Phytochemical, antioxidant, enzyme inhibitory, thrombolytic, antibacterial, antiviral and in silico studies of Acacia jacquemontii leaves

2.2.1 Extraction of antioxidant enzymes from leaves

The freshly collected leaves had been washed using distilled water and air dried to remove surface water. Then sample leaves were packed in airtight plastic bags and stored in a refrigerator for enzymatic antioxidant, studies. The 0.3 g of fresh leaves were ground in a 2 mL volume of 50 mM cold phosphate buffer (pH 7.8) solution to obtain fresh leaves sample extract. After that, the sample was centrifuged at 15,000 × g at 4 °C for 20 min. Results were exhibited as units of enzyme per gram of fresh sample (units/g f. wt.). After the separation of extract from residues by filtration, the supernatant layer of liquid was used to determine enzymatic antioxidant activity (Güneş et al., 2019).

2.2.2 Antioxidant enzyme assays

To determine the superoxide dismutase (SOD) activity was performed by following the method reported in the literature (Li et al., 2015). Enzyme activity (one unit) was defined as the quantity of enzyme which causes 50 % inhibition of photochemical reduction of NBT. The peroxidase activity (POD) was determined by using a procedure described in the literature (Jameel et al., 2021). POD activity was equal to one unit when the change in absorbance occurred at 0.01/minute. Catalase (CAT) activity was measured following Jameel et al., (2021). For the determination of enzyme activity, a change of absorbance at 0.01/minute was one unit of enzyme activity per gram of fresh leaves of the plant. Ascorbate peroxidase (APX) activity was determined through an already-established method (Dixit et al., 2001). The rate of oxidation of ascorbic acid had been measured by noting the decline in absorbance at 290 nm after every 30 s.

2.2.3 Total antioxidant capacity (TAC) of fresh sample

A phosphomolybdenum assay was followed to do this study as reported in the literature with slight modifications (Osman et al., 2022). The molybdate reagent contained 1 mL each of 0.6 M sulfuric acid, 28.0 mM sodium phosphate, and 4.0 mM ammonium molybdate and made up the volume to 50 mL by adding distilled H₂O. After extraction of leaves of A. jacquemontii by homogenization, following, the already reported method with some modifications (Pellegrini et al., 2006), the supernatant layer of the extract was taken (100 µL extract) and poured into a test tube, which was already contained 3 mL of distilled water and 1 mL of molybdate reagent. The test tube was incubated at 95 °C for 90 min. After that, the temperature of the test tube was allowed drop till it reached room temperature, taking a time of 20–30 min, and the absorbance of this reaction mixture was measured at 695 nm. Mean values were noted after three independent samples and results were reported in micromoles equivalents of Trolox (as a standard in this study) per gram of fresh leaves weight of sample along with standard deviation (mmol TE/g f. wt).

2.5.1 Radical scavenging activities

The radical scavenging antioxidant potential of extracts was measured through 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. DPPH radical scavenging assay was performed by the already reported method (Shahzad et al., 2022) with some modifications. The absorbance of the test solution was measured by setting the wavelength of 517 nm with the help of the BioTek Synergy HT microplate reader. The results of the experiment were written as Trolox equivalents per gram of dried extract (mg TE/g dried extract). For the ABTS assay, we adopted a previously reported procedure and results were recorded as mg of Trolox equivalent in one gram of extract (mg TE/g extract).

2.5.2 Reducing power assays

The reducing capacities of extracts were analyzed by utilizing CUPRAC (cupric-reducing antioxidant capacity) and FRAP (ferric-reducing antioxidant power). CUPRAC assay was performed by following the already described procedure. For the FRAP assay, we followed the mentioned protocol in the literature (Ghalloo et al., 2022), and Trolox was used as standard in this assay.

2.5.3 Total antioxidant capacity (TAC)

A volume of 0.30 mL of the sample solution (1 mg/mL in methanol) was combined with 3.0 mL of molybdate reagent solution (4.0 mM ammonium molybdate, 0.60 M sulfuric acid and 28.0 mM sodium phosphate), and incubated for 90 min at 95 °C. Finally, the absorbance was recorded at 765 nm against a blank solution (0.30 mL methanol and 3.0 mL reagent solution). The results were expressed as millimoles of Trolox equivalents per gram of dry extract (mmol TE/g extract) (Ahmed et al., 2022).

2.8.1 Bacterial strains for study

The Bacillus subtilis ATCC1692, Micrococcus luteus ATCC 4925, Staphylococcus epidermidis ATCC 8724, Bacillus pumilus ATCC 13835, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Bordetella bronchiseptica ATCC 7319, and Pseudomonas aeruginosa ATCC 9027 were the Gram-positive and Gram-negative strains of bacteria investigated in this analysis. The strains were obtained from the Bahawalpur Drug Testing Laboratory (DTL). The candidate strains were able to survive in a variety of environments including extreme conditions.

2.8.2 Preparation of inoculums

The bacterial inoculum of each strain was prepared from the previously preserved cultures (24 h old). Afterwards, a few colonies of each strain were taken and added to test tubes with 10 mL of previously sterilized nutrient broth medium. Then test tubes were incubated at 37 °C overnight. The bacterial colonies were diluted to a cell density of 10⁵ CFU/mL (Ahmad et al., 2019).

2.8.3 Micro-dilution method

Minimum inhibitory concentration (MIC) was determined by the microtitre broth dilution method by following the guidelines and procedures reported in the literature after slight modifications (Dahibhate et al., 2022). The MIC assay was performed on methanolic extract (MEAJ) and n-hexane fraction (HEAJ). Serial dilution of both samples was done for the determination of MIC. Stock solutions of both samples were prepared in Eppendorf tubes by dissolving respective samples in dimethylsulphoxide (DMSO) and made a final concentration of 64 mg/mL. Then the serial dilutions from this stock solution were prepared in the range of 32 mg/mL to 0.25 mg/mL using Mueller–Hinton broth in a 96-well microplate. The bacterial inoculum containing a cell density of nearly 10⁵ CFU/mL was prepared from a 24 h freshly cultured plate, and a volume of 100 μL of this bacterial suspension was inoculated into each well. A negative control well (without sample and standard) and a positive control well were also studied for each strain of bacteria. The microtiter plates were incubated for 24 h at 37 °C. Then a volume of 40 μL of a 0.40 mg/mL solution of INT was added to each well (to indicate the bacterial growth). The microtiter plates were incubated again, at 37 °C for 30 min and the MIC values were visually observed and reported. The MIC was the lowest concentration of each sample showing no visible growth (observed first clear well). The experiment was repeated to confirm the activity, and the values were taken as duplicates. Co-amoxiclav was used as a standard antibacterial drug (starting conc. 0.10 mg/mL in DMSO) to observe the sensitivity of the experimental strains and to compare the results of samples, and DMSO (without sample/standard) was used to perform the experiment for negative control. The final concentrations for these experiments ranged from 25.00 μg/mL to 0.19 μg/mL.

2.9.1 Inoculation of viruses in chicken embryonated eggs (cultivation of viral strains)

Chicken embryonated eggs were used as a medium for inoculation. All viral strains were cultured in embryonated eggs of chicken (7–11 days old), obtained from the poultry farm, established by the government of Pakistan in Model Town A, Bahawalpur. A 5 cc syringe was used to inoculate the viral strains through the chorioallantoic route. After incubation at 37 °C for 48–72 h, allantoic fluid was dropped in Eppendorf to preserve at 4 °C and subjected to haemagglutination (HA) and indirect haemagglutination (IHA) to measure the titers of the virus. The highest titer score of viral strains was obtained which was 1024 for AIV (Avian Influenza Virus; H3N2) and NDV (Newcastle Disease Virus). For IBDV (Infectious Bursal Disease Virus) and IBV (Infectious Bronchitis Virus), viruses the highest titer score was 2048. These titers were used as a control in this assay (Andleeb et al., 2020).

2.9.2 Heamagglutination (HA) test procedure

First of all, a 1 % RBCs solution was prepared using chicken blood. After that, we took a 96-well round bottom microtiter plate and added 50 µL of phosphate buffer solution to each well. 50 µL of the viral sample or allantoic fluid was added in the first column of wells and serially diluted to the 11th well. Negative control (phosphate buffer solution only) was left in the 12th well. 50 µL of 1 % RBCs solution was poured into each well. Then, the sample extract (1 mg/mL in DMSO) of 50 µL volume was added to each well except for negative control. Incubated, the plate for 2–3 h at 37 °C. Uniform reddish colors showed positive results, and red dots at the bottoms of the wells showed negative results. The highest dilution number was the HA titer which show a positive result (Harazem et al., 2019). The experiment was used for testing the titer of NDV, IBV, IBDV and H9N2 strains of the virus.

2.10.1 Molecular docking studies for tyrosinase inhibition

Structures of tyrosinase with the highest resolution were obtained from the protein data bank as pdb format. Structures of selected ligands and kojic acid were downloaded from the PubChem database for docking studies. The structures of receptors and ligands were prepared for docking by using AutoDock Tools-1.5.6, Discovery Studio 2021 Client, and open babel GUI. All the water molecules in the structures were removed. Polar hydrogen and Kollman charges were added.

The molecular docking of selected and prepared ligands (phytochemicals and kojic acid), and receptors was performed with AutoDock vina of PyRx software (Abdulfatai et al., 2019). The maximum binding energies and binding interactions between ligands and receptor molecules were determined. The minimum energy position represents the maximum binding affinities. Results of receptor-ligand interactions were made visible with Discovery Studio 2021 Client.

2.10.2 ADMET prediction studies

Measuring the absorption, distribution, metabolism, excretion and toxicity (ADMET) are the essential tools for any compound before its selection as a drug candidate. The online web tool Swiss ADME ( https://www.swissadme.ch/index.php) was used to obtain the characteristics related to absorption and distribution of the major compounds identified from GC–MS and depicting equal to or lower score of binding energies than kojic acid (standard for tyrosinase inhibition). Excretion assessment of these compounds was done by using the online web tool pkCSM ( https://biosig.unimelb.edu.au/pkcsm/prediction), while toxicity was predicted by using Protox-II ( https://tox-new.charite.de/protox_II/).

3.2.1 Antioxidant potential estimation of fresh leaves of A. jacquemontii

The antioxidant potential of fresh leaves of A. jacquemontii was determined, through antioxidant enzymes activity and TAC (Table 3). This activity was done to authenticate our selection of the plant with good antioxidant potential, so that it may be further investigated as a source of functional food components. Plants with good activity in fresh form may also have better activities in dried form (Kozłowska et al., 2021). However, the dried plant extracts were frequently investigated by researchers for bioactive phytochemicals and their biological effects. Moreover, plants having good antioxidant enzyme activities have good health-promoting effects (Khalid Rai et al., 2017). From the finding of good TAC of fresh sample ranks our selection of plant as a food source for good health, and a potential candidate for detailed investigations. So, it was obvious to proceed with the present research by extracting dried plants in bulk, with an organic solvent for more detailed investigations through various phytochemical and biological evaluations. All these experiments were designed in search of a potential source for functional foods and nutraceutical constituents. Organic solvents (especially methanol) have the capacity to extract various phytochemicals of biological importance and also increase the yield of bioactive secondary metabolites (Truong et al., 2019), so, further research for extraction with methanol is designed and also the extraction with a non-polar solvent is considered. Then, the antioxidant activities of dried extract were performed to reveal the antioxidant potential of the plant and its relation with phytochemical constituents.

3.2.2 Antioxidant activities of dried plant extract

Phenolic and flavonoid compounds are ascribed as “high-level natural antioxidants” and are responsible for the antioxidant properties of food plants and also increase their medicinal value by contributing to other biological activities (Kalaivani and Mathew 2010). As we found a high concentration of phenolic and flavonoid contents in A. jacquemontii, the antioxidant potential of MEAJ and HEAJ extracts was evaluated through four diverse assays, which include two radical scavenging (DPPH and ABTS), and two reducing capacity methods (FRAP and CUPRAC). The results were expressed as mg Trolox equivalents per gram of the dried extract along with standard deviation (mg TE/g of dry extract) and listed in Table 4. Radical scavenging activities revealed that MEAJ extract has high antioxidant potential (DPPH, 154.04 ± 2.47 and ABTS, 122.36 ± 0.80 mg Trolox/g of dry extract) as compared to HEAJ extract (DPPH, 97.06 ± 1.61 and ABTS, 74.37 ± 0.49 mg Trolox/g of dry extract). Further, a similar pattern of activity was observed for reducing power as MEAJ extract showed greater antioxidant activity (FRAP, 453.18 ± 5.9 and CUPRAC, 1389.97 ± 5.32 mg Trolox/g of dry extract) when compared with HEAJ (FRAP, 217.8 ± 3.10 and CUPRAC, 610.49 ± 4.91 mg Trolox/g of dry fraction) of A. jacquemontii. Moreover, the total antioxidant capacity (TAC) of MEAJ was also higher (4.36 ± 1.12 mmol TE/g dry extract) as compared to HEAJ (1.10 ± 0.5 mmol TE/g dry fraction) which may due to the presence of higher phenolic and flavonoid contents in MEAJ. These findings also supported the previous study in which strong antioxidant activity was observed from ethyl acetate extract of leaves of A. jacquemontii. They found that the extract showed 69.470 % inhibition of DPPH compared with the standard drug antioxidant (ascorbic acid; 78.90 %) at the same concentration (1 mg/mL) (Awan et al., 2021). Previous research on the leaves extracts of a closely related species A. nilotica also claimed that the high antioxidant potential of plant leaves was due to the presence of a high concentration of phenolic and flavonoid contents in addition to saponins and terpenoids (Saratale et al., 2019). The presence of a high concentration of terpenoids, along with other bioactive phytochemicals in the HEAJ fraction reflected the antioxidant activity of that sample (Mohandas and Kumaraswamy 2018). According to our findings, A. jacquemontii has a lot of potential as an antioxidant agent for the development of new functional foods and nutraceuticals.

3.2.3 Tyrosinase inhibition and thrombolytic activity

Various Acacia species have previously demonstrated effective tyrosinase inhibition (Casas et al., 2020, Zheleva-Dimitrova et al., 2021), thus we investigated A. jacquemontii for tyrosinase inhibition activity. The MEAJ extract showed 71.69 % inhibition against tyrosinase, which was comparable to the standard (kojic acid 83 %), While HEAJ (n-hexane fraction) exhibited a 57.83 % inhibition (Table 5). Tyrosinase inhibition is found to be closely connected to plant flavonoids content and plant materials containing more flavonoids contents show stronger tyrosinase inhibition (Shimizu et al., 2000). So, in the case of A. jacquemontii, the MEAJ extract contained more flavonoid contents, which may be responsible for better tyrosinase inhibition. As the A. jacquemontii extracts demonstrated a very high reducing power so it may also affect the chelating activity of copper at the active site of the enzyme, which prevents the binding of copper ions to oxygen and leads to the irreversible deactivation of the tyrosinase enzyme (Ortonne and Ballotti 2000, Yaar and Gilchrest 2004). Our results were also supported by previous findings from a similar specie A. nilotica that was found to be a potent inhibitor of tyrosinase enzyme (Opperman et al., 2020), and other investigations also show that methanolic extract of A. nilotica was more effective tyrosinase inhibitor compared with other tested species of Acacia (Zheleva-Dimitrova et al., 2021). So, our study may be helpful for designing herbal products and pharmaceuticals, in order to develop skin-lightening agents.

Now a day, another big problem is a failure in hemostasis, which causes thrombus (blood clot) formation which may cause a partial or complete blockage in small vessels of the circulatory system. This vascular blockage may lead to catastrophic thrombotic illnesses such as acute myocardial or cerebral infarction, which can ultimately become the cause of death (Keziah and Devi 2018). To dissolve thrombus, thrombolytic drugs such as streptokinase, alteplase, anistreplase, urokinase and tissue plasminogen activator are routinely used. The majority of them are synthetic and have adverse effects (Li et al., 2020). Therefore, there is a crucial need to look into indigenous sources for new, safer and more effective therapies that lead to good thrombolytic action. In this investigation, we used various subject’s samples A-E for thrombolytic activity (Table 6) and found that MEAJ extract has shown significant activity ranging from 56.22 ± 1.01 to 57.15 ± 1.41 % which is higher than the HEAJ fraction (41.8 ± 1.21 to 43.1 ± 0.69 %), Whereas streptokinase showed the thrombolytic activity varying from 82.5 to 84.1 %. This activity could be attributed to the many phytochemicals present in these extracts. According to the above findings, A. jacquemontii may be used as a source of natural thrombolytic medicines and herbal products can be formulated from it.

3.2.4 Antibacterial activity

The bacterial strains employed in this study are frequently drug-resistant or isolated from clinical specimens, implying that they are responsible for human infections (Mukerji et al., 2017). Both extracts of A. jacquemontii showed antibacterial activity in a concentration-dependent manner and we observed the antibacterial activity of MEAJ (methanolic extract) was higher, as compared to the HEAJ (hexane fraction) as presented in Table 7. Our findings were in line with the previously conducted study on a similar species (Jabaka et al., 2019). Who reported that more polar fractions of A. nilotica had the highest inhibition against Bacillus subtilis, Escherichia coli and Staphylococcus epidermidis). Our analysis also showed that MEAJ had the strongest inhibitory effect on B. subtilis (MIC; 0.5 mg/mL) whereas MIC of the standard drug was 0.5 µg/mL. The results of antibacterial activity were supported by the previous study in which the ethanolic extract of the stem bark of A. jacquemontii was found to be active against tested bacteria. They reported that the extract of this plant was highly active against Bacillus species. The zone of inhibition for Bacillus cereus was 15.4 ± 0.02 mm, and Bacillus pumilus showed 16 ± 0.01 mm (Choudhary et al., 2009). Our results were also consistent with another study conducted on Acacia species that found the highest zone of inhibition against B. Subtilis (Thambiraj and Paulsamy 2015). The antibacterial activity of HEAJ may be due to the presence of terpenoids in high concentrations. Moreover, phytochemicals like terpenoids, unsaturated and saturated fatty acids, and coumarins also contribute to the activity of n-hexane fraction (Adhoni et al., 2016) and similar bioactive metabolites were also identified from HEAJ extract, in our study by GC–MS technique (Table 2). Moreover, the higher antibacterial activity of the methanolic extract was correlated with the possible synergistic effect of phenolic and flavonoid contents and we found a good quantity of them in our experiment as shown in Table 1 (Neto et al., 2017). So, the methanolic extract if further investigated, may provide natural antibacterial ingredients for nutraceutical products and the pharmaceutical industry.

3.2.5 Antiviral activity

The antiviral study of the methanolic and n-hexane extracts of A. jacquemontii was tested against four different viruses and significant results were obtained against all the tested viruses as shown in Table 8, and the titer score is directly proportional to the number of viral particles, so presenting efficacy of extract concerned viral growth (Musaddiq et al., 2020). In the present study, both extracts MEAJ and HEAJ displayed good antiviral activity against all four viruses, including avian influenza virus (AIV) H9N2, infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV), and very less viral titer growth of virus was found as presented in Table 9. The HEAJ extract shows weaker antiviral activity with a viral titer 12 (IBV virus) whereas MEAJ expressed good activity with a viral titer 08 (IBV virus). These findings were also consistent with prior research, which found that the hydroalcoholic extract of Acacia nilotica (Linn) leaves had good antiviral action against Peste des petits ruminant’s virus (PPRV), a single-stranded RNA virus with a negative sense (Ahmad et al., 2014). Another deadly virus is IBDV, due to the non-availability of antiviral brands in the market. However, several researchers have found evidence of the use of medicinal herbs to combat the fatal IBDV virus (Pant et al., 2012). IBDV is also called HIV for poultry (HIV causes AIDS in humans), as it causes immunosuppression (Fauci 2003). The recent global pandemic of COVID-19, which was triggered by SARS-CoV-2, has been devastating to communities all over the world. It is critical to investigate all possibilities to develop a much-needed therapeutic medication for SARS-CoV-2, as there are no specific medications to treat COVID-19 in the current pandemic (Stasi et al., 2020). So the antiviral study against IBV virus, which has similar characteristics to the coronavirus (Wang et al., 2020), may also be helpful to develop antiviral agents. According to our results, A. jacquemontii has a high potential as an antiviral agent or could pave the path for the development of novel antiviral compounds from this plant to combat viral infections.

3.3.1 Molecular docking studies for tyrosinase inhibition

Molecular docking is the computer-aided procedure describing the interaction of ligand and receptor molecules that interact together in 3-dimensional spaces. It is a key technique in computer-aided drug designing and structural biology (Varela et al., 2020). Compounds are chosen as ligands were eight bioactive moieties from the library of GC–MS (Ladokun et al., 2018). The selection criteria were that the compounds should have a maximum peak area and belong to a major class of bioactive phytochemicals, and vitamin E was also selected for its frequent use in cosmetic formulations and skincare products. Kojic acid was selected as a standard ligand to compare the activities of phytochemicals for tyrosinase inhibition. The binding energies (kcal/mol) score of the ligands was obtained and given as, 1-naphthalenecarboxylic acid (-7.8), lanosterol (-7.3), squalene (-6.7), (E,E)-7,11,15-Trimethyl-3-methylene-hexadeca-1,6,10,14-tetraene (-6.5), ar-turmerone (-6.4), 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- (-6.3), geranyllinalool (-6.0), 3-Cyclohexene-1-methanol, alpha,alpha,4-trimethyl-, 1-acetate (-6.0). All these docked compounds had higher binding affinities with tyrosinase active sites as they revealed lower binding energies compared with kojic acid (-5.9 kcal/mol). Herein, the binding energy of 4-(hydroxymethyl) cyclohexane carboxaldehyde (-5.9 kcal/mol) was equal to the binding energy of kojic acid, and the only compound which showed less binding energy than kojic acid was 1,4-Eicosadiene (-4.2 kcal/mol). The higher negative value of binding energy from the docking score (lower binding energy) indicates a higher binding affinity of the ligand with the tyrosinase active site. Further, the binding efficacy of the ligand may be explained by the type of bonding involved in the interaction of ligand molecules with amino acids of the target receptor (Ashraf et al., 2021). Among various types of bonds, van der Waals and alkyl bonding had been shown by all ligands. Conventional hydrogen bonding was present among the amino acids of the tyrosinase active site and two compounds (ar-turmerone, and 4-(hydroxymethyl) cyclohexane carboxaldehyde), and kojic acid, while the carbon-hydrogen bond was formed by 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- with the GLU 232 residue of tyrosinase active site. Unfavorable acceptor–acceptor interaction was shown by ar-tumerone and kojic acid with active site residues. Various π-π interactions were depicted by ar-turmerone, and standard ligand (kojic acid), and turmerone. This data revealed that the tyrosinase inhibitory potential of HEAJ extract of A. jacquemontii may be due to the presence of the docked phytochemical ligands. Results of interactions of ligands from this docking study for tyrosinase inhibition were given below in Table 9, which included binding energies of ligands and details of interacting amino acids of the active site. Diagrammatic presentations, showing the ligand-receptor interaction are given in Fig. 2. So, comparing the significant results of our experiment on HEAJ extract (HEAJ, 57.83 % while kojic acid, 83 % inhibition) with the results of our computational study, it was concluded that docked compounds may be responsible for tyrosinase inhibition because they were contributing the major portion of extract. So HEAJ extract may also be considered for the isolation of compounds with tyrosinase-inhibiting effects in future studies to develop natural products for skin, pharmaceutical and cosmeceutical formulations.

Close

Fig. 2

Diagrammatic presentation (two-dimensional) of interaction between selected ligands and amino acid residues of tyrosinase enzyme with; A = 1-naphthalenecarboxylic acid, B = ar-turmerone, C = 3-Cyclohexene-1-methanol, alpha, alpha, 4-trimethyl-, 1-acetate, D = kojic acid (used as a standard).

Export to PPT

3.3.2 ADME prediction studies

Absorption, distribution, metabolism, and excretion (ADME) were predicted by different parameters obtained by using online predictors and presented in Table 10. The six major compounds were evaluated for their pharmacokinetic parameters and all the compounds showed high absorption through the gastrointestinal tract. The studied compounds also show good permeation parameters of skin because the more negative values of log_p, the less permeable will be the molecule for the skin (Daina et al., 2017). Geranyllinalool showed the properties as a substrate for P‐glycoprotein, which is a mechanism of efflux used by cancer cells leading to drug resistance, while other compounds were not acting as substrates in this prediction study. Lanosterol showed the highest volume of distribution (0.66 log L/kg), and 4-(hydroxymethyl) cyclohexane carboxaldehyde depicted the lowest volume of distribution (-0.016 log L/kg). All the compounds were predicted to cross the blood–brain barrier except geranullinalool, and lanosterol.

Furthermore, the employed tool also predicted the interaction of compounds with five major cytochromes (CYP) isoforms, which play a crucial role in the metabolism and excretion of the majority of drugs available in the market and it was predicted that the compounds do not inhibit the CYP2D6, CYP2C19, and CYP3A4 enzymes. CYP1A2 and CYP2C9 were inhibited by 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- and geranyllinalool. So 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl- and geranyllinalool were the compounds which may observe the first-pass metabolism and can also interact with other drugs (Chen et al., 2020). Total clearance of the compounds was tested including hepatic and renal clearance which predicted that all the tested compounds showed inadequate values of total clearance. According to another parameter of excretion, it was found that the compounds were non-substrate of OCT2 (Organic Cat‐ ion Transporter 2), and the substrates of this transporter may cause adverse interactions with the inhibitors of OCT2 (Hassan et al., 2021).

3.3.3 In silico toxicity prediction study

In silico toxicity was evaluated by using the parameters of organ toxicity and the median lethal dose was also predicted along with the toxicity class of the compounds and it was found that 1-naphthalenecarboxylic acid is hepatotoxic (active toxicity prediction) and belongs to class-4 in toxicity with a median lethal dose of 390 mg/kg, while lanosterol predicted to be immunotoxic. All the other compounds were found to be safe with inactive prediction, however, their probability score varied which suggested their comparative inactivity for organ toxicity. The detail of the prediction scores is given in Table 11.