LC–MS/MS-QTOF analysis of Anodendron parviflorum (Roxb.) leaves extract and exploring its antioxidant, antimicrobial, and cytotoxic potential

Background Traditional medicine claims that Anodendron parviflorum has benefits for treating various human diseases. The present study seeks to understand better the phytochemical and LC–MS/MS-QTOF profiling of A. parviflo-rum ’s ethanolic extract and to investigate the properties of the different solvents of A. parviflorum for anti-inflammatory, antioxidant, antimicrobial, toxicity, and cytotoxic effects. Results The quantitative methods exhibited higher total phenolics (327.16 ± 2.4 mg GAE/g dw), total flavonoid (109.82 ± 1.9 mg QE/g dw), and total alkaloid (14.13 ± 0.09%) content in ethanol extract. In contrast, a higher total extraction value (22.8 ± 0.6%) and total terpenoid (57.23 ± 0.06 mg LL/g dw) content was shown in the methanol extract of A. parviflorum . LC–MS/MS-QTOF analysis of its ethanolic extract revealed a notable occurrence of phenols and flavonoids. The ethanolic extract of A. parviflorum exhibited significant antioxidant activities with lower IC 50 values in DPPH, phosphomolybdenum, and metal chelating and reducing power assay. The methanolic extract of A. parvi-florum had the more significant anti-inflammatory property (94.55 ± 0.1%) in the bovine serum albumin assay. The extracts also demonstrated a higher inhibition zone against pathogenic bacteria. The ethanolic extract of A. parviflo-rum demonstrated substantial cytotoxicity against A549 cells.


Background
The conventional herbal medicines are acknowledged by the WHO as a vital part of primary healthcare, and approximately 11% of the 252 medications are plantbased.The need for medicinal herbs is rising rapidly globally, with several in-depth studies on plants and their possible therapeutic properties [1].Traditional treatments heavily rely on phytocompounds of plants for improved therapy, and it is crucial to identify and standardize these substances through cumulative pharmacognostic investigations [2].These bioactive compounds are used to treat various human immunological, neurological and metabolic ailments.As a result, with an expanding population comes an increase in the utilization of native plants in commercial medicine [3].In general, allopathic medicines contain a wide range of severe adverse reactions which may contribute to significant health issues.Instead, natural medicines from herbs and plant extracts are highly effective and non-toxic if administered in the correct dosage.They offer a long-term, sustainable alternative due to their renewable source of origin [4,5].
Plant extracts have several pharmacological characteristics; the most common applications are beverages, decoctions, or other homemade treatments.Pharmaceutical products, including tablets, tinctures, powders, and other forms, are made using either crude plant extracts or standardized fractions of the plants [6].Drinking sage (Salvia officinalis) tea has been shown to prevent the initial stages of colon carcinogenesis, and its extract with cholinergic qualities has been reported to enhance cognitive function in young adults [7,8].The esophageal, gastric, and duodenal ulcers were almost wholly cured after consuming ten weeks of neem bark extract (30-60 mg) in human subjects [9].Oral administration of citrus and rosemary extracts lowered ROS generation in keratinocytes (HaCaT) and human subjects, thereby preventing damage to cellular DNA [10].According to reports, amaranth extract increases the nitrite and nitrate rates in the body for at least eight hours after a single oral dose; thereby, it can enhance the effectiveness of healthy individuals performing intense physical activity or sports [11].Orally administered Artemisia absinthium herb suppresses TNF-α and has a significant impact on the clinical signs and consequent depression of Crohn's disease in patients [12].Thus, different plant extracts are engaged in treating various human diseases.
Cancer is an aggressive illness, becoming one of the major global health issues due to insufficient early detection technologies and appropriate medication.Despite substantial advancements in medical research, cancer continues to be a mystery, particularly in possibilities for treatment perspective [13].Due to the harmful adverse reactions of conventional chemotherapy, plants are receiving much interest as alternative sources to enhance the treatment of cancer.Herbs are a significant source of antitumor medicines, with > 60% of approved anticancer drugs [14].The development of cytotoxic agent resistance is a considerable issue with cancer treatment therapies.Plant-based natural compounds have demonstrated great promise as drug repositories to combat multidrug resistance [15].Natural and readily accessible medications produced from plants are typically taken orally as a component of their diet.Drugs derived from plants are being developed for medicinal use with excellent outcomes in clinical studies.They are in great demand because of their harmful impact on malignant cells and their non-hazardous effects on healthy cells [16].
The predominance of organic and semisynthetic compounds for treating infectious conditions is increasing daily, concerning the number of bacterial strains resistant to antibiotic medications.It still presents a hazard to both industrialized and developing nations.Antibacterial drug resistance caused by gram-positive bacteria and some gram-negative bacteria is a vital global issue.These bacteria can cause bacterial infections, for which a few potent antimicrobial treatments are on the market [17].The problem of antibiotic resistance has caused an increase in focus on biologically active phytoconstituents or secondary metabolites isolated from plant species.These substances can provide a new, powerful source of antibacterial and antifungal abilities and are commonly used in herbal medicine [18,19].
Scientists are constantly interested in extracting different plant parts and characterizing their functional elements and antibacterial, anticancer, and antioxidant activities.Experts from diverse fields are investigating the contribution of antioxidants in cancer therapeutics and whether antioxidants extracted from plants are promoters or suppressors of cancer [20].Plant chemistry is vibrant and includes many semi-polar molecules, notably significant secondary metabolites readily separated and identified by LC-MS techniques.Therefore, LC-MS-based techniques are anticipated to be particularly important in the field of plants to elucidate different bioactive compounds [21].Quadrupole time of flight (QTOF) can be combined with an effective analytical technique known as LC-MS/MS to detect the unknown phytocompounds in various plants [22].
The family Apocynaceae has an extensive range of bioactive compounds, including lactones, phenols, lignans, hydrocarbons, terpenoids, flavonoids, phenols, and glycosides.These phytochemicals are notable for different biological applications, including cytotoxicity, antidiabetic, antibacterial, antioxidant, and anti-inflammatory activities [23].Anodendron parviflorum or Anodendron paniculatum (A.parviflorum) is an endemic climber under the Apocynaceae family.It is widespread in India, Bangladesh, Burma and Sri Lanka.In Indian traditional medicine, the roots of A. parviflorum are used to treat cough and vomiting and its latex is utilized to treat centipede and snake bites [24,25].According to tribal folklore, A. parviflorum is a significant medicinal herb that can treat many illnesses.It was listed as an endangered or vulnerable species by the International Union for Conservation of Nature (IUCN) on the list of medicinal herbs in Andhra Pradesh [26,27].In a study, the isolated phytoconstituents from A. paniculatum aerial parts include ursolic acid, bis-(2-ethylhexyl) phthalate, esculenic acid, desmosterol, vanillin, (E)-phytol and stigmasterol, as well as active elements of triterpene ester, termed anopaniester and cycloartenol.Among these compounds, ursolic acid and desmosterol exhibited significant cytotoxicity against MKN-7 and LU-1 cell lines, whereas other compounds showed considerable anticancer effects on these cell lines [28].The aerial part of the methanolic extract showed a substantial antibacterial impact against Vibrio cholera [26].
Anodendron parviflorum is considered to be a potent medicinal plant in traditional medicine.However, there were few reports on the phytochemical screening and bioactive components of A. parviflorum.There has never been a scientific study to confirm the various biological applications of the different solvent fractions of A. parviflorum.So, the current work aims to elucidate the phytochemical profiling, LC-MS/MS-QTOF profiling of ethanolic extract of A. parviflorum to reveal the various phytoconstituents, and to examine the hemolytic, antiinflammatory, thrombolytic, antioxidant, antimicrobial, and cytotoxic properties of different solvent fractions of A. parviflorum.

Plant collection and extraction
The A. parviflorum leaves (Voucher No: VIT/ BT/2023/08) were obtained from FRLHT, Bangalore.The sample was authenticated by Dr. N. M. Ganesh Babu, Heading Centre of Herbal Gardens, TDU, Bangalore.The plant samples were shade-dried for four days.Twenty grams of coarsely pulverized samples were added each to 100 mL of various solvents, such as water, methanol, ethanol, ethyl acetate, and chloroform and kept for cold maceration at 37 °C for 24 h at 120 rpm in an incubated shaker (MaxQ ™ 4000 Benchtop, Thermo Scientific, India).The extracts were then filtered using Whatman No. 1 filter paper and concentrated using a rotary evaporator (Lelesil Innovative Systems, India).The dried extract was collected and stored at 4 °C for further experiments [29].

Total extraction value (TEV) estimation
The cold maceration method was used to extract the dry powdered plant material with various solvents, including water, methanol, and ethanol.About two grams of the plant sample was added with 30 mL of various solvents, incubated at 37 °C for 24 h and kept for intermittent shaking.The mixtures were filtered and transferred into pre-weighed petri plates.The resulting extracts were dried out by letting the filter completely evaporate the solvent [32].The total extraction value was obtained by the formula,

Total phenolic content (TPC) estimation
The overall phenol content of the A. parviflorum was quantified using the protocol [33] with some alterations.The standardization plot of gallic acid served as the control.A combination of 100 µL test sample, 400 µL distilled H 2 O, and 100 µL Folin-Ciocalteu mixture was subsequently diluted with 500 µL of sodium carbonate (7%) and incubated at ambient temperature for half an hour.At 765 nm, the absorbance was noted (Jenway 7415 Scanning UV/Visible Spectrophotometer, USA).The TPC was calculated using the following formula and shown in mg/g gallic acid equivalent (GAE) of dry weight (dw).The standard curve's formula was y = 0.0031x + 1.4688 (R 2 = 0.9916).
where 'TPC' stands for total phenolic content in mg GAE/g dw, 'c' is gallic acid concentration (mg/mL), 'V' for extract volume (mL), and 'm' for extract mass (g).

Total flavonoid content (TFC) estimation
The colorimetric aluminum chloride test assessed the overall flavonoid amount of A. parviflorum [34].The process involved adding plant extracts (1 mL) to distilled H 2 O (4 mL), and then sodium nitrite (5%; 0.3 mL) and aluminum chloride (10%;0.3mL) were mixed and kept for 6 min.Furthermore, 1 M NaOH (2 mL) was subjected.At 510 nm, the absorbance was determined.The TFC was evaluated through a quercetin calibration curve and was given as mg/g Quercetin Equivalent (QE) of dry weight.The standard curve's formula was y = 0.0294x + 0.5003 (R 2 = 0.9866).
where 'TFC' stands for total flavonoid content in mg QE/g dw, 'c' is quercetin concentration (mg/mL), 'V' for extract volume (mL), and 'm' for extract mass (g).

Total terpenoid content (TTC) estimation
The overall terpenoid of the various solvent fractions of A. parviflorum leaves was estimated through the protocol of [35].About 1.5 mL of chloroform was supplemented to 500 µL of the different test samples, thoroughly agitated, and incubated for 3 min.It was then mixed with 100 µL of concentrated sulfuric acid and incubated at 37 °C for two hours in the absence of light.The supernatant was removed, and methanol (1.5 mL) was added to the resulting precipitate.The absorbance was determined at 538 nm.The TTC was determined as mg/g linalool equivalent (LL) of dried extract.The standard curve's formula was y = 0.0204x−0.3033(R 2 = 0.9855).
where 'TTC' stands for total terpenoid content in mg LL/g dw, 'c' is linalool concentration (mg/mL), 'V' for extract volume (mL), and 'm' for extract mass (g).

Total alkaloid content (TAC) estimation
A non-spectrophotometric approach was used to quantify alkaloids with minor modifications [36].Dried plant extracts (10 mg) were mixed with 7.5 mL of acetic acid (10% in ethanol) and incubated for 4 h.After incubation, the sample was filtered and concentrated using a water bath.Subsequently, the mixture was precipitated by gradually adding a drop of concentrated ammonium hydroxide (NH 4 OH).The reaction mixture was allowed to settle down, the precipitate was decanted into a new Eppendorf tube (1.5 mL), and it was diluted with NH 4 OH; the Total terpenoid content (TTC) = cV /m residue obtained was measured to determine the percentage of alkaloids in the plant extracts by the formula,

LC-MS/MS-QTOF analysis
The LC-MS/MS-QTOF study identifies all the phytochemical contents of the ethanolic extract of A. parviflorum.Agilent Technologies 6550 Mass QTOF, USA, was employed in both positive and negative modes.Nitrogen (N) was employed as a drying, collision, and nebulizing gas.A complete scan mode was attained between 126-1200 amu with the drying gas flow frequency adjusted to 13 L/min; the capillary temperature was fixed to 250 °C, and the nebulizer pressure was at 35 psi.The mobile phase A has formic acid (0.1%), while the mobile phase B has acetonitrile (95%).The ion source's parameters values of octapole radio frequency peak voltage, fragmentor, Vcap, and skimmer were all set to 750 V, 175 V, 3500 V, and 65 V, respectively.Bioactive compound identification was done using Mass Hunter software version B.05.01 (B5125.3)(LC/MS Data Acquisition for 6200 TOF/6500 series).

Radical scavenging assays DPPH study
The reactive oxygen species (ROS) mitigating abilities of various A. parviflorum solvent extracts were assessed using the DPPH assay [37].The five distinct concentrations (20-100 ug/mL) of each sample were diluted to 1 mL using the appropriate solvents, and DPPH methanolic dilution (1 mL) was supplemented.The combination was incubated for 30 min in the absence of light.The wavelength at 517 nm was used to assess the DPPH radical reduction.The absorbance reduction percentage of the plant samples in comparison to the control was employed to estimate the antioxidant activity, where A Control is controls' optical density and A Sample is samples' optical density.

Phosphomolybdenum assay
The phosphomolybdenum method assesses the A. parviflorum's antioxidant ability.The assay depends on the shift from Mo (VI) to Mo (V) by the test sample and the green color development in an acidic environment.A reagent mixture comprising H 2 SO 4 (0.6 M), ammonium molybdate (4 mM) and Na 3 PO 4 (28 mM) was supplemented with 1 mL of various extracts at 25-100 µg/ mL and the tubes were kept for 90 min at 95 °C.The % yield = (Wt. of the plant extract/Wt. of dried plant sample) * 100 % radical mitigating activity = OD Control − OD Sample /OD Control * 100 wavelength was noted at 695 nm [38].An antioxidant for reference was ascorbic acid.The formula below gives the radical scavenging percentage,

Metal chelating assay
The Fe 2+ chelating property of the A. parviflorum extracts was calculated by a procedure [38] with minor changes.Four different concentrations of test samples (25, 50, 75 and 100 µg/mL), ferrous sulfate (0.1 mM), and ferrozine (0.25 mM) was thoroughly stirred and kept at 37 °C for 10 min.At 562 nm, the wavelength was noted.The samples' ability to chelate ferrous ions is determined by,

Reducing power assay
The reducing power experiment performed based on the standard procedure with subtle changes [39].The varying concentrations (20,40,60,80, and 100 µg/mL) of different extracts were administered with 150 µL of 0.2 M PBS (pH 6.6) and 150 µL of potassium ferricyanide (1%).The combination was kept at 50 °C for 20 min.It was stirred with trichloroacetic acid (150 µL) and spun at 3000 rpm for 10 min at an ambient temperature.To 100 µL of supernatant, 150 µL of distilled H 2 O and 50 µL of FeCl 3 (0.1%).The control was ascorbic acid, and at 700 nm, absorbance was measured.The outcome suggests that a higher absorbance of the sample corresponds to a rise in reduction potential.

Anti-inflammatory assay
The bovine serum albumin (BSA) test was performed to assess the anti-inflammatory activity of A. parviflorum's various solvent extracts [40].The several extracts of A. parviflorum and the reference standard aspirin of four different concentrations (100-400 μg/mL) was supplemented with 1% BSA.After 20 min of incubation at 37 °C, it was kept at 57 °C for 15 min.PBS served as the blank.The wavelength was estimated at 660 nm.The chelating activity percentage was assessed by,

Hemolytic activity
Hemolytic assay was performed on A. parviflorum extracts using a protocol with minor changes [41].In accordance with the protocols of the institutional ethical committee for studies on human subjects (VIT/IECH/ XIII/2023/07), healthy participants were chosen to obtain human blood samples.Erythrocytes were obtained as supernatant by centrifuging blood samples for 5 min at 10,000 rpm.PBS was used to wash the erythrocyte pellet.The resulting suspension (200 µL) was added with 100 µL of sample dosages (25-100 µg/mL), incubated for an hour at 37 °C, and then centrifuged for 6 min (6000 rpm) at room temperature (RT).The wavelength was determined at 540 nm.The positive control was Triton X-100, whereas the negative control was dimethyl sulfoxide.The results were expressed as a below formula,

Thrombolytic activity
The human blood was obtained from healthy participants using venipuncture and placed in separate pre-weighed (W1) clean 1.5 mL micro centrifuge tubes for 45 min at RT.After the blood clots, the blood serum was drained, and the weight (W2) of the clot containing tube was measured to evaluate the weight of the clot.Various test samples of 100 µL were added to the tube having a clot.The sterile distilled H 2 O was employed as a negative control and streptokinase served as a positive control.After 90 min at 37 °C incubation, the clot disruption of each tube was monitored, the liquid was extracted, and tubes (W3) were measured for the change in mass after clot lysis.The clot lysis percentage was determined by comparing the weight (W3) measured before and following the lysis process (W4) [42].

Antibacterial activity
Petri plates with nutrient agar media were used in the well-diffusion method to calculate the zone of inhibition [43].Different bacterial strains, including grampositive bacteria Staphylococcus aureus (S. aureus; ATCC-3160), and gram-negative bacteria Proteus vulgaris (P.vulgaris; ATCC-426), Escherichia coli (E.coli; ATCC-25923) and Pseudomonas aeruginosa (P.aeruginosa; ATCC-8027), were grown on media containing Petri plates and stored at 37 °C for 24 h.The agar plates were inoculated with these four pathogenic bacteria using a spread plate method.Five (7.0 mm diameter) wells were punched with a cork borer in each plate.The different concentrations (25,50,75, and 100 µL) % lysis = OD Sample − OD Neg.control / OD Pos.control − OD Neg.control * 100 % Clot lysis = Clot weight after lysis (W4)/Clot weight (W3) * 100 of methanol, ethanol and aqueous extracts were introduced into a separate well for every strain.Streptomycin (50 µg/mL) was used as a control.The plates were allowed to fully diffuse for one hour before being incubated for 24 h at 37 °C, and the inhibitory zone diameter was noted in millimeters.

Antiproliferative assay
The A549 cells were cultured in the appropriate medium to attain 70% confluency of cells.The A. parviflorum extracts were added to cells at several dosages (25-100 µg/mL) and incubated for 24 h, while DMSO was employed as a control.Each well was supplemented with MTT solution (5 mg/mL) and incubated for 4 h.Subsequently, DMSO (200 µL) was supplemented.The wavelength was determined at 570 nm [44].

Statistical data
The findings were demonstrated as the average ± SD of three repeats.A two-way ANOVA and Tukey's test were employed in the statistical studies to examine significant differences (p < 0.05) using GraphPad Prism, version 10.0.0 (GraphPad Software, Boston, USA).

Preliminary screening
The initial qualitative findings of several solvent extracts of Anondendron parviflorum leaves are summarized in Table 1.According to the Wagner, Mayer, and Hager test, the presence of alkaloids was found in the plant sample's ethanolic, methanolic, and aqueous extracts.In the Salkowski test, the appearance of a brown ring signifies the occurrence of steroids and triterpenoids in ethanol, methanol, and aqueous extract.Most extracts tested positive for cardiac glycosides in the Killer-Killani method, and the study proved the occurrence of cardiac glycosides by producing a red color in the chloroform and ethanolic extract.The ferric chloride and lead acetate tests exhibited the existence of phenols and tannins.In contrast, the alkaline reagent test confirmed the presence of flavonoids with a yellow color appearance in ethanol, methanol, and aqueous extracts.The denser foam formation in ethanol and aqueous fractions indicated the presence of saponins in the froth test.The production of an emerald green % cell viability = OD Sample /OD Control * 100 color in the copper acetate test showed the occurrence of diterpenes in ethanolic and methanolic extract.The test for quinones was positive in ethanol, methanol, and aqueous extracts.

Quantitative analysis
The total extraction values (TEV) revealed a significant percentage value of 22.8 ± 0.6% in methanolic extract, followed by aqueous and ethanolic plant extract.The TPC estimation in three solvent fractions revealed that the ethanolic extract had a significant phenol amount (327.16 ± 2.4 mg GAE/g) than the methanolic and aqueous extracts.The TFC was higher in ethanolic extract at about 109.82 ± 1.9 mg QE/g, followed by methanolic and aqueous extracts.The methanolic extract of A. parviflorum exhibited a higher total terpenoid content (TTC) of 57.23 ± 0.06 mg LL/g dw than the other samples.The entire alkaloid content (TAC) showed a greater percentage yield of 14.13 ± 0.09% in ethanolic extract, followed by aqueous and methanolic extracts.The results of quantitative analysis, including TEV, TPC, TFC, TTC, and TAC values, are shown in Table 2.

DPPH study
The investigation of the free radical scavenging ability of the solvent extracts derived from A. parviflorum at various concentrations (20-100 μg/mL) was assayed by DPPH method.The leaf extracts displayed a dosedependent activity (Fig. 2a).The ethanol extract demonstrated greater antioxidant efficacy with an associated IC 50 value of 23.13 ± 0.4 µg/mL followed by methanol and aqueous extracts with IC 50 values of 41.78 ± 1.8 and 64.15 ± 0.9 µg/mL, respectively, in comparison with the ascorbic acid (13.56 ± 0.4 µg/mL) (Table 5).

Phosphomolybdenum assay
The fundamental principle of the phosphomolybdenum assay is that plant extracts with antioxidants substances can convert Mo (VI) to Mo (V), thereby elucidating its antioxidant ability [45].The outcomes of this study demonstrated that IC 50 values of A. parviflorum's ethanol extract (52.04 ± 1.6 µg/mL) were more significant than methanol (84.5 ± 1.4 µg/mL) and water extract (102.97 ± 2.1 µg/mL) in Mo (VI) to Mo (V) reduction.The standard ascorbic acid demonstrated IC 50 values of 22.43 ± 1.2 µg/mL in the reduction process (Table 5).The results revealed the dose-dependent reduction ability of different solvent extracts of A. parviflorum (Fig. 2b).

Metal chelating assay
In this experiment, the primary factor determining the metal chelating abilities of plant samples is the existence of electron-donating substances facilitating the conversion of Fe 3+ (ferric cyanide) to Fe 2+ (ferrous).As the concentration was elevated, there was a notable rise in activity, as observed thereby indicating the reducing capability of the extracts (Fig. 2c).Notably, the ethanol extract showed more antioxidant ability with the IC 50 value of 35.48 ± 1.2 μg/mL than the methanol and aqueous extracts with the IC 50 value of 68.01 ± 2.4 and 80.95 ± 2.1 μg/mL, respectively.In comparison, the IC 50 value of EDTA was 17.53 ± 0.2 μg/mL (Table 5).

Reducing power assay
The substances with reducing ability are associated with potassium ferricyanide (Fe 3+ ) to produce potassium ferrocyanide (Fe 2+ ), which subsequently interacts with FeCl 3 to create a ferric-ferrous complex [46].Among the three extracts, the methanol exhibited significant reducing power ability with a lower IC 50 value of 76.19 ± 1.2 μg/mL than the ethanol extract (83.26 ± 0.9 µg/mL) and aqueous extract (134.25 ± 0.6 µg/mL).The control showed an IC 50 value of 32.47 ± 1.2 μg/mL, respectively (Table 5).The findings demonstrated, if higher the absorbance value, the higher the reduction potential (Fig. 2d).The polyphenols/bioactives present in the A. parviflorum ethanolic extract may be responsible for its potential antioxidant activity (Fig. 3).

Anti-inflammatory assay
A simple alternative to assess the anti-inflammatory abilities of medicinal herbs is by performing protein denaturation method with BSA.The breaking of the disulfide, hydrophobic, electrostatic, and hydrogen bonds that protect the 3D form of proteins is due to denaturation mechanism [47].The ability to reduce inflammation rises with various doses of aspirin, aqueous, ethanol, and methanol extracts of A. parviflorum at 100, 200, 300, and 400 µg/ mL.At maximum dosage, the methanol extract of the plant sample had the greater anti-inflammatory capacity   4).

Hemolytic activity
The methanol, ethanol, and aqueous extract of A. parviflorum were tested for their hemolytic effect against human RBCs.The hemolytic activity is reported as a percentage of lysis.At 100 μg/mL, the amount of lysis of aqueous, ethanol, and methanol extract was determined to be 2.56 ± 0.1%, 3.25 ± 0.1%, and 6.1 ± 0.2%, respectively.Triton X-100, the positive control, showed 99 ± 0.3% lysis.The extracts demonstrated a low level increase in hemolytic activity that was dosage-dependent (Fig. 5).

Antibacterial studies
The antimicrobial ability of A. parviflorum was tested on harmful bacteria.The inhibitory properties of the various extracts of A. parviflorum against the microbes were measured by the inhibitory zone surrounding the diffusion pore (Fig. 7a, b and c).At maximum concentration, the aqueous extract showed a higher inhibition zone of 25 ± 0.2 mm against S. aureus and P. vulgaris, followed by P. aeruginosa (21 ± 0.4 mm) and E. coli (20 ± 0.4 mm).
The ethanol extract showed an excellent inhibitory zone of 23 ± 0.5 mm against P. vulgaris, followed by S. aureus (20 ± 0.6 mm), P. aeruginosa (20 ± 0.5 mm), and E. coli (20 ± 0.5 mm).The methanol extract exhibited a higher inhibitory zone of 21 ± 0.4 mm against S. aureus and E. coli, followed by 20 ± 0.6 mm of P. aeruginosa and 12 ± 0.3 mm of P. vulgaris.In our results, the higher concentration of various extracts significantly inhibited all pathogenic bacteria.In contrast, the decreasing concentration of extracts had marginally effective antimicrobial properties (Fig. 8).

MTT assay
The cytotoxicity effects of several solvent extracts of A. parviflorum were tested against A549 cell lines.The control wells did not exhibit any noticeable effect on cell proliferation.The A. parviflorum ethanolic extract demonstrated cell death in a dose-dependent pattern, having an IC 50 value of 72.11 ± 2.4 µg/mL.The methanolic and aqueous extracts revealed substantially raised IC 50 values of 105.3 ± 1.4 and 92.1 ± 2.6 µg/mL, respectively (Fig. 9).

Discussion
The medicinal plants are a rich source of bioactive compounds.Previous findings reported the presence of phenols, flavonoids, terpenoids, and alkaloids in the methanolic plant extract of A. parviflorum [26].In the qualitative analysis, the methanol, ethanol, and aqueous extracts of A. parviflorum leaves have more potential for extracting more bioactive compounds than other solvent extracts.Consequently, ethanol, methanol, and ethanol extracts of A. parviflorum were selected for quantitative analysis.In the literature, plant polyphenolic compounds can modify biological responses, enhancing the immune system and shielding cells from the damaging effects of free radicals.Both phenols and flavonoids were extensively linked to the prevention and treatment of cancer [48,49].In various studies, the methanolic extract of A. parviflorum aerial plant parts exhibited higher phenolic content and flavonoid content of 25.53 ± 1.89 mg GAE/g and 20.62 ± 1.99 mg QE/g than the solvent extracts of chloroform, ethyl acetate and petroleum ether respectively [26].Our findings demonstrated that the ethanolic leaf extract of A. parviflorum exhibited a remarkable amount of phenols and flavonoids, and the methanolic plant extract showed higher terpenoid content.Extraction yield indicates its potential therapeutic use.Numerous advantages of plant-based bioactive chemicals are being investigated for possible biological applications.Several in vitro epidemiological investigations on glucosinates, alkaloids, saponins, alkaloids, polyphenols, sterols, flavonoids, and salicylates have shown positive outcomes [50].In studies, deacetyl-ganoderic acid F showed an excellent anti-inflammatory property and a strong curative potential for conditions linked to inflammation in the brain both in in vitro (BV-2 cell line) and in vivo conditions (mice model) [51].Catechins showed strong antiviral, anti-inflammatory, antibacterial, antiallergenic, and anticancer properties.Additionally, it improves the usefulness of nutritious food products and biocosmetics by increasing their distribution and uptake into the skin and body [52].Rutin demonstrated the highest free radical scavenging capabilities and a potent inhibitory effect on the growth curve and bacteria biofilm formation [53].Quizalofop-P-Ethyl (QPE) is a typical phenoxy herbicide in agriculture for weed control when tested against the meristem cells (root) of Allium cepa [54].In an investigation, substances with derivatives of aurone demonstrated promising antibacterial activity against resistant gram-positive infections [55].The bacterial pathogens' growth is inhibited by epigallocatechin gallate, and the Bcl-2 family proteins bind firmly with epitheaflagallin 3-O-gallate, suggesting that the latter is a potential functional dietary component with significant anticancer properties on in vivo animal model (BALB/c mice) [56,57].The 2,4,6-triethyl-1,3,5-trioxane belongs to the trioxanes class and was found to possess antimalarial activity, and 2E-decenedioic acid exhibited nematocidal activity [58,59].The proliferation of the colon cancer cell line (caco-2) was significantly reduced by the increased quercetin found in Olea eurpaea fruit extracts [60].Phenolic substances have also demonstrated favorable effects in regulating the metabolism of various cancer cell lines [50].Similarly, the various bioactive compounds in the ethanolic extract of A. parviflorum shown in LC-MS/MS-QTOF analysis may possess substantial therapeutic applications.
The phenolic substances of plants are regarded as potential electron donors due to their ability to contribute to antioxidant effects actively through their hydroxyl groups.These compounds exhibited ROS inhibition, peroxide breakdown, and reduced oxidative stress [4].In earlier reports, the Mammea suriga aqueous extract showed a significant reduction ability with lower IC 50 values of 111.51 ± 1.03 μg/mL.The Cordia dichotoma methanolic extract revealed a higher absorbance of 0.291 at maximum concentration [61].The methanol extract of Psidium guajava demonstrated a more significant reducing potential of 2.165 ± 0.013 absorbance at 500 μg/mL concentration than petroleum ether, chloroform and aqueous extracts [62].The antioxidant capabilities of plant polyphenols are related to their chemical structure, which is precisely the number and position of the hydroxyl groups.These phenolic compounds have strong antioxidant properties and consequently lower the risk of serious illnesses, including cancers, persistent inflammation, heart issues, and several degenerative conditions, by reducing the oxidation of low-density lipoproteins, erythropoiesis, and thrombus formation.They may predominantly serve as anticarcinogens, metal chelators, and antimicrobial agents [63].In studies, the leaf extracts had anti-inflammatory properties as they demonstrated the ability to lower the quantity of fibroblasts and collagen synthesis, which are required to create granulation tissues [64].Thus, the current study on A. parviflorum leaf extracts validated its traditional use in certain inflammatory and painful diseases.
In studies, human RBCs have shown no toxicity or reduced toxicity when treated with aqueous leaf extracts from Calotropis gigantea, Elaeocarpus ganitrus, and Aerva lanata, both alone and in combinations [65].The ripened wild fig fruits in different solvent extracts showed hemolytic activity of 2-3% lysis [66].In analogous to our findings, the hemolysis rates of aqueous, ethanol, and methanol extract of Acacia nilotica were determined to be 2.6 ± 0.5%, 3.7 ± 0.4%, and 4.6 ± 0.2%, respectively [67].The thrombolytic property of the various fractions of Centella asiatica ranged between 17.58 ± 0.23% and 43.94 ± 0.62%.The plant's phenolic concentration corresponds to the strongest thrombolytic activity [68].A phytochemical investigation showed the occurrence of bioactive substances in the crude extract of the plant, suggesting that these phytochemicals might cause thrombolytic effects.The sources of medicinal plants may significantly impact the development of affordable and efficient medications for cardiovascular health [69].
In studies, the methanolic extract of the A. parviflorum demonstrated a higher zone of inhibition against Streptococcus pyrogens (19.5 ± 0.5 mm), Pseudomonas aeruginosa (14.0 ± 0.5 mm), Vibrio cholerae (20.0 ± 0.0 mm), and Staphylococcus aureus (17.8 ± 0.45 mm) than petroleum ether, ethyl acetate, and chloroform extract [26].In our Fig. 8 Antibacterial activity of A. parviflorum aqueous, ethanol, and methanol extract findings, the different extracts exhibited substantial antibacterial activity with a higher zone of inhibition against P. aeruginosa, S. aureus, P. vulgaris, and E. coli.Furthermore, a variety of distinct bioactive chemicals found in aqueous, ethanolic, and methanolic plant extracts have been found to have the ability to impact various target sites in opposition to bacterial cells [19,70].The phenolic molecule para-trifluoromethylphenol is made up of trifluoromethyl groups.The FDA approves trifluoromethyl group-containing medications which are frequently used for treating malaria, cystic fibrosis, cancer, and infectious diseases of microbes [71].The compound 1-(2-furanyl)-1-propanone from plant samples possesses antibacterial activity [72].Quercetin inhibits the growth of several viruses, fungi, and gram-positive and gram-negative bacteria.Quercetin inhibits the development of various drug-resistant microbes and helps prevent biofilm formation [73][74][75].Maritimetin showed significant antimicrobial efficacy against S. aureus [76].Soysaponins ability to suppress β-lactamase activity has increased the antibacterial efficacy of β-lactam medicines and demonstrated antimicrobial and insecticidal activities [77,78].These plant compounds bind to the proteins and enzymes in the cell membrane of microbes, disrupting it and releasing a flow of protons into the outside of the cell, which either causes the cell to break down or may prevent the enzymes needed for the synthesis of amino acids [79].
Plant sample phenolic substances are well-known to act as chemopreventive agents in cancer treatment [80].The compound 2-methoxy-3-(1-methyl propyl) pyrazine belongs to pyrazine derivatives in which pyrazines are identified as anticancer agents against different enzymes, including inhibitors of protein kinase, mitotic kinase, checkpoint kinase, A549 lung cancer cell, and rapamycin [81].Quercetin can slow down the growth of several human malignancies due to its specific proapoptotic actions on tumor cells [82].A previous study reported that ganoderic acid F has significant cytotoxic activity against HeLa cell lines [83].Catechins obtained from various plants have antitumor, antiangiogenic, and antioxidant properties, and they possess chemopreventive properties as they trigger apoptosis, inhibit cell proliferation, trigger killer caspases, and block oncogenic transcription factors [84].Rutin functions as a chemotherapeutic and chemopreventive drug, and its anticancer property can be attributed to its ability to inhibit angiogenesis and metastasis, induce apoptosis or autophagy, and limit cell proliferation [85].A class of aurones that includes the compound aureusidin has been shown to have anticancer properties [86].Soyasaponins exhibited anticancer activity against Ehrlich ascites carcinoma [87], breast cancer [88], and colon cancer [89].In earlier reports, the compounds that were isolated from A. parviflorum, specifically ursolic acid and demosterol, demonstrated a noteworthy inhibitory action against LU-1 and MKN-7 cell lines with IC 50 values ranging from 30.89 ± 3.60 to 44.37 ± 5.40 μg/mL [28].In our results, the ethanolic extract of A. parviflorum exhibited significant cytotoxicity against A549 cell lines with lower IC 50 values.The presence of phytochemicals that changed the redox balance necessary for the A549 cell's survival may be the basis of the extracts of A. parviflorum's anticancer mechanism.This process may induce or inhibit ROS levels in A549 cells [44].

Conclusion
The endemic plant A. parviflorum was thoroughly studied for the first time to identify its bioactive compounds and different biological activities.LC-MS/ MS-QTOF studies of A. parviflorum ethanolic extract demonstrated potent phytoconstituents.Among all the extracts, the ethanol extract of A. parviflorum showed promising results in exhibiting biological activities.Further investigations are required to isolate bioactive compounds from the plant and exploring their effective therapeutic use.In vivo investigations must be focused on determining these phytocompounds' bioavailability and bio-accessibility for pharmaceutical applications.

Fig. 1
Fig. 1 LC-MS/MS-QTOF base peak chromatogram of ethanol extract of A. parviflorum in a positive ionization mode and b negative ionization mode

Table 3
Compound list of ethanol extract of A. parviflorum in positive ionization mode S.

Table 4
Compound list of ethanol extract of A. parviflorum in negative ionization mode S.

Table 5
IC 50 values of A. parviflorum extracts for various antioxidant activities