BIBECHANA Vol. 20, No. 3, December 2023, 236–247 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 Phytochemistry, Biological Activities, and Chemical Profiling of Berberis asiatica Keshab Bhattarai, Devendra Dhakal, Indira Pandey Bimala Subba, Khaga Raj Sharma∗ Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal, ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract This study focused on chemical profiling and assessed the total phenolic and flavonoid content as well as the antioxidant and antibacterial effect of the medicinal plant Berberis asiatica. The results revealed that this plant has high concentrations of TPC (Total phenol content) and TFC (Total flavonoid content) of 37.686±2.728 mg GAE/g and 115.568±8.012 mg QE/g, respectively. The DPPH free radical scavenging assay demonstrated strong inhibition, with an IC50 of 205.7± 5.353µg/mL, and also showed robust antibacterial properties against Staphy- lococcus aureus and Klebsiella pneumoniae with a zone of inhibition (ZOI) of 14 mm and 19 mm, respectively. The extract exhibited an excellent inhibitory potential against S. aureus and K. pneumoniae with a MIC (Minimum inhibitory concentration) of 0.39 mg/mL and 3.125 mg/mL respectively, indicating significant inhibitory action. Furthermore, the MBC (Mini- mum bactericidal concentration) for both S. aureus and K. pneumoniae was found to be 6.25 mg/mL, emphasizing the extract’s consistent bactericidal effectiveness against these bacteria. These findings underscore the potential utility of the methanolic extract of Berberis asiatica as a natural antibacterial agent. GC-MS analysis of hexane fraction indicates the plant is rich in secondary metabolites, specifically 2,2-dimethyl-3-pentanol, 2-methyl-2-pentanol, 2,5- dimethyl-4-hydroxy-3-hexanone, 3-hexanol, 4-methyl-2-pentanol are identified. Overall, this study highlights the importance of plant-based natural products as potential sources of an- tioxidants and antibacterial agents that contribute to the future drug development process. Keywords Berberis asiatica, antibacterial activity, minimum inhibitory concentration (MIC), minimum bacte- ricidal concentration (MBC), Staphylococcus aureus, Klebsiella pneumoniae Article information Manuscript received: August 17, 2023; Accepted: September 14, 2023 DOI https://doi.org/10.3126/bibechana.v20i3.57711 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 236 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v20i3.57711 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 237 1 Introduction Traditional medicines rely on natural products to treat diseases, and modern pharmaceutical medi- cations are often derived from natural sources [1]. In recent years, there has been an assessment of plant-derived natural products as promising can- didates for drug development [2]. The medicinal plant serves as a valuable and organic reservoir of bioactive elements that play a significant role in contemporary medicine [3]. Various plant-derived natural products, including alkaloids, phenols, and flavonoids, have been discovered with capabilities to induce apoptosis, and migration and exhibit po- tential against cancer, oxidation, diabetes, and in- flammation [2, 4]. Renewed interest and focus on medicinal plant research have been triggered by the rise in side effects, the emergence of new intestinal pathogens, and the increasing problem of microbial resistance [5]. In this research, we examined the antioxidant, antibacterial, and alpha-amylase inhibition proper- ties of Berberis asiatica, a medicinal plant grown in Nepal belonging to the family Berberidaceae. Berberis plants of the Berberidaceae family are ex- tensively distributed across the globe, comprising almost 550 species [6]. Genus Berberis is globally recognized in traditional medicine for its potent medicinal properties. Its roots and bark are em- ployed as antipyretic, anti-periodic, and diaphoretic [7]. Barberry fruits, due to their significant rich- ness in anthocyanin content, are consumed as an antidiabetic drug [8]. Among many species of Berberis, Berberis asiatica is widely used in tra- ditional medicinal practices to cure diabetes and infectious diseases [9]. Berberis asiatica, commonly known as Chutro in Nepali, is a wild shrub found in Nepal between ele- vations of 1200 to 2500 meters. It thrives in shaded, rocky slopes, north-facing hillsides, and drier re- gions. This erect, spiny shrub grows up to 4 meters tall, sporting yellow bark. Its leaves are simple, al- ternate, leathery, dark green on top, and grayish underneath, usually with 2-5 spiny teeth along the margins. The shrub produces pale yellow flowers in flat-topped clusters and dark purple berries with a sweet and sour taste [10, 11]. Traditionally, differ- ent parts of Berberis asiatica were used for medical purposes. Root and bark have extensive use in in- digenous medicine for treating various ailments, in- cluding eye and ear diseases, rheumatism, jaundice, diabetes, fever, stomach disorders, skin disease, and malarial fever [9]. These root and stem bark are integral to a renowned Ayurvedic medicine, con- taining several alkaloids such as berberine, palma- tine, jatrorrhizine, columbamine, berbamine, oxy- berberine, and oxyacanthine [12]. These secondary metabolites play an important role in treating dif- ferent diseases and disorders in the human body. It was reported that berberine plays a vital role in controlling diabetes by inhibiting digestive enzymes alpha-amylase and alpha-glucosidase [13]. Oxidation, a fundamental metabolic procedure present in all life forms, holds particular significance in humans as it underpins the production of energy. However, these biochemical reactions create free radicals such as reactive oxygen species (ROS), po- tentially amplifying oxidative stress and causing po- tential harm to essential biological components like lipids, proteins, and DNA [14, 15]. As time passes, the harmful effects of ROS become more prominent and are associated with aging and age-related ill- nesses like heart diseases, cancer, brain disorders, diabetes, and other chronic diseases [16]. Further- more, diabetes represents a significant metabolic syndrome with abnormally high blood glucose lev- els (hyperglycemia), and oxidative stress is closely associated with this condition, especially in indi- viduals with type 2 diabetes [17, 18]. Type 2 di- abetes mellitus is a multifaceted and diverse col- lection of metabolic disorders marked by increased serum glucose levels, primarily resulting from defi- ciencies in both the function of insulin and its se- cretion [19]. Roughly, 90 to 95 % of diabetes cases are attributed to type 2 diabetes [20]. To manag- ing diabetes, one of the main approaches involves controlling postprandial hyperglycemia through the inhibition of the carbohydrate hydrolyzing enzyme alpha-amylase, responsible for breaking down long- chain carbohydrates like starch, amylase, and amy- lopectin into glucose, and another is by reducing ox- idative stress [14,20,21]. Hence, a novel and promis- ing drug is required in modern times to effectively manage diabetes and oxidative stress. As a result, there has been a concentrated effort to discover novel antidiabetic compounds from plants utilized in traditional medicine [22]. Furthermore, medic- inal plants offer a valuable source of antimicrobial agents [23]. Several plants belonging to Berberis are renowned for their antibacterial properties [24, 25]. Notably, Berberis asiatica has generated particu- lar attention as a source of novel compounds with remarkable antibacterial activity. Taking into ac- count the aforementioned factors, this study was undertaken to explore the antioxidants, antibacte- rial, and α-amylase inhibition properties using in vitro assays, aiming to contribute significant in- sights into the holistic understanding of Berberis asiatica. 2 Materials and Methods 2.1 Chemicals All chemicals and reagents utilized in this study were of analytical grade. Gallic acid, Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 238 Folin-Ciocalteu’s reagent, quercetin, 2,2-diphenyl- 1-picrylhydrazyl (DPPH), acarbose, α-amylase from porcine pancreases, 2-chloro-4-nitrophenyl- -D-maltotrioside (CNPG3) were demanded from Sigma-Aldrich. 2.2 Plant Collection and Preparation of Extract The whole plant of the Berberis asitica was col- lected from the Arghakhanchi district of Nepal growing at an altitude of 1810 m in October 2021 based on ethnobotanical approaches. The plant was verified in the National Herbarium and Plant Lab- oratories Godavari, Kathmandu, Nepal. To pre- pare the plant extract, freshly collected whole plant parts (leaves, bark, and roots) were washed with tap water to remove the contaminants, and the col- lected samples were dried and ground into a fine powder. For the extraction process, methanol was used as a solvent. Due to high volatility, lower boil- ing point, and strong polarity, methanol is an ideal choice for the extraction process [26,27]. Moreover, methanol is renowned for its ability to yield higher extraction efficiency as compared to other solvents and its efficacy in extracting a diverse range of com- pounds, including antibacterial compounds, antiox- idants, polyphenols, flavonoids, alkaloids, and ter- penoids [28–32]. Furthermore, methanol is cost- effective and readily available, making it an easily manageable choice as a preferred extraction solvent. 75 grams of powder was then mixed with 300 mL methanol in a conical flask and meticulously shaken to completely mix the powder in the solvent. Then the conical flask was covered with aluminum foil and left to undergo cold percolation for 72 hours at room temperature. Subsequently, the mixture was filtered using a muslin cloth, and filtrate was col- lected in a beaker. This process was repeated for subsequent 2 times to get a higher amount of the ex- tract. The filtrate was concentrated using a rotary evaporator at a temperature of 400C, and concen- trated solution was kept in the water bath for fast evaporation of the solvent. The crude extract was stored in the refrigerator at 4 0C for the subsequent evaluation of various biological activities, following the standard procedures. 2.3 Phytochemical Analysis The recently prepared raw extracts of B. asiat- ica were subjected to qualitative tests to analyze the presence or absence of various plant secondary metabolites including alkaloids, flavonoids, steroids, terpenoids, reducing sugars, saponins, tannins, car- diac glycosides, anthraquinones. For qualitative phytochemical analysis, a series of tests were car- ried out according to the protocols of [33,34]. 2.4 Total Phenolic Content (TPC) To determine the TPC, the Folin-Ciocalteu phenol assay was employed [35,36]. The dry methanolic ex- tract was prepared at a concentration of 5 mg/mL in 30% DMSO. A sample (20 µL) of the plant ex- tract and a standard solution of gallic acid (rang- ing from 10 to 80 µg/mL) was mixed with Folin- Ciocalteu phenol reagent (100 µL) and sodium car- bonate (80 µL). After a 30-minute incubation, the absorbance was measured at 765 nm using a mi- croplate reader (Synergy LX, Bio-Tek, Instruments, Inc., USA). The TPC was calculated using a stan- dard curve and expressed as milligrams of gallic acid equivalent (mg GAE) per gram of plant extract. 2.5 Total Flavonoid Content (TFC) To estimate the Total Flavonoid Content (TFC), the aluminum chloride (AlCl3) procedure was em- ployed [37]. A 20 µL of the stock solution of the plant extract and 130 µL of the standard quercetin solution (ranging from 10 to 80 µg/mL) were loaded in 96-well plates. The standard quercetin solution was prepared by diluting a stock solution of 0.1 mg/mL. Subsequently, to maintain the volume of 110 µL, distilled water was added to the plant ex- tract in triplicate. Additionally, 60 µL ethanol, 5 µL 10% AlCl3, and 5 µL potassium acetate were added to each well. After a 30-minute incubation at room temperature, the absorbance was measured at 415 nm using a microplate reader (Synergy LX, Bio-Tek Instruments, Inc., USA). The TFC was calculated using a standard curve and expressed as milligrams of quercetin equivalent (mg QE) per gram of plant extract. 2.6 Evaluation of Antioxidant Activity To evaluate the free radical scavenging activity, the DPPH radical scavenging assay was employed [38,39]. A solution of 0.1 mM DPPH was prepared, and a stock solution of quercetin was also prepared. Quercetin served as the positive control, while 50% DMSO was used as the negative control. The pos- itive control, negative control, and plant samples were loaded into separate wells of a 96-well plate in triplicate. Subsequently, the DPPH reagent was added to each well, followed by a 30-minute incu- bation period in darkness. After incubation, the absorbance at 517 nm was measured using a mi- croplate reader. Percentage inhibition of the tested sample was calculated using the following formula. %inhibition = [Abs. of control-Abs. of sample] Abs. of control ×100 Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 239 2.7 α−AmylaseInhibitionActivity To assess the inhibitory activity of α-amylase, an in vitro assay was conducted using CNPG3 as the substrate [40]. Plant samples of various concentra- tions (20 L) were mixed with α-amylase (80 L at a final concentration of 1.5 U/mL) and incubated at 37 °C for 15 minutes. The reaction was initiated by adding 100 L of the substrate, CNPG3 (final con- centration of 0.5 mM), prepared in the buffer. The experiment was performed in a 50 mM phosphate buffer. After incubation, the absorbance was mea- sured at 405 nm using a microplate reader. DMSO (30%) was used as the negative control, while acar- bose served as the standard. The inhibition per- centage was calculated using the provided formula. %inhibition = [Abs. of control-Abs. of sample] Abs. of control ×100 2.8 Determination of Antibacterial Activ- ity The antibacterial activity of the extract was eval- uated using the agar well diffusion method on Mueller-Hinton Agar (MHA) plates [41]. The test organisms gram-positive Staphylococcus au- reus (ATCC 25293), and gram-negative bacteria Escherichia coli (ATCC 25922), Salmonella Ty- phi (ATCC 14028), and Klebsiella pneumoniae (ATCC 13883) were cultured in Mueller-Hinton broth. These tested bacteria Staphylococcus au- reus and Escherichia coli were obtained from clini- cal isolation whereas Salmonella typhi was isolated from the liver of the four-week-old chicken. These cultured bacteria were incubated at 37 0C until the turbidity reached a standardized 0.5 McFarland unit. The MHA plates were then uniformly inoc- ulated with the microbial suspension. A stock so- lution of the plant extract at a concentration of 5 mg/mL was prepared. Using a sterile cork borer with an 8 mm diameter, three wells were created on the agar surface. Each well was filled with 50 µL of the plant extract, along with a positive con- trol consisting of neomycin at a concentration of 1 mg/mL, and a negative control consisting of 50% DMSO. These controls were included on each plate. After allowing approximately 15 minutes for the ex- tracts and controls to diffuse into the agar at room temperature, the plates were incubated at 370C for 18-24 hours. Following the incubation period, the plates were examined for the presence of clear zones of inhibition (ZOI) surrounding the wells. The di- ameter of the ZOI was measured in millimeters us- ing a ruler, providing a quantitative assessment of the antimicrobial activity of the plant extract. 2.9 Determination of Minimum Inhibitory Concentration (MIC) Determination of the minimum inhibitory con- centration (MIC), which represents the smallest amount of compounds needed to inhibit or kill mi- croorganisms in vitro, can be achieved through the broth microdilution method [42]. In this method, the methanolic extract of the plant exhibiting sig- nificant antibacterial activity was subjected to a two-fold serial dilution. Under aseptic conditions, a sterile 96-well plate was filled with 100 µL of Mueller Hinton broth in each well. The extract was double diluted by adding equal volumes (100 µl) of the extract to MHB to get a series of concen- trations ranging from 25 mg/mL to 0.012 mg/mL in plates. Next, 5 µL of the prepared bacterial in- oculums adjusted to 0.5 McFarland Standard was added in each dilution. The microplate was incu- bated at 37 oC for 12 to 18 hours. After incubation, 10 µL of a 0.003% resazurin solution was added to wells, followed by an additional incubation period of 2 to 3 hours. Any color change from purple/blue to pink or colorless was recorded as a positive result. In this test neomycin solution was taken as posi- tive control and DMSO solution was placed as neg- ative control. The MIC value was determined as the lowest concentration at which no color change oc- curred, indicating the inhibition of bacterial growth by the plant extract. 2.10 Determination of Minimum Bacteri- cidal Concentration (MBC) MBC was determined by subculturing the MIC cul- tures on suitable agar plates. The sterile nutrient agar plate was taken in which solution from the microplate was loaded by streaking with the help of an inoculating loop. The solutions of the well showing MIC value and above were loaded in the plate. The plates were incubated at 37ºC for 18-24 hours. Then plates were examined for the growth of microorganisms. The tubes with a minimum con- centration of extract in which the growth was com- pletely checked were noted as the MBC of the plant extract [10]. MBC is complimentary to MIC and it is the lowest concentration of antibacterial agent that has a reducing capacity of viability of initial bacterial inoculum up to 99.9%. 2.11 GC-MS Profiling of Chemical Com- pounds In this study, for the chemical characterization hex- ane fraction was fractionated from the methano- lic extract and this fraction was analyzed us- ing Gas Chromatography-Mass Spectrometry (GC- MS) with an Agilent 7890 GC instrument and an Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 240 Agilent 5975C inert MSD with Triple-Axis Detec- tor. The analysis employed a gas carrier of helium at a flow rate of 1 mL/min. The sample injection volume was set at 2 µL, and the temperature pro- gram consisted of an initial temperature of 320C, followed by a ramping rate of 50C/min up to 2300C, with a final hold time of 15 minutes. The aux- iliary heater was set to 2300C to ensure optimal conditions for efficient analysis. These carefully op- timized analytical conditions provided valuable in- sights into the chemical composition of the hexane fraction, facilitating the identification and quantifi- cation of its compounds. These findings enhance our understanding of the chemical profile of B. asi- atica and its potential therapeutic applications. 2.12 Statistical Analysis All the experiments were performed in triplicate and data were presented in ± standard error of the mean. The TPC, TFC, antioxidant assay, and en- zyme inhibition were calculated by Microsoft Excel 2007. The IC50 value was calculated using Graph- Pad Prism software. 3 Results and Discussion 3.1 Percentage Yield The percentage yield was calculated to measure the abundance of secondary metabolites in the whole plant sample used in the study. The percentage yield of B. asiatica was found 10.66%, which is shown in Table 1. 3.2 Phytochemical Screening of Crude Ex- tract Preliminary phytochemical analysis was performed for the analysis of the presence and absence of differ- ent secondary metabolites. The result of the phy- tochemical screening of the methanolic extract of Berberis asiatica is shown in Table 2. 3.3 Total Phenolic and Flavonoid Content The total phenolic content (TPC) and total flavonoid content (TFC) were estimated by con- structing a calibration curve taking different con- centrations of gallic acid and quercetin, against the absorbance. TPC was expressed as milligrams of gallic acid equivalent per gram (mg GAE/g), while TFC was expressed as milligrams of quercetin equivalent per gram (mg QE/g). The total pheno- lic content of the methanolic extract of B. asiatica was determined to be 37.686 ± 2.728 mg GAE/g, while the total flavonoid content was estimated as 115.568 ± 8.012 mg QE/g. 3.4 Antioxidant Potential At a concentration of 1000 g/mL, the crude extracts of B. asiatica demonstrated significant inhibitory activity of 89.588% against the DPPH free radical. As a result, further dilution of the samples was per- formed to determine the IC50 value. The methano- lic extract of B. asiatica exhibited noteworthy rad- ical scavenging activity, with an IC50 value of 205.7 ± 5.353 g/mL. This result was compared to the IC50 value of the standard quercetin, which was found to be 6.29 ± 1.02 g/mL. Table 1: Physical characteristics and percentage yield of plant extract. Plant Color of Extract Weight of Dry Sample (g) Weight of Extract (g) B. asiatica Yellow 75 8 Table 2: Phytochemical screening of whole plant extract. Phytochemicals Methanolic extract of Berberis asiatica Alkaloids + Flavonoids + Tannins - Saponins + Terpenoids + Steroids + Glycosides + Reducing sugars + Coumarins + Polyphenols + Volatile oils - where + represents the presence of metabolites and - represents absence. Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 241 Table 3: Total phenolic and flavonoid content in methanolic extract of Berberis asiatica. Whole Plant Extracts TPC (mg GAE/g) TFC (mg QE/g) Berberis asiatica 37.686 ± 2.728 115.568 ± 8.012 3.5 In Vitro α-Amylase Inhibition Activ- ity The crude methanolic extracts of B. asiatica ex- hibited an inhibitory activity of 37.732% against α- amylase at a concentration of 500 g/mL. Therefore, additional dilution of the samples was not carried out to determine the IC50 value. According to the literature, it has been observed that plants contain secondary metabolites that possess properties sim- ilar to metformin, which are known to aid in the control of diabetes [43]. 3.6 Antibacterial Activity The methanolic extract of B. asiatica, at a concen- tration of 50 mg/mL, exhibited varying inhibitory effects against different bacteria. Against S. aureus, moderate inhibitory activity was observed, with a zone of inhibition measuring 14 mm. However, no inhibitory effect was detected against E. coli and S. typhi. In contrast, the extract displayed a strong inhibitory activity against K. pneumoniae, as ev- idenced by a substantial zone of inhibition mea- suring 19 mm. These results indicate that the methanolic extract of B. asiatica possesses antibac- terial properties, particularly against S. aureus and K. pneumoniae while showing no inhibitory effect on E. coli and S. typhi. 3.7 Minimum Inhibitory Concentration The methanolic extract of Berberis asiatica demon- strated significant antibacterial activity against two types of bacteria, namely S. aureus (gram-positive) and K. pneumoniae (gram-negative). To determine the potency of the extract in inhibiting the growth of these microorganisms in vitro, the Minimum In- hibitory Concentration (MIC) values were deter- mined using the two-fold serial broth microdilution method. MIC is expressed in terms of mg/mL. The MIC shown by the methanolic extract of Berberis asiatica and the standard antibiotic neomycin (pos- itive control) against the test bacteria are summa- rized in Table 6. The results indicate that the methanolic extract of Berberis asiatica exhibited excellent inhibitory activity against S. aureus, with a MIC value of 0.39 mg/mL. Although the methanolic extract also demonstrated inhibitory activity against K. pneu- moniae, it was less potent than the positive control Neomycin. The MIC value for K. pneumoniae was 3.125 mg/mL for the plant extract, whereas it was 0.0019 mg/mL for Neomycin. 3.8 Minimum Bactericidal Concentration MBC is the lowest concentration of antibacterial agent that has a reducing capacity of viability of ini- tial bacterial inoculum up to 99.9%. MBC is com- plimentary to MIC. Methanolic extract of Berberis asiatica exhibited minimum bactericidal concentra- tion for S. aureus was 6.25 mg/mL and for K. pneu- moniae was 6.25 mg/mL. The results showed that the plant extract exhibited the same MBC for S. aureus and K. pneumoniae. 3.9 Chemical Constituents Profiling by GC-MS Analysis The GC-MS analysis of the hexane extract of Berberis asiatica revealed the presence of 35 com- pounds. The GC-MS chromatogram is shown in Figure 2. The NIST08 MS library was employed to compare and identify the chemical constituents. These compounds consist of ketones, alkanes, cy- cloalkanes, alcohol, and amines along with aromatic compounds. The profiling of chemical compounds in the hexane extract of asiatica with their reten- tion time (RT), molecular weight (MW), and rel- ative area percentage (%) are presented in Table 8. Table 4: Antioxidant (IC50 ) of methanolic extract of Berberis asiatica and standard quercetin. Samples IC50 (µg/mL) Berberis asiatica 205.7 ± 5.353 Quercetin (Standard) 6.29 ± 1.02 Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 242 Figure 1: Antibacterial activity, MIC, and MBC determination of plant extract. Figure 2: GC-MS chromatogram of hexane fraction of Berberis asiatica. 3.10 Pharmacological Importance of GC- MS Detected Compounds Among the detected compounds by GC-MS, some of those compounds having the alcohol and ke- tone functional groups have been reported to pos- sess pharmaceutical significance owing to their bi- ological activities. The compound 2,5-dimethyl- 4-hydroxy-3-hexanone is reported to have poten- tial therapeutic effects such as antioxidant, an- timicrobial, anti-inflammatory, antidiabetic, anti- cancer, and also effective against Alzheimer’s dis- ease.. The compound 2-methyl-2-pentanol has been demonstrated to induce apoptosis in cancer cells, enhance the immune response to viral infections, and exhibit antimicrobial, antifungal, and antiviral activity. The compound 4-methyl-2-pentanol has been reported to act as an acetylcholinesterase in- hibitor and demonstrate antibacterial and antifun- gal properties. Furthermore, it has shown effective- ness in the treatment of Alzheimer’s disease and other neurological disorders. Other identified com- pounds like 2,2-dimethyl-3-pentanol and 3-hexanol also demonstrated antifungal, antibacterial, and an- titumor activities [44]. The compound 3-hexanol can serve as an agonist for the GABA-A recep- tor, which plays a role in regulating neurotransmit- ters in the brain. Meanwhile, in vitro studies of 2,2-dimethyl-3-pentanol have shown that this com- pound can inhibit the growth of various cancer cell lines, including those associated with breast, lung, and colon cancers [44]. The chemical profiling of the present study reported that the plant B. asi- atica is the source of potential natural compounds that could be used in treating different diseases. 4 Discussion A limited number of studies have been conducted to explore the bioactive metabolites of plants in Nepal. Being a landlocked country, Nepal boasts remark- able biodiversity in its flora, resulting in a high den- Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 243 Figure 3: Structures of some chemical compounds detected from GC-MS analysis of hexane fraction of Berberis asiatica. Table 5: Zone of Inhibition shown by methanolic extract of Berberis asiatica against bacterial strain. Bacterial strain Zone of inhibition (mm) of plant extract Zone of inhibition (mm) of positive control S. aureus 14 13 E. coli - 16 K. pneumoniae 19 18 S. typhi - 15 sity and diversity of plant species. For this research, various readily available Berberis species were col- lected and confirmed to be Berberis asiatica . The primary objective of this study was to evaluate the inhibitory effects of Berberis asiatica on α-amylase, various Gram-positive and Gram-negative bacte- ria, as well as the antioxidant properties of plants. In earlier investigations, the methanolic extract of Berberis asiatica exhibited a percentage yield of 6.8% [10]. However, in the current study, the per- centage yield was found to be higher at 10.66%. The variation in secondary metabolites abundance can be ascribed to the distribution of the selected plant across different geographical locations, thereby af- fecting its overall availability of these compounds. In the previous study, the total phenolic con- tent (TPC) and total flavonoid content (TFC) were examined in different parts of Berberis asiatica , including leaves, bark, and roots. The TPC val- ues were recorded as 132.63 mg GAE/g, 52.240 ± 0.119 mg/100g, and 59.076 ± 0.219 mg/100g for leaves, bark, and roots, respectively [45]. Similarly, the TFC was found to be 31.36 mg QE/g, 1.735 ± 0.396 mg QE/g, and 1.576 ± 0.197 mg QE/g for leaves, bark, and roots, respectively [45]. In the present study, a notable difference in TPC and TFC was observed in Berberis asiatica compared to the previously reported results. The TPC value for this plant was reported as 37.686 ± 2.728 mg GAE/g, and the TFC was recorded as 115.568 ± 8.012 mg QE/g. The plant sample collected in the present study showed higher contents of secondary metabolites which impart a significant effect on the biological activity, particularly in terms of antioxi- dant and antidiabetic properties. In previous research, the antioxidant activity of Berberis asiatica was investigated, and the IC50 value for the root and stem bark extracts was de- termined to be 102.31 and 120.7 µg/mL, respec- tively [46]. However, in the current study, a no- table difference was observed, showing a significant radical scavenging activity with an IC50 of 205.7 ± 5.353 µg/mL. The variation in the IC50 value of Berberis asiatica indicates that the antioxidant potential can be influenced by multiple factors, in- cluding the extraction methods, a sample collected geographical area, and the timing of sample col- lection. Apart from exhibiting poor inhibition of Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 244 Table 6: Minimum inhibitory concentration of methanolic extract of Berberis asiatica against test bac- teria. Bacteria Berberis asiatica MIC (mg/mL) Positive control (Neomycin) MIC (mg/mL) S. aureus 0.39 0.0039 K. pneumoniae 3.125 0.0019 Table 7: Minimum bactericidal concentration (MBC) of methanolic extract of Berberis asiatica against test bacteria. Bacterial strain Berberis asiatica MBC (mg/mL) Positive control (Neomycin) MBC (mg/mL) S. aureus 6.25 0.015 K. pneumoniae 6.25 0.0078 alpha-amylase in the current study, Berberis asi- atica is well-documented in some literature for its anti-diabetic properties. Previous research suggests that the plant’s blood glucose and lipid regulatory effects can be attributed to berberine, a significant alkaloid present in Berberis asiatica . This com- pound has been found to enhance insulin sensitivity by regulating the secretion of adipokines [47]. The results of the current study demonstrate that the methanolic extract of Berberis asiatica ex- hibited significant inhibition in the growth of two bacterial strains, one gram-positive (S. aureus) and one gram-negative (K. pneumoniae), with a zone of inhibition (ZOI) of 14 mm and 19 mm, re- spectively. This finding indicates that the plant extract possesses potent antibacterial properties, likely due to the presence of specific antibacte- rial agents. However, in the previous study, the crude aqueous extract of Berberis asiatica stem bark demonstrated a zone of inhibition in the disk diffusion method. Among the Gram-negative bac- teria, Escherichia coli, Klebsiella pneumoniae, and Salmonella paratyphi-A exhibited a zone of inhi- bition of 8 mm [48]. This outcome suggests that our study has exhibited a notably robust antibac- terial property when compared to the previous re- search. The results of the current study revealed that the methanolic extract of B. asiatica displayed a minimum inhibitory concentration (MIC) of 0.39 mg/mL for S. aureus and 3.125 mg/mL for K. pneumoniae. Notably, the plant extract demon- strated better inhibition for S. aureus compared to K. pneumoniae. The literature also reported sim- ilar findings, where the methanolic stem bark ex- tract of Berberis asiatica exhibited a MIC of 312.5 µg/mL for S. aureus [10], which aligns with the MIC value obtained in the present study. How- ever, for K. pneumoniae, the aqueous stem bark extract of Berberis asiatica reported in the liter- ature exhibited a MIC of 2500 µg/mL, which is higher than the MIC value obtained in the present study [48]. Furthermore, the methanolic extract of the Berberis asiatica plant exhibited an MBC of 6.25 mg/mL for both S. aureus and K. pneumo- niae, indicating the same bactericidal concentration for both bacterial strains. In comparison, the litera- ture reported a methanolic stem bark extract with an MBC of 0.78125 mg/mL for S. aureus, which differs and shows the potential activity of the plant sample used in this study [10]. The difference in the biological activities and the richness of secondary metabolites in the plant may be attributed to the part of the plant sample used and the geographical variation of plant growth. In the chemical profiling, different secondary metabolites were identified by GC-MS analy- sis which includes, 3,3-dimethylpentane, cyclo- hexane, 2-methylhexane, 3-methylhexane, 1,3- dimethylcyclopentane, 1,2-dimethylcyclopentane, Heptane, methylcyclohexane, 2,2-dimethyl-3- pentanol, 2-methyl-2-pentanol, 2,5-dimethyl-4- hydroxy-3-hexanone, 3-hexanol. The previous study reported some of the compounds like berber- ine, palmatine, jatrorrhirine, oxyacanthine, oxyber- berine, tetrahydropalmatine, and columbamine [7]. The GC-MS profiling in this study shows some different compounds with a richness of bioactive compounds like alkaloids, phenolic, and flavonoid compounds highlighting the pharmaceutical poten- tial of the plant and may be a good source of such compounds in the future drug development process. The abundance of secondary metabolites may be affected due to climatic conditions, soil type, tem- perature, and geographical locations [49]. Although the same plant species with nearby environmental conditions may vary in chemical compositions [50]. Increasing the altitude contribute significantly to chemical composition, at higher altitude plant pos- sess a higher amount of phenolic and flavonoid com- pounds [41]. However, at lower altitudes, there is a greater abundance of volatile organic compounds in the plants [41]. These variations in chemical composition according to altitude can be ascribed to discrepancies in the biological activity of plant extract. Therefore, current research results show some different potential results as compared to the previous one. Keshab Bhattarai et al./ BIBECHANA 20 (2023) 236-247 245 Table 8: Major chemical compounds detected by GC analysis in hexane fraction. S.N. Name of Compound Retention Time (Rt) Area % Mol. Wt. 1 3,3-dimethyl pentane 2.354 0.78 100.2 2 Cyclohexane 2.479 86.25 84.16 3 2-methyl hexane 2.518 4.14 100.2 4 3-methyl hexane 2.632 3.45 100.2 5 1,3-dimethyl cyclopentane 2.758 0.40 98.19 6 1,2-dimethyl cyclopentane 2.850 0.53 98.19 7 Heptane 3.036 0.55 100.2 8 Methylcyclohexane 3.494 0.93 98.186 9 2,2-dimethyl-3-pentanol 10.542 0.28 116.2 10 2-methyl-2-pentanol 10.662 0.84 102.17 11 2,5-dimethyl-4-hydroxy-3-hexanone 11.252 0.58 144.21 12 3-hexanol 12.135 0.16 102.17 13 4-methyl-2-pentanol 12.490 0.18 102.174 5 Conclusion In conclusion, the current study on Berberis asiat- ica from Nepal has provided valuable insights into its bioactive properties. The plant extract demon- strated significant inhibition of α-amylase and ex- hibited potent antibacterial activity against both gram-positive (S. aureus) and gram-negative (K. pneumoniae) bacteria. Additionally, the extract showed promising antioxidant potential. More- over, there were variations in the total phenolic and flavonoid content of the plant when compared to previous findings. These differences could be attributed to various factors, such as plant parts used, extraction methods, and environmental con- ditions. Chemical profiling revealed the presence of various secondary metabolites in the plant, and some of these compounds were also reported in the literature, further confirming the plant’s potential medicinal value. This study highlights the rich bio- diversity of Nepal’s flora and the importance of ex- ploring and understanding the bioactive properties of its plant species. 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Introduction Materials and Methods Chemicals Plant Collection and Preparation of Extract Phytochemical Analysis Total Phenolic Content (TPC) Total Flavonoid Content (TFC) Evaluation of Antioxidant Activity -Amylase Inhibition Activity Determination of Antibacterial Activity Determination of Minimum Inhibitory Concentration (MIC) Determination of Minimum Bactericidal Concentration (MBC) GC-MS Profiling of Chemical Compounds Statistical Analysis Results and Discussion Percentage Yield Phytochemical Screening of Crude Extract Total Phenolic and Flavonoid Content Antioxidant Potential In Vitro -Amylase Inhibition Activity Antibacterial Activity Minimum Inhibitory Concentration Minimum Bactericidal Concentration Chemical Constituents Profiling by GC-MS Analysis Pharmacological Importance of GC-MS Detected Compounds Discussion Conclusion