Novel Phytosterol with Antimicrobial Potential of Digera muricata Mart. from Chittorgarh Region, India

Digera muricata Mart, a plant having therapeutic characteristics that has been utilised traditionally, belongs to the Amaranthaceae family, and a promising source of specific natural products utilized as antioxidant, prophylactic, antimicrobial, anthelmintic, anti-diabetic, and allelopathic agent. In the present study, a biologically active phytosterol was isolated from Digera muricata Mart. The isolated compound was characterized by 13C, 1H NMR, FTIR, and HRMS. Characterization of the isolate was done by antimicrobial assay, and molecular docking. The antimicrobial potential of the isolated phytosterol (50 µl) against Streptococcus pyogenes was found to be maximum (ZOI-20.0 ± 1.0), followed by Streptococcus agalactiae (ZOI-11.3±1.5) , Candida albicans (ZOI-09.0 ± 1.0), Klebsiella pneumonia (ZOI-8.6 ± 1.5) and Escherichia coli (ZOI-8.6 ± 1.5). The molecular docking results indicate that the phytosterol binds to the receptor 1AI9 at the 32 th and 58 th positions; 1KZN receptor at the 76 th position, the 5L3J receptor at the 46 th (ASN) and 136 th (ARG) position; 7WIJ receptor at the 419 th (ARG) and 582 th (ASP) and 585 th (ASN) positions.


INTRODUCTION
Attention has been drawn to natural products based on plants as sustaining medicinal and health ailments due to the cost-effective treatment of chemically synthesized drugs, their toxicities in the sustainability of individual medical aid and development, and the exploitation of plant-mediated new medicines.Some reports on recent investigations and economic expenses, medicinal plants will ultimately continue to have a crucial role in developing as a health aid.These bioactive compounds based on therapeutic plants have a essential role in medical treatment and play a twin role in the innovation of such drugs as they are the backbone for designing such medicine, a natural blueprint for creating novel molecules or; natural products engaged in combating many diseases. 1Therefore, these medicinal plants are acquired for the isolation of such compounds to gain knowledge about their underlying mode of action with reduced toxicity and cost-effective features. 2The enhanced risk and toxicities of chemotherapeutics and the occurrence of multiple drug resistance mutants in pathogenic bacteria forced us to search for such novel drugs having antimicrobial potential. 2,3

About experimental plant
As part of the family Amaranthaceae, Digera muricata Mart (D. muricata) grows in the wilderness and is a consumable plant.It is found across India, especially Rajasthan, Maharashtra and Andhra Pradesh. 4 D. muricata have many names, including Cancalisoppu, Latamouri or Gungutiya, Latmahuria, Lesua, False Amaranth, Latmahuria, Kunanjara, and Aranya.D. muricata is an annual medicinal plant growing 20-70 cm tall with long, narrowed, and pointed leaf stalks.Flowers grow to 30 cm in height and are spikelike racemes. 5There is typically a greenish-white colour in fruit, with white flowers mixed with pink, carmine, or red.August and September are the months when flowers bloom.The tender shoots and foliage of D. muricata are utilised both medicinally and as a vegetable in the local community for the treatment of gastrointestinal diseases, diabetes, constipation, and bowel issues.Urinary discharges can be treated with flowers and seeds.The application of leaf paste prevented the local production of pus.The mother is given a boiled root infusion after childbirth for induced lactation. 6D. muricata is also used as an expectorant and mild astringent. 7,8. muricata is regarded as a laxative and a cooling astringent to the bowels in Ayurveda.Fungal infections were treated with the extract of the leaves of the plant. 9People's health is directly benefited by phytochemicals found in traditional foods. 10A study of D. muricata treatment provides evidence that it has antioxidant properties, is protective against CCl 4 -induced toxicity, and may help treat diseases caused by free radicals. 11ignificant antiproliferative activity against MG-63 cell lines has been shown by the ethanolic leaf extract of D. muricata. 12Recently, two compounds with herbicidal properties were isolated from D. muricata. 13During the growing season, the roots of D. muricata contain sugars and phenols, while the leaves contain proteins, starches, and fats.In addition, the leaf extract has antimicrobial properties against Escherichia coli and Fusarium oxysporum.Kidney stones can be treated with leaf extract. 14For a variety of male hormones in rats, CCl 4 disturbances were reversed by D. muricata. 15y use of this herb as a substitute for secondary infertility. 16D. muricata has a well-documented history of being an effective antibacterial. 17he study was intended to extract, isolate and structurally characterize bioactive compound and its antimicrobial potential.

Collection and verification of plant
The herb was gathered from Rajasthan's Chittorgarh district.The collected plant material was identified and deposited in Botanical Survey of India, Jodhpur, Rajasthan.

I s o l at i o n o f co m p o u n d u s i n g co l u m n chromatography Column packing
To create an admixture, 16 g of ethyl acetate extract was combined with silica gel (60-120 mesh).The admixture combined with hexane was placed onto a column with a 2.4-inch diameter.Hexane was used as the starting solvent, followed by ethyl acetate to elute the column.

Fractionation of the extract
60 to 120 mesh size activated Silica gel used to fill the glass column.The extract was added to the column and gradually eluted by using 1000 ml of hexane of the ratio 100:0 v/v, 1200 ml of hexane: ethyl acetate mixture in the proportion of 95:5 v/v, 900 ml of hexane:ethylacetate in the proportion of 90:10 v/v, 900 ml of hexane:ethylacetate in the proportion of 85:15 v/v and 800 ml of hexane:ethylacetate in the ratio of 80:20, 400 ml hexane:ethylacetate in the proportion of 75:25 v/v, 800 ml of hexane:ethylacetate in the proportion of 70:30 v/v, 200 ml of ethyl acetate in the proportion of 0:100 v/v.

Thin-layer chromatography (TLC)
Concentrated fraction was placed onto the 20X20 cm activated silica gel TLC plates.The plates were created using various hexane:ethyl acetate ratios, including 90:10, 75:25, 50:50, and 25:75.The plate was subjected to vanillin and iodine fumes in order to identify any spots.A single spot denoted a pure compound with excellent purity and yield was selected for spectral analyses, and structures will be clarified.

Structure elucidation
High-Resolution Mass Spectrometry (HRMS) and Nuclear magnetic resonance (NMR) was exploits to draw the structures of the isolated compound.Thermo Scientific's Evolution 160 spectrophotometer was used to record UV spectra, while Perkin-Elmer's FTIR model 1600 spectrophotometer was used to record IR spectra.Tetramethylsilane (TMS) served as the internal standard while spectra of NMR were captured in CDCl 3 using a Bruker AMX 500 spectrometer at 500 MHz.Relative to the TMS internal standard, chemical shifts are expressed in ppm, and scalar coupling constants are described in Hz (J).

Antimicrobial susceptibility test Growth conditions of microorganisms
In the current investigation, test microorganisms included Streptococcus agalactiae (ATCC13813), Escherichia coli (MTCC730), Klebsiella pneumoniae (MTCC432), and Streptococcus pyogenes (MTCC1924).In nutrient broth, the bacterial strains were resurrected.Each bacterium received a fresh inoculum for the antibacterial test, which was performed at 37°C for 24 hours.The nutrient broth was then added to the cell suspension to modify the turbidity until it was close to the 1.5 × 10 8 CFU/ml McFarland 0.5 standard.
In the current investigation, Candida albicans (MTCC7315) was utilised as a test fungus.
It was kept at 4°C on potato dextrose agar (PDA) slants before usage.

Antibacterial susceptibility assay
The conventional disc diffusion technique was used to test the antibiotic sensitivity of bacterial strains to two antibiotics with potencies of 10 μg per disc, including gentamycin, and tetracycline. 18Using the disc diffusion technique, the antibacterial test of isolated compound was conducted.Swabs of the test microorganisms were diluted to a concentration of 100 µl of diluted inoculum (1.5X10 8 CFU/ml) and added to plates of sterile Mueller Hinton agar (pH 7.2). 1 mg of isolated compound dissolved in DMSO used as stock.From stock 40 µl was added to the 4 mm diameter agar plate well.Pure DMSO used as negative control.

Antifungal susceptibility assay
Candida albicans inoculum was suspended in 5 ml of potato dextrose broth and cultured at 37°C for a day.The experiment was used to evaluate the antifungal activity using disc diffusion principle. 18The inoculum was applied to the PDA medium with a glass spreader aseptically.Separately, 2.5 mm radius paper discs soaked with 40 µl from the stock was put on the infected plates' surface and kept at 37°C in the incubator for 24 hours.The diameter of the inhibitory zone was used to gauge the antifungal activity.Dimethyl sulfoxide (DMSO) was used as a negative control, and fluconazole served as the positive control.All experiments were run in triplicate.

Minimum inhibitory concentration
The Clinical and Laboratory Standards Institute (CLSI) recommendations and Eloff's microtitre broth dilution procedure 19 were followed to ascertain the isolated compound's Minimum Inhibitory Concentration (MIC).Initially, isolated compound was prepared by dissolving it in dimethylsulphoxide (DMSO) to achieve a final concentration of 1 mg/ml.
A bacterial suspension with around 5X10 5 CFU/mL was produced using a 24-hour culture plate and 100 µl of this suspension was introduce into every well of the microplates.For each strain, sterile environment and growth was controlled.The microtiter plates were incubated for 24 hours at 37°C for bacterial species and 48 hours for fungal strains.Following incubation, 40 µl of a 0.4 mg/ml solution of iodonitrotetrazolium chloride was added to every well as a microbial growth indicator.The plates were then incubated at 37°C for 30 min for bacteria and 24 h for fungi, and MIC values were visually determined.The minimum inhibitory concentration was noted as having the least amount of extract with no visible growth, while the MIC value was identified as the concentration completely inhibiting bacterial/ yeast growth (the first clear well).
To assess microorganism sensitivity, A negative control test utilizing only DMSO was also conducted and positive control experiments were performed for bacterial strains, gentamycin was used at a starting concentration of 0.10 mg/ml in sterile water, and for fungal strains, fluconazole was used at a starting concentration of 0.10 mg/ml in DMSO and water.Final concentrations for these experiments ranged from 0.1 mg/ml to 0.001 mg/ ml.

Molecular docking study
The aim is to explain the behavior of small molecules within the binding site of target proteins.Employing the molecular docking technique, this investigation seeks to comprehend essential biological processes by reproducing the atomic-level interaction with a tiny chemical and a protein. 20Using AutoDock, phytosterol was docked towards the binding site of the proteins.By setting the scaling factor for the nonpolar atoms to 0.8, the flexibility of the proteins was controlled.Default conditions were set for all other parameters.Docking scores were used to measure the binding affinity of protein/ligand complexes.

Collection and verification of Plant
Collected herb was identified as D. muricata and deposited in Botanical Survey of India, Jodhpur, Rajasthan.

I s o l at i o n o f co m p o u n d u s i n g co l u m n chromatography
Fractions were collected from column chromatography and concentrated by the rotary evaporator.A pure portion was collected and used for further characterisation at the Hexane:Ethyl acetate (70:30) concentration.

Thin-layer chromatography (TLC)
Isolated pure compounds showing good purity and good yield was taken for spectrum studies and used for structure elucidation.

MIC of the isolated compound
MIC outcomes for the isolated compounds and fractions are depicted in Table 2.The isolated compound exhibits higher antibacterial potential against Streptococcus pyogenes with MIC of 8 mg/ ml as compared to the gentamycin i.e., 4 mg/ml.MIC for Candida albicans found to be 20 mg/ml as compared to the fluconazole i.e., 6 mg/ml.

Molecular docking 1AI9_C3
The results indicate that the isolated phytosterol binds to the receptor 1AI9 at the  32 nd and 58 th position, respectively.GLU at 32 th position makes a substrate binding site, while THR at 58 th position makes a binding site for NADP+.Both the amino acid forms a helix structure in the protein.The isolated molecule interacts with the side chains of both amino acids, with a docking score of -5.869.The docking score indicates a good interaction between the molecule and the receptor, while gentamycin and streptomycin bind more strongly to the receptor, with docking score of -7.15 and -9.338, respectively (Table 3, Figure 6).

1KZN_C3
The results indicate that the isolated phytosterol binds to the receptor 1KZN at the 76 th position.ARG at 76 th position makes an ATPase domain region.Two different oxygen atoms   3, Figure 7).

5L3J_C3
The results indicate that the isolated phytosterol binds to the receptor 5L3J at the 46 th (ASN) and 136 th (ARG) positions, respectively.The isolated molecule interacts with the amino acid's side chains, with a docking score of -3.627.The docking score of the isolated compound is  less than gentamycin (-6.238) and streptomycin (-4.507).Besides, all three molecules compound, Gentamycin, and Streptomycin interacts with the 5L3J rectors at the same position 46 th amino acid residue ASN but the binding affinity of compound is less (Table 3, Figure 8).

7WIJ_C3
The results indicate that the isolated phytosterol binds to the receptor 7WIJ at the 419 th (ARG) and 582 th (ASP) and 585 th (ASN) positions, respectively.The isolated molecule interacts with the side chains of all three amino acid, with a docking score of -4.507.The docking score of compound is less than the docking score of gentamycin (-5.052) and streptomycin (-8.896) (Table 3, Figure 9).

DISCUSSION
Digera muricata Mart. is a plant with a long history of traditional medicinal use in various cultures.Recently, there has been growing interest in exploring its bioactive compounds for potential therapeutic applications.One such compound that has garnered attention is a novel phytosterol found in Digera muricata Mart., which exhibits promising antimicrobial properties.The antimicrobial potential of phytosterols extracted from the leaf of Annona squamosa, Adenocalymma alliaceum, and Amaranthus tricolor was examined.These phytosterol was isolated, purified, characterized, and assessed for their antibacterial effects on both gram-positive bacteria, like Staphylococcus aureus, Staphylococcus albus, and Streptococcus viridians, and gram-negative bacteria, including Escherichia coli, Pseudomonas pyocyanea, and Klebsiella. 21he study by Monu et al. in 2008 also investigated the antimicrobial potential of phytosterols in milk. 22urthermore, a bacteriostatic experiment involving phytosterols derived from pumpkin seeds revealed significant antagonistic actions against E. coli, Bacillus subtilis, Staphylococcus aureus, and Salmonella.Complete inhibition of bacterial at a dosage of 3.0 mg/mL, proliferation was seen. 23Numerous phytochemicals contained in D. muricata have been shown to have potent antiinflammatory and powerful free radical scavenging effects. 24D. arvensis (a synonym of D. muricata) demonstrates notable antibacterial activity against Ralstonia solanacearum (previously known as Pseudomonas solanacearum), with a mean zone of inhibition measuring 6.14 mm. 25 P re s e nt ev i d e n c e f ro m st u d i e s demonstrating the antimicrobial activity of the novel phytosterol.The discovery of a novel phytosterol with antimicrobial potential in Digera muricata Mart.opens up exciting possibilities for harnessing its health-promoting effects.The antimicrobial properties of this phytosterol hold promise for addressing oxidative stress-related conditions, and further research may unveil its full therapeutic potential.Integrating this compound into functional foods and pharmaceuticals could offer innovative strategies for promoting health and preventing diseases associated with oxidative damage.However, ongoing exploration is necessary to fully recognize its mechanisms of action, assess safety, and improve its applications for human health.

CONCLUSION
The isolated compound was characterized by 13C, 1H NMR, FTIR, and HRMS and subjected to antimicrobial assay, and molecular docking study.The antimicrobial potential of the isolated phytosterol against Streptococcus pyogenes was found to be maximum (ZOI-20.0 ± 1.0).The molecular docking results indicate that the phytosterol binds to the receptor 1AI9 at the 32 th and 58 th positions; 1KZN receptor at the 76 th position, the 5L3J receptor at the 46 th (ASN) and 136 th (ARG) position; 7WIJ receptor at the 419 th (ARG) and 582 th (ASP) and 585 th (ASN) positions.This study showed that isolated phytosterols might exploits as potential antibacterial agents.
The novel phytosterol isolated from Digera muricata Mart.represents a promising avenue for the development of antimicrobial agents.Its unique properties and demonstrated efficacy against various microorganisms make it a valuable candidate for further exploration.However, more research is required to know its mechanisms, assess its safety, and optimize its potential applications in clinical settings.The discovery of this novel phytosterol opens up new possibilities for combating microbial infections and addressing the challenges of antibiotic resistance Escherichia coli (strain K12) Protein -DNA gyrase subunit B Gene -gyrB AA -804 c) 5L3J: Escherichia coli Protein-DNA Gyrase b in complex with benzothiazole-based inhibitor Gene-gyrB, Z5190, ECs4634 AA-378 d) 7WIJ -Macrophomina phaseolina (strain MS6) (Charcoal rot fungus) Protein -geranylgeranyl diphosphate synthase AA -698

Figure 3 .
Figure 3. C-13 NMR of the isolated compound

*
More docking score indicates a more binding affinity with the receptor molecule in the isolated molecule interact with the side chains of both amino acids, with a docking score of -3.95, which is more than the docking score of gentamycin (-3.068) and less than the docking score of streptomycin (-7.039).More docking scores indicate more binding affinity with the receptor (Table

Figure 6 .
Figure 6.Isolated phytosterol binds to the receptor 1AI9 at the 32 nd and 58 th position

Table 1 .
Antimicrobial activity of the isolated compound