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Original article
10 2022
:15;
104133
doi:
10.1016/j.arabjc.2022.104133

Phytochemical profiling, in vitro biological activities, and in-silico molecular docking studies of Typha domingensis

, , , , , , , , , ,
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
b Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11495, Saudi Arabia
c Department of Pharmacognosy, College of Pharmacy, Prince Sattam bin Abdul Aziz Unversity, Alkharj 11942, Saudi Arabia
d Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
e Department of Pharmacology, Faculty of Pharmacy, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
f Government College of Technology Bahawalpur, Bahawalpur 63100, Pakistan
g Quaid-e-Azam College of Pharmacy, Quaid-e-Azam Educational Complex, Sahiwal, Punjab, Pakistan

⁎Corresponding authors. kashifur.rahman@iub.edu.pk (Kashif-ur-Rehman Khan), hati@ksu.edu.sa (Hanan Y. Aati),

Received: , Accepted: ,
© The Author(s) 2022
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

A comparative study between methanolic extract and n-hexane fraction of Typha domingensis (Typhaceae) was conducted for the evaluation of phytochemical potential, in vitro biological activities, and in-silico molecular docking studies. The phytochemical composition was estimated by total phenolic and total flavonoid contents, and by GC–MS analysis. Several biological activities were performed such as antioxidant assays (ABTS, FRAP, DPPH, & CUPRAC), enzyme inhibition activity (Tyrosinase, Acetylcholinesterase & Butyrylcholinesterase), thrombolytic activity, and antimicrobial activity (antibacterial & antiviral) to evaluate the medicinal importance of Typha domingensis. The results of the comparative study showed that methanolic extract has more total phenolic and total flavonoid contents (95.72 ± 5.76 mg GAE/g, 131.66 ± 7.92 mg QE/g, respectively) as compared to n-hexane fraction which confirms its maximum antioxidant potential (ABTS 114.31 ± 8.17, FRAP 116.84 ± 3.01, DPPH 283.54 ± 7.3 & CUPRAC 284.16 ± 6.5 mg TE/g). In the case of in vitro enzyme inhibition study and thrombolytic activity, better results were observed for methanolic extract. Almost similar antimicrobial patterns were observed for both methanolic extract and n-hexane fraction of Typha domingensis. The major bioactive phytochemicals identified by GC–MS were further analyzed for in-silico molecular docking studies to determine the binding affinity between ligands and the enzymes. The docking study indicated that most of the bioactive compounds showed a better binding affinity with enzymes as compared to the standard compounds (kojic acid & galantamine). The results of this study recommended that Typha domingensis has promising pharmaceutical importance and it should be further analyzed for the isolation of bioactive phytochemicals which may be useful for the treatment of several diseases.

Keywords

Molecular docking
Phytochemical Screening
Methanolic extract
Typha domingensis
Biological activities
PubMed
1

1 Introduction

Phytochemicals such as phenolic compounds and flavonoids, commonly known as antioxidants are the major source of lead compounds and drugs (Carlson 2015). Many studies have reported that phytochemicals from medicinal plants are used for their potential applications in nutraceutical, cosmetic, pharmaceutical, and health-promoting e.g., antioxidant, antibacterial, antiviral, and anti-cancer effects, and also in neurological disorders (Dahibhate et al., 2019). The pharmaceutical and nutraceutical sector can benefit from understanding the antioxidant properties of plant extract/fractions to find new natural sources of antioxidants for supplements and therapeutic drugs. The literature review of antioxidant activity indicates that wild leaf plants with higher contents of phenolic compounds and flavonoids are an important source of natural antioxidants (Xiang et al., 2019). The pollens of Typha domingensis are a good source of bioactive secondary metabolites such as tannins, glycosides, alkaloids, saponins, polyphenols, and flavonoids (Karbon and Alhammer 2020).

T. domingensis, commonly named southern cattail belongs to the Typhaceae family, having a single genus. The T. domingensis, a familiar plant, is found in a variety of wetland ecosystems, including swamps, marshes, and lakeshores. This is spread in tropical and subtropical areas. The plant has a diverse morphology and is found in various countries throughout the world. In the Turkish traditional medicine system, the flowers of this plant are used for wound healing (Akkol et al., 2011). The leaves have analgesic, astringent, dehydrating, antioxidant, diuretic, and hemostatic effects (Duke and Ayensu 1985, Islam et al., 2015). It is used to treat nosebleeds, hematemesis, hematuria, uterine bleeding, dysmenorrhea, postpartum abdominal pain and gastralgia, scrofula, and abscesses. It is contraindicated for pregnant women (Yeung 1995). The pollens of T. domingensis are eaten in Pakistan owing to properties like antipyretic, increase flow of urine, and the treatment of injuries (Sardar et al., 2014). The people of Al Ahwar, the southern part of Iraq, use the powder of pollen to increase male fertility, named Viagra of Al Ahwar (Al-Kalifawi et al., 2017). It was observed that T. domingensis is efficacious in decreasing bacterial contamination and this plant also reduces enterobacteria, found in the intestine of humans which is responsible for the development of many diseases (He et al., 2020). The leaves of the plant have antimicrobial potential against gram-positive and gram-negative bacteria (Al-Kalifawi et al., 2017). The aqueous ethanol extract of T. domingensis showed bronchodilator, spasmolytic, and vasodilatation effects (Imran 2020) and it has also cytotoxic activity against breast cancer (Karbon and Alhammer 2020). Different solvent extractives of T.angustifolia revealed significant thrombolytic activity as compared to streptokinase as standard (Akkol et al., 2011). The leaves of T.angustifolia have revealed significant nootropic activity and they have also the potential for the treatment of Alzheimer's disease (Kumar et al., 2014).

The current study aimed to evaluate the bioactive phytochemicals, biological activities, and in-silico molecular docking studies of methanolic extract and n-hexane fraction of T. domingensis. The bioactive phytochemical screening was assessed by preliminary phytochemical, total polyphenol contents (total phenolic and total flavonoid contents), and GC–MS analysis. The biological activities were analyzed by antioxidant assays (ABTS, FRAP, DPPH, and CUPRAC), enzyme inhibition (tyrosinase, acetylcholinesterase, and butyrylcholinesterase) thrombolytic and antimicrobial activities (antibacterial and antiviral). The bioactive phytochemicals identified by GC–MS were also analyzed for binding with enzymes by molecular docking technique. To the best of our knowledge, there is no such comparative study between methanolic extract and n-hexane fraction of T. domingensis has ever been conducted.

2

2 Materials and methods

2.1

2.1 Sample collection and plant identification

The collection of mature plants was carried out in March 2019 from Multan, Pakistan, identified by the Herbarium Department of Botany, Life science Faculty, The Islamia University of Bahawalpur, and the botanical sample was submitted in the herbarium with reference number 412.

2.2

2.2 Extract preparation

The air-dried powdered plant material (4 kg) was soaked in 80% aqueous methanol (16 L) for 7 days at room temperature with occasional stirring. An aqueous methanol solvent system was used due to its efficient extraction of phenolics and flavonoids (Justine et al., 2019). It was initially filtered with muslin cloth followed by filtration through Whatman-1 filter paper. The filtrates were subjected to a rotary evaporator (Heidolph, Germany) under reduced pressure at 40 °C for concentrating the extract. This concentrated extract was further air-dried. The dried methanolic extract was dissolved in distilled water to form a uniform solution for liquid–liquid fractionation. This solution was fractionated with the n-hexane solvent by using a separating funnel. The fraction was further concentrated with the rotary evaporator at 40 °C, air-dried, and stored for further analysis (Ghalloo et al., 2022).

2.3

2.3 Phytochemical analyses

2.3.1

2.3.1 Preliminary phytochemical analysis

The sample solutions of T. domingensis were analyzed for their primary and secondary metabolites to confirm the presence of various primary metabolites, such as carbohydrates, amino acids, proteins, and secondary metabolites, such as alkaloids, tannins, phenols, saponins, steroids, glycosides, and resins, according to standard methods described in the literature (Geetha and Geetha 2014).

2.3.2

2.3.2 Estimation of total phenol contents

For total phenolic contents, 25 µL sample solution (1 mg/mL) was mixed with diluted 10 µL Folin-Ciocalteu reagent (1:9, v/v) and shaken vigorously. After 3 min, 75 µL sodium carbonate solution (1%) was added and the absorbance of the sample was measured at 760 nm after a 2 h incubation at 37 °C with a BioTek synergy HT microplate reader. The standard curve of gallic acid was plotted accordingly. The same procedure was repeated for negative control replacing fraction solutions with buffer solutions. Results were exhibited as milligrams of gallic acid equivalents (mg GAE/g DE) (Boeing et al., 2014).

2.3.3

2.3.3 Estimation of total flavonoid contents

The aluminum chloride method was used to measure the total flavonoid content. The solution was made by mixing 0.5 mL of plant extract/fraction solution (1 mg/mL), 2 mL of distilled water, and 0.15 mL of 5% sodium nitrite in a test tube. After 5 min of incubation at room temperature, 0.15 mL of 10% aluminum chloride was added and the solution was allowed to incubate for another 5 min. Then 1 mL of a 4% NaOH solution was added and diluted in 5 mL of pure water. At room temperature, the final solution was vortexed and incubated for 15 min. The standard used for the estimation of flavonoids was quercetin. A blank was made by mixing the sample solution (0.5 mL) with methanol (1 mL) but without AlCl3. The 250 µL volume of the mixture was taken in the 96-well plates and the absorbance was measured at the 420 nm wavelength with a BioTek HT synergy microplate reader (Obeng et al., 2020).

2.3.4

2.3.4 Gas Chromatography-Mass spectrometry analysis (GC–MS)

The methanolic extract and n-hexane fractions were analyzed by GC–MS Agilent (6890 series and Hewlett Packard, 5973 ground sensor). Blown barriers were removed on an HP-5MS column (30 m length × 250 µm diameter × 0.25 µm film thickness). GC–MS spectroscopy involves an electron ionization system that uses energy-intensive electricity (70 eV). The Helium gas was used as a carrier at the flow of 1.0 mL/min. The injector was operated at 250 °C and the oven temperature was set in such a way, 50 °C for 5 min, then gradually increased to 250 °C at 100 °C/min, and lastly to 3000 °C for 10 min at 70 °C/min. The identification was made using a standard scanning method ranging from 35 to 600 m/z, and the bioactive compounds were tentatively identified by the NIST 2011 library (Djouahri et al., 2013).

2.4

2.4 Bioactivity assays

2.4.1

2.4.1 Antioxidant activities

Radical scavenging (DPPH and ABTS) and reducing potency (FRAP and CUPRAC) assays were used to assess antioxidant activity. The sample solutions were made at 1 mg/mL in methanol.

2.4.1.1
2.4.1.1 Free Radical Scavenging (DPPH) Activity

The DPPH solution (0.267 mM 40 µL) was combined with 10 µL of the sample solution and incubated for 30 min before the absorbance was measured at 517 nm using a BioTek synergy HT microplate reader. The results were given in milligrams of Trolox equivalent per gram of dry extract (Marinova and Batchvarov 2011).

2.4.1.2
2.4.1.2 Radical Cation Scavenging (ABTS) Activity

The reaction of a 7 mM ABTS solution with 2.45 mM potassium persulfate resulted in the formation of the ABTS+ radical cation. 1 mL of sample solution was mixed with 2 mL of ABTS+ solution, and after 30 min, the absorbance was measured at 734 nm with a BioTek HT synergy microplate reader. Results are expressed as mg Trolox equivalent per gram of dry extract (He et al., 2012).

2.4.1.3
2.4.1.3 Cupric Ion Reducing (CUPRAC) Method

This technique was carried out by mixing a 0.5 mL sample and mixing it with CuCl2 (1 mL, 10 mM), nectarine (1 mL, 7.5 mM), and NH4Ac buffer (1 mL, 1 M, pH 7.0). The mixture was incubated at room temperature for 30 min. Then, the absorption of the mixture was measured at 450 nm with a BioTek Synergy HT microplate reader. Results were expressed in the mg of Trolox equivalent per gram of the dry extract (Apak et al., 2004).

2.4.1.4
2.4.1.4 Ferric Reducing Antioxidant Power (FRAP) Method

In this process, 0.1 mL of sample solution was added to a 2 mL reagent in acetate buffer (0.3 M, pH 3.6), 2,4,6-tris (2-pyridyl) - s-triazine (TPTZ) (10 mM) in 40 mM HCl, and ferric chloride (20 mM) in a 10: 1: 1 ratio (v /v /v) and the absorbance was measurements at 593 nm after 30 min with BioTek Synergy HT microplate reader. The results were presented in the mg of Trolox equivalent per gram of the dry extract (Benzie and Szeto 1999).

2.4.2

2.4.2 Enzyme inhibition activities

2.4.2.1
2.4.2.1 Tyrosinase Inhibition Activity

The volume of 20 μL of 0.1 M potassium phosphate buffer having pH 6.8 and 40 μL extract/fraction solution (1 mg/mL) was mixed. Tyrosinase enzyme 40 μL (200 units/mL) was added and incubated for 15 min. The substrate l-DOPA 100 μL was added to the incubated mixture. This solution was further incubated for 20 min at room temperature. The absorbance was measured at 450 nm with a BioTek synergy HT microplate reader. The same procedure was adopted for negative control by adding 40 μL of buffer solution instead of extract/fraction. The kojic acid was used as the standard compound in tyrosinase inhibition activity (Grochowski et al., 2019).

2.4.2.2
2.4.2.2 Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) Inhibition Assay

For AChE and BChE inhibition activity analysis, after 15 min of incubation at room temperature of the reaction mixture which is composed of 50 µL solution of the extract/fraction (1 mg/mL), 125 µL DTNB (3 mM), and 25 µL enzymes solution (0.265 U/mL for AChE or 0.026 U/mL for BChE) in Tris-HCl. The buffer having pH 8.0 was incubated for 15 min at ambient temperature. The volume of 5 µL acetylthiocholine iodide or butyrylthiocholine chloride (15 mM) as substrates were mixed into the incubated mixture. Then absorbance of the final solution was measured at 405 nm after 15 min. Also, blank solution (without extract/fraction or standard) was prepared in the same way and analyzed according to this approach. The galantamine was used as a standard agent in this assay (Pohanka et al., 2011).

2.4.3

2.4.3 Thrombolytic activity

The 5 mL blood samples of healthy volunteers (n = 05) with no history of antidepressants were used (according to guidelines approved by the ethical committee). The volume of 5 mL sterile water was added to the streptokinase vial (15,00,000 i.u) and stirred. The standard drug for thrombolytic activity was streptokinase injection.

The volume of 0.7 mL Blood samples was added to the previously weighed eppendorf tubes. The blood was incubated for clotting purposes, and the serum was removed once the clot had formed. These eppendorf tubes were weighed after removing serum. In these eppendorf tubes, 100 mL of sample solution (1 mg/mL) or standard was mixed and stored for 30 min. Finally, the clot's liquid part was removed and weighed. The weight difference was computed, and the weight loss was reported as a percentage of lytic activity of sample solutions or a standard. Instead of fractions solution, water was used as a negative control, and the same method was followed. After deducting the value of negative control activity, the results were calculated (Elnager et al., 2014).

2.4.4

2.4.4 Antimicrobial activity

2.4.4.1
2.4.4.1 Antibacterial Activity

Eight bacterial strains including Bacillus subtilis ATCC 1692, Micrococcus luteus ATCC 4925, Staphylococcus epidermidis ATCC 8724, Bacillus pumilus ATCC 13835, and Staphylococcus aureus ATCC 6538 (Gram-Positive) Escherichia coli ATCC 25922, Bordetella bronchiseptica ATCC 7319, and Pseudomonas aeruginosa ATCC 9027 (Gram-negative) bacteria were obtained by the DTL Bahawalpur (Punjab Pakistan). Inoculums were made by mixing a few colonies of the relevant bacteria from the 24-hour old cultures into a sterile nutrient broth medium of 10 mL. The turbidity was adjusted to 0.5 McFarland, which is equivalent to 108 CFU/mL of cell density (Zaman et al., 2016).

The procedure described in the literature with slight alteration was followed (Manyasree et al., 2018). In an autoclave, Petri dishes and nutrient agar media were sterilized. Agar nutrient was placed into Petri dishes and allowed to solidify in a laminar flow hood. Bacterial cultures were streaked on the agar surface, followed by the development of four 5 mm diameter holes in each petri dish. Using a micropipette, 60 μL of co-amoxiclav (1 mg/mL) and extract/fraction solutions (20, 10, and 5 mg/mL) were added to wells. All of these Petri plates were incubated for 18–24 h at 37 °C in an incubator. The zones of inhibition were evaluated after incubation to determine the antibacterial activity. The results were calculated by averaging three tests.

2.4.4.2
2.4.4.2 Antiviral activity

The four viral strains Avian Influenza virus H9N2 (AIV), New Castle Disease virus Lasoota (NDV), an Infectious bursal disease virus (IBDV), and Infectious bronchitis virus (IBV) were used in this antiviral analysis. All the viral strains were analyzed against methanolic extract and n-hexane fraction through In Ovo antiviral activity. Sterile eggs were acquired from the government-owned poultry farm, Model Town A Bahawalpur, and all viral strains mentioned above were inoculated in the chorioallantoic fluid of 9–11 days chick embryonated (CE) eggs. The eggs were sterilized by candling before and after inoculations. The broader ends of eggs were further swabbed with 70% alcohol for sterilization and transferred into cleaned trays in biosafety cabinet II. The sterile wide broader ends of eggs were pierced with a sterile needle. Then viral inoculum is injected through the sterile disposable 1 cc syringe. After the inoculation, a hole was wrapped with molten wax, and eggs were incubated at 37 °C. The eggs were harvested 72 h post-inoculation and allantoic fluids were collected and subjected to two types of assays. Haemagglutination (HA) or Indirect Hemagglutination (IHA) in case of IBDV test to check the titer of each virus. The whole activity was done in biosafety cabinet type II. Serial passages of viral strains were performed to increase titer before taking the antiviral analysis of the methanolic extract and n-hexane fraction of T. domingensis.

The first step is to perform a hemagglutination (HA) test. 20 mL of Alsever's solution is poured into a test tube and 5 mL is injected into the blood of the chickens. The volume of 10 mL of blood was injected at 4000 rpm for 7 min. This process was repeated three times to optimize and find the best solution. 1% RBC was prepared by adding 10 µL of packaged R.B.C to 1 mL of PBS solution (pH 7.4) in an eppendorf tube. The tube was gradually discarded to prevent rain (Musaddiq et al., 2020).

2.5

2.5 Molecular docking

Molecular docking is a useful tool in the development of molecular biology and computer-aided design. The goal of ligand–protein binding is to predict biological activity. For molecular docking, different tools auto Dock vina software, MGL Tools, Discovery Studio, PyRx, and Babel were used. The receptor molecule which was downloaded from the protein data bank was further prepared with the Discovery Studio. Ligand molecules were prepared with the Babel. These prepared receptors and ligands were uploaded in Vina which was embedded in PyRx. Finally, docking was performed with Vina. The results were visualized with Discovery Studio.

2.6

2.6 Statistical analysis

Each test was performed three times. Results were expressed as mean ± S.D. One-way ANOVA was performed with IBM SPSS Statistics version 20. P-value < 0.05 was considered significant.

3

3 Results and Discussion:

The manufacture of novel medications for the treatment of various ailments relies on a phytochemical analysis of medicinal plants (Viji and Murugesan 2010). Anticancer, antibacterial, antiviral, antidiabetic, antidiuretic, skin disorders, and anti-inflammation properties are found in medicinal plants (Gnanaraja et al., 2014).

T. domingensis is of commercial interest in research institutions and the pharmaceutical sector. We anticipate that the essential phytochemical qualities discovered in the research of the native T. domingensis plant will benefit in the fight against a variety of diseases in this region.

3.1

3.1 Phytochemical analysis of T. domingensis

Secondary metabolites are often species-specific and can be used in a variety of situations. However, most secondary metabolites are derived as by-products or intermediates of primary metabolism. Secondary metabolites are mainly produced under regulated stress for a specific function, such as defense against diseases and herbivores, enhanced tolerance to abiotic stressors, insect and mammal attraction for fertilization and/or seed dissemination, or repellence of undesirable feeders (Böttger et al., 2018). Alkaloids, flavonoids, phenols, saponins, steroids, and tannins are among secondary metabolites (Mungole and Chaturvedi 2011). The qualitative phytochemical screening of methanolic extract and n-hexane fraction was carried out for different constituents. Table.1 revealed the presence of carbohydrates, proteins, glycosides, terpenes, steroids, resins, tannins, and phenols while amino acid, saponin, resins, and alkaloids were absent. In the literature, it was observed that methanol extract showed the presence of alkaloids, phenols, flavonoids, tannins, saponins, and steroids.

Table 1 Preliminary phytochemical screening for methanolic extract and n-hexane fraction of T. domingensis.
Sr.no Constituents Test TDm TDnh
1 Carbohydrates Molish test + +
2 Amino acid Ninhydrin – –
3 Protein Biuret + –
4 Tannins and Phenols (a) FeCl3
(b) Lead Acetate		 +
+		 +
+
5 Saponin Frothing – –
6 Alkaloids (a)Dragendorff
(b)Mayers
(c)Wagner		 +
-
-		 +
-
-
7 Glycosides Borntrager + –
8 Steriods Salkowki + +
9 Resins Acetic Acid + +

“TDm” methanolic extract, “TDnh” n-hexane fraction “-“Absent “+” Present.

3.1.1

3.1.1 Polyphenolic quantification (total phenolic and flavonoid contents)

Due to their antioxidant and other health-promoting characteristics, phenolic compounds are prevalent in most medicinal plants and constitute an important part of the human diet (Pandey and Rizvi 2009). The total phenolic and flavonoid content of methanolic extract and n-hexane fraction were analyzed as shown in Fig. 1 and Table 2.

(A) “TPC” Total phenolic contents (mg GAE/g DE) of T. domingensis, (B) “TFC” Total Flavonoid Contents (mg QE/g DE) of T. domingensis.
Fig. 1
(A) “TPC” Total phenolic contents (mg GAE/g DE) of T. domingensis, (B) “TFC” Total Flavonoid Contents (mg QE/g DE) of T. domingensis.
Table 2 TPC, TFC, DPPH, ABTS, CUPRAC, and FRAP values for extract/fraction of T. domingensis.
Extract/Fraction name TPC (mg GAE/g extract.) TFC (mg QE/g extract.) DPPH (mg TE/g extract.) ABTS (mg TE/g extract.) CUPRAC (mg TE/g extract.) FRAP (mg TE/g extract.)
TDm 95.72 ± 5.76 131.66 ± 7.92 283.54 ± 7.3 114.31 ± 8.17 284.16 ± 6.5 116.84 ± 3.01
TDnh 65.91 ± 3.63 110.25 ± 6.57 140.53 ± 4.67 75.12 ± 5.02 140.14 ± 3.01 84.28 ± 4.01

All the experiments were performed in triplicates (n = 3) “TDm” methanolic extract, “TDnh” n-hexane fraction, “TPC” total phenolic contents, “TFC” total flavonoid contents, “DPPH” 2,2-Diphenyl-1-Picrylhydrazyl, “ABTS” 2, 2'-Azino- Bis (3- Ethylbenzthia zoline-6-Sulfonic acid, “CUPRAC” Cupric Ion Reducing Antioxidant Capacity, and “FRAP” Ferric Reducing Antioxidant Power Assay.

The total phenolic contents (TPC) observed in the methanolic extract of T. domingensis was 95.72 ± 5.76 mg GAE/g DE while in n-hexane extract was 65.91 ± 3.63 mg/g DE. Previously reported a TPC value of 401.46 ± 5.77 mg GAE/g DE in methanolic extract of fruits of T. domingensis (Chai et al., 2015). This very significant difference between the TPC values revealed that there are different phytochemicals in plants or this difference may be due to different habitats of the plant. Because phenolic molecules are extremely reactive to free radicals, measuring them is also a good indicator of antioxidant activity (Ammar et al., 2014).

The total flavonoid contents (TFC) revealed by methanolic extract and n-hexane fraction of T. domingensis were 131.66 ± 7.92 and 110.25 ± 6.57 mg QE/g DE respectively. Chai et al. 2014 reported a TFC value of 103.38 ± 5.29 mg QE/g DE in an aqueous extract of fruits of T. domingensis (Chai et al., 2014). The results of the present study and reported in the literature revealed a significant difference because of different parts of plants and different solvents used for extraction and this difference may be also due to different atmospheric conditions.

3.1.2

3.1.2 Gas Chromatography-Mass Spectrometry (GC–MS)

Similarly, to have detailed knowledge about phytochemical composition, the GC–MS analysis of the methanolic extract and n-hexane fraction was performed and resulted in the tentative qualitative identification of numerous phytochemicals in the methanolic extract and n-hexane fraction, (Figs. 2 and 3, Tables 3 and 4). This tentative identification of the bioactive phytochemicals was performed by using the NIST 11 library database. The detailed list of these tentatively identified phytochemicals (with their tentatively identified names, molecular formula, molecular weight, retention time, % peak area, quality index, chemical class, and reported biological activity) in methanolic extract and n-hexane fraction are listed in Tables 3 and 4 has been taken which have quality index more than 90% respectively and the GC–MS spectra for both extract and a fraction showing different peaks of the tentatively identified compounds are shown in Figs. 2 and 3. The majority of these compound classes include benzene derivatives, fatty acids, alkanes, aldehydes, steroids, and terpenes, among others. The major tentatively identified bioactive phytochemicals were benzene, o-xylene, 8,11-octadecadienoic acid, 9,12,15-octadecatrienoic acid, phytol, N-acetoacetyl-deacetylcolchicine, sitosterol, phenol, 2,5-bis(1,1-dimethylethyl) phenol, tetradecanoic acid, octadecane, eicosane, n-hexadecanoic acid, Octadec-9-enoic acid, 1,2-Benzenedicarboxylic acid, Vitamin E and many other minor compounds.

GC–MS spectra of the methanolic extract.
Fig. 2
GC–MS spectra of the methanolic extract.
GC–MS spectra of n-hexane fraction of.
Fig. 3
GC–MS spectra of n-hexane fraction of.
Table 3 Metabolic profile of methanolic extract by GC–MS.
R.T. Compound Name Molecular formula Molecular weight Relative area (%) Similarity index Chemical class Biological Activity
3.06 Ethylbenzene C8H10 106.16 2.00 91 Benzenoids Antimicrobial (Wang et al., 2013)
3.13 Benzene C6H6 78.11 15.62 97 Aromatic hydrocarbon Anti-inflammatory (Firke and Bari 2015)
3.37 o-Xylene C8H10 106.16 7.59 97 Hydrocarbon Catalytic (Wu et al., 2019)
10.42 2,4-bis(1,1-dimethylethl) phenol C17H30 278.5 0.92 94 Phenol derivative Antibacterial, Antifungal, Anticancer, Antioxidant (Devi et al., 2021)
11.29 1-Heptadecene C17H34 238.45 0.26 94 Alkene Antifungal (Yoon et al., 2011)
12.77 Methyl tetradecanoate C15H30O2 242.4 0.77 97 Fatty Acid Antimicrobial (Kalsoom et al., 2020)
13.55 Tetradecanoic acid C14H28O2 228.37 0.44 94 Fatty Acid Antioxidant (Sokmen et al., 2014)
14.82 9-Hexadecenoic acid C16H30O2 254.41 0.88 99 Fatty Acid Antimicrobial (Ubaid et al., 2016)
15.52 8,11-Octadecadienoic acid C18H32O2 280.4 9.38 99 Fatty Acid Antifungal (Khan and Javaid 2020)
17.35 9,12,15-Octadecatrienoic acid C18H32O2 278.4 13.38 99 Fatty Acid Antimicrobial, Anticancer, Antiinflamatory (Malathi and Ramaiah 2017)
17.35 8-Octadecenoic acid C18H34O2 282.5 1.06 99 Fatty Acid Antioxidant (Farhan et al., 2021)
17.51 Phytol C20H40O 128.17 1.33 91 Acyclic Diterpenoids Antioxidant, Anticancer (Ran and Han 2014)
17.67 Heptadecanoic acid C17H34O2 270.45 0.97 97 Fatty Acid Antioxidants (Ashley et al., 2014)
17.89 Oleic Acid C18H34O2 282.47 1.97 95 Fatty Acid Apoptotic (Van Evera 1998)
21.90 Phenol C6H5OH 94.11 0.67 93 Phenolic compounds Antimicrobial, Antioxidant (Amrani-Allalou et al., 2021, Morais et al., 2022)
23.39 N-Acetoacetyl-deacetylcolchicine C20H23NO5 357.4 1.98 91 Alkaloid derivative Anti-tubulin (Brossi 1991)
35.86 Sitosterol C29H50O 414.7 3.09 90 Lipids Analgesic, anti-inflammatory (Brossi 1991)
Table 4 Metabolic profile of n-hexane fraction by GC–MS.
R.T. Compounds Molecular formula Molecular weight(g/mol) Relative area (%) Similarity index Chemical Class Biological Activity
9.05 Tetradecane C14H30 198.39 0.36 98 Alkane Antimicrobial, Antipyretic, Bronchitis, Tuberculosis (Banakar and Jayaraj 2018)
10.22 Pentadecane C15H32 212.42 0.19 96 Alkane Antibacterial (Uma et al., 2010)
10.44 Phenol, 2,5-bis(1,1-dimethylethyl C14H22O 206.32 0.54 95 Phenol Antimicrobial, Antioxidant (Nyaberi et al., 2017)
11.11 Dodecanoic acid C12H24O2 200.31 0.28 99 Fatty Acid Antimicrobial (Chionis et al., 2016)
12.10 Hexadecane C16H34 226.41 0.36 98 Alkane hydrocarbon Antimicrobial (El-Sheshtawy and Doheim 2014)
12.38 Tridecanoic acid C13H26O2 214.34 0.15 95 Long-chain fatty acid Antimicrobial (Chowdhury et al., 2021)
12.51 Heptadecane C17H36 240.47 0.33 97 Alkane Antibacterial (Uma et al., 2010)
12.79 Tridecanoic acid C13H26O2 241.34 0.38 96 Long-chain fatty acid Antipersister (Jin et al., 2021)
13.36 Tetradecanoic acid C14H28O2 228.37 1.03 95 Long-chain fatty acid Antimicrobial (Abubakar and Majinda 2016)
13.67 Octadecane C18H38 254.49 1.76 97 Alkane Antioxidant, Antimicrobial (Jasim 2015)
13.92 Pentadecanoic acid C15H30O2 242.39 0.35 98 Palmitic acid Antioxidant (Adeyemi et al., 2017)
14.35 Octadecane, 2-methyl- C19H40 268.5 0.45 93 Alkane Antimutagenic (Lawal et al., 2015)
14.77 Nonadecane C19H40 268.51 0.30 96 Alkane Antimutagenic (Lawal et al., 2015)
14.87 9-Hexadecenoic acid C16H30O2 254.40 0.51 99 Omega-7 fatty acid Antifungal (Hameed et al., 2018)
15.71 Dibutyl phthalate C16H24O4 278.34 1.41 91 Phthalates Antifungal (Ahsan et al., 2017)
16.14 Eicosane C20H42 282.54 4.83 99 Alkane Antioxidant, Antimicrobial (El-Shahaby et al., 2019)
16.31 n-Hexadecanoic acid C16H32O2 256.4 1.78 99 Fatty Acid Antioxidant, Lubricant, Antiandrogenic, 5- alpha-reductase inhibitor (Lawal et al., 2015)
16.60 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propionic Acid C17H26O3 278.39 0.70 96 Phenyl propanoid Enzyme catalyst (Milaeva et al., 2006)
17.12 Cyclotetradecane C14H28 196.37 0.33 91 Cyclic Alkane Antimicrobial (Chuah et al., 2018)
17.66 9,12,15-Octadecatrienoic acid C18H30O2 278.43 7.71 99 Fatty Acid Antimicrobial (Abker 2021)
18.81 Octadec-9-enoic acid C18H34O2 282.46 12.07 95 Fatty Acid Anti-hyperglycaemic (Kapoor et al., 2019)
19.20 14-Methyl-8-hexadecyn-1-ol C17H32O 252.4 1.20 90 Aliphatic hydrocarbon Antimicrobial, Anticancer (Ahuchaogu et al., 2018)
19.43 Pentadec-7-ene C15H30 210.4 0.55 90 Alkene Antioxidant, Antimicrobial
19.60 E,E,Z-1,3,12-Nonadecatriene-5,14-diol C19H34O2 294.5 0.59 95 Alcohol Antiviral (Tassakka et al., 2021)
19.76 Docosane C22H46 310.60 0.98 98 Alkane Antifungal (Bierer et al., 1995)
19.90 Decanoic acid C10H20O2 172.26 0.46 92 Fatty Acid Antiseizure, Antim (Chang et al., 2016, Shen et al., 2021)
20.25 Octadecane C18H38 254.49 2.17 96 Alkane Antineoplastic Anticancer (Talbaoui et al., 2020)
20.81 Pyridine-3-carboxamide C6H8N2O 140.14 1.09 91 Organoheterocyclic compound Antibacterial (Narramore et al., 2019)
21.79 Tetracosane C24H50 338.65 4.72 97 Alkane Antimicrobial, Antioxidant (Asha et al., 2017)
22.23 2-Dodecen-1-yl(-)succinic anhydride C16H26O3 266.38 0.54 93 Carboxylic anhydride Antioxidant (Marzocchi et al., 2018)
22.62 Octacosane C28H58 394.77 2.69 98 Alkane Antimicrobial (Chelliah et al., 2017)
22.97 22-Tricosenoic acid C23H44O2 352.6 0.50 90 Fatty Acid Antimicrobial (Selvan and Velavan 2015)
24.06 1,2-Benzenedicarboxylic acid C8H6O4 166.14 5.69 98 Benzenoid Antimicrobial, Anticancer, antitumor, Antioxidant, chemopreventive (Ezhilan and Neelamegam 2012)
25.05 1-Eicosene C20H40 280.53 0.92 95 Hydrocarbon Antimicrobial (Naser et al., 2019)
25.90 Triacontane C30H62 422.82 0.68 96 Alkane Antibacterial, Antidiabetic, Antitumor (Mallick and Dighe, 2014a, 2014b)
26.89 9-Hexacosene C26H52 364.7 0.68 96 Alkene Antinociceptive, Analgesic, Antiinflammatory (Naser et al., 2019)
27.38 Hentriacontane C31H64 436.85 2.03 99 Alkane Antiinflammatory, Antitumor, Antimicrobial (Khajuria et al., 2017)
28.58 Nonacosane C29H60 408.6 0.46 99 Alkane Antimicrobial (Palic et al., 2002)
28.78 Piperine C17H19NO3 285.34 0.40 97 Piperine derivatives Depression, Analgesic, Antipyretic, anti-inflammatory, Antioxidant (Okwute and Egharevba 2013)
30.06 Triacontane C30H62 422.82 1.47 99 Alkane Antitumor, Antidiabetic, Antibacteria (Mallick and Dighe, 2014a, 2014b)
30.63 Stigmasta-5 C29H48O 412.7 0.74 95 Steroids derivative Antibacterial (Joycharat et al., 2013)
31.62 Stigmastan-3,5-diene C29H48 396.7 1.00 99 Phenanthrene Antimicrobial (Sánchez-Pérez et al., 2009)
32.22 Vitamin E C29H50O2 430.71 0.41 98 Methylated phenol Antioxidant (Vrolijk et al., 2015)
33.49 Triacontane C30H62 422.82 0.84 99 Alkane Antitumor, Antibacterial, Antidiabetic (Amudha et al., 2018)

3.2

3.2 In vitro biological activities

3.2.1

3.2.1 Antioxidants activity (mg Trolox Eq. Per gram of dry extract) of T. domingensis

Natural antioxidants have brought a lot of interest in recent years due to their beneficial role in many ailments associated with free radicals (Sen and Chakraborty 2011). Antioxidants of natural origin are gaining popularity because they can protect the human body from free radicals without causing toxicity (Altemimi et al., 2017). Consumption of antioxidants has long been linked to a lower risk of a variety of diseases in which oxidative stress may play a role, particularly chronic diseases like cardiovascular diseases, cancer, neurological disorders, and skin diseases. Natural antioxidants derived from plant components are currently gaining popularity as a replacement for synthetic antioxidants. This is because natural antioxidants are safer than synthetic antioxidants because they occur naturally in plant foods, and they are more desirable than their synthetic derivatives, thus there is a lot of interest in identifying natural antioxidants from plants (Gulcin 2020). The literature review of antioxidant activity indicates that wild leaf plants with higher contents of phenolic compounds and flavonoids are an important source of natural antioxidants (Xiang et al., 2019). Plants that have been shown to have high antioxidant activity can be considered to prevent toxic oxidation in food chemicals or drugs for treating cancers (Amadi et al., 2019). According to the literature, two-thirds of all plant species have therapeutic and antioxidant potential (Al Rashdi et al., 2021) (Fig. 4).

Antioxidant potential for methanolic extract “TDm” and n-hexane fraction “TDnh” of T. domingensis.
Fig. 4
Antioxidant potential for methanolic extract “TDm” and n-hexane fraction “TDnh” of T. domingensis.

Antioxidant activity for the methanolic extract and n-hexane fraction of T. domingensis (Table 2 and Fig. 5) was determined by 2,2′-Azino-Bis(3-Ethylbenzthia zoline-6-Sulfonic acid (ABTS), Ferric Reducing Antioxidant Power Assay (FRAP), 2,2-Diphenyl-1-Picrylhydrazyl (DPPH), and Cupric Ion Reducing Antioxidant Capacity (CUPRAC). The antioxidant potential determined by ABTS, FRAP, DPPH, and CUPRAC methods revealed that methanolic extract exhibited maximum activity (114.31 ± 8.17, 116.84 ± 3.01, 283.54 ± 7.3, 284.16 ± 6.5 respectively) While n-hexane extract has ABTS, FRAP, DPPH, and CUPRAC (75.12 ± 5.02, 84.28 ± 4.01, 140.53 ± 4.67, 140.14 ± 3.01 respectively) antioxidant activity. The antioxidant potential in different extracts of fruit of T. domingensis by the DPPH method was previously reported (Chai et al., 2015). The antioxidant activity of this plant extract and fraction is attributed due to phenolic and flavonoid contents because of their ability to donate hydrogen atoms to free radicals for denaturation of these free radicals (Aryal et al., 2019). These results suggest that both methanolic extract and n-hexane of T. domingensis are excellent sources of antioxidants.

“A” Tyrosinase inhibition activity “KA” kojic acid (standard), “B” Acetylcholinesterase inhibition activity “GA” galantamine (standard) “C” Butyrylcholinesterase inhibition, galantamine (standard).
Fig. 5
“A” Tyrosinase inhibition activity “KA” kojic acid (standard), “B” Acetylcholinesterase inhibition activity “GA” galantamine (standard) “C” Butyrylcholinesterase inhibition, galantamine (standard).

3.2.2

3.2.2 Enzyme inhibition activity of T. domingensis

Enzyme inhibitors are of clinically prodigious value in various areas of disease management. The tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibition activity of methanolic extract and n-hexane fraction of T. domingensis were analyzed.

3.2.2.1
3.2.2.1 Tyrosinase Inhibition Activity

Hyperpigmentation and browning of the skin are two common unwanted effects in humans, and tyrosinase is the enzyme involved in these effects humans (Roselan et al., 2021). Tyrosinase is known as polyphenol oxidase, and it is a copper-containing multifunctional enzyme, found in both plants and animals (Seo et al., 2003). Due to the high demand for tyrosinase inhibitors, researchers are working hard to identify, isolate, and synthesize novel moieties for use in the food, cosmetics, and pharmaceutical industries (Masum et al., 2019).

The results revealed that methanolic extract showed maximum tyrosinase inhibition activity (74.51 ± 3.49%) as compared to the n-hexane fraction (65.32 ± 2.64%) of T. domingensis (Table 5 and Fig. 5). The %age inhibition of tyrosinase expressed by kojic acid was (85.58 ± 0.85%). The tyrosinase inhibition and antioxidant potential of plant extracts are dependent upon the presence of polyphenolic compounds (Nerya et al., 2004). This activity was the first time performed on any part of T. domingensis.

Table 5 Enzyme % Inhibition of methanolic extract and n-hexane fraction of T.domingensis.
SAMPLES Tyrosinase Inhibition Acetylcholinesterase Inhibition Butyrylcholinesterase Inhibition
TDm 74.51 ± 3.49 68.25 ± 0.13 62.62 ± 1.43
TDnh 65.32 ± 2.64 43.72 ± 1.23 29.53 ± 1.17
Reference 85.58 ± 0.85a 97.11 ± 1.26b 72.88 ± 2.61b

Each experiment was performed in triplicates (n = 3), “TDm” methanolic extract and “TDnh” n-hexane fraction.

a Kojic acid.
b Galantamine.

3.2.2.2
3.2.2.2 Acetylcholinesterase and Butyrylcholinesterase Inhibition Activity

Cholinesterase inhibitors are agents that enhance cholinergic function by increasing the amount of acetylcholine in cholinergic synapses in the brain (Khan et al., 2018). Acetylcholine is the most common neurotransmitter in the human body, with roles in both the peripheral and central nervous systems. Cholinesterase inhibitors are most commonly used to treat dementia in Alzheimer's disease patients. Cholinesterase inhibitors have been demonstrated to have a minor impact on cognitive symptoms associated with dementia (Anand and Singh 2013). Numerous plant extracts have been analyzed for their potential role to counteract cognitive disorders and other neurological diseases (Chonpathompikunlert et al., 2010).

The methanolic extract revealed maximum acetylcholinesterase and butyrylcholinesterase inhibition activity (68.25 ± 0.13 and 62.62 ± 1.43% respectively) as compared to inhibition results of n-hexane extract (43.72 ± 1.23 and 29.53 ± 1.17%). The acetylcholinesterase and butyrylcholinesterase inhibition activities shown by galantamine were 97.11 ± 1.26 and 72.88 ± 2.61% respectively (Table 5 and Fig. 5). The cholinesterase inhibition activity is directly related to flavonoid contents in the plant extracts (Khan et al., 2018). The potential of T. domingensis for the neurological disorder was first time evaluated.

3.2.3

3.2.3 Thrombolytic activity

Thrombolysis, also known as thrombolytic therapy, is a procedure that dissolves blood clots, improves blood flow, and prevents tissue and organ damage (Wardlaw et al., 2014). Thrombolytic therapy is used in patients with acute pulmonary embolism (PE) to eliminate the embolic burden efficiently and improve cardiorespiratory hemodynamics. Thrombolytic agents function by activating plasminogen, and it is subsequently transformed to plasmin, which is an enzyme that breaks down fibrin strands (Voros 2017). Five samples of blood were taken and were observed through thrombolytic activity by the use of Streptokinase as a standard. The current study revealed that the n-hexane fraction showed higher thrombolytic activity than methanolic extract (Table 6). Previously the aqueous, methanol, and chloroform extracts of leaves of T. angustifolia revealed 51.76 ± 2.5, 58 ± 2.32, and 18 ± 1.84% clot lysis activity respectively (Umesh et al., 2014). Methanolic extract of the entire plant of T. elephantina was analyzed for thrombolytic activity and serial concentrations of plant extracts were used (2, 4, 6, 8, 7, and 10 mg/mL) among these concentrations 4 mg/mL exhibited maximum thrombolytic activity (Singh et al., 2020).

Table 6 Thrombolytic Activity (%age lysis) of methanolic extract and n-hexane fraction.
Plant Extract and Fraction Blood Sample 1 Blood Sample 2 Blood Sample 3 Blood Sample 4 Blood Sample 5
TDm 60.06 60.82 60.78 60.07 60.89
TDnh 66.62 66.96 67.27 67.58 67.41
SK (standard) 79.07 79.15 79.33 78.52 78.65

“TDm” methanolic extract, “TDnh” n-hexane fraction, and “SK” streptokinase.

3.2.4

3.2.4 Antimicrobial activity

3.2.4.1
3.2.4.1 Antibacterial activity

Eight bacterial strains (Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Bacillus pumilus, Staphylococcus aureus, Escherichia coli, Bordetella bronchi septica, and Pseudomonas aeruginosa) were used to check the antibacterial potential of methanolic extract and n-hexane fraction of T. domingensis and co-amoxiclav was used as a standard antibacterial agent for this assay. Both methanolic extract and n-hexane fraction revealed good antibacterial results (Table 7). Three different Conc. (5, 10, and 20 mg/mL) were used and observed zone of inhibition. This antibacterial potential was concentration-dependent. At higher concentrations (20 mg/mL), the zone of inhibition was very significant except for Pseudomonas aeruginosa (gram-negative). The findings of this assay suggest that these test samples are efficacious against most gram-positive bacteria and some gram-negative bacteria. Al-Kalifawi et al., 2017 determined the antimicrobial potential of some aqueous, methanol, chloroform, and petroleum ether extracts of T. domingensis (Al-Kalifawi et al., 2017). Various compounds in the GC–MS profile of methanolic extract and n-hexane fraction have reported antimicrobial and antibacterial potential. These compounds include 2,4-bis(1,1-dimethylethl) phenol (Yoon et al., 2011), 9,12,15-Octadecatrienoic acid (Malathi and Ramaiah 2017), Heptadecane (Uma et al., 2010), Tetradecanoic acid (Abubakar and Majinda 2016), and Triacontane (Mallick and Dighe, 2014a, 2014b).

Table 7 Antibacterial activity of methanolic extract and n-hexane fraction of the T.domingensis.
Strain Zone of inhibition (mm) of Standard (Co-Amoxiclav) (Conc. 1 mg/mL) Conc. of sample (mg/mL) Zone of Inhibition of TDm (mm) Zone of Inhibition of TDnh (mm)
Bacillus subtilis 23 5 NA NA
10 8 14
20 13 18
Micrococcus luteus 20 5 5 NA
10 11 13
20 18 18
Staphylococcus epidermidis 18 5 6 NA
10 11 14
20 20 17
Bacillus pumilus 21 5 7 NA
10 12 8
20 18 13
Staphylococcus aureus 21 5 NA NA
10 10 13
20 17 18
Escherichia coli 24 5 6 NA
10 9 15
20 15 19
Bordetella bronchiseptica 25 5 NA NA
10 8 11
20 13 16
Pseudomonas aeruginosa 6 5 NA NA
10 NA 8
20 8 13

3.2.4.2
3.2.4.2 Antiviral Activity of T. domingensis

Due to the lack of antiviral agents and the emergence of new viral diseases, there is a demand for the screening of newer antiviral therapies. Four viral strains were used namely Influenza A viruses (IAVs), Infectious bronchitis virus (IBV), Newcastle disease virus (NDV), and Infectious bursal disease virus (IBDV). The results were expressed as HA titre count. Both methanolic and n-hexane extracts showed highly strong results against these tested viral strains (Table 8). Many GC–MS profiled compounds of both methanolic extract and n-hexane fraction have reported antimicrobial and antiviral activities such as E, E, Z-1,3,12-Nonadecatriene-5,14-diol (Tassakka et al., 2021), Dodecanoic acid (Chionis et al., 2016), Octadecane (Jasim 2015), Eicosane (El-Shahaby et al., 2019), and Hentriacontane (Khajuria et al., 2017).

Table 8 Antiviral activity of T.domingensis.
Strain Name Control HA titre count treated with TDm HA titre count treated with TDnh
IAVs 2048 04 04
IBV 1024 00 08
NDV 2048 00 08
IBDV 1024 08 02

HA titre 0–8: highly strong, 16–32: strong, 64–128: moderate, 256–2048: not active (Musaddiq et al., 2020).

“TDm” methanolic extract, “TDnh” n-hexane fraction, “IAVs” Avian Influenza A viruses, “IBV” Infectious bronchitis virus, “NDV” New Castle Disease virus, and “IBDV” Infectious Bursal Disease Virus.

3.3

3.3 Molecular docking studies

In addition, further improvements can be made to in vitro research methods for the rapid screening of enzyme inhibitors using molecular modeling. Therefore, a combination of bioinformatics simulation and in vitro analysis will be useful for the evaluation of extract and fraction for their biological activities (Sabe et al., 2021). Molecular docking is the study of how two or more molecular structures (such as drugs and enzymes or proteins) fit together. In a simple definition, docking is a molecular modeling technique used to predict how proteins (enzymes) interact with small molecules (binders or ligands) (Rudnitskaya et al., 2010). A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein–ligand interactions and binding will also facilitate the discovery, design, and development of drugs. In the binding of enzymes and ligands, binding affinity has prime importance. The least the binding affinity, the more will be better binding between the ligands and enzyme. The binding affinity in the positive (+) symbol represents that the ligand and the enzyme have no interaction. To estimate the binding affinities and binding interactions at the active sites, the molecular docking of major GC–MS-identified compounds was performed. The standard drugs utilized in these assays were also docked with respective enzymes. Piperine resulted in the best binding affinity (-10.2 kj/mol) for tyrosinase and the binding affinity for kojic acid (standard) was −5.4 kj/mol. The 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propionic acid showed the least binding affinity against both acetylcholinesterase and butyrylcholinesterase (-9.9 and −10.8 kj/mol respectively) while the binding affinity of galantamine (standard) for these both enzymes were −7.6 and −8.4 kj/mol respectively. The ligands interacting with weak intermolecular forces, like van der waals forces revealed better binding affinity than the ligands binding with conventional hydrogen bonding. In the docking results represented in the 2D figures (Figs. 6–8), it is clear that there is the least number of amino acids which are interacting with conventional hydrogen bonding than van der waals forces. This indicated that our ligands have better binding affinities with the enzyme (Table 9).

3D and 2D Interaction of tyrosinase with ligands. “A” Vitamin E, “B” Piperine, and “C” Kojic acid.
Fig. 6
3D and 2D Interaction of tyrosinase with ligands. “A” Vitamin E, “B” Piperine, and “C” Kojic acid.
3D and 2D Interaction of acetylcholinesterase with ligands “A” 3-(3,5-Di-tert-butyl-4-hydroxyphenyl) propionic Acid, “B” Vitamin E, and “C” Galantamine.
Fig. 7
3D and 2D Interaction of acetylcholinesterase with ligands “A” 3-(3,5-Di-tert-butyl-4-hydroxyphenyl) propionic Acid, “B” Vitamin E, and “C” Galantamine.
3D and 2D Interaction of butyrylcholinesterase with ligands “A” Sitosterol, “B” 3-(3,5-Di-tert-butyl-4-hydroxyphenyl) propionic Acid, and “C” Galantamine.
Fig. 8
3D and 2D Interaction of butyrylcholinesterase with ligands “A” Sitosterol, “B” 3-(3,5-Di-tert-butyl-4-hydroxyphenyl) propionic Acid, and “C” Galantamine.
Table 9 Molecular docking of tyrosinase (TYR), acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) with different ligands representing binding affinity and interacting amino acids.
Sr.No Compounds Name TYR (Binding affinity) Interacting Amino Acid Residues AChE (Binding affinity) Interacting Amino Acid Residues BChE (Binding affinity) Interacting Amino Acid Residues
1. Vitamin E −9.1 Gly46,Asn57,Ala59,Met61,Phe65,His69,Thr156, Glu158,Ala159,Glu195,Phe197,Gly200,Pro201,Asn205Met215,Val218,Ala221,Phe227, −8.5 Tyr72,Asp74,Tyr124,Trp286,His287,Leu289,Gln291,Glu292,Ser293,Val294,Phe295,Phe297,Tyr337,Phe338,Tyr341 −8.4 Asp70,Trp82, Gly115,Gly116, Gly117,Thr120, Gly121,Leu125, Tyr128,Glu197, Pro285,Ala328, Phf329,Tyr332, Trp430,Met437, His438,Tyr440
2. 3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propionic Acid −5.6 Trp41,Arg75, Arg78,Trp269, Asn270,Thr271, Asp275,Asp275 −9.9 Tyr72,Asp74,Thr75,Leu76,Tyr124,Trp286,Leu289,Ser293,Val294,Phe295,Arg296Phe297,Phe338,Typ341 −10.8 Asp70,Gly78,Ser79,Asn83,Trp112,Tyr114,Gly115,Gly116,Thr120,Tyr128,Glu197,Ala328,Tyr332,Trp430,Met437,His438,Gly439,Tyr440,Ile442
3. Sitosterol −8.3 Asp36,Ile39, Ala40,Phe48, His49,Ile139, Gly143,Pro219,Tyr267, --9.7 Ala377,Leu380, Tyr382,Asp404,Gln527,Ala528, Phe531, −9.3 Asn68,Asp70,Trp82Thr120,Gln119,Pro285,Ser287,Asn289,Ala328,Phe329,Try332,Phe329,His438,Tyr440,
4. Pyrrolidine 2,5-Cyclohexadiene-1,4-dione −8.5 Asp36,Ile39, Lys47,Phe48, His49,Pro52, Ile139,Asp140 Glu141,Gly143, Tyr267 −7.4 Ala412,Gln413, Gly416,Arg417,Tyr503,Ala505,Gln508,Ala526,Gly529,Ala530,Asn533, −6.9 Trp82,Gly115, Gly116,Glu197, Ala328,Phe329, Tyr332,His438, Gly439,Tyr440
5. Piperine −10.2 Asn57,Ala59,Met61,Phe65,His69,Glu195,Phe197,Leu73,Ans205,Val218,Phe227His230,,Ala232,Val234,Asp235, −7.9 Ala377,Leu380, Tyr382,Thr383, Asp384,Ala397Asp404,Gln527Ala528,Phe531, Phe535 −6.8 Gly78,Trp82, Gly116,Gly117, Gln119,Thr120, Pro285,Leu286, Ser287,Val288, Ala328,Phe329, His428,Trp430, Met437,Gly439,Tyr440
6. N-Acetoacetyl-deacetylcolchicine −6.2 Trp41,Arg75, Glu71,Agr78, Trp269,Pro268, Asn270, −8.4 Ala377,Val378, Leu380,Tyr382,Thr383,Gln527, Ala528,Phe531 −9.2 Asp70,Gly78, Ser79,Asn83, Gly115,Gly116, Gly117,Thr120, Gly121,Tyr128, Glu197,Ala199, Trp231,Tyr322, Phe329,Phe398, His438
7. Phytol −7.9 Asp36,Ile39, Ala40,Ala44, Lys47,Phe48, Pro52,Ile139, Glu141,Gly143,Tyr267 −6.9 Ala377,Val378, Leu380,Tyr382, Gln527,Ala528, Phe531,Phe535 −6.5 Trp82,Tyr114, Gly115,Gly116, Gly117,Thr120, Glu197,Ala199, Trp231,Leu286, Ala328,Phe329, Tyr332,Phe398, His438
8. 9,12,15-Octadecatrienoic acid −7.7 Asp36,Ile39, Ala40,Lys47, Phe48,His49, Pro52,Ile139, Gln142,Gly143, Tyr267 −6.8 Ala377,Val378, Leu380,Tyr382, Gln527,Ala528, Phe531,Phe535 −6.9 Trp82,Gly115, Gly116,Gly117, Ala199,Trp231, Pro285,Leu286, Val288,Phe329, Phe398,His438, Gly439
9. 2-Dodecen-1-yl(-)succinic anhydride −7.4 Ilu39,Ala40, Gly43,Lys47, Phe48,Ile139, Tyr267, −6.9 Ala377,Val378, Leu380,Tyr382,Thr383,Gln527, Ala528,Phe531, Phe535 −7.0 Gly78,Trp82, Gly116,Gly117, Ala199,Trp231, Leu286,Ser287, Val288,Ala328, Phe329,Phe398,Trp430, His438
10. Standard −5.4a Tyr38,Ile39,Trp41,Gly43,Ala44,Ala45,Gly46,Asn57,Ala59,Trp68,His69,Tyr72,Val218,Pro222,Phe227 −7.6b Tyr72,Asp74,Trp296,Leu289,Glu292,Ser293,Val294,Phe295,Arg296,Phe297,Phe338,Tyr341 −8.4b Asp70,Gly78, Ser79,Trp82, Gly116,Thr120, Glu197,Ala328, Tyr332,Trp430, Met437,His438,Gly439,Tyr440, Ile442
a Kojic Acid.
b Galantamine.

4

4 Conclusion

The current comparative study was focused on phytochemical profiling and in vitro biological activities for methanolic extract and n-hexane fraction of T. domingensis. The methanolic extract exhibited the maximum polyphenolic contents (TPC and TFC), antioxidant potential and tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibition activity. The GC–MS analysis of the methanolic extract and n-hexane fraction resulted in the tentative identification of several secondary metabolites. The enzyme inhibition activity for methanolic extract and n-hexane fraction of T. domingensis was further confirmed by in-silico molecular docking studies. n-Hexane fraction resulted in better thrombolytic activity than methanolic extract. Methanolic extract and n-hexane fraction of T. domingensis exhibited significant and almost similar antibacterial and antiviral activity against the tested strains. The phytochemical and biological potential of this plant highlighted its value for further isolation of bioactive compounds which is currently in progress.

Author contributions

Conceptualization, R.D. and B.A.G.; methodology, K.U.R.; software, H.T., and A.E.S.; validation, H.Y.A., and J.H.A.; formal analysis, A.B.; investigation, M.H.; resources, H.Y.A; data curation, M.A.; writing—original draft preparation, R.D. and B.A.G.; writing—review and editing, B.A.G And R.D.; visualization, S.A.; supervision, K.U.R, And S.A.; project administration, J.H.A.; funding acquisition, H.Y.A. And J.H.A. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are grateful to the King Saud University, Riyadh, Saudi Arabia for funding this study through Project number RSP2022R504.

Institutional review board statement

All the trials were carried out following the NIH guidelines and were approved by the Department of Pharmaceutical chemistry’s concerned committee (1009/AS & RB/12/07/2021).

Informed consent statement

Not applicable.

Acknowledgments

The authors are thankful to Researchers Supporting Project number (RSP2022R504), King Saud University, Riyadh, Saudi Arabia. As well, the authors are grateful to Dr. Saeed Ahmed, Chairperson Department of Pharmaceutical Chemistry for his technical support, suggestions, and laboratory facilities.

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