BIBECHANA Vol. 21, No. 2, August 2024, 113-123 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 Phytochemical analysis and biological activities on solvent extracts of two traditionally used medicinal plants Sangita Pakka, Akash Budha Magar, Deepa Shrestha, Tinky Sharma, Khaga Raj Sharma∗ Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu ∗Corresponding author. Email: khaga.sharma@cdc.tu.edu.np Abstract The plants Buddleja asiatica and Buddleja paniculata are two closely related species of which the former has widespread uses in traditional medicine while the latter remains relatively unexplored. The present study focused on the analysis of phytoconstituents and the estimation of different biological activities of extracts and semi-purified fractions. The ethyl acetate stem fraction and methanolic extract of the leaf of B. asiatica displayed the highest phenolic contents of 390.98 ± 5.32 and 383.72 ± 7.31 mg GAE/g. TFC was measured as 207.33 ± 0.34 and 138.58 ± 0.53 mg QE/g for ethyl acetate leaf and stem fraction of B. asiatica. The highest antioxidant activity was displayed by crude stem extract of B. paniculata with IC50 35.65±0.61 µg/mL in DPPH assay followed by ethyl acetate stem fraction of B. asiatica with IC50 36.17±0.92 µg/mL. The DCM leaf fraction and stem fraction of B. asiatica were found active against gram-negative and gram-positive bacteria. The median lethal concentration (LC50) in brine shrimp assay ranged from 346.96 ± 25.39 in hexane fraction of leaf to 2719.32 ± 706.5 µg/mL in crude methanol extract of B. paniculata. The present study showed these medicinal plants are rich in an important class of organic compounds that play a significant role in the cure of bacterial infection which ultimately supports pharmaceutical research. Keywords Buddleja asiatica, Buddleja paniculata, antimicrobial, antioxidant, phytochemical, toxicity. Article information Manuscript received: December 30, 2023; Revised: January 23, 2024; Accepted: February 3, 2024 DOI https://doi.org/10.3126/bibechana.v21i2.61208 This work is licensed under the Creative Commons CC BY-NC License. https://creativecommons. org/licenses/by-nc/4.0/ 1 Introduction Plants have a huge importance in both tradi- tional medicine and the modern health care sys- tem. The biological activities and therapeutic properties of medicinal plants are the results of specific or synergistic interactions of their sec- ondary metabolites. These compounds are not required for central processes of growth and de- velopment in plants, but they are important for defense, signaling, symbiosis, metal trans- port, and competition [1]. They are synthesized through various biosynthesis pathways in plants [2]. About 200,000 plant secondary metabolites have been isolated and characterized with many more expected in the future [3]. Specific sec- 113 http://nepjol.info/index.php/BIBECHANA khaga.sharma@cdc.tu.edu.np https://doi.org/10.3126/bibechana.v21i2.61208 https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 114 ondary metabolites may be exclusive to a par- ticular species or group of species. The quali- tative and quantitative composition of metabo- lites may vary within or between species and in- fluenced by environmental stresses like nutrition, drought, temperature, and light [4]. Genus Buddleja belonging to the family Scro- phulariaceae includes more than 100 species that grow in temperate and drier regions of Asia, Africa, and America [5, 6]. These plants are mostly shrubs or small trees that can grow up to 5 m. Buddleja species are used to treat bronchial complaints, liver diseases, and wounds and they are known to possess antimicrobial, antihyperglycemic, antioxidant activities, seda- tive functions, and, analgesic potential [6]. Al- though Buddleja has been used as a diuretic and topical antiseptic in traditional medicine, its phytochemical investigation has been some- what neglected [7]. Buddleja asiatica (Bhim- senpati) and Buddleja paniculata (Narayanpati) are two species of the genus Buddleja found in the Kathmandu Valley, Nepal. B. asiat- ica is reported to have antihepatotoxic, anti- inflammatory, analgesic, antipyretic, hypoten- sive, hypoglycemic, antimicrobial, neuroprotec- tive, and anticataract properties [8]. It possesses chemical compounds like monoterpenoids, diter- penes, triterpenes, flavonoids, phenylpropanoids, and steroids [6]. A novel compound buddlin has been isolated from the plant [9]. B. asiatica is used as an additive in the preparation of fermen- tation starter culture in Arunachal Pradesh, In- dia [10]. The leaf juice is used for treatment of gastrointestinal issues, skin conditions [11], abor- tifacient, cure for loss of weight [12], and, bever- age fermentation [13]. Root paste with rice water is used as a toner in Burma [14]. In Laos, the same procedure is applied to make a base for a medicine taken during childbirth, and leaves are used to treat headaches [15]. Ullah et al., (2014) evaluated the cytotoxic and phytotoxic activities of B. asiatica which showed that the plant is safe for human consumption [16]. The leaves of B. paniculata are used by the indigenous Chepang community of Nepal in fermentation [17]. While many scholars have evaluated the biological ac- tivities and medicinal properties of B. asiatica, closely related species B. paniculata remains un- explored. The present work is a comparative study of qualitative and quantitative phytochem- ical analysis, antioxidant and antimicrobial, and cytotoxicity in methanol extracts and fractions of B. asiatica and B. paniculata. Reactive oxygen species (ROS) of both free radical and non-radical nature are produced in- side the cell during mitochondrial oxidative phos- phorylation or interaction with exogenous sources such as xenobiotic compounds [18]. They are in- volved in many signaling pathways and influence cell proliferation, necrosis, apoptosis, and gene expression in animal and host defense by pro- grammed cell death that prevents the spread of invading pathogens in plants [19]. The steadily forming prooxidants (ROS) are consumed by antioxidant molecules of enzymatic and non- enzymatic nature and a prooxidant-antioxidant equilibrium is maintained [20]. The imbalance in this equilibrium causes oxidative stress dur- ing which the highly reactive free radical can attack and cause structural damage to impor- tant biomolecules such as DNA, proteins, car- bohydrates, lipids, and enzymes [21]. Oxidative stress promotes and assists development of vari- ous health issues including neurodegenerative dis- eases, diabetes, cancer, and cardiovascular dis- eases [22]. Plant extracts and metabolites with rich antioxidant activity can prevent or delay the oxidation of substrate biomolecules and ex- ert various health-promoting and therapeutic ef- fects. Although the exact mechanisms by which a given herbal medicine exerts its pharmacological effects are not established, most such medicinal plants are known to contain significant antioxi- dant activity [23]. Adverse or side effects of synthetic drugs kill approximately 100,000 people in the USA each year and they are also responsible for about 8% of total hospital admission cases in the country [24]. Although plant-based traditional medicines and secondary metabolites have far fewer side ef- fects or toxicity than modern synthetic drugs due to their natural origin, they can potentially be toxic [23]. 2 Materials and Methods 2.1 Plant Collection and Identifica- tion Leaf and stem of B. asiatica and B. paniculata were collected from Dakshinkali 1, Champadevi, Lalitpur, and Bhaktapur 1, Dudhapati, Bhakta- pur, Nepal respectively in September 2021. The herbariums of the plant specimens were prepared and were identified at the Central Department of Botany, Tribhuvan University, Kathmandu, Nepal, having voucher no. TUCH-210070 and TUCH-210071 of B. asiatica and B. paniculata respectively. The photographs of fresh plants and herbarium are given in Figure 1 and traditional uses of plants are listed in Table 1. 2.2 Extraction and Fractionation he collected plant samples were cleaned and shade-dried up to a constant weight that was not attained. Then, the plant samples were ground to powder using a mechanical grinder. 100 g of the powder was soaked with 300 mL of reagent-grade methanol solvent inside a conical flask. After 72 hours, the content in the flask was filtered us- ing muslin cloth followed by Whatman no 1 filter Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 115 Figure 1: (a) Fresh plant sample B. asiatica (b) herbarium of plant sample B. asiatica (c) Fresh plant sample of B. paniculata (d) herbarium of plant sample B. paniculata. paper. The filtrate was concentrated at a con- trolled temperature (40 C) and reduced pressure in a rotatory evaporator. The process was re- peated after intervals of 48 hours and then 24 hours for maximum and complete extraction of phytochemicals. Table 1: The plant samples used in the study, voucher number, and traditional medical uses S.N. Scientific name Identification no. Altitude/plant growing Plant parts Traditional Uses 1 Buddleja asiat- ica TUCH-210070 Altitude-2180m Leaves and stems Fermentation [10, 13] Coordinates- 27°38’ 57" N 85°17’ 58" E Gastrointestinal issues, skin condition [11] Abortifacient, cure for weight loss [12] Toner [14] Childbirth, headache [15] 2 Buddleja panic- ulata TUCH-210071 Altitude-1996m Leaves and stems Fermentation [17] Coordinates- 27°40’ 08" N 85°26’ 32" E 2.3 Separation of Semi-purified Sol- vent Fractions A series of reagent-grade solvents with increas- ing polarity (hexane, DCM, ethyl acetate, water) were used to separate the different solvent frac- tions of methanol extract. Differences in the po- larities of these solvents will result in the separa- tion of plant secondary metabolites into different solvent fractions. Thus, it will help isolate the active compound in future works. 20 g methanol extract was dissolved in 50 mL of distilled wa- ter in a separating funnel and an equal volume of hexane was added. The contents were shaken vigorously at first and then left undisturbed un- til a clear upper layer of hexane and lower layer of water formed and then both layers were col- lected in separate beakers. The hexane layer was concentrated in the rotary evaporator to obtain a hexane soluble fraction. The aqueous layer was subjected to further fractionation with DCM and ethyl acetate for the separation of DCM and ethyl Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 116 acetate soluble fractions. 2.4 Phytochemical Analysis The phytochemical analysis of extracts and frac- tions was performed by following the procedures described by Harborne (1998) and Mallikharjuna et al. (2007) [25, 26]. The preliminary qualitative analysis for the presence and absence of secondary metabolites was carried out by the color differen- tiation method adopting separate protocols. 2.5 Estimation of Total Phenolic Content (TPC) Phenolic content was measured using the Folin- Ciocalteu reagent method with slight modifica- tions [27]. 20 µL each of different concentrations of standard (10 µg/mL to 100 µg/mL gallic acid solutions in methanol) and plant extracts (500 µg/mL in 50% DMSO) were loaded in separate bores of a 96 well plate in triplicates. Then, 100 µL Folin-Ciocaleu phenol reagent and 80 µL of Na2CO3 were added to each bore, and the well plate was placed in the dark. After 30 min- utes, absorbance was taken at 765 nm using a microplate reader (Synergy LX, BioTek, Instru- ments, Inc., USA) with Gene 5 software. TPC was calculated using the regression equation ob- tained from the gallic acid calibration curve and expressed as mg of gallic acid equivalent per gram dry extract or fraction (mg GAE/g). 2.6 Estimation of Total Flavonoid Content (TFC) AlCl3 colorimetric method as described by Zhishen et al., (1999) was used to determine to- tal phenolic content [28]. 130 µL of different con- centrations of the standard (15.4 µg/mL to 154 µg/mL quercetin in methanol) and 20 µL of plant extracts (500 µg/mL in 50% DMSO) were loaded in the bores of a 96-well plate in triplicates. 110 µL of distilled water was added to each bore con- taining plant extract. Then, 60 µL of ethanol, 5 µL of AlCl3, and 5 µL of CH3COOK were added to each bore, and the well plate was placed in the dark. After 30 minutes, absorbance was taken at 415 nm using the microplate reader. TFC was calculated using the regression equation from the quercetin calibration curve and expressed in terms of milligrams of quercetin equivalent per gram dry extract or fraction (mg QE/g). 2.7 Antioxidant Activity DPPH assay was employed to determine the an- tioxidant activity of plant extracts and fractions by following standard protocol [29]. 100 µL each of different concentrations of plant extracts and fractions (15.625 µg/mL to 500 µg/mL in 50% DMSO) were loaded in triplicates to the bores of a 96-well plate. 100 µL of 0.1 mM DPPH solu- tion was added to each bore. The reaction mix- ture was placed in the dark for 25 minutes and absorbance was taken at 517 nm using the mi- croplate reader. Quercetin was used as standard and 50% DMSO was used as control. The re- lationship given below was used to calculate the percentage of radical scavenging. GraphPad Prism 9 software was used to calcu- late the concentration of plant extract that scav- enges 50% of the available free radical (IC50). 2.8 Antibacterial Assay The agar well diffusion method was used to mea- sure the antibacterial activity in extracts and fractions [30]. The types of bacteria and their ATCC numbers are given in Table 2. Overnight incubated broth cultures of test organisms were prepared in nutrient broth me- dia. The concentration of bacteria was diluted and maintained at 0.5 McFarland standard (106-8 CFU/mL). 100 µL of inoculum was spread on Muller Hinton Agar (MHA) plates and wells hav- ing 7 mm of diameter were bored on the plates. 20 µL of 25 mg/mL (in DMSO) plant extracts were added to the bores in triplicates. Then, the plates were incubated at 37 C, and the zone of inhibition (ZOI) was measured after 24 hours. 100% DMSO was used as control and 1 mg/mL Ampicillin was used as standard. Table 2: Bacterial strain, type, and ATCC number of test organisms Bacterial strain Type ATCC Staphylococcus aureus Gram-positive 25923 Bacillus subtilis Gram-positive 35021 Escherichia coli Gram-negative 25922 Klebsiella pneumoniae Gram-negative 700603 2.9 Antifungal Activity The agar well diffusion method was used to mea- sure the antifungal activity in plant extracts [30]. Overnight incubated broth culture of test organ- isms Fusarium solani (ATCC 11712) was pre- pared in nutrient broth media and concentration Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 117 was maintained at 0.5 McFarland standard. 100 µL of inoculum was spread on potato dextrose agar (PDA) plates and wells having 7 mm of di- ameter were bored. 20 µL of 25 mg/mL plant fraction solutions were added in triplicates to those bores. The plates were incubated at 37 C and ZOI was measured after 48 hours. 100% DMSO was used as control and 20 mg/mL cyclo- heximide was used as standard. 2.10 Toxicity The toxicity of plant fractions was measured by using the brine shrimp assay [31]. 2 mL of each of the different concentrations of plant fractions (10, 100, 1000 µg/mL in methanol) was added in triplicates to test tubes. The solvent was evap- orated using a water bath. The leftover residue in the test tube was dissolved using 5 mL of ar- tificial seawater followed by the addition of 10 brine shrimp nauplii. The number of live nauplii was counted after 24 hours. The concentration of plant extracts lethal to 50% of the organism (LC50) was calculated from the percentage mor- tality versus concentration curve. 100% methanol was used as a control. 2.11 Statistical Analysis All the experiments were performed in triplicates. Values were presented as mean ± standard er- ror (SE). Comparisons were made using one-way ANOVA followed by Tukey’s test performed with SPSS version 29 software. Values with p < 0.05 were considered statistically different. 3 Results 3.1 Phytochemical Analysis The results of qualitative phytochemical analysis of the crude extracts are given in Table 3. ALC, B. asiatica crude leaf extract; ASC, B. asiatica crude stem extract; PLC, B. paniculata crude leaf extract; PSC, B. paniculata crude stem extract; ‘+’, present; ‘-‘, absent. Important phytochemicals such as polyphe- nols, flavonoids, and quinones are observed in the leaf and stem extracts of both plants. Al- kaloids were present only in leaf extracts and ter- penoids were exclusive to the stem. The absence of coumarin in PLC and glycoside in PSC was recorded. Table 3: Qualitative phytochemical analysis of crude extracts Group of compounds ALC ASC PLC PSC Polyphenols + + + + Alkaloids + - + - Coumarins + + - + Glycosides + + + - Quinones + + + + Flavonoids + + + + Terpenoids - + - + ALC, B. asiatica crude leaf extract; ASC, B. asiatica crude stem extract; PLC, B. paniculata crude leaf extract; PSC, B. paniculata crude stem extract; ‘+’, present; ‘-‘, absent. 3.2 Phytochemical Analysis The total phenolic content and flavonoid content were measured using the Folin-Ciocalteu phenol reagent method and aluminum chloride colori- metric method respectively and the results are provided in Table 4. The observed phenolic con- tent ranged from 39.5 ± 1.18 in PLC to 390.98 ± 5.32 mg GAE/g in ASE. The descending order of TPC in crude extracts and solvent fractions is 390.98 ± 5.32 (ASE) > 383.72 ± 7.31 (ALC) > 374.32 ± 5.72 (ASD) > 295.57 ± 0.54 (ASC) > 271.58 ± 5.83 (PSC) > 177.05 ± 7.91 (PLD) > 131.34 ± 3.82 (ALE) > 93.6 ± 3.97 (ALD) > 60.39 ± 3.48 (PLH) > 39.5 ± 1.18 mg GAE/g (PLC). The measured amount of flavonoid con- tent ranged from 13.44 ± 0.18 in PLD to 207.33 ± 0.34 mg QE/g in ALE. The descending order of TFC in crude extracts and fractions is 207.33 ± 0.34 (ALE) > 138.58 ± 0.53 (ASE) > 43.33 ± 0.14 (ALC) > 42.48 ± 1.09 (ASC) > 40.58 ± 0.58 (PLC) > 37.22 ± 0.19 (PSC) > 30.96 ± 0.18 (PLH) > 15.41 ± 0.44 (ALD) > 13.89 ± 0.32 (ASD) > 13.44 ± 0.18 mg QE/g (PLD). TPC and TFC values are graphically presented in Figure 2 and Figure 3. 3.3 Antioxidant Properties The antioxidant activity of the plant extract was measured using DPPH assay. Concentration- dependent increments in the percentage of free radical scavenging are observed (Figure 4). The concentration of plant extract or fraction that scavenges 50% of available free radical (IC50) Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 118 Table 4: TPC, TFC, and half maximal inhibitory concentration (IC50) in DPPH assay of plant extracts and fractions Plant extracts and fractions TPC (mg GAE/g) TFC (mg QE/g) IC50 (/) ALC 383.72 ± 7.31a,b 43.33 ± 0.14a 51.91 ± 1.09 ALE 131.34 ± 3.82 207.33 ± 0.34 139.70 ± 2.30 ALD 93.6 ± 3.97 15.41 ± 0.44b,c 119.91 ± 0.41 ASC 295.57 ± 0.54c 42.48 ± 1.09a,d 71.86 ± 1.67 ASE 390.98 ± 5.32a,d 138.58 ± 0.53 36.17 ± 0.92a ASD 374.32 ± 5.72b,d 13.89 ± 0.32b,e 83.59 ± 1.57 PLC 39.5 ± 1.18e 40.58 ± 0.58d 374.7 ± 0.55 PLD 177.05 ± 7.91 13.44 ± 0.18c,e 177 ± 0.72 PLH 60.39 ± 3.48e 30.96 ± 0.18 315.3 ± 0.45 PSC 271.58 ± 5.83c 37.22 ± 0.19 35.65 ± 0.61a Quercetin # # 3.28 ± 0.89 ALE, B. asiatica ethyl acetate leaf fraction; ALD, B. asiatica dichloromethane leaf fraction; ASE, B. asiatica ethyl acetate stem fraction; ASD, B. asiatica dichloromethane stem fraction; PLD, B. paniculata dichloromethane leaf fraction; PLH, B. paniculata hexane stem fraction; * positive control; #, value not measured; Values are the mean ± SE (n=3); values marked by the same letter within the same column are not significantly different at p < 0.05. ranged from 35.65±0.61 in PSC to 315.30±0.45 µg/mL in PLC (Table 4). The decreasing or- der of antioxidant activity is 35.65±0.61 (PSC) > 36.17 ± 0.92 (ASE) > 51.91 ± 1.09 (ALC) > 71.86 ± 1.67 (ASC) > 83.59 ± 1.57 (ASD) > 119.91 ± 0.41 (ALD) > 139.70 ± 2.30 (ALE) > 177 ± 0.72 (PLD) > 315.3 ± 0.45 (PLH) > 315.30±0.45 µg/mL (PLC). The IC50 values for extract and fractions were higher than 3.28 ± 0.89 µg/mL recorded for quercetin. Figure 2: Total phenolic content (mg GAE/g) of plant extracts and solvent fractions. Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 119 Figure 3: Total flavonoid content (mg QE/g) of plant extracts and solvent fractions. Figure 4: Percentage radical scavenging versus concentration (µg/mL) of plant extracts and solvent frac- tions. 3.4 Antimicrobial Properties The antibacterial and antifungal potential ex- hibited by the crude extracts and the solvent fractions is shown in Table 5. The antibac- terial activities of plant extracts were tested against an American-type culture collection of gram-positive and gram-negative bacteria. The dichloromethane fraction of the stem of B. asiat- ica displayed ZOIs of 8.33 ± 0.67 mm against S. aureus and 8.33 ± 0.33 mm against K. pneumo- niae. Similarly, the dichloromethane fraction of the leaf of the plant displayed a ZOI of 8.67 ± 0.33 mm against S. aureus. Other extracts and solvent fractions did not display significant ZOI. ZOI of 27.5 ± 0.25 mm, 13 mm, 16.5 ± 1.25 mm, and 22 ± 0.5 mm were recorded against S. aureus, B. subtilis, E. coli, and K. pneumoniae for Ampi- cillin used as positive standard. Plant extracts and solvent fractions were found to be inactive against fungal species F. solani but the cyclohex- imide used as a standard displayed a ZOI of 17.5 mm. The photographs of Petri plates from the antimicrobial assay are given in Figure 5. ALC = B.asiatica crude extract, ASC = B. Asiatica crude stem extract, PLC = B. Pan- iculata crude leaf extract, PSC = B. panic- ulata crude stem extract, ALE = B. asiatica Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 120 Table 5: Zone of inhibition (ZOI) shown by the plant extracts and solvent fractions against bacterial and fungal strains Plant extracts and fractions ZOI (mm) of plant extracts and fractions S. aureus B. subtilis E. coli K. pneumoniae F. solani ALC - # - - - ALE - - - - # ALD 8.33 ± 0.33a - - - - ASC - # - - # ASE - - - - - ASD 8.33 ± 0.67a - - 8.67 ± 0.33a # PLC - # - - # PLD - - - - # PLH - # - - - PSC - # - - - Ampicillin* 27.5 ± 0.25 13 16.5 ± 1.25 22 ± 0.5 # Cycloheximide* # # # # 17.5 Values are the mean ± SE (n=3); * positive control; -, no significant ZOI; #, value not measured; a p < 0.05 versus ampicillin. ethyl acetate leaf fraction, ALD = B. asiatica dichloromethane leaf fraction, ASE = B. asiat- ica ethyl acetate stem fraction, PLD = B. pan- iculata dichloromethane leaf fraction, PLH = B. paniculata hexane stem fraction; 3.5 Toxicity The results of the toxicity analysis of the crude extracts and the solvent fractions of the plants are displayed in Table 6. The assay revealed the non-toxic to mildly toxic nature of plant extracts and the solvent fractions. Values are the mean ± SE (n=3); a p < 0.05 versus PSC. The LC50 value ranged from 346.96 ± 25.39 in PLH to 2719.32 ± 706.5 µg/mL in PSC. The descending order of toxicity can be presented as 346.96 ± 25.39 (PLH) > 353.68 ± 83.62 (ASE) > 472.95 ± 51.36 (PLD) > 540.46 ± 39.87 (ASD) > 824.48 ± 103.54 (ALE) > 971.34 ± 253.48 (ALC) > 1074.83 ± 115.76 (ASC) > 1362.93 ± 393.21 (ALD) > 1366.7 ± 258.69 (PLC) > 2719.32 ± 706.5 µg/mL (PSC). Table 6: Half maximal lethal concentration (LC50) shown by plant extracts and solvent fractions against brine shrimp nauplii Plant extracts and fractions LC50 (µg/mL) ALC 971.34 ± 253.48a ALE 824.48 ± 103.54a ALD 1362.93 ± 393.21 ASC 1074.83 ± 115.76a ASE 353.68 ± 83.62a ASD 540.46 ± 39.87a PLC 1366.7 ± 258.69 PLD 472.95 ± 51.36a PLH 346.96 ± 25.39a PSC 2719.32 ± 706.5 Values are the mean ± SE (n=3); a p < 0.05 versus PSC. 4 Discussion The observed results were supported by previ- ous studies as the presence of phenols, flavonoids, glycosides, and terpenoids in the leaf of B. asi- atica was reported by Sai et al., (2019) and Nafees et al., (2022) [13, 32]. Among differ- ent plant secondary metabolites, phenolics are a large group of phytochemicals that includes a di- verse family of compounds such as phenolic acids, flavonoids, phenylpropanoids, quinones, tannins, lignins, and hydroxycinammic acids [33]. Among Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 121 them, flavonoids are involved in pigmentation, signaling, plant growth and development, and plant defense mechanisms [34]. The significance of phenolics in the modern drug discovery process is illustrated by the fact that 17 out of 29 small molecules approved by the Federal Drug Admin- istration, USA in 2020 contained phenol moieties [35]. The crude methanol extracts and solvent frac- tions of B. asiatica and B. paniculata are found rich in phenolic and flavonoid contents but signif- icant variations were observed between the two plant species and their crude extracts and frac- tions. Crude extracts of B. asiatica possessed higher phenolic content than crude extracts of B. paniculata. Stem extracts and fractions contained more phenolics than their corresponding leaf ex- tract. The concentration of phenolics was higher in more polar solvent ethyl acetate 131.34 ± 3.82 (ALE), 390.98 ± 5.32 (ASE) than in less polar sol- vent dimethyl chloride 93.6 ± 3.97 (ALD), 374.32 ± 5.72 mg GAE/g (ASD). Similarly, more po- lar fraction PLD contained higher phenolic com- pounds than PLH. The ability of a more polar sol- vent to extract a higher amount of phenolic con- tent is also mentioned by Khanal et al., (2022) in bark extracts of Beilschmiedia roxburghiana [36]. Sai et. al., (2019) reported a TPC of 127.48±1.58 mg GAE/g for 80% ethanol extracts of B. asiatica leaves [13]. The biological activities of phenolics, flavonoids, alkaloids, and terpenoids in the plant should be responsible for the traditional use of B. asiatica in gastrointestinal issues, childbirth, headache, skin conditions, weight loss, cancer, and diabetes [8, 11, 12, 15]. Similar to phenolic contents, the flavonoid content was found to be higher in crude ex- tracts of B. asiatica than in B. paniculata. Also, the concentrations of flavonoids were higher in crude extracts and fractions of leaves than in stems. This is expected as high concentrations of flavonoids are reported in UV-exposed mesophyll cells that seem to act as protection for chloroplast from photo-oxidative damage [37]. More polar ethyl acetate contained significantly higher TFC 207.33 ± 0.34 (ALE), 138.58 ± 0.53 (ASE) than less polar dimethyl chloride 15.41 ± 0.44 (ALD), 13.89 ± 0.32 mg QE/g (ASD). Sai et al., (2019) reported a TFC of 648.42±2.88 µg/mL for 80% ethanol extract of the leaf of B. asiatica [13, 33]. Extracts and fractions with higher phenolic and flavonoid content displayed more antioxidant activity. Phenolic compounds donate hydrogen atoms from their hydroxyl group and form sta- ble, unreactive antioxidant radicals [38]. The antioxidant activity of such compounds depends upon the number and position of the hydroxyl group [39]. In addition to phenolics, El-Sayed et al., (2008) isolated non-phenolic antioxidant com- pounds from the methanol extract of the leaf of B. asiatica [40]. Previous scholars have reported IC50 values of 3.04±0.04 µg/mL for 80% ethanol extract and 16.28 µg/mL for methanol extract of the leaf of B. asiatica [13, 40]. The present and past studies confirm the presence of high an- tioxidant activity in B. asiatica and B. panicu- lata. Antioxidant compounds present in medic- inal plants prevent oxidation, which is the ini- tial step of many neurodegenerative diseases and cancers [41]. They also reduce the risk of dia- betes and cardiovascular diseases [22]. High an- tioxidant activity in B. asiatica and B. paniculata could be responsible for the traditional uses of the plants as anticancer, antidiabetic, and neuropro- tective medicine [42, 43]. The plant is also used for the treatment of rheumatism in China [16]. The significant antioxidant activity in B. asiatica and B. paniculata makes them valuable medicinal plants. Only the dichloromethane fractions of the leaf and stem of B. asiatica displayed significant ZOI. Both plants do not possess significant activity against B. subtilis, E. coli, and F. solani in the present study. The lack of ZOI against K. pneu- moniae for methanol extract of B. asiatica in the present study is in agreement with the study con- ducted by Ali et al., (2011) in which no ZOI was observed against K. pneumoniae for methanolic extract, hexane, and ethyl acetate fractions of whole plant, but the ZOIs of 15 and 5 mm ob- served against E. coli and B. subtilis was dif- ferent from the present study [12]. The essen- tial oil of the whole plant was reported to be ac- tive against bacterial species E. coli, B. subtilis, and S. aureus and, fungal species F. solani [44]. The accumulation and concentration of plant sec- ondary metabolites depend upon plant growth and development stage, genetics, and environ- mental stress such as radiation, drought, temper- ature, and salinity [45]. The antimicrobial ac- tivity of plant extract and fractions is subject to change with the presence or absence of specific metabolites, their concentration, and synergistic interactions. Crude extracts of B. asiatica were more toxic than that of B. paniculata. The LC50 value of 971.34 ± 253.48 µg/mL for ALC was higher than the reported value of 469.63 µg/mL for ethanol extract of B. asiatica leaves [16]. The same study also reported higher toxicity in leaf than bark ex- tracts in agreement with higher toxicity in ALC than ASC observed in the present study. A good correlation between acute toxicity and the results of the Brine shrimp lethality assay has been es- tablished in previous studies [46]. Thus, the ab- sence of toxicity means it is potentially safe for humans. 5 Conclusion Stem and leaf of B. asiatica and B. paniculata dis- played the presence of secondary metabolites such as polyphenols, flavonoids, quinones, alkaloids, coumarins, glycosides, and terpenoids. Plant ex- tracts and solvent fractions of both plants are rich in phenolics and flavonoids and they also Sangita Pakka et al./ BIBECHANA 21 (2024) 113-123 122 display significant antioxidant activity. The con- centration of phenolics and flavonoids was found higher in polar solvents than in less polar ones. There is a weak correlation between phenolic and flavonoid concentration against antioxidant ac- tivity. The dichloromethane fraction of the leaf and stem of B. asiatica was found active against both gram-positive and gram-negative bacterial strains. Both plants were found inactive against the fungal species F. solani. The plant ex- tracts and solvent fractions displayed low toxi- cities against the brine shrimp nauplii. Thus, the plants are safe for use as a source for isolating nat- ural antioxidants rich in secondary metabolites. The crude extracts of B. asiatica displayed higher TPC and TFC along with the potential antibac- terial activity and toxicity than crude extracts of B. paniculata, however, both plants are found rich in phytochemicals and biological activity. Fur- ther work on isolation, purification, and char- acterization of bioactive compounds from both plants may lead to the discovery of novel ther- apeutic agents. In this way, the findings of the present study support the use of these two medic- inal plants as traditional medicine against infec- tious diseases and diabetes for many years by the people of Nepal. Acknowledgment The authors would like to acknowledge the Cen- tral Department of Botany, Tribhuvan Univer- sity, Kathmandu, Nepal for the identification of plants. 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