Chemical Characterisation, Antidiabetic, Antibacterial, and In Silico Studies for Different Extracts of Haloxylon stocksii (Boiss.) Benth: A Promising Halophyte

The objective of the study is to evaluate the chemical characterisation, and biological and in silico potential of Haloxylon stocksii (Boiss.) Benth, an important halophyte commonly used in traditional medicine. The research focuses on the roots and aerial parts of the plant and extracts them using two solvents: methanol and dichloromethane. Chemical characterisation of the extracts was carried out using total phenolic contents quantification, GC-MS analysis, and LC-MS screening. The results exhibited that the aerial parts of the plant have significantly higher total phenolic content than the roots. The GC-MS and LC-MS analysis of the plant extracts revealed the identification of 18 bioactive compounds in each. The biological evaluation was performed using antioxidant, antibacterial, and in vitro antidiabetic assays. The results exhibited that the aerial parts of the plant have higher antioxidant and in vitro antidiabetic activity than the roots. Additionally, the aerial parts of the plant were most effective against Gram-positive bacteria. Molecular docking was done to evaluate the binding affinity (BA) of the bioactive compounds characterised by GC-MS with diabetic enzymes used in the in vitro assay. The results showed that the BA of γ-sitosterol was better than that of acarbose, which is used as a standard in the in vitro assay. Overall, this study suggests that the extract from aerial parts of H. stocksii using methanol as a solvent have better potential as a new medicinal plant and can provide a new aspect to develop more potent medications. The research findings contribute to the scientific data of the medicinal properties of Haloxylon stocksii and provide a basis for further evaluation of its potential as a natural remedy.


Introduction
Herbs have been the foundation of nearly all medicinal therapy since the prehistoric era until the era of synthetic drugs in the 19th century [1]. Traditional medicines are used as an alternative therapy to treat human and animal diseases in developing countries. studies for both aerial parts and root extracts of H. stocksii. The study will provide meaningful insights into the screening of the Haloxylon stocksii plant as a new medicinal plant.

Phytochemical Composition
The phytochemical characterisation of different solvent extracts of H. stocksii was determined by their total phenolic contents (TPC), total flavonoid contents (TFC), gas chromatography-mass spectrometry (GC-MS) analysis, and liquid chromatography-electron spray ionization mass spectrometry (LC-ESI-MS 2 ) screening.

Total Phenolic Contents (TPC)
The TPC was detected at the maximum level in the aerial methanolic extract and least in the roots dichloromethane extract (Table 1). Results are expressed as mean ± standard deviation. "AMHS" aerial parts methanolic H. stocksii extract; "ADHS" aerial parts dichloromethane H. stocksii extract; "RMHS" roots methanolic H. stocksii extract; and "RDHS" roots dichloromethane H. stocksii extract. "GAE/g extract" Gallic acid equivalent per gram of extract, "QE/g extract" Quercetin equivalent per gram of extract, the superscripts a, b, c, and d represent significant difference (p < 0.05).

Total Flavonoid Contents (TFC)
The TFC was determined in the methanolic and dichloromethane extracts of both parts, i.e., in aerial and root parts ( Table 1). The TFC was significantly different in all the sample extracts. The highest TFC was exhibited by methanolic aerial parts (99.19 ± 1.14 mg of quercetin equivalent per gram of extract, mg QE/g) and lowest in the roots dichloromethane extract (54.65 ± 0.65 QE/g of extract).

Antibacterial Activity
In the current study, the antibacterial activity of the Haloxylon stocksii plant was determined by measuring its antibacterial activity against pathogenic bacteria. These bacteria include Bacillus subtilis, Bacillus pumilus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Escherichia coli, Bordetella bronchispetica, and Micrococcus luteus, which are the main pathogenic and spoilage organisms. The results of the antibacterial activity of various extracts of Haloxylon stocksii are shown in Table 5. Results are expressed as mean ± standard deviation. "ZI" zone of inhibition in mm; "Std" standard co-amoxiclav; "AMHS" aerial parts methanolic extract; "ADHS" aerial parts dichloromethane extract; "RMHS" roots methanolic extract; and "RDHS" roots dichloromethane extract.

Antidiabetic Activities
The potential of H. stocksii to alleviate diabetes was investigated in vitro by analysing its ability to inhibit two key enzymes involved in the disease, namely α-amylase and α-glucosidase. Both parts (aerial and roots) of the plant were found to exhibit significant inhibitory activity against these enzymes, indicating their potential as antidiabetic agents. Figure 1 and Table S2 shows the antidiabetic potential of this plant.

Antibacterial Activity
In the current study, the antibacterial activity of the Haloxylon stocksii plant was determined by measuring its antibacterial activity against pathogenic bacteria. These bacteria include Bacillus subtilis, Bacillus pumilus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Escherichia coli, Bordetella bronchispetica, and Micrococcus luteus, which are the main pathogenic and spoilage organisms. The results of the antibacterial activity of various extracts of Haloxylon stocksii are shown in Table 5. Results are expressed as mean ± standard deviation. "ZI" zone of inhibition in mm; "Std" standard co-amoxiclav; "AMHS" aerial parts methanolic extract; "ADHS" aerial parts dichloromethane extract; "RMHS" roots methanolic extract; and "RDHS" roots dichloromethane extract.

Antidiabetic Activities
The potential of H. stocksii to alleviate diabetes was investigated in vitro by analysing its ability to inhibit two key enzymes involved in the disease, namely α-amylase and α-glucosidase. Both parts (aerial and roots) of the plant were found to exhibit significant inhibitory activity against these enzymes, indicating their potential as antidiabetic agents. Figure 1 and Table S2 shows the antidiabetic potential of this plant.

In silico Evaluation
In silico evaluation was done by performing molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), and toxicological studies.

In silico Evaluation
In silico evaluation was done by performing molecular docking, ADME (Absorption, Distribution, Metabolism and Excretion), and toxicological studies.

Molecular Docking (MD) for α-Amylase
MD of GC-MS identified molecules was carried out for α-amylase receptors. In order to find out docking score, binding affinity, and interaction of different compounds at the active sites of α-amylase receptors. Amongst those, the interaction with best docked molecules in terms of binding affinity are represented in Table 4. The docking score for phenol, 2,2 -methylenebis[6-(1, 1-dimethylethyl)-4-(1-methylpropyl) and γ-sitosterol with α-Amylase receptors was (−8.5) and (−8.7), respectively. These findings reveal the potent role of Haloxylon stocksii in α-amylase inhibition. The details of binding affinities and interactive forces of ligands with receptors are given in Table 6 and Figure 2.

Molecular Docking (MD) for α-Glucosidase
MD was done with α-glucosidase receptors to find out the binding score and binding interactions at active sites. For MD, molecules tentatively identified by GC-MS were selected. The binding scores for phenol, 2,2 -methylenebis[6-(1, 1-dimethylethyl)-4-(1methylpropyl), and γ-sitosterol were −8.1 and −8.9, respectively. This reveals the key role of Haloxylon stocksii in α-glucosidase inhibition. The details of binding interactions, binding forces, and docking scores are given in Table 7 and

Molecular Docking (MD) for α-Glucosidase
MD was done with α-glucosidase receptors to find out the binding score and binding interactions at active sites. For MD, molecules tentatively identified by GC-MS were selected. The binding scores for phenol, 2,2′-methylenebis[6-(1, 1-dimethylethyl)-4-(1methylpropyl), and γ-sitosterol were −8.1 and −8.9, respectively. This reveals the key role of Haloxylon stocksii in α-glucosidase inhibition. The details of binding interactions, binding forces, and docking scores are given in Table 7 and Figure 3.

Lipinski Rule of Five
Lipinski's rule of five is a rule that evaluates the chemical compounds having pharmacological or biological properties that may act as orally active drugs in human beings. The definition of the Lipinski rule is that the compounds should follow the following criteria with only exception allowed of the following criterion: 1.
The hydrogen bond donors should not be more than 5.

2.
The hydrogen bond acceptors should not be more than 10.

3.
The molecular mass should not be more than 500 daltons.

4.
Lipophilicity should not exceed the value of 5.
In silico study of best-docked compounds disclosed that all the compounds have <5 hydrogen bond donors and <10 hydrogen bond acceptors. The molecular mass of all chemical compounds evaluated is less than 500 daltons. The lipophilicity value of all the observed compounds follow the rule except eicosanoic acid, methyl ester and phthalic acid, bis(7-methyloctyl) ester (Table 9 and Figure 4). So, with these observations, all the compounds can be used as oral drugs.

Toxicity Study
During the in silico study, the toxicity profile of chemical compounds was evaluated by determining LD50, toxicity class, hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity (Table 10). The toxicity findings were that all the compounds have ≥ 4 toxicity levels. This toxicity level indicates that all the compounds are slightly toxic or practically non-toxic. All the compounds except standard acarbose do not cause hepatotoxicity. Three compounds, namely phthalic acid, bis(7-methyloctyl) ester, and din-octyl phthalate, exhibited properties of carcinogenicity. γ-Sitosterol and acarbose displayed immunotoxicity. Only 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-exhibited mutagenicity and no chemical compound showed cytotoxicity.

Toxicity Study
During the in silico study, the toxicity profile of chemical compounds was evaluated by determining LD 50 , toxicity class, hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity (Table 10). The toxicity findings were that all the compounds have ≥ 4 toxicity levels. This toxicity level indicates that all the compounds are slightly toxic or practically non-toxic. All the compounds except standard acarbose do not cause hepatotoxicity. Three compounds, namely phthalic acid, bis(7-methyloctyl) ester, and di-noctyl phthalate, exhibited properties of carcinogenicity. γ-Sitosterol and acarbose displayed immunotoxicity. Only 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-exhibited mutagenicity and no chemical compound showed cytotoxicity.

Discussion
Screening of plants used for medicinal purposes has remained one of the most valuable approaches towards the development and provision of new drug candidates. The findings disclosed that there was significant difference (p < 0.05) among the extraction methods as well as the different plant parts, i.e., in aerial parts and roots. Briefly, it was found that AMHS samples had significantly (p < 0.05) high TPC and TFC compared to RMHS, which indicates that the methanol extract of aerial parts has higher TPC and TFC compared to that of the roots. Our results regarding higher TPC and TFC in aerial parts are in line with the literature that also revealed that phenolic compounds were significantly higher in aerial parts as compared to roots [45]. The difference in TPC between the roots and aerial parts could be due the presence of carotenoids in the aerial parts that are missing in the roots. Further, the effectiveness of different extraction methods was evaluated. It was observed that TPC and TFC were higher in the methanol extraction method compared to the dichloromethane extraction method. In addition, the higher TPC and TFC levels in the aerial parts indicate the presence of polyphenolic secondary metabolites, which may be a result of the environmental stress factors that the aerial parts face. This could be the reason that aerial parts face more stress against the environmental factors, which produce more antioxidant compounds as compared to roots, agreeing well with previous studies [46,47]. Peerzada, Khan et al. determined the TPC and TFC (262.6 ± 7.49 mg GAE/g and 79.86 ± 6.02 mg QE/g, respectively) in methanolic extract of the whole plant [48]. These high values may be due to difference in habitat of plant or due to synergistic effect of both aerial parts and roots.
GC-MS analysis is a prevailing analytical technique used in the identification of the components present in plant extracts [49]. This technique is particularly useful in the field of natural product chemistry, where plant extracts are commonly used as a source of bioactive secondary metabolites [50]. One of the main advantages of GC-MS analysis of plant extracts is its high sensitivity and specificity. The technique can detect and identify components present in trace amounts, allowing for the detection of compounds that may have important biological activities even at low concentrations [51]. Additionally, mass spectrometry is used for the characterisation of the molecular weight and structure of the identified components, which is critical for understanding their biological activities and potential therapeutic applications.
Liquid chromatography-mass spectrometry (LC-MS) profiling of plant extracts is an effective analytical approach for identifying the numerous chemical components found in plant extracts. It gives a thorough breakdown of the extract's phytochemical makeup, including flavonoids, alkaloids, terpenoids, and other bioactive substances. The identification of unidentified chemicals in the extract via LC-MS analysis can also aid in the discovery of novel natural products with potential medicinal uses. In general, LC-MS profiling of plant extract is a useful tool for characterising chemicals obtained from plants, and it can help create new medications and functional food ingredients. The LC-MS screening resulted in the identification of amines, aromatic ketones, phenyl amines, flavonoids, alkaloids, organophosphate esters, fatty acids, and glycerolipids.
Antioxidant properties are of great importance as they determine the suitability and applicability of these plants for medicinal use. Researchers are seeking new plants to assess their effectiveness, as they believe that these are the best alternatives, with the fewest side effects, to medicines. Plant extracts have been recognized as a rich source of antioxidants, which have played a critical role in the prevention of various illnesses, including cancer, cardiovascular diseases, diabetes, and neurodegenerative disorders [52]. The antioxidant property of plant extracts is primarily due to their content of polyphenolic compounds, likely flavonoids, phenolics, and tannins [53]. Numerous studies have reported the antioxidant properties of plant extracts from various sources, including roots, vegetables, and aerial parts [54,55]. Overall, plant extracts are a promising source of antioxidants that could have potential health benefits, although further studies are needed to better understand their bioavailability and potential therapeutic applications. A study conducted by Yaseen et al. (2020) investigated the total antioxidant activity of the acetone, chloroform, acetic acid and propranolol extracts of Haloxylon stocksii [56]. The literature review of some bioactive phytoconstituents, identified with GC-MS and LC-MS, possess antioxidant activities. These bioactive secondary metabolites include triacontanoic acid [57], octadecanoic acid [44], γsitosterol [58], and fraxetin [59]. There is no antioxidant comparative study of the aerial and roots part of H. stocksii available in literature by utilising methanol and dichloromethane as extraction solvents. This comparative study is the first one that reports the antioxidant potential of H. stocksii for different parts and with different extraction methods.
Type 2 diabetes (T2D) is a prevalent health condition worldwide that results in high blood sugar levels and abnormal carbohydrate metabolism. This disease is a significant contributor to illness and death, and it also places a substantial economic burden on societies [68]. The H. stocksii extracts were evaluated in vitro for antidiabetic activity (α-amylase and α-glucosidase inhibition). The aerial parts extracts were more effective compared to that of the roots. When we compare the two solvents, the methanol extract was more effective as compared to the dichloromethane extract. The higher antidiabetic potential of the methanolic extraction method could be due to the higher TPC of this method. Truong et al. demonstrated that the methanolic extraction method gives maximum extraction yield, highest phenolics and flavonoid contents, and antioxidant activity as compared with ethanol, chloroform, acetone, and dichloromethane extraction methods [69]. In addition, higher polyphenols are found to be effective in diabetes management. The antidiabetic potential of this plant is attributed due to the presence of some bioactive phytocompounds which were identified with GC-MS and LC-MS, such as β-sitosterol [51]; phenol; 2,2 -methylene bis[6-(1,1-dimethylethyl)-4-(1-methylpropyl) [55]; hordenine [70]; and piperine [63]. The compounds having good antioxidant activities are also responsible for antidiabetic activity [56]. The aqueous extract of Chenopodium botrys exhibited α-amylase and α-glucosidase inhibition activity [71]. Atriplex halimus L. significantly increases body weight and decreases blood glucose and hepatic levels in the rats [72]. The results of in vitro antidiabetic activities of plants endorse that oral administration of these extracts, especially aerial parts extracts in non-insulin-dependent diabetes, can reasonably reduce postprandial blood glucose levels; however, future studies, especially in vivo studies, are necessary to confirm their effectiveness.
In silico studies of Haloxylon stocksii have been gaining attention due to their costeffectiveness and ability to provide insights into the potential biological activities of the plant's bioactive compounds. Several studies have used computational approaches such as molecular docking, ADME, and toxicological studies to predict the binding affinity and activity of Haloxylon stocksii's bioactive compounds with various therapeutic targets [55]. In silico studies have identified several compounds with potential antidiabetic, anti-inflammatory, and anticancer activities, such as stigmasterol and β-sitosterol. These studies have also helped to elucidate the mechanism of action of the plant's bioactive compounds and have guided further experimental studies [73]. Therefore, future studies should aim to combine in silico and in vitro/in vivo experiments to further validate the potential therapeutic activities of Haloxylon stocksii's bioactive compounds.
Overall, these studies suggest that Haloxylon stocksii could be a potential source of bioactive compounds, natural antioxidants, antibacterial, and antidiabetic and that further research is needed to better understand the bioactive compounds present in the plant extract and their potential therapeutic applications.

Plant Identification and Sample Collection
The roots and aerial parts of the plant were collected from sandy cliffs of Tehsil Choti Zarieen District Dera Ghazi Khan in August 2020 Punjab, Pakistan. The plant was identified as Haloxylon stocksii by Zafar Ullah Zafar, Institute of Pure and Applied Biology, Bahauddin Zakariya University Multan, Pakistan (BZU). Voucher no. http://www.theplantlist.org/ tpl1.1/record/tro-50307719 (accessed on the 20 May 2020) was given to that plant.

Extract Preparation of the Plant Material
Firstly, the plant was washed, and shade-dried in two separate portions (i.e., aerial parts and roots) for 6 weeks at 25 • C. Both the plant parts were pulverized roughly to a coarse powder and the extraction of both powdered materials was done by simple maceration procedure separately in dichloromethane (DCM) and methanol (the ratio between powder and solvent was 1:3). Then, filtration was done after 24 h. This procedure was repeated four times with both these solvents. DCM and methanol extracts were dried under reduced pressure in a rotary evaporator (Buchi, Flawil, Switzerland). The DCM extract of roots (50.0 g) and aerial parts (30.8 g) were collected in sample bottles and assigned codes RDHS and ADHS, respectively. Methanol resulted in the yield of roots extract (70.4 g) and aerial parts extract (52.0 g). These dried extracts were collected in the sample bottles and assigned codes RMHS and AMHS, respectively.

Estimation of Total Phenolic Content
The determination of total phenolic contents (TPC) and total flavonoid contents (TFC) were done using the Folin-Ciocalteu (FC) and aluminium trichloride colorimetric methods, respectively [44].

GC-MS Analysis
GC-MS screening was done with an Agilent 6890 series and Hewlett Packard 5973 ground sensor. The HP-5MS column (Santa Clara, CA, USA) with 30 m length × 250 µm diameter × 0.25 µm film thickness was used. The temperature of the injection was set at 220 • C up to 240 • C, while the temperature of the oven was programmed from 60 • C to 246 • C at a rate of 3 • C/min. Pure He (helium) gas was used as a carrier. A volume of 1.0 µL of the reconstituted sample extract was injected. The temperature was set between 50 • C and 150 • C at a rate of 3 • C/min, and then raised to 300 • C at a rate of 10 • C/min. The identification of bioactive compounds was performed by tentatively identifying peaks using the NIST 2011 library scanning ranging from 35 to 600 m/z [74] The roots and aerial parts of the Haloxylon stocksii plant were evaluated for antioxidant potential through different antioxidant assays which include the determination of radical scavenging activity and reducing power. Trolox was used as standard, and results were expressed as milligrams of Trolox equivalents per gram of dry extract (mg TE/g extract). Control was prepared by the same procedure without adding plant extract.

Radical Scavenging Activity
Aerial parts and roots of the plant were analysed for the evaluation of scavenging potential and were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. The procedures for above mentioned assays were in line with the literature [75].

DPPH Assay
For this radical scavenging assay, the volume of 50 µL stock solutions of the extracts of both parts of the plants were mixed with 200 µL of DPPH solution (0.267 mM). After, these mixtures were incubated in a 96-well microtiter plate at 25 • C for 30 min (in darkness). The absorbance of samples was measured by a microplate reader (BioTek Synergy HT, Winooski, Vermont, USA) at wavelength 517 nm. The blank solution was also assessed according to this procedure.

ABTS Assay
For this reducing power assay, stock solutions of the extracts of both parts of the plants were prepared and diluted with ABTS + solution until absorbance reached 0.700 ± 0.02 at 734 nm. The volume of 100 µL of extract solutions were mixed with 200 µL of ABTS + solution in 96-well microplate. This mixture was incubated at 25 • C for 30 min. After this, absorbance of both solutions was measured at wavelength 734 nm. The control solution was also assessed according to this procedure.

Reducing Power Assays
Both parts of the H. stocksii plant were assessed for their reducing capacity by utilising direct reducing antioxidant Cupric Reducing Antioxidant Capacity (CUPRAC) and Ferric Reducing Antioxidant Power (FRAP). Both mentioned assays were performed according to the procedures mentioned in the literature [76].

CUPRAC Assay
For performing the CUPRAC assay, stock solutions of the extracts of both parts of the plant were prepared and added to reaction mixture containing neocuproine (200 µL, 7.5 mM), cupric chloride (200 µL, 10 mM), and ammonium acetate buffer (200 µL, 1 M, pH 7.0)]. After this, both solutions were incubated at 25 • C for 30 min, and absorbance was measured at 450 nm. The control solution was also assessed according to this procedure.

FRAP Assay
For performing the FRAP assay, stock solutions of the extracts of both parts of the plants were prepared and added to acetate buffer in 40 mM HCl and ferric chloride (20 mM) in a final concentration with the ratio of 10:1:1 (v/v/v). Absorbance was measured at 593 nm after the incubation for 30 min at 25 • C. Furthermore, a blank solution was also assessed according to this procedure.

Antibacterial Activities
Antibacterial activities of the Haloxylon stocksii plant aerial parts and roots were assessed against seven bacterial stains. Three Gram-negative stains (Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 25922, and Bordetella bronchiseptica ATCC 7319) and four Gram-positive strains (Bacillus subtilis ATCC 1692, Bacillus pumilus ATCC 13835, Staphylococcus epidermidis ATCC 8724, and Micrococcus luteus ATCC 4925) of bacteria. These strains were obtained from Drug Testing Laboratory, Lahore (DTL). Co-amoxiclav was used as standard in this assay. The 24 h old cultures of all seven strains were taken, prepared inoculum by taking few colonies of each bacterium and adding them in test tubes containing broth medium. Tubes were incubated overnight, and colonies were diluted to a cell density of 10 6 CFU/mL (colony forming unit).

Disc Diffusion Method
This method was used to estimate the antibacterial activity of common infectioncausing bacterial strains from the aerial parts and roots of the Haloxylon stocksii plant. The results were measured as zones of inhibition. Solutions were prepared in 10% of DMSO. These solutions were sterilised by filtering with a sterile membrane filter. After this Petri dishes with Mueller Hinton agar were prepared and inoculated with the seven bacterial strains. Positive and negative control discs were made using 10% DMSO and co-amoxiclav (10 µg/disc), respectively. These Petri dishes were incubated at 37 • C for 24 h. Triplicate experiments were performed to minimize the error. The zone of inhibition was measured in millimetres (mm), and greater than 6 mm was considered for antibacterial activity [52].

In vitro Antidiabetic Potential
For the estimation of the in vitro antidiabetic potential in aerial parts and root extracts of H. stocksii, α-amylase and α-glucosidase inhibition assays were performed.
• α-Amylase inhibitory assay: The α-amylase inhibition assay of methanolic and DCM extracts of aerial parts and roots was performed according to the method described in the literature with minor changes [73]. The volume of 0.5 mL extracts of both parts was mixed with 0.5 mL of α-amylase solutions with 0.02 M of sodium phosphate buffer (pH 6.9) this mixture was kept for incubation at 25 • C for 10 min, and then the reaction was terminated by adding 1 mL of the colouring agent dinitrosalicylic acid. After this, these test tubes were heated in a warm bath at 100 • C for 5 min. After cooling the reaction mixture till 25 • C, it was diluted with 10 mL of deionized water, and the absorbance of blank and control samples was measured at 540 nm. Acarbose was used as a standard drug. The following equation measured the inhibition of α-amylase.
% inhibition o f α − amylase = Abs Control − Abs Sample /(Abs Control ) × 100 • α-Glucosidase inhibitory assay: For determination of the antidiabetic potential crude methanolic and DCM extracts of both aerial and root, parts were also analysed using α-glucosidase inhibitory assay [13]. For this analysis, 1 mL of each extracted part was mixed with 100 µL of phosphate buffer of pH 5.9. Then, 50 µL of enzyme α-glucosidase was added and kept at 37 • C for 10 min for incubation. The volume of 50 µL of substrate, i.e., p-nitrophenyl-α-D-glyco-pyranoside (pNPG) was added to the reaction mixture and the mixture was kept for incubation for a further 30 min at 35 • C. After complete incubation, the absorbance of all samples was measured at 405 nm. The % inhibition was measured by the following formula.

In-Silico Evaluation
In silico evaluation was performed by molecular docking, ADME, and toxicity studies.

Molecular Docking Study
Molecular docking plays a key role in computer-aided drug designs and the development of molecular biology. For molecular docking in the current study, various software tools were employed, such as Autodock Vina, MGL Tools, PyRx, Babel and Discovery Studio. The enzymes α-amylase (PDB DOI: 10.2210/pdb1SMD/pdb, accessed on 7 August 2022) and α-glucosidase (PDB DOI: 10.2210/pdb5ZCB/pdb, accessed on 7 August 2022) were downloaded from Protein Data Bank [77]. The enzymes were prepared by Discovery Studio (Discovery Studio 2021 client). The GC-MS identified compounds along with acarbose (standard drug) were used as ligands and the SDF format of these ligands was downloaded from PubChem. Open Babel was used for the preparation of Ligand. Both prepared receptor and ligands were loaded to vina, embedded in PyRx. These were placed in the active area of the enzyme and the evaluation of outcomes was carried out by using Discovery Studio Visualizer [10].

Conclusions
The exploration of new plants for drug development is a promising approach in the search for more effective therapies. In this study, Haloxylon stocksii was evaluated for its potential in various parameters including polyphenol contents, antioxidant, antibacterial, and antidiabetic properties. The antidiabetic potential of methanolic extracts was better than that of dichloromethane extracts. The results demonstrate that aerial parts extracts are more effective against-Gram-positive bacteria and root extracts showed more activity against Gram-negative bacteria. These findings suggest that H. stocksii may have significant potential for drug development, particularly in the treatment of infections and metabolic diseases; however, further investigation, including in vitro and in vivo cytotoxicity assays to evaluate the safety of the identified compounds is necessary to fully validate these find-ings before considering H. stocksii as a viable candidate for drug development. Nonetheless, these results may represent a promising milestone in the medical industry's search for novel and effective therapies.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.