BIBECHANA Vol. 22, No. 2, August 2025, 131-141 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 Estimation of phytochemicals, antioxidant, antimicrobial, and brine shrimp lethality activities of Valeriana jatamansi Jones Indra Ojha, Khaga Raj Sharma∗ 1Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract Herbal medicine has been widely utilized in traditional medicine for treating various ailments. This study aimed to quantify the phenolic, flavonoid, and tannin content in different plant extracts, assess their antioxidant capacity, and evaluate their antimicrobial efficacy. Among the extracts tested, methanol exhibited the highest total phenolic content (TPC) at 166.5 ± 2.0 mg GAE/g and total flavonoid content (TFC) at 103.87 ± 5.47 mg QE/g, whereas hex- ane displayed the lowest TPC (16.03 ± 2.22 mg GAE/g) and TFC (8.27 ± 3.88 mg QE/g). The methanolic extract demonstrated the strongest antioxidant activity with an IC50 of 103.3 ± 1.53 g/mL, compared to the hexane extract's IC50 of 524.7 ± 0.89 µg/mL. Antimicrobial testing revealed that aqueous, ethanolic, and hexane extracts effectively inhibited Klebsiella pneumoniae with a zone of inhibition of 17 mm, comparable to the positive control neomycin (24 mm). The ethanolic extract showed substantial minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against K. pneumoniae and Staphy- lococcus aureus (3.12 mg/mL and 6.25 mg/mL, respectively), relative to the control (0.0078 mg/mL and 0.015 mg/mL). Additionally, cytotoxicity assays showed LC50 values of 1905.46 µg/mL for the ethanolic and 2137.96 µg/mL for the methanolic extract. These findings suggest that the studied plant extracts, particularly methanolic and ethanolic, have significant bioactive potential, positioning them as promising sources for future drug development against infectious diseases. Keywords Valeriana jatamansi, TPC, TFC, DPPH, antimicrobial, MIC, MBC, toxicity Article information Manuscript received: November 8, 2024; Revised: March 16, 2025; Accepted: March 17, 2025 DOI https://doi.org/10.3126/bibechana.v22i2.71378 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Medicinal plants have long played a critical role in human health, providing therapeutic compounds that have been used since ancient times to man- age and treat diseases. These plants, which contain bioactive compounds in various organs, serve either as direct therapeutic agents or as essential precur- sors for drug synthesis. A distinction exists between medicinal plants that have been scientifically vali- dated for their therapeutic effects and those consid- 131 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v22i2.71378 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 132 ered medicinal but lack thorough scientific study, underscoring the need for continued research into these natural resources [1, 2]. Natural materials, including plant parts, ani- mal products, and microorganisms, have a histori- cal foundation in medicine, with fossil evidence in- dicating that humans have utilized plants for medic- inal purposes for at least 60,000 years. The chemi- cal diversity found in natural compounds has de- veloped over millions of years, providing an ar- ray of biologically active substances with potential pharmacological effects [3]. Phytochemicals, the bioactive constituents produced by plants, are es- sential metabolites that contribute to various func- tions such as plant growth, pollination, and defense against pathogens. These compounds are not only crucial for plant survival but also play an impor- tant role in protecting plants from environmental stressors, including disease and UV radiation [4,5]. Phytochemicals can be classified into primary and secondary metabolites. Primary metabo- lites, like proteins, carbohydrates, and nucleic acids, are indispensable for basic plant functions, while secondary metabolites, including phenolics, flavonoids, and terpenoids, arise from more special- ized metabolic pathways. These secondary com- pounds are often associated with defensive func- tions, including antibacterial, antifungal, and an- tiviral properties, making them of great interest for therapeutic use [6,7]. Antioxidants, flavonoids, and phenolic compounds found in medicinal plants help combat oxidative stress, which is linked to degen- erative diseases such as cancer, atherosclerosis, and gastric ulcers [8, 9]. One notable medicinal plant is Valeriana jata- mansi Jones, a perennial herb from the Valeri- anaceae family, native to the Himalayan region and traditionally valued for its broad spectrum of medicinal properties [10, 11]. Distributed from Afghanistan to China, V. jatamansi Jones has been used to treat various conditions, including neurological disorders, insomnia, and bacterial in- fections. Its bioactive profile includes valepotriates, lignans, sesquiterpenoids, flavones, and other phy- tochemicals, supporting its application in ailments such as epilepsy, skin diseases, and obesity [12–14]. In addition to its historical significance in tradi- tional medicine, studies have highlighted the plant’s anti-inflammatory, sedative, antioxidant, and neu- roprotective effects [15]. Despite its longstanding use, comprehensive studies examining the phytochemical composition and biological properties of the aerial parts of V. jatamansi Jones remain limited. This study aims to fill this research gap by analyzing the phytochem- ical constituents and evaluating the biological ac- tivities of the aerial parts of V. jatamansi Jones, contributing to a deeper understanding of its ther- apeutic potential. 2 Materials and Methods 2.1 Chemicals For this study, high-purity analytical-grade solvents-methanol, ethanol, ethyl acetate, dichloromethane, and hexane- were sourced from Merck and Fischer Scientific to ensure optimal extraction conditions. EDTA disodium salt di- hydrate was supplied by SRL, while Merck pro- vided additional reagents including sodium chlo- ride, boric acid, and fused calcium chloride. Key materials such as the Folin-Ciocalteu (FC) reagent, Resazurin, and microbial culture media including Mueller Hinton Broth, Nutrient Agar, and Mueller Hinton Agar were obtained from HieMedia and LOBA CHEMI Pvt. Ltd. The use of high-quality reagents and culture media was essential for achiev- ing precision in both the phytochemical analyses and microbiological assays. 2.2 Plant Collection and Identification The aerial parts of Valeriana jatamansi Jones were collected from the Far Western region of Nepal (Sayal Gaupalika-02, Doti). Details such as the scientific name, local name, plant parts used, and ethnomedicinal applications are summarized in Ta- ble 1. The specimen was authenticated by research officers at the Herbarium Center, where it was as- signed the voucher code 01KATH162457. A visual representation of the V. jatamansi Jones medicinal plant is provided in Figure 1. Figure 1: Aerial parts of V. jatamansi Jones Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 133 Table 1: Description of V. jatamansi Jones Scientific name V. jatamansi Jones Local name Sugandhawal Family Valerianaceae Parts used Aerial parts Traditional uses Curing blood diseases, burning sensation, cholera, skin disease, throat troubles, and ulcers. Reference [16] 2.3 Preparation of Extract After collection, the plant material was thoroughly cleaned, followed by shade drying to preserve its phytoconstituents. It was then finely powdered in the grinding mill, ensuring a uniform consistency for further use. Approximately ten grams of V. jatamansi Jones powder was dissolved in 200 mL of six different solvents, ranging in polarity from more polar to less polar: water, methanol, ethanol, ethyl acetate, dichloromethane (DCM), and hex- ane. The content kept in maceration was shaken every 24 hours for three days. The contents were then filtered, and the filtrate was dried using a rota- tory evaporator, maintaining a temperature of 40- 45 °C. 2.4 Qualitative Phytochemical Analysis Qualitative phytochemical screening was conducted using standard protocols [17–19] to identify var- ious metabolites present in V. jatamansi Jones. The screening targeted a range of phytochemicals, including glycosides, flavonoids, alkaloids, pheno- lic compounds, terpenoids, steroids, carbohydrates, saponins, tannins, fixed oils, and lipids. This anal- ysis provided an overview of the bioactive con- stituents within the plant extract. 2.5 Estimation of Total Phenolic Content (TPC) The total phenolic content (TPC) of the plant ex- tracts was determined using the Folin-Ciocalteu col- orimetric method, as described by Lu et al. [20]. In triplicate, 96-well plates were prepared with 20 L of plant extract, 100 L of 10% Folin-Ciocalteu reagent (diluted 1:10), and 80 L of 1M Na2CO3. The reaction mixture was incubated for 30 minutes at room temperature until a blue color developed. Absorbance was then measured at 765 nm using a spectrophotometer. TPC was quantified as mil- ligrams of gallic acid equivalent per gram of extract dry weight (mg GAE/g), based on a gallic acid stan- dard curve ranging from 7.5 to 100 g/mL. 2.6 Estimation of Total Flavonoid content (TFC) The total flavonoid content of the plant extracts was assessed using the aluminum chloride method as described by Ahmed et al. [21]. In triplicate, 20 L of plant extract, 100 L of distilled water, and 60 L of ethanol were added to 96-well plates. This was followed by the addition of 10 L of a 10% aluminum chloride (AlCl3) solution and 10 L of 1M potassium acetate (CHCOOK) solution. The reaction mix- ture was kept to incubate at room temperature for 30 minutes. Absorbance was then measured at 415 nm using a spectrophotometer. The total flavonoid content was written as milligram of quercetin equiv- alent per gram of extract dry weight (mg QE/g), with quantification based on a standard calibration curve for standard quercetin having the value from 7.5 to 100 g/mL. 2.7 Evaluation of Antioxidant Activity The positive control, quercetin, was serially diluted from the original concentration of 20 g/mL down to 0.625 g/mL, while the crude plant extract was diluted from 640 g/mL to 5 g/mL. In triplicate, 100 L of each diluted plant extract and the positive control were added to a 96-well plate. The initial absorbance was recorded at 517 nm. Following this, each well received 100 L of DPPH reagent, and the mixture was incubated for 30 minutes. The final ab- sorbance was measured again at 517 nm. Methanol and 50% DMSO served as negative controls. This formula quantifies the ability of the extracts to scav- enge free radicals, providing insight into their an- tioxidant potential [22, 23]. Radical scavenging capacity = A control−A sample A control × 100 % Where, Acontrol = absorbance of control, Asample = absorbance of sample Inhibitory concentration (IC50) was calculated using GraphPad Prism (version 8.0.2.263). 2.8 Evaluation of Antimicrobial Activity Antibacterial activity was assessed using the agar well diffusion method on Mueller Hinton Agar (MHA) plates [24, 25]. The test microorganisms, including Shigella sonnei (ATCC 25931), Staphy- lococcus aureus (ATCC 43300), Klebsiella pneumo- niae (ATCC 700603), and Escherichia coli (ATCC 25312), were cultivated in Mueller Hinton Broth (MHB) and incubated at 37 ºC for 24 hours. The turbidity of the broth was adjusted to a 0.5 Mc- Farland standard to ensure uniform bacterial den- sity. Using a cork borer, wells were created in the agar plates, which were then filled with 50 L of the plant extract. Negative control wells received 50% DMSO, while positive control wells were filled with Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 134 50% neomycin. After allowing for 15 minutes of dif- fusion, the Petri dishes were incubated for 18 to 24 hours at 37 °C. Following incubation, the zones of clearance were observed and measured to evaluate the antibacterial efficacy of the plant extract. 2.9 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined following the standard procedure de- scribed by Sarker et al. [26]. The bacterial inocu- lum was prepared to a final concentration of 106 CFU/mL by diluting the 0.5 McFarland turbidity culture in Mueller Hinton Broth (MHB) at a ratio of 1:100. A 5 L inoculum was then introduced into each well of a 96-well plate. Neomycin served as the positive control in this assay. After sealing the plate with a sterile lid, it was incubated at 37 °C for 20 to 24 hours. Following incubation, 0.003% resazurin was added to each well, and the plates were incu- bated for an additional 3 to 4 hours at 37 °C. Wells that remained blue indicated no bacterial growth, while those that turned pink signified the presence of bacterial growth. The lowest concentration of the extract at which bacterial growth was inhibited was recorded as the MIC. To ascertain the MBC, the contents of the wells were streaked onto nutri- ent agar plates, which were subsequently incubated at 37 °C for over 18 hours. The absence of bacte- rial colonies on the agar plates indicated the mini- mum bactericidal concentration of the crude plant extracts. 2.10 Brine Shrimp Lethality Assay (BSLA) The toxicity of the plant extracts was assessed us- ing a standard procedure [27]. The pH of the arti- ficial seawater was adjusted to a range of 8 to 8.5 by adding 1M NaOH. Various concentrations of the plant extract were prepared, including 1000, 800, 500, 250, 100, and 10 µg/mL. Each test tube was filled with 4 mL of the prepared seawater solution. In duplicate, 10 nauplii were added to each test tube along with 500 V. µL of the respective plant extract. Potassium dichromate solution served as the positive control, while artificial seawater acted as the negative control. After 24 hours of incuba- tion, the number of dead nauplii in each test tube was counted. The percentage of nauplii mortality was calculated using the following formula: % mortality = Number of dead nauplii/Total number of nauplii× 100 Y= mx + c is the linear equation that may be found using the Probit value table, where Y is the Probit value at 50% mortality, m is variable, and c is the intercept. Calculating the lethal concentra- tion (LC50). This method provided insight into the potential toxicity of the plant extracts on aquatic organisms. 2.11 Statistical Analysis Data collection and analysis from the Gen5 Mi- croplate Reader were conducted using Microsoft Excel. The results for total phenolic content (TPC) and total flavonoid content (TFC) were reported as mean ± standard deviation. For antioxidant activ- ity, the findings were expressed as mean ± standard error of the mean (SEM). The inhibitory concen- tration (IC) values were computed using GraphPad Prism software (version 8.0.2.263). This analytical approach ensured a comprehensive evaluation of the data obtained from the experiments. 3 Results 3.1 Qualitative Phytochemical Analysis The results of qualitative phytochemical screening of the extract of V. jatamansi Jones(in different solvents) are shown in Table 2. Table 2: Qualitative phytochemical screening of plant extracts Phytochemicals Test Plant ex- tracts Alkaloids Dragendorff’s test - Carbohydrates Molish’s test + Reducing sugars Fehling 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 3.2 Total Phenolic Content (TPC) The phenolic content of the various solvent extracts of Valeriana jatamansi is summarized in Table 3. Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 135 The methanolic extract exhibited the highest to- tal phenolic content (TPC) value of 166.5 ± 2.0 mg GAE/g, while the hexane extract displayed the lowest TPC value of 16.03 ± 2.22 mg GAE/g, as in- dicated in the table below. These results highlight the significant variation in phenolic compound con- centrations among the different solvent extracts. Table 3: TPC values for various solvent extracts Plant Extracts TPC (mg GAE/g) Aqueous 93.9 ± 3.89 Methanol 166.5 ± 2.0 Ethanol 105.66 ± 5.42 Ethyl acetate 74.55 ± 3.62 Dichloromethane 34.18 ± 2.74 Hexane 16.03 ± 2.22 3.3 Total Flavonoid content (TFC) The flavonoid content of the various plant extracts is presented in Table 4 below. The methanolic ex- tract demonstrated the highest total flavonoid con- tent (TFC) value of 103.87 ± 5.47 mg QE/g, while the hexane extract exhibited the lowest TFC value of 8.27 ± 3.88 mg QE/g. These findings indicate a significant variation in flavonoid concentrations across the different solvent extracts. Table 4: TFC of different solvent extracts of V. jatamansi Jones Plant Extracts TFC (mg QE/g) Aqueous 94.63 ± 6.11 Methanol 103.87 ± 5.47 Ethanol 93.87 ± 3.41 Ethyl acetate 54.93 ± 5.61 Dichloromethane 30.69 ± 4.41 Hexane 8.27 ± 3.88 3.4 Antioxidant Activity The antioxidant activity of the plant extracts was assessed using the DPPH assay, with the IC values for the different solvent extracts of Valeriana jata- mansi summarized in Table 5. The methanolic ex- tract exhibited the lowest IC value of 103.3 ± 1.53 V µg/mL, indicating a strong antioxidant activity. In contrast, the hexane extract showed a significantly higher IC50 value of 524.7 ± 0.89 V µg/mL, re- flecting lower antioxidant capacity compared to the other extracts. These results highlight the vary- ing effectiveness of different solvents in extracting antioxidant compounds from the plant. The inhi- bition of radical scavenging activity is displayed in Figure 2. Table 5: Antioxidant potential (IC50) values of dif- ferent solvent extract Plant Extract IC50 (g/mL) Aqueous 104.3 ± 0.41 Methanol 103.3 ± 1.53 Ethanol 155.7 ± 0.34 Ethyl acetate 161.3 ± 1.14 Dichloromethane 323.8 ± 0.14 Hexane 524.7 ± 0.89 Quercetin (Standard) 3.49 ± 2.29 Note: Quercetin = Standard antioxidant Figure 2: A plot showing % inhibition against the concentration of (a) aqueous (b) methanol, (c) ethanol, (d) ethyl acetate, (e) dichloromethane, (f) hexane extract, and (g) standard quercetin 3.5 Antibacterial Activity The antibacterial activity of the plant extracts was evaluated using the agar well diffusion method, tar- geting both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Shigella sonnei, Kleb- siella pneumoniae, and Escherichia coli). The re- sults of the antibacterial tests are presented in Ta- Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 136 ble 6 and illustrated in Figure 3. Among the ex- tracts, the aqueous, ethanolic, and hexane extracts exhibited the highest zone of inhibition against Klebsiella pneumoniae, measuring 17 mm. Con- versely, the aqueous extract demonstrated limited antimicrobial activity against Escherichia coli, with a zone of inhibition of only 9 mm. These find- ings suggest varying levels of antibacterial efficacy among the different extracts against the tested mi- croorganisms. For ease of interpretation, the an- tibacterial activities are displayed in the bar dia- gram in Figure 4. Table 6: Zone of Inhibition (ZOI) of plant extracts against different bacteria Plant Ex- tracts Bacteria ZOI of Sample (mm) ZOI of Positive Control (Neomycin) (mm) Aqueous Klebsiella pneumoniae 17 24 Escherichia coli 9 22 Shigella sonnei 15 24 Staphylococcus aureus 15 23 Methanol Klebsiella pneumoniae 15 24 Escherichia coli 12 22 Shigella sonnei 13 24 Staphylococcus aureus 12 23 Ethanol Klebsiella pneumoniae 17 24 Escherichia coli 13 22 Shigella sonnei 12 24 Staphylococcus aureus 11 23 Ethyl acetate Klebsiella pneumoniae 15 24 Escherichia coli 12 22 Shigella sonnei 15 24 Staphylococcus aureus 13 23 Dichloromethane Klebsiella pneumoniae 14 24 Escherichia coli 15 22 Shigella sonnei 11 24 Staphylococcus aureus 10 23 Hexane Klebsiella pneumoniae 17 24 Escherichia coli 14 22 Shigella sonnei 11 24 Staphylococcus aureus 11 23 Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 137 Figure 3: Antibacterial test slides against differ- ent bacterial strains, KP = Klebsiella pneumo- niae, Met = Methanol, SS = Shigella sonnei, Eth = Ethanol, SA = Staphylococcus aureus, EA = Ethyl acetate, E. coli = Escherichia coli, DCM = Dichloromethane, Hex = Hexane, Aqu = Aqueous Figure 4: Bar diagram showing antibacterial ac- tivity (ZOI in mm) by different solvent extracts against K. pneumoniae, E. coli, S. sonnei, and S. aureus. 3.6 MIC and MBC The study revealed that the ethanolic plant ex- tract exhibited stronger antimicrobial effects than the methanolic extract, demonstrated by lower min- imum bactericidal concentration (MBC) values for both K. pneumoniae (Gram-negative) and S. au- reus (Gram-positive). Specifically, the minimum inhibitory concentration (MIC) and MBC values for both plant extracts against K. pneumoniae and S. aureus were 3.12 mg/mL and 12.5 mg/mL for the methanolic extract, and 3.12 mg/mL and 6.25 mg/mL for the ethanolic extract as shown in Ta- ble 7. This suggests that the ethanolic extract was more effective at a lower concentration. In con- trast, the positive control, neomycin, showed far lower MIC and MBC values of 0.0039 mg/mL and 0.0078 mg/mL. This indicates that while the plant extracts do exhibit antimicrobial activity, they are much less potent compared to neomycin. However, the ethanolic extract still presents potential for an- timicrobial applications due to its relatively lower MBC in comparison to the methanolic extract. The photographs of the experimental results are shown in Figure 5 and Figure 6. Figure 5: Well-Plates showing MIC values against (a) K. pneumoniae and (b) S. aureus, Met = Methanol, PC = Positive control, Eth = Ethanol, and NC = Negative Figure 6: Petri plates showing MBC of plant ex- tract against K. pneumoniae and S. aureus, KP = Klebsiella pneumoniae, Met = Methanol extract, SA = Staphylococcus aureus, Eth = Ethanol extract 3.7 Toxicity Analysis The lethality of the plant extracts, measured by the LC50 values, showed that the ethanolic extract had the lowest LC50 at 1077.78 µg/mL, indicating greater toxicity to the brine shrimp nauplii than the methanolic extract, which had the highest LC50 at 1905.46 µg/mL Table 8 and 9. In this bioassay, 50% DMSO served as a negative control, in which all ten-brine shrimp nauplii survived, demonstrat- ing that it had no toxic effects. Conversely, potas- sium dichromate, used as a positive control, proved highly toxic, as all ten nauplii exposed to it were dead. These findings suggest that the ethanolic ex- tract is more toxic to brine shrimp compared to the methanolic extract, with the positive and negative controls confirming the validity of the assay. Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 138 Table 7: MIC and MBC values for plant extract Plant Extracts Klebsiella pneumoniae Staphylococcus aureus MIC (mg/mL) MBC (mg/mL) MIC (mg/mL) MBC (mg/mL) Methanol 3.12 12.5 3.12 12.5 Ethanol 3.12 6.25 3.12 6.25 Positive control 0.0078 0.015 0.0039 0.0078 Table 8: The number of survived nauplii after treatment with methanolic and ethanolic extracts and their percentage mortality Plant Extract Concentration (g/mL) Total no. of survived nauplii % Mortality Methanolic extract 10 25 16.67 100 23 23.33 250 21 30 500 20 33.33 800 16 46.67 1000 15 50 Ethanolic extract 10 28 6.67 100 23 23.33 250 21 30 500 20 33.33 800 18 40 1000 17 43.33 Table 9: The toxicity of plant extracts for both methanolic and ethanolic extracts Plant Extracts Linear Regression Equation LC50 (g/mL) Methanolic 5.0 = 0.4554x + 3.4823 2137.96 Ethanolic 5.0 = 0.6306x + 2.9269 1905.46 This table highlights the dose-dependent ef- fect of both methanolic and ethanolic extracts on brine shrimp mortality, with the methanolic extract demonstrating slightly higher toxicity than the ethanolic extract at higher concentrations. How- ever, the LC50 values demonstrate the ethanol ex- tract was found toxic than the methanolic extract. 4 Discussion The study confirms that the methanolic extract of Valeriana jatamansi Jones is particularly rich in phenolic (166.5 ± 2.0 mg GAE/g) and flavonoid (103.87 ± 5.47 mg QE/g) compounds, which were significantly higher than those in other extracts (aqueous, ethanol, ethyl acetate, dichloromethane, and hexane). Phenolic compounds, known for their redox properties, contribute to antioxidant activity by acting as reducing agents, hydrogen donors, and singlet oxygen quenchers [28]. In previous research, the methanolic extract of V. jatamansi Jones roots exhibited a TPC of 187.13 ± 6.8 mg GAE/g and an IC50 value of 78 ± 2.9 g/mL. However, in this study, the IC50 for the methanolic extract was slightly higher at 103.3 ± 1.53 µg/mL, and the aqueous ex- tract had an IC50 of 104.3 ± 0.41 µg/mL, showing improvement from its previous IC50 of 154 ± 4.6 µg/mL [29]. These variations from climatic and en- vironmental factors affect antioxidant capacity, to- tal phenolic content (TPC), and total flavonoid con- tent (TFC) [29–31]. The collection site has a cold temperature, organic-rich soil, moderate water sup- ply, and minimal light exposure. These conditions influence the biosynthesis of secondary metabolites, as supported by various studies. Environmental changes can affect the production of certain medic- inal plant species because the concentration of sec- ondary metabolites (SMs) in these plants is influ- enced by factors such as climate and ecological con- ditions [32]. Plant metabolism and life depend on light because of photosynthesis. Plants must there- fore be able to detect the many light spectra found in solar radiation to survive [33]. Additionally, a wide variety of secondary metabolites in the intri- cate biochemical interaction are affected by light at varying intensities [34]. Temperature changes sig- Indra Ojha and Khaga Raj Sharma/ BIBECHANA 22 (2025) 131-141 139 nificantly influence plant growth and the metabolic pathways involved in signaling, physiological con- trol, and defense responses. When photosynthesis is disrupted due to extreme conditions, the tem- perature of the primary meteorological factors can greatly affect the composition of secondary metabo- lites [35]. The Pearson correlation coefficients pro- vide valuable insights into the relationships between Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Antioxidant Activity (AA), allowing us to better understand how these fac- tors interact with one another. The strong posi- tive correlation between TPC and TFC (r = 0.923, p = 0.009) suggests that phenolics and flavonoids might share similar biosynthetic pathways. This relationship is statistically significant at the 0.01 level. However, the negative correlations between TPC-AA (r = -0.835, p = 0.038) and TFC-AA (r = -0.922, p = 0.009) indicate that other factors may influence antioxidant activity beyond just phenolic and flavonoid contents. TPC and AA relationship is statistically significant at the 0.05 level, whereas, TFC and AA relationship is statistically significant at the 0.01 level. Regarding antimicrobial activity, previous work reported that the methanolic extract of V. jata- mansi Jones achieved a zone of inhibition (ZOI) of 19 mm against E. coli and 21 mm against Pseu- domonas [36]. In this study, the ethanolic and hexane extracts demonstrated the highest ZOI of 17 mm, nearing the efficacy of neomycin, the pos- itive control. Ethyl acetate extract also showed moderate inhibition, with a 15 mm ZOI against K. pneumoniae and S. sonnei. Besides pheno- lics and flavonoids, other compounds like vitamins, carotenoids, saponins, enzymes, and minerals may also contribute to antimicrobial activity [37]. The study further investigated the antibacte- rial potency of methanolic and ethanolic extracts against K. pneumoniae and S. aureus, with MIC and MBC values of 3.12 mg/mL and 6.25 mg/mL, respectively, for the ethanolic extract, and 3.12 mg/mL and 12.5 mg/mL for the methanolic ex- tract. These results are consistent with findings from earlier research, demonstrating that V. jata- mansi Jones extracts possess robust antibacterial properties against these pathogens [38]. 5 Conclusion In conclusion, Valeriana jatamansi demonstrates significant medicinal potential due to its diverse phytochemical composition, which includes high levels of phenolics, flavonoids, and other bioac- tive compounds. Among the solvent extracts, the methanolic extract exhibited the highest total phe- nolic content (TPC) and total flavonoid content (TFC), correlating with its strong antioxidant ca- pacity. In contrast, the hexane extract displayed the lowest TPC and TFC, alongside the weakest antioxidant potential. Antimicrobial activity as- says revealed that the aqueous, ethanolic, and hex- ane extracts effectively inhibited K. pneumoniae, with the ethanolic extract showing superior min- imum inhibitory concentration (MIC) and mini- mum bactericidal concentration (MBC) values, in- dicating a potent antimicrobial effect. Addition- ally, the ethanolic extract’s lower lethal concentra- tion (LC50) compared to the methanolic extract suggests enhanced toxicity, further highlighting its bioactivity. The comprehensive phytochemical and biological profile of V. jatamansi underscores its value as a promising candidate for drug discovery. Its diverse therapeutic properties make it a viable source for the development of new antimicrobial and antioxidant agents. Future studies are essential to fully explore the pharmacological potential and underlying mechanisms of action of V. jatamansi Jones, paving the way for its integration into mod- ern medicinal applications. 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Rahimi-Moghaddam, and M. Yazdani. An- tioxidant, cytotoxic and antibacterial activ- ity of Rosmarinus Officinalis l. essential oil against bacteria isolated from urinary tract in- fection. Eur. J. Integr. Med., 38:101192, 2020. Introduction Materials and Methods Chemicals Plant Collection and Identification Preparation of Extract Qualitative Phytochemical Analysis Estimation of Total Phenolic Content (TPC) Estimation of Total Flavonoid content (TFC) Evaluation of Antioxidant Activity Evaluation of Antimicrobial Activity Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Brine Shrimp Lethality Assay (BSLA) Statistical Analysis Results Qualitative Phytochemical Analysis Total Phenolic Content (TPC) Total Flavonoid content (TFC) Antioxidant Activity Antibacterial Activity MIC and MBC Toxicity Analysis Discussion Conclusion