Description of A. nepalensis D. Don Scientific Name Local Name Family Parts Used Traditional Uses Reference A. nepalensis D. Don Utis Betulaceae Leaf, Bark The plant parts have been used to treat wound healing, against dysentery, diarrhea, inflammation, and headaches. [21] 2.3 Preparation of Extract The bark and leaves parts of A. nepalensis D. Don were washed thoroughly, shade dried, ground into finely divided powder, and immersed in six differ- ent solvents, ranging in polarity from more polar to less polar they are; water > methanol > ethanol > ethyl acetate, DCM > hexane. Afterward, each filtrate was dried in a water bath that was kept at a temperature of up to 40 ºC. Finally, they were collected in glass vials and kept at 4 ºC for further analysis. The yield percentage was calculated by using the formula: Yield percentage of crude extract = Weight of dried crude extract Weight of sample × 100 (1) 2.4 Qualitative Phytochemical Analysis The phytochemicals present in the crude plant ex- tracts were qualitatively analyzed by following stan- dard protocol [22–24]. The numerous phytochemi- cals, including glycosides, flavonoids, alkaloids, phe- nolic compounds, terpenoids, steroids, carbohy- drates, saponins, tannins, fixed oils, and lipids, were screened as metabolites. 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 as described by Lu et al. [25]. 20 µL of plant extract, 100 µL of 10 % FC reagent (1:10), and 80 µL of 1M Na2CO3 were loaded in a 96-well plates in triplicates. The reaction mixture was allowed to in- Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 96 cubate at room temperature for half an hour before a deep blue colour was observed. Ultimately, a spec- trophotometer was used to measure absorbance at 765 nm. The standard curve was generated which is the standard curve for gallic acid (7.5-100 µg/mL) and the total phenolic content (TPC) was measured in milligrams of gallic acid equivalent (mg GAE/g) per gram of extract dry weight. 2.6 Estimation of Total Flavonoid Content (TFC) The total flavonoid content was estimated by using aluminium chloride method as described by Ahmed et al. [26]. 20 µL of plant extract, 100 µL of dis- tilled water, and 60 µL ethanol followed by 10 µL of 10% AlCl3 solution and 10 µL (1M) CH3COOK solution were loaded in 96-well plates in triplicates. The reaction mixture was incubated for 30 minutes at room temperature. Then, absorbance was taken at 415 nm with the help of a spectrophotometer. A quercetin (10-100 µg/mL) standard calibration curve was created, and measured in milligrams of quercetin equivalent per gram of the extract’s dry weight (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 standard protocol [25]. 96-well plates were filled with 10 µL of plant extract and different tannic acid concentrations (7.5-100 µg/mL). The first reading was taken at 725 nm using a microplate reader after adding 70 µL of distilled water and 50 µL of 10% FC reagent. After taking an initial reading, 70 µL of 35 % Na2CO3 was loaded. Finally, the 96-well plate was incubated for 30 minutes and consequently, its final absorbance was taken at 725 nm. The TTC was measured as mg TA/g. Calibration curves were created by plotting the graph of absorbance on the y-axis and the standard concentration on the x-axis. These curves were then used to calculate the concentrations of phenolics, flavonoids, and tannins. R2, the coefficient of de- termination, was determined to be between 0.9753 and 0.9997 when the data were fitted linearly. 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 5 µg/mL from 160 µg/mL concentrations but positive control quercetin was serially diluted up to 0.625 µg/mL from 20 µg/mL concentrations. 100 µL of plant extracts and positive control were loaded in 96-well plates in triplicates. After that, the initial reading was taken at 517 nm. Following this, 100 µL of DPPH reagent was loaded into every well and incubated for half an hour. Finally, the final ab- sorbance was taken at 517 nm. Since 50% DMSO and methanol were used as a negative control. The following formula was used to evaluate rad- ical scavenging activity: Radical scavenging capacity =( Acontrol −Asample Acontrol ) × 100 (2) 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 Hin- ton Agar (MHA) plates [29–31]. Mueller Hinton Broth (MHB) was used for the growth of test mi- croorganisms, ATCC 25931 Shigella sonnei, ATCC 43300 Staphylococcus aureus, ATCC 700603 Kleb- siella pneumonae and ATCC 25312 Escherichia coli respectively and it was incubated at 37 ºC for 24 hrs and its turbidity was maintained by using 0.5 McFarland. 50 µL of plant extract, 50% DMSO as the negative control, and 50% neomycin as the positive control were loaded into each well created by a cork borer. The Petri dishes were then incu- bated for 18-24 hours at 37 °C after being left for 15 minutes to allow for diffusion. After incubation, the zone of clearance was observed and measured. 2.10 Determination of Minimum In- hibitory Concentration (MIC) and Minimum Bactericidal Concentra- tion (MBC) The minimum inhibitory concentration (MIC) and minimum bactericidal concentration were deter- mined by following the standard protocol as de- scribed by Sarker et al. [32]. The 0.5 McFarland turbidity culture in MHB was diluted 1:100 to give the bacterial inoculum a final concentration of 106 CFU/mL. 5 µL of bacteria were injected into ev- ery well of 96 well plates. A common medication called neomycin served as the positive control. The plate was covered with a sterile lid and incubated for 20-24 hrs at 37 °C. The microtiter plate wells were filled with 0.003% resazurin, and the mix- ture was incubated for 3 to 4 hours at 37 °C. The colour of the wells with bacterial growth changed to pink, whereas the wells without infection stayed blue. The extract’s minimum inhibitory concentra- tion (MIC) was found to be the lowest at which bac- terial growth is inhibited. By streaking the contents Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 97 of the wells onto nutrient agar plates and incubating them at 37 °C for more than 18 hours consequently, the MIC and MBC of the crude plant extracts were determined. 2.11 Brine Shrimp Lethality Activity (BSLA) The toxicity of plant extracts was determined by following a standard protocol [33]. The artificial sea salt water was prepared by maintaining a pH of around 8 to 8.5 by adding 1M NaOH. The plant ex- tract was diluted in various concentrations such as 1000, 800, 500, 250, 125, 100, and 10 µg/mL. Then, 4 mL of artificial sea salt water was filled in each test tube. After that, 500 µL of sample, and 10 nauplii were added to each test tube in triplicate. Artifi- cial sea salt water served as the negative control, while potassium dichromate solution was employed as the positive control. The number of dead nau- plii in a test tube was counted after 24 hours, and the mortality of nauplii percentage was determined using the following formula: % mortality = Number of dead nauplii Total number of nauplii × 100 Using the Probit value table, the linear equa- tion can be obtained as Y = mx+c, where Y is the Probit value at 50% mortality, m is 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 Gen5 Microplate reader for Data Collection and Analysis software was used for result process- ing, followed by Microsoft Excel. The data were reported for TPC, TFC, and TTC as the mean ± standard deviation. For antioxidants, the data were reported as mean ± standard error mean. In- hibitory concentration (IC50) was calculated by us- ing GraphPad Prism (version 8.0.2.263). One-way ANOVA using Tukey’s test was used for compar- isons, P < 0.05 values were regarded as statistically significant. 3 Results 3.1 Percentage Yield The yield percentage of crude extracts of bark was found to be higher in methanolic extract (19.17 %) followed by ethanol (9.23 %), ethyl acetate (3.56 %), hexane (2.81 %), aqueous (2.80 %), and DCM (2.45 %). Similarly, In the case of leaves, methano- lic extract had a higher percentage of yield (10.78 %) followed by ethyl acetate (10.18 %), DCM (8.92 %), ethanol (5.82 %), and hexane (5.42 %) (Figure 4). Figure 4: Yield percentage of bark and leaf crude extracts 3.2 Qualitative Phytochemical Analysis The results of qualitative phytochemical screening of the bark and leaf extract (in different solvents) are shown in Table 2. Table 2: Qualitative phytochemical screening of plant extracts. Phytochemicals Test Bark extract Leaf extract Alkaloids Dragendorff’s test - - Carbohydrates Molish’s test + + Reducing sugars Fehling’s test - - Glycosides Borntrager’s test + + Amino acids Xanthoproteic test + + Flavonoids Alkaline reagent test + + Phenols FeCl3 test + + Tannins Braymer’s test + + Terpenoids Salkowski’s test + + Anthraquinones Borntrager’s test + + Phytosterols Salkowski’s test + + Note: - (+) = present, (-) = absent Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 98 3.3 Total Phenolic Content (TPC) The ethanolic bark extract exhibited the maximum TPC of 300.6± 3.12 mg GAE/g. The TPC of hex- ane bark extract was found to be the lowest which is 53.67±3.59 mg GAE/g. The TPC of bark extracts ranges from 300.6±3.12 mg GAE/g to 53.67±3.58 mg GAE/g (Table 3). The TPC of leaf extracts ranges from 254.6±5.58 mg GAE/g to 73.93±3.63 mg GAE/g. Methanolic leaf extract had the high- est TPC which is 254.6± 5.38 mg GAE/g and hex- ane extract of the leaf had the lowest TPC which is 73.93 ± 3.63 mg GAE/g respectively. Ethanolic bark extract had the highest TPC than of methano- lic leaf extract. The calibration curve is shown in Figure 5. Figure 5: Gallic acid standard calibration curve. Table 3: TPC for various solvent extracts Extraction sources Solvent type Total phenolic content (mg GAE/g) Bark Aqueous 101.8± 3.41 Bark Methanol 292.2± 1.74 Bark Ethanol 300.6± 3.12 Bark Ethyl acetate 124.47± 4.20 Bark DCM 121.53± 1.22 Bark Hexane 53.67± 3.59 Leaves Methanol 254.6± 5.58 Leaves Ethanol 152.47± 2.89 Leaves Ethyl acetate 201.13± 3.45 Leaves DCM 88.47± 2.57 Leaves Hexane 73.93± 3.63 3.4 Total Flavonoid Content (TFC) DCM extract of bark had the highest TFC which is 75.99 ± 4.72 mg QE/g and hexane extract had the lowest TFC which was 50.09 ± 6.31 mg QE/g. TFC of bark extract ranges from 75.99 ± 4.72 mg QE/g to 50.09 ± 6.31 mg QE/g (Table 4). The methanol leaf extract had the highest TFC which is 137.21 ± 4.67 mg QE/g and hexane extract had the lowest TFC 13.12 ± 1.84 mg QE/g. The TFC of leaf extract ranged from 137.21± 4.67 mg QE/g to 13.12 ± 1.84 mg QE/g. Among all the extracts, the methanolic leaves extract had the highest TFC, which is 137.21 ± 4.67 mg QE/g than that of bark extracts. The calibration curve is shown in Figure 6. Figure 6: Quercetin standard calibration curve. Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 99 Table 4: TFC of bark and leaf extracts Extraction sources Solvent types Total flavonoid content (mg QE/g) Bark Aqueous 62.97± 1.84 Bark Methanol 60.40± 2.78 Bark Ethanol 65.24± 2.24 Bark Ethyl acetate 61.76± 1.39 Bark DCM 75.99± 4.72 Bark Hexane 50.09± 6.31 Leaves Methanol 137.21± 4.67 Leaves Ethanol 104.33± 1.39 Leaves Ethyl acetate 111.15± 3.87 Leaves DCM 35.24± 2.24 Leaves Hexane 13.12± 1.84 3.5 Total Tannin Content (TTC) The maximum TTC was found to be 120.09± 3.15 mg TA/g, in the ethanolic bark extract. Hex- ane extract of bark had the lowest TTC which is 8.27 ± 0.45 mg TA/g. The TTC of bark extract ranged from 120.09± 3.15 mg TA/g to 8.27± 0.45 mg TA/g. The TTC of the methanolic leaf extract was found to be 43.72±3.96 mg TA/g, whereas the lowest TTC was found in ethyl acetate leaf extract which is 8.42± 1.31 mg TA/g (Table 5). The TTC ranges from 43.72±3.96 mg TA/g to 8.42±1.31 mg TA/g in leaf extracts. Among all solvent extracts, ethanolic bark extract had the highest TTC, and hexane extract of bark had the lowest TTC. The calibration curve is shown in Figure 7. Figure 7: Tannic acid standard calibration curve Table 5: Total tannin content of bark and leaf extracts. Extraction sources Solvent type Total tannin content (mg TA/g) Bark Aqueous 74.03± 5 Bark Methanol 86.15± 4.73 Bark Ethanol 120.09± 3.15 Bark Ethyl acetate 28.88± 3.03 Bark DCM 71± 6.01 Bark Hexane 8.27± 0.45 Leaves Methanol 43.73± 3.96 Leaves Ethanol 28.42± 1.72 Leaves Ethyl acetate 8.42± 1.31 Leaves DCM 15.85± 0.69 Leaves Hexane 9.79± 0.95 3.6 Antioxidant Potential Hexane and ethyl acetate extracts had the highest IC50 which is > 500 µg/mL and 247.9±1.73 µg/mL respectively while ethanolic extract had the lowest which is 17.55±1.17 µg/mL (Figure 8). The ethano- lic extract had more antioxidant potential than all crude bark extracts. The IC50 of the standard quercetin compound was found to be 3.43 ± 1.61 µg/mL. Which is five times less than ethanolic ex- tract. The methanolic extract of the leaf had the lowest IC50 24.03 ± 1.59 µg/mL among all leaf ex- tracts. However, the hexane and ethyl acetate ex- tracts of the leaf exhibited the highest IC50 which are > 500 µg/mL and 464.7 ± 0.22 µg/mL respec- tively. The methanolic leaf extract had greater an- tioxidant potential and the hexane extract had the lowest antioxidant potential. The IC50 of methano- lic leaf extract was eight times greater than the IC50 of standard quercetin. Compared to the crude leaf Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 100 extracts, the ethanolic extract of bark had the high- est antioxidant potential. The percent inhibition against the different concentrations of the plant ex- tract is shown in Figure 13. Figure 8: Bar diagram showing antioxidant poten- tial (IC50) of bark and leaf crude extracts in differ- ent solvents at various concentrations. Figure 9: A plot of % inhibition against the concen- tration of (a) quercetin (b) methanolic and ethano- lic bark extracts (c) DCM, ethyl acetate & aqueous bark extracts (d) methanol, ethanol, ethyl acetate, and DCM leaf extracts The IC50 values of various extracts of bark and leaf and standard quercetin as a reference com- pound are shown in Table 6. Table 6: The antioxidant capacity (IC50) of the aqueous, methanol, ethanol, ethyl acetate, DCM, and hexane crude extracts of bark and leaf. Part Plant extracts IC50 (µg/mL) Bark Aqueous 62.16± 3.66 Bark Methanol 58.06± 0.22 Bark Ethanol 17.55± 1.17 Bark Ethyl acetate 247.9± 1.73 Bark DCM 87.16± 0.87 Bark Hexane > 500 Bark *Quercetin 3.43± 1.61 Leaf Methanol 24.03± 1.59 Leaf Ethanol 42.07± 2.17 Leaf Ethyl acetate 464.7± 0.22 Leaf DCM 55.44± 1.70 Leaf Hexane > 500 Leaf *Quercetin 3.43± 1.61 Note: - *Quercetin = positive control 3.7 Antimicrobial Activity The antimicrobial activity in terms of the zone of inhibition of various crude extracts of bark and leaf against K. pneumoniae, E. coli, S. sonnei, and S. aureus respectively are shown in Table 7. The ethanolic bark extract had a strong antibacterial ac- tivity with ZOI of 20 mm, 15 mm, 14 mm, and 19 mm respectively against Escherichia coli, Shigella sonnei, Staphylococcus aureus, and Klebsiella pneu- moniae. This was quite close to the positive control, namely Neomycin 24 mm for Staphylococcus aureus and Shigella sonnei and 28 mm for E. coli and Kleb- siella pneumoniae respectively. Among all extracts of bark, DCM extract shows very potent activity against E. coli with ZOI 21 mm (Neomycin control 28 mm). However, hexane extract had no zone of inhibition against all four bacteria which is 9 mm. This was far from the ZOI of positive control which are 25 mm for Klebsiella pneumoniae, 22 mm for E. coli, 20 mm for Shigella sonnei, and 24 mm for Staphylococcus aureus respectively. The methanolic, ethanolic, and ethyl acetate Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 101 extracts of leaves show good antibacterial activity against Klebsiella pneumoniae, E. coli, and Shigella sonnei with ZOI 21, 11, and 15 mm respectively (Neomycin positive control 25 mm and 24 mm). The DCM and hexane extracts of leaves had less po- tent antimicrobial activity against Klebsiella pneu- moniae, E. coli, Shigella sonnei, and Staphylococcus aureus respectively. The ZOI shown by the bark and leaf extracts of the plant is shown in Figures Figure 10 and Figure 11. 3.8 MIC and MBC The MIC and MBC of ethanolic bark and methano- lic leaf extracts are illustrated in Table 8. The methanolic leaf extract and the ethanolic bark ex- tract were employed for the evaluation of MIC and MBC against Klebsiella pneumoniae and Staphy- lococcus aureus, respectively. The ethanolic bark extract shows minimum inhibitory concentration (MIC) against Staphylococcus aureus and Kleb- siella pneumoniae, which are 3.12 mg/mL and 1.56 mg/mL, respectively. The MBC of ethanolic bark extract against Klebsiella pneumoniae and Staphy- lococcus aureus were found to be 3.12 mg/mL and 6.25 mg/mL, respectively. The methanolic leaf extract shows MIC 1.56 mg/mL and 0.78 mg/mL against Staphylococcus au- reus and Klebsiella pneumoniae, respectively. The MBC of the methanolic leaf extract against Kleb- siella pneumoniae and Staphylococcus aureus was found to be 1.56 mg/mL and 3.12 mg/mL, respec- tively. The methanolic leaf extract exhibited the lowest MIC and MBC against Klebsiella pneumo- niae, which are 0.78 and 1.56 mg/mL, respectively. In conclusion, methanolic leaf extract had the low- est MIC and MBC against Klebsiella pneumoniae. The MIC and MBC of methanolic leaf and ethanolic bark extracts against Klebsiella pneumo- niae and Staphylococcus aureus were evaluated us- ing nutrient agar plates, and the captured images are displayed in Figure 12. Table 7: ZOI shown by crude bark and leaf extracts against Klebsiella pneumoniae, Escherichia coli, Shigella sonnei, and Staphylococcus aureus. Part Plant Extracts Bacteria Used ZOI shown by the sample (mm) ZOI shown by the positive control Neomycin (mm) Bark Methanol Klebsiella pneumoniae 17 28 Escherichia coli 18 28 Shigella sonnei 14 24 Staphylococcus aureus 12 24 Ethanol Klebsiella pneumoniae 19 28 Escherichia coli 20 28 Shigella sonnei 15 24 Staphylococcus aureus 14 24 Ethyl acetate Klebsiella pneumoniae 9 25 Escherichia coli 9 22 Shigella sonnei 14 20 Staphylococcus aureus 13 24 DCM Klebsiella pneumoniae 11 28 Escherichia coli 21 28 Shigella sonnei 12 20 Hexane Klebsiella pneumoniae 9 25 Escherichia coli 9 22 Shigella sonnei 9 20 Staphylococcus aureus 9 24 Leaves Methanol Klebsiella pneumoniae 21 25 Escherichia coli 9 22 Shigella sonnei 14 24 Staphylococcus aureus 9 24 Ethanol Klebsiella pneumoniae 13 26 Escherichia coli 11 24 Shigella sonnei 9 20 Staphylococcus aureus 9 20 Ethyl acetate Klebsiella pneumoniae 9 25 Escherichia coli 9 22 Shigella sonnei 15 24 Staphylococcus aureus 9 24 DCM Klebsiella pneumoniae 9 26 Escherichia coli 9 24 Shigella sonnei 9 20 Hexane Staphylococcus aureus 9 20 Klebsiella pneumoniae 9 26 Escherichia coli 9 24 Shigella sonnei 9 20 Staphylococcus aureus 9 20 Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 102 Table 8: MIC and MBC of methanolic leaf and ethanolic bark extracts against Klebsiella pneumoniae and Staphylococcus aureus. Plant Extracts Klebsiella pneumoniae Staphylococcus aureus MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL) Ethanol-B 1.56 3.12 3.12 6.25 Methanol-L 0.78 1.56 1.56 3.12 Positive Control 0.003 0.007 0.007 0.015 Figure 10: ZOI shown by the bark extract against the organism used (a) Klebsiella pneumoniae (b) Escherichia coli (c) Shigella sonnei. Figure 11: ZOI shown by the leaf extracts against the microorganisms (a) K. pneumoniae, (b) E. coli, (c) S. sonnei, and (d) S. aureus Figure 12: MIC and MBC shown by (a) ethanol- bark extract, (b) methanol-leaf extract, (c) ethanol bark extract (d) methanol leaf extract (e) positive control against Klebsiella pneumoniae, and Staphy- lococcus aureus 3.9 Toxicity Analysis The percentage mortality shown by the plant ex- tract against the Brine shrimp nauplii is displayed in Table 9. The ethanolic bark extract showed the lowest LC50 of 80.08 µg/mL. The methanolic leaf extract had the highest LC50 of 468.81 µg/mL. The potassium dichromate served as the positive control and 50% DMSO served as the negative control. All nauplii were dead in the positive control (10 nau- plii), and the same number of nauplii survived in the 50% DMSO solution. The LC50 of crude plant extracts. Figure 13: A plot Log C against Probit for the determination of LC 50 of ethanolic bark and methanolic leaf extract 4 Discussion The Genus Alnus is an alder plant mostly found in Nepal’s mountainous regions, it is also found in China, India, and Korea. The majority of its quite extensive applications are for the treatment of fever, diarrhoea, dysentery, inflammatory diseases, skin diseases, elephantiasis, stomachaches, and wound healing. Numerous biological activities, including antidiabetic, antioxidant, hepatoprotective, and an- tifungal effects, are performed by A. nepalensis D. Don. In previous studies, 423.49 ± 0.89 mg GAE/g was the TPC of methanolic leaf extract and 408.88 ± 2.08 mg GAE/g was the flavonoid content [34]. However, in this study, 254.6 ± 5.58 mg GAE/g TPC and 137.21 ± 4.67 mg QE/g TFC respectively. Methanolic extract of the leaf contains maximum phenolic and flavonoid levels. TPC and TFC of bark extracts were compared with Alnus nitida. Ac- cording to a previous study on Alnus nitida (Bark), methanolic bark extract had TPC 631.5 ± 1.7 mg GAE/g and ethanolic bark extract had TPC 607 ± 1.97 mg GAE/g respectively. Similarly, 221.5 ± 2.5 mg QE/g was the reported TFC of methanolic extract of this plant [35]. However, in this study, the ethanolic bark extract had a higher phenolic level which is 300.6 ± 3.12 mg GAE/g. However, the flavonoid level was higher in the DCM extract which is 75.99 ± 4.72 mg QE/g. Methanolic leaf ex- Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 103 tract had maximum TPC and TFC which is 254.6 ± 5.58 mg GAE/g and 137.21 ± 4.67 mg QE/g respec- tively. In comparison with both bark and leaf dif- ferent extracts, ethanolic bark extract contained a high quantity of phenolics. But, methanolic leaf ex- tract had a high amount of flavonoids. The overall phenolic and flavonoid contents of medicinal plants might vary depending on factors such as geographi- cal range, plant collecting time, and climate fluctu- ation. Phenolics and flavonoids were directly cor- related with antioxidant properties. High amounts of phenolics and flavonoid phytoconstituents corre- lated with antioxidant properties. Phenolic compounds are directly correlated with tannins. Ethanolic bark extract had a high TTC which is 120.09 ± 3.15 mg TA/g. In the case of leaves, methanolic extract had a high TTC which is 43.73 ± 3.96 mg TA/g. In the previous study, methanolic leaf extract had IC50 which is 4.838 µg/mL [34]. However, in this study, IC50 of methanolic leaf extract was found as 24.03 ± 1.59 µg/mL. Ethanolic bark extract had IC50 39.5 ± 2.11 µg/mL in the previous study [35]. In this study, IC50 of ethanolic bark extract found as 17.55 ± 1.17 µg/mL. In comparison with leaves and bark, ethanolic bark extract possesses good antioxidant potential. TPC and TTC values in bark extract were higher in ethanolic extract which was posi- tively correlated with the IC50 of ethanolic bark extract. The climatic conditions and other environ- mental factors, antioxidant capacity, TPC, TFC, and TTC were considerably different [36]. Plant extracts are more effective antioxidants because they have a special functional group of secondary metabolites [37,38]. A prior study depicts that methanolic leaf ex- tract of A. nepalensis D. Don was subjected to the antimicrobial assay against A. baumannii with ZOI 14.66 mm and P. mirabilis with ZOI 15.50 mm [34]. However, the methanolic leaf extract of this plant in this study possesses better antimicro- bial activity against Klebsiella pneumoniae with ZOI 21 mm. No antimicrobial assay was carried out for the A. baumannii and P. mirabilis bac- teria. Instead, this antimicrobial assay was car- ried out against Klebsiella pneumoniae, Escherichia coli, Shigella sonnei, and Staphylococcus aureus re- spectively. Methanolic leaf extract had ZOI 14 mm against Shigella sonnei. Ethanolic leaf extract of this medicinal plant possesses 13 mm ZOI against K. pneumoniae. 15 mm was the ZOI of ethyl ac- etate extract against Shigella sonnei. None of the remaining leaf extracts inhibit bacterial growth. In bark, ethanolic bark extract had good antimicro- bial activity against E. coli with ZOI 20 mm, and K. pneumoniae with ZOI 19 mm. DCM extract had maximum ZOI which is 21 mm against E. coli. Hexane has no inhibition against subjected bacteria. Ethanolic extract of bark and methano- lic extract of leaf showed good antimicrobial activ- ity against Klebsiella pneumoniae and Staphylococ- cus aureus respectively. Not only phenolics and flavonoids but other phytochemicals such as vita- mins, carotenoids, Saponins, enzymes, and miner- als showed antimicrobial activity [39, 40]. Ethano- lic bark extract and methanolic leaf extract of this medicinal plant showed good antimicrobial ac- tivity against K. pneumoniae and S. aureus, so, these samples were subjected to determine MIC and MBC. The ethanolic bark and methanolic leaf extract medicinal plant had good MIC and MBC which are 1.56 mg/mL and 3.12 mg/mL for bark and for leaf 0.78 mg/mL and 1.56 mg/mL respec- tively against K. pneumoniae. In comparison to ethanolic bark extract, methanolic leaf extract had potent MIC and MBC against K. pneumoniae. A lethality experiment was conducted on ethanolic and methanolic extracts of bark and leaves and LC50 of ethanolic bark extract was found to be 80.08 µg/mL and LC50 for methanolic leaf extract was found to be 468.81 µg/mL. In comparison be- tween them, ethanolic bark extract had a potent lethal activity as compared to methanolic leaf ex- tract. Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 104 Table 9: The number of survived nauplii after treatment with methanolic leaf and ethanolic bark extracts and their percentage mortality Plant Extract Concentration (g/mL) Total No. of Survived Nauplii % Mortality Ethanol-Bark Extract 10 24 20 100 18 40 125 16 46.66 250 3 90 500 8 73.33 800 3 90 1000 6 80 Methanol-Leaf Extract 10 22 26.66 100 18 40 125 16 46.66 250 24 20 500 18 40 800 12 60 1000 8 73.33 Table 10: LC50 of ethanolic bark and methanolic leaf plant extracts Plant Extracts Linear Regression Equation LC50 (g/mL) Ethanol-B 5.0 = 1.0244x+ 3.05 80.08 Methanol-L 5.0 = 0.4499x+ 3.79 468.81 5 Conclusion The present study highlights Alnus nepalensis D. Don as a rich source of bioactive phytochemi- cals, including carbohydrates, glycosides, amino acids, flavonoids, phenols, tannins, terpenoids, anthraquinones, and phytosterols. Among the tested extracts, the ethanolic bark extract exhibited the highest total phenolic content (TPC) among bark extracts, while the methanolic leaf extract had the highest TPC among leaf extracts. The dichloromethane (DCM) bark extract showed the highest total flavonoid content (TFC) among bark extracts, whereas methanolic leaf extract had the highest TFC among leaf extracts. Similarly, tan- nin content was highest in the ethanolic bark and methanolic leaf extracts. Overall, bark extracts demonstrated higher phenolic and tannin content, while leaf extracts were richer in flavonoids. In terms of bioactivity, the ethanolic bark ex- tract exhibited the strongest antioxidant potential among the bark extracts, whereas the methanolic leaf extract showed the highest antioxidant activity among leaf extracts. Hexane extracts of both bark and leaves demonstrated the weakest antioxidant potential. The ethanolic and DCM extracts of bark exhibited good antimicrobial activity against Es- cherichia coli, whereas the methanolic leaf extract showed significant antimicrobial activity against Klebsiella pneumoniae. Furthermore, the methano- lic leaf extract demonstrated superior MIC and MBC values compared to the ethanolic bark ex- tract. The ethanolic bark extract exhibited higher toxicity, as indicated by a lower lethal concentra- tion, compared to the leaf extract. These findings suggest that A. nepalensis D. Don possesses signif- icant antioxidant, antimicrobial, and toxicological properties, making it a promising candidate for the discovery of natural antibacterial compounds. Fur- ther research is required to explore its pharmaco- logical potential and underlying mechanisms, par- ticularly in the development of novel therapeutic agents. Abbreviations • DMSO: Dimethyl sulfoxide • GAE/g: Gallic acid equivalent per gram • QE/g: Quercetin equivalent per gram • TA/g: Tannic acid equivalent per gram • TPC: Total phenolic content • TFC: Total flavonoid content • TTC: Total tannin content • IC50: Half-maximum inhibitory concentra- tion • DPPH: 2,2-diphenyl-1-picrylhydrazyl • ZOI: Zone of inhibition Dipak Raj Jaishi et al./ BIBECHANA 22 (2025) 93-107 105 • MIC: Minimum inhibitory concentration • MBC: Minimum bactericidal concentration • 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. 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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 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Brine Shrimp Lethality Activity (BSLA) Statistical Analysis Results Percentage Yield Qualitative Phytochemical Analysis Total Phenolic Content (TPC) Total Flavonoid Content (TFC) Total Tannin Content (TTC) Antioxidant Potential Antimicrobial Activity MIC and MBC Toxicity Analysis Discussion Conclusion