Mechanistic Studies of Dinoxin B withanolide, A Herbal Antibiotic from Datura inoxia against Staphylococcus aureus

 

Chandni Tandon1, Ruby George1, Preeti Bajpai2, Priti Mathur1*

1Amity Institute of Biotechnology, Amity University, Uttar Pradesh, Lucknow Campus - 226028, India.

2Zoology Department, Mahatma Gandhi Central University Motihari, Bihar, India.

*Corresponding Author E-mail: pmathur@lko.amity.edu.

 

ABSTRACT:

In view of urgent need for effective herbal antibiotics, we have isolated, characterized, an effective antibacterial compound Dinoxin B withanolide from leaves of Datura inoxia to investigate its antibacterial potential against Staphylococcus aureus. The bactericidal efficacy of Dinoxin B withanolide was assessed on Staphylococcus aureus, using transmission and scanning electron microscopy. Its effect on DNA and protein was analyzed by the electrophoresis technique. In vitro and in vivo studies were performed to evaluate the cytotoxicity and anti-inflammatory nature of the compound.  Electron microscopic study showed that Dinoxin B withanolide damaged bacterial cell wall and membrane. It also causes degradation to DNA and protein, resulting in bacterial death. This compound was found to be non-toxic even at higher concentrations. Treatment of Balb/c mice revealed the significant suppression of T cells by the Dinoxin B withanolide. In vivo and in vitro, studies confirm that Dinoxin B withanolide could be used as herbal antibiotics with bactericidal, anti-inflammatory, low toxicity and good shelf life.

 

KEYWORDS: Datura inoxia, Staphylococcus aureus, Dinoxin B withanolide, Cytotoxicity assay, Immuno-modulatory potential.

 

 


INTRODUCTION: 

Antibiotic resistance necessitates the development of novel treatment approaches1,2. As medicinal phytocompounds are important natural resources for future drug discovery3, we have isolated and characterized Dinoxin B withanolide (dBw) a potentially broad-spectrum antibacterial compound from ethanolic leaf extract of D. inoxia. This compound was identified by a characteristic mass spectrum generated by LC-MS and data from the literature and published4. The fraction was found to be a mixture of six major phytoconstituents, which were known as dBw and their aglycone with the molecular formula C34H49O11 and exact mass calculated as 633.3269 (Figure 1). Vermillion et.al., 2011; identified with NMR spectra and evaluated the cytotoxic activity of this compound5.

 

Figure 1: Structural and molecular formula of dBw from the ethanolic fraction of D. inoxia leaf. Identification was done by characteristic mass spectrum developed by LC-MS and observed as a mixture of six major phytoconstituents; dBw and their aglycone4

 

Methicillin-resistant and other Staphylococcus bacterium are immune to some antibiotics, including beta-lactam antibiotics, making infections more difficult to treat6. MRSA infections can be very severe and are the most common in all bacterial infections. Resistant strains have emerged through natural selection, horizontal gene transfer, and became resistant to beta-lactam antibiotics7. In recent years, Multidrug-resistant S.aureus infections have increased global morbidity and mortality8,9. There is an urgent need for effective antibiotics, with a detailed study on the mode of action10. The encouraging results of our previous experiments prompted us to further investigate the comprehensive mechanism of action of dBw against S. aureus to explicate its potential, as mechanistic studies of natural anti-bacterial compounds are not sufficiently studied worldwide. This research also intended to bring awareness of the therapeutical importance of D. inoxia and to preserve this plant from getting massively destroyed.

 

MATERIALS AND METHODS:

Test organism and plant:

Collection, preservation, extraction and purification of dBw from D. inoxia, procurement and preparation of S. aureus done from similar methods of our previous work4. All experiments were performed at 12.5µg/ml.

 

Treatment of bacterial cells with Dinoxin B withanolide:

S. aureus was treated according to Tandon et. al.,11. The cells were inoculated in nutrient broth and incubated for 10hr at 370C12. Then the fraction containing compound was added to the suspension, followed by centrifugation, after incubation at 370C for six hours, for further studies13.

 

SEM studies:

Treated and untreated cells were fixed with few drops of glutaraldehyde (2.5%) prepared in 0.1M sodium phosphate buffer (pH 7.4) for 2-4 hrs at 4°C. It was then washed 3 times using 0.1 M phosphate buffer, each of 15 min at 4°C. One percentage of osmium tetroxide was used as post fixative for 2 hrs at 4°C. The fixed samples were dehydrated using 30%, 50%, 70%, 80%, 90%, 95%, and 100% acetone. The bacterial suspension was mounted on the aluminum stubs using carbon adhesive tape, which was attached to metal stubs. These stubs were coated with gold-palladium in a sputter coater (JFC 1600; JEOL, Tokyo, Japan) at 20 mA and viewed in a scanning electron microscope (JSM 6490 LV; JEOL, Tokyo, Japan) at 15kV.

 

TEM studies:

Treated and untreated cells were fixed with few drops of glutaraldehyde (2.5%) prepared in 0.1M sodium phosphate buffer (pH 7.4) for 2-4 hrs at 4°C. It was then washed 3 times using 0.1 M phosphate buffer, each of 15 min at 4°C. 1% osmium tetroxide was used as a post fixative for 2 hrs at 4°C. The fixed samples were dehydrated using 30%, 50%, 70%, 80%, 90%, 95%, and 100% acetone followed by infiltration and embedding in Epon-Araldite. Ultra-thin sections of the sample were then cut and stained with uranyl acetate and lead citrate and viewed in a transmission electron microscope (Thermo Scientific Spectra 200) at 60kV.

 

Bacterial DNA extraction and agar gel electrophoresis:

This study was performed according to the pre-described protocol14. Cells of S. aureus were prepared and treated as defined above. The total bacterial DNA was extracted using the standard bacterial DNA isolation method15. The cells were centrifuged until a compact pellet was formed. The pellet was dissolved in 467 µl TE buffer {10 mM Tris-Cl, 1mM EDTA (pH 8)}. The dissolved pellet was further treated with 30µl of 10% SDS and 3 µl of 0.020g/ml Proteinase K. The suspension was incubated for 1 hr at 370C. Equal volumes of the above suspension were added to phenol and chloroform (1:1) and centrifuged at 5000rpm for 5mts. The upper layer was transferred in a fresh tube and mixed with 100 µl of 3M sodium acetate (pH 5.2). The mixture was added with an equal volume of chilled isopropanol and incubated overnight at 40C. After brief centrifugation, the pellet formed was dissolved in 70% ethanol. After further centrifugation, the pellet was air-dried and resuspended in TE buffer. Agarose gel electrophoresis of an equal amount of treated and untreated bacterial DNA was performed using 0.8% Agarose gel containing 0.5µg/ml of EtBr solution and the gel was visualized in Gel Documentation System.

 

Bacterial Protein extraction and SDS-PAGE:

The assay was performed following the process described by Sánchez et al., 200316, with some modifications. The preparation and treatment of susceptible bacterial cells were done as described above. After centrifugation, cells were dissolved in 0.1 M phosphate buffer solution (pH 7.4). Total protein was extracted by the freeze-thaw lysis method. This process was repeated 3-4 times for efficient lysis of cells. After cell lysis, 50µl of the cell suspension was dissolved with 10µl of SDS-PAGE loading buffer containing 0.2% bromophenol blue, 60% glycerol, 7.5% β-mercaptoethanol (BME), 0.3% SDS, and 300mM Tris 6.8. The mixture was then loaded (containing an equal amount of protein in treated and untreated cells) on SDS-PAGE gel prepared with 5% stacking gel and 10% separating gel. The gel was allowed to run at 100V until the dye front reaches the bottom of the gel followed by Coomassie Brilliant Blue (CBB) staining. It was then destained and photographed over white background. A Broad range Protein Marker of 3.5 to 205.0kDa, procured from the Banglore Genei lab.

 

Shelf Life of compound:

The agar well diffusion method17 was carried out to determine the shelf life of fractions containing dBw. It was employed on MHA plates after timed intervals of 6, 12, 18, and 24 months of storage. The plates were inoculated with the most susceptible bacterial suspension. Wells of 6 mm size were made on these plates by using a sterile cork borer. Each well was inoculated with 100µl of the purified fraction containing dBw. These plates were then incubated at 370C for 24 hrs. After incubation time, results were recorded in terms of zone of inhibition.

 

In vivo assessment of the immune modulatory potential of dBw:

a)Animal maintenance and dosage schedule:

Four to six weeks of old Balb/c mice (Male) with a nearby weight of 18-20gm were procured from the animal house facility of CSIR-CDRI, Lucknow. The animals were maintained in standard conditions with a temperature of 22±1oC, 12hr light and dark cycle, and could access food and water ad libitum. As per standard protocol, animals were acclimatized to laboratory conditions for 7 days before. This study was observed by the Institutional Animal Ethical Committee (IAEC) of Integral University, Lucknow under the endorsement number 22/12/IAEC/SPS/SOA and followed CPCSEA guidelines. The animals were divided into three groups and administered with the standard drug, dBw, and double-distilled water as ad libitum. The dosage and treatment schedule are given in Table 1. After 15 days, the mice were sacrificed after anaesthetization as per guidelines.The CFSE labeling method was adapted from a previously described protocol18. The splenocytes were cultured in 24-well plates at 1X106 per well in the presence of, Con A (0.1µg/ml), standard drug Levamisole (10-7M), and dBw (100µg/ml). Un-stimulated CFSE labeled cells were defined as controls. Cell incubation was done for 3 days at 370C and 5% CO2. The cells were then stained with CFSE and incubated for 20mts at 370C. Analysis was done using a BD flow cytometer with 488nm excitation and emission filters appropriate for fluorescing.

 

Cytotoxicity Assay:

The toxicity of fractionated ethanolic extract containing dBw was determined using MTT assay as described earlier by Mao et al., 201319. Murine alveolar macrophage cell line (J774A.1) was procured from the cell repository of National Center for Cell Science, Pune, India, and maintained in RPMI-1640 medium supplemented with 10% FBS (v/v) and 1% antibiotic-antimycotic solution (v/v) in standard humidified conditions constituted by 5% CO2 at 37°C. Briefly, murine macrophage J774 cells were seeded at a density of 104 cells/well in a 96-well plate and allowed to adhere overnight under standard conditions. Post adherence, media in each well was replaced with media containing different concentrations of D. inoxia extract (125µg/ml) and the plate was further incubated for 24, 48, and 72h. After incubation, the media in each treated group was decanted, MTT dye (5mg/ml; 10µl) was added to each well and the plate was incubated for 4h in humidified conditions. Subsequently, 100µL of Dimethylsulfoxide was added to each well and the plate was incubated for an additional 30 min at room temperature to allow solubilization of formazan crystals formed (if any). Finally, the absorbance of solubilized formazan crystals was recorded at 490 nm using a microplate reader (Bio-Rad, USA).

 

Percent viability was calculated by the given formula:

 

Percent Viability = E/C x 100, Where E is the absorbance of treated cells and C is the absorbance of untreated cells.

 

Statistical Analysis:

Experiments were conducted in triplicates and mean ± standard deviation was used to interpret all data. Graph Pad Prism software version 10 was used to examine statistical differences. Standard errors of the mean values were symbolized with the error bar.

 

RESULTS:

Electron microscopic studies by SEM and TEM:

Electron microscopic studies were conducted to understand the mechanism of action especially on cell shape and structure and bactericidal effect of dBw4. SEM images of untreated cellsshowed normal morphology, giving their spherical and regular shape while treated cells were found damaged and irregular (Figure 2). In the TEM micrograph (Figure 3), a cell transformed from its typical spherical shape to an irregular shape. We find a very interesting observation that cell wall and cell membrane damaged at specific points, while other part remains intact. It is suggested here that there are specific entry points of dBw which leads to the degradation of other metabolites and resulting in cells death.


 

Table 1: Treatment protocol of dBw on Balb/c mouse for assessment of T cell proliferation by CFSE Labeling. The animals were divided into three groups and administered with the standard drug, dBw, and double-distilled water as ad libitum.

Sl. No

Group

Number of animals

Treatment

Dose mg/kg

Route of Administration

Duration (days)

1

Control

10

DDW

-

Oral

15

2.

Standard

10

Levamisole

2.5 mg/kg

Oral

15

3

extract

10

dBw

300

Oral

15


 

Figure 2: SEM Micrograph of Untreated (Control) and Treated S. aureus. Untreated cells showed normal morphology, with regular shape, while after treatment, cells became irregular and damaged.

 

 

Figure 3: TEM Micrograph of Untreated (control) and Treated S. aureus. Treated cells show distorted cell walls giving rise to the leakage of cell substances resulting in cell death.

 

Study of DNA by Agarose gel electrophoresis:

Extracted DNA from treated and untreated bacteria, allowed to run on agarose gel electrophoresis. An intact DNA band was observed in Lane 1 of figure 4, whereas DNA from treated bacteria, appeared as a smear, which indicates that the dBw degrades the DNA (Figure 4, lane 2).

 

Figure 4: Agarose Gel Electrophoresis of Bacterial DNA (S. aureus). Lane 1- Control DNA and Lane 2- Treated DNA. Agarose gel electrophoresis was conducted to observe the DNA degrading ability of dBw.

 

Study of protein by SDS-PAGE:

Total protein content from both treated and untreated S. aureus was extracted and loaded in wells of SDS-PAGE. A Broad range Marker (Figure 5, lane 1) of 3.5 to 205.0KDa from Banglore genni wasallowed to run with Protein (lane1). Gel image showed (Figure 5) protein bands from untreated S. aureus were strong and clear (Lane 2). Seven different Sharp bands from 97.4Kda to 29.0 kDa were observed, suggesting different proteins present in S. aureus. 205 kDa light band, 97.4Kda sharp band, 66 kDa sharp, three light bands between 43 to 66 kDa were observed with some light bands from 29 kda to 43 kDa. There were also light bands between 14.3 kDa to 29 kDa. Whereas protein bands werefound as smeared in treated bacteria. Smeared appearance reveals the degradation of proteins (Figure 5, lane 3). These results suggest that the compound degrade cellular proteins by entering through the damaged cell wall and cell membrane resulting in cellular death

 

Figure 5: SDS-PAGE was implemented to identify the protein degrading ability of the dBw. Lane 1- Broad Range Marker, Lane 2- Untreated Bacterial Protein, and Lane 3- Treated Bacterial Protein in smeared appearance.

 

Shelf life of dBw by agar well diffusion assay:

The shelf life of dBw was studied continuously for two years by storing it in a refrigerator (40C) and determined against S. aureus using agar well diffusion assay. The result was recorded in terms of ZOI after every 6 months of storage. dBw showed remarkable inhibitory activity against the tested strain at every studied time interval. There is no significant change even after 24 months in which percentage of decrease (6.89, 13.79, 17.24, 20.68%, in 6,12,18 and 24 months respectively), is very mild, and that too without any preservative (Figure 6). These results indicate good stability of dBw, suggesting its ability as an herbal antibacterial medication with significant shelf life.

 

Figure 6: Shelf life of dBw against S aureus (ATCC 25923) by using agar well diffusion assay. The outcomes were recorded in terms of Zone of Inhibition after every 6 months of storage

Study of the anti-inflammatory nature of dBw was assessed by labeling the solenocytes of experimental animals by CFSE:

The anti-inflammatory nature of dBw was assessed by labeling the solenocytes of experimental animals by CFSE. Results (Figure 7) revealed that mitogen Concavalin A stimulation induced the significant T cell in all the experimental groups. However, the stimulation by dBw demonstrated the suppression of T lymphocytes in treated animals indicating the anti-inflammatory potential.

 

 

Figure 7: CFSE labeling of Balb/c mice splenocytes in vitro stimulated with ConA and plant extract. The cells were seeded at the concentration of 1x105 cells/well and stimulated with mitogen concavalin A and dBw. The incubation period lasted for three days at 37°C with 5% CO2. The cells were thereafter stained CFSE to monitor the T cell suppression induced by plant extract.

 

The cytotoxic effect was investigated on J744 cell lines by using MTT assay:

The cytotoxic effect (Figure 8) of dBw at a concentration ranging from 50 to 400μg/ml was investigated on J744 cell lines by using MTT assay20. J774 is a murine macrophage cell line obtained from a spontaneous tumor established in a female BALB/c mouse. It consists of adherent slow-migrating monocyte-macrophages with the ability to kill foreign cells21 Cytotoxicity activity of dBw was evaluated after 24, 48, and 72hrs incubation. The compound showed mild toxicity at higher concentrations, with increasing time intervals. Toxicity was insignificant at the highest concentrations of 400µg/ml, where cell viability was more than 80%. These results confirm the therapeutic applicability of dBwdue to its very low toxic effect.

 

Figure 8: Dose and Time-Dependent Effect of dBw on Cell Viability as Determined by MTT assay.The 1 × 106 cells/ml were inoculated in 96-well culture plates and then incubated with increasing concentrations of the fraction containing compound (50μg, 100μg, 200μg, 300μg, and 400μg) for 24, 48 and 72 hrs at 37°C in a CO2 incubator.

 

DISCUSSION:

The result of electron microscopy studies conducted by using SEM (Figure 2) and TEM (Figure 3) suggested that cells treated with dBw were found distorted, with the damaged cell wall and the cell membrane. This also leads to the degradation of other metabolites and resulting in cells death. Evidence of this phenomenon we found in the results of Agarose Gel electrophoresis (Figure 4) and SDS-PAGE (Figure 5). In a similar study, Xu et al., 201622 revealed that natural compound degrades bacterial DNA and protein. Our docking results23confirm the above described “specific points” as Penicillin Binding Protein. Docking results showed a high affinity of dBw to membrane proteins, specifically to PBP. We find a similarity between dBw and ampicillin for binding score and molecular interaction. This predicts dBw showing a similar mechanism as of ampicillin, a beta-lactam antibiotic. Notably, as hydroxyl groups promote antibacterial interactions, the involvement of more hydroxyl groups in docking interactions promotes the therapeutic potentiality of dBw. The presence of unsaturated lactone ring and glycosidic group, the exemplified features of dBw promote its binding affinity. Shelf-life (Figure 6) studies showed that dBw is stable up to 24 months without any preservative at 40C. These results suggesting the ability of dBw, as an antibacterial medication with significant shelf life. Shelf-life results could give a solution for chemical-based antibiotics, with a shorter life span. The main purpose of identifying the shelf life of pharmaceutical products is to ensure the efficacy and quality of active compounds present in them.Anti-inflammatory studies by using the CFSE technique (Figure 7) showed that there is suppression of T lymphocytes in treated animals, indicating the anti-inflammatory potential of dBw. A recently published report by Tan et al.24 indicates the presence of anti-inflammatory withanolides in Datura leaf extracts projecting towards the potential of dBw as a novel anti-inflammatory therapeutic agent. MTT assay results (Figure 8) confirm the therapeutic applicability of dBw due to its insignificant toxic effect. We have already defined by our article by George et al 202123 that Computer-aided selection of compounds helps to minimize the synthetic and biological testing efforts in drug design25, sothe study of Drug likeness of dBw by Lipinski’s Rule was carried out using Pre ADMET Pharmaceutical and pharmaco dynamics evaluation on dBw using software tools revealed its drug-likeness and effectiveness as an oral drug. With an 82.57% of absorption rate the compound could be better absorbed from the intestinal tract upon oral administration. With higher Plasma Protein Binding affinity it can freely cross membranes and bind to the intended molecular target26,27. Physiochemical and pharmacokinetic properties with non-mutagenicity along with lesser CNS side effects highlights its drug-likeness.

 

CONCLUSION:

Medicinal plants being used as a rich source of bioactive and therapeutic compounds since early civilization. This study proved Dinoxin B withanolide (dBw) as an effective herbal antibiotic due to its safety and efficacy. dBw degrades bacterial DNA and protein which results in cell death. Possible entry point is through Penicillin Binding Protein. This study supports our bioassay results and the bactericidal nature of dBw. The anti-inflammatory ability, low toxicity and good shelf life suggesting it to be an antibacterial drug candidate. Since natural active compounds have a more complex structure, they are less likely to be affected by bacterial resistance. There is further need for detailed animal studies and human trials to make it effective antibiotic for human welfare.

 

ABBREVIATION:

dBw, Dinoxin B withanolide; MDR, Multidrug-Resistant; MRSA, Methicillin-resistant Staphylococcus aureus; LC-MS, Liquid Chromatography-Mass Spectrometry; SEM, Scanning Electron Microscopy; TEM, Transmission electron microscopy; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MTT, 3(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; CFSE, Carboxy-fluorescein -succinyl- ester; DDW, Double Distilled water; PBP, Penicillin Binding Protein.

 

CONFLICT OF INTEREST

The authors have no conflicts of interest regarding this investigation.

 

ACKNOWLEDGMENTS:

The authors are thankful to Pro-Vice-Chancellor, Amity University Uttar Pradesh, Lucknow; Dr. Manodeep Sen, Associate Professor, Ram Manohar Lohia Institute of Medical Science, Lucknow; Dr. Atin Singhai, Associate Professor, KGMU Lucknow; Dr. Mukesh Kumar from BBAU Lucknow; for providing the necessary facilities for experiments.

 

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Received on 21.02.2022            Modified on 23.10.2022

Accepted on 26.04.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4505-4511.

DOI: 10.52711/0974-360X.2023.00734