Phytochemical derivatives and secondary metabolites rich Rhizophora mucronata as an active anti-oxidant and anti-bacterial agent against multi drug resistant bacteria

2.1 Chemical and glass wares

Basic chemicals of this work were used in this research was purchased from Suresh Scientific & Co. In addition, some solvent and media were procured from Merck India PVT limited, Sub organization of Ponmani, Tiruchirappalli, Tamil Nadu, India. Detection and identification of phytochemicals and anti-oxidant using various chemicals were chosen by Suresh Scientific & Co, Tiruchirappalli, Tamil Nadu, India.

2.2 Preparation of R. mucronata samples

Initially, look out and carefully pluck the fresh, undamaged, well grown R. mucronata leafs for detection of phytochemicals and anti-oxidant property from Muthupet Mangrove Region, Thiruvarur District, East Coast of Tamil Nadu, India. The surface contaminant was removed by tap water and followed by distilled water. Followed by alcohol wash made to remove surface contamination and free floating microbes which are abundantly present in surface of the plant. To finalize the powder formation, the dried samples were ground and taken safe and use for extraction (Rajivgandhi et al., 2021).

2.3 Extraction of crude extract preparation

Initially 1:1 ratio concentration, about 1 Kg of R. mucronata powder was mixed with 1L of ethanol solution to keep in soxhlet instrument with 100 ℃ with 12 h incubation. After incubation, the process was monitored and cools the process when the colour of grey or green turn to yellow, clear yellow in requested time interval. It may be taken 12–15 h for complete the extraction process. Then, the cooled solvent separated in upper portion and mixed R. mucronata leaves were settling down separately in bottom of the flask. Both the layers of the samples were separately collected in1, 000 mL beaker using what man No.1 filter paper as a filtrate material. After, the liquid phase of the organic phase was dried in hot air oven at 45 ℃ and finally use rotary evaporator to complete dry the samples. The yield of the dried product was detected universal formula which is available in previously reported evidence (Nur et al., 2018; Ukwubile et al., 2020).

2.4 LC-MS analysis of phytochemical and anti-oxidant properties of dried extract

Based on the NIST library comparison, the extracted R. mucronata extract was performed by LC-MS spectroscopy for detect the alkaloids, flavonoid, phenols, bioactive materials and other organic materials. The interpretation was followed by previously published articles of Jamal et al. (2021). The program was set at 60-120℃ for 5–30 min respectively and also, the 200–300 ℃ of constant temperature was also set, and crosscheck the injector coupled detector was attached with this instruments. After set these manually, the capillary column was checked thoroughly which it fixed automatically or not for 30 m × 200 μm × 0.30 μm. Manually set the flow rate that it maintains 1 mL/min and gas carrier of 30 cm/s with linier velocity was made. Finally, the sample was put in in the respective tray and run the LC-MS machine for detect the chemical molecules of R. mucronata extract. After scanning, the result was checked with NIST library with retention time and occupation percentage of the extract.

2.5 Detection of phenol and flavonoid content

For detection of the phenols and flavonoid content availability in R. mucronata extract was performed by UV-spectroscopy reading analysis and followed by universal method of folin-ciocalteu’s (Varaprasadham et al., 2010). 1:5 ratio of R. mucronata extract and 10 fold dilution of folin-ciocalteu’s reagent were taken together in 100 mL test tube. Followed by addition of Na₂ CO₃ into the same mixture tubes before the previous reaction was done in tube and shaken gently to mix equally. The mixture of the sample was shaken vigorously to mix each other using shaking incubator with 1 h time interval. Then, the mixture tube was performed to scanned by UV–vis spectroscopy for detection of presented phenols and flavonoid contents of the R. mucronata extract. For working control, the various concentration of 10–100 ug/mL of gallic acid was used, as same as without extract of the gallic acid was acted as a negative control. After O.D value, the result was checked that the 1ug gallic acid was equal to conversion of 1 mg tested sample or not.

Further, the flavonoid derivatives of R. mucronata content were detected by colorimetric analysis performed by earlier report of Atamgba et al. (2015). As like phenolic detection, the 100 mL tube was filled by 200 µl of extract and 75 µl of sodium nitrate and mixed slightly for generate the initial process. Followed by addition of 1: ratio of AlCl₃ and 1% potassium iodide combination was taken into the same tube, which added drop by drop through wall of the tubes gradually. The mixture sample was kept in shaking incubator at 1 h for complete mixture of the sample. Then, the sample was performed by analysis of O.D value using colorimeter at 520 nm. For flavonoid content detection, the rutin was performed as a control and without Avicennia sp. extract of the rutin maintained for negative control. As same as the 1 ug of rutin concentration equal to 1 mg of the sample or not was calculated.

2.6 Anti-oxidant effect of R. mucronata extract

The extracted R. mucronata extract was utilized in Phosphor-molybdenum method for detection of their anti-oxidant activity based on the procedure of recent report of Trouillas et al. (2003). Each two tube were taken with 5 mL of sodium phosphate and ammonium molybdate, and then addition of 5 mL H₂SO₄ into the available tubes and it were mixed thoroughly. In one tube, the sample mixture of each 2 mL was taken and put in water bath at 100 ℃ for 1 h. The shaking water bath facility is good for the solution mixture and then cools after incubation. Then, 1 mL of the cooled solution was taken in new tube and mixed with 10 µl of R. mucronata extract and maintained at room temperature 15 min time interval. Between the time interval, the anti-oxidant property was formed and it monitored by spectrophotometer with the O.D value of 610 nm. For anti-oxidant activity, ascorbic acid as a positive control and it used to comparison of available anti-oxidant property of R. mucronata. Finally, the result was considered as 1 µg ascorbic acid is equal to 1 mg of R. mucronata extract.

2.7 Free radical scavenging method by using DPPH

To check the amount of free radical that present in the R. mucronata extract was detected by DPPH assay followed previously published article procedure of Tlili and Sarikurkcu (2020). The sterile tubes were filled by 150 µl of DPPH solution and various concentrations (10–100 µg/mL) of R. mucronata extract. As same as 5ul butylated hydroxyl toluene with DPPH without addition of extract was taken in separate tube for control. All the experimental tubes were maintained at room temperature for 1 h and formed scavenging properties were scanned by UV-spectrometer at O.D value of 610 nm. The anti-oxidant activity result was interpreted based on the control tube result using available formula, $$\text{Percentage}\text{of}\text{scavenged}\text{activity}={\text{Control}}_{\text{OD}}-{\text{Test}}_{\text{OD}}/{\text{Control}}_{\text{OD}}\times 100.$$

2.8 Bacterial inactivation using agar well diffusion method

The disruption of bacterial growth in the muller hinton agar plate was observed with zone formation by agar well diffusion method followed by Khalid et al. (2021). Briefly, the 24 h matured K. pneumoniae and A. baumannii were spread on the fresh muller hinton agar plate and wells were punched on the agar surface. Then, 50–250 µg/mL of R. mucronata extract was added into the wells and maintained at 37 ℃ for 24 h incubation. After incubation, the zones around the wells were calculated to confirm the anti-bacterial ability of R. mucronata extract. The zones were calculated in diameter.

2.9 Microbroth dilution method

The confirmation of anti-bacterial ability of R. mucronata extract was further validated by microbroth dilution method to detect the effective concentration that has highest inhibition of tested K. pneumoniae and A. baumannii (Ramachandran et al., 2019). Shortly, the nutrient broth was prepared freshly and added into the separate 96-well polystyrene plates which have the quantity of 100 µg/mL concentration. Followed by both the pathogens with 10 µg/mL were added into in respective 96-well plates. Subsequently, 25–250 µg/mL concentration of R. mucronata extract was treated in all the wells except control wells as filled fresh broth with pathogens only. Then, all the plates were maintained at room atmosphere for 24 h time interval. Finally, the lowest concentration of the R. mucronata extract inhibit the bacteria in high level was noted and fixed as a minimum inhibition concentration and this concentration was used for further invitro experiments.

2.10 Outer morphology inactivation of R. mucronata extract

The outer morphology of K. pneumoniae and A. baumannii was disrupted by the R. mucronata extract through minimum inhibition concentration was proved by scanning electron microscope, the procedure was followed by earlier report of Famuyide et al. (2020). Initially, 48 h matured cultures of K. pnumoniae and A. baumannii were fixed by 4% glutaraldehyde using cover glass and allowed 6 h for complete fixation. After complete fixation, the cover glass was treated by dehydration using alcohol in the preparation of 30–100%, each process conducted with each 10 min time interval. Finally, the dehydrated cover glass was washed by n-butanol and treated by t-butanol until complete fixation into the pathogens. Then, the samples were put on deep freezer for 6 h. After the time interval, the samples were taken out and cool, then seen under scanning electron microscope under 40 × magnifications (Zeiss, Japan).

3.1 Phytochemical screening of R. mucronata extract

Based on the LC-MS result, we have confirmed that the R. mucronata extract has more contents of phenols, terpens, alkaloids, flavonoids, essential oils, organic materials, some hydrocarbons and bioactive metabolites (Fig. 1). Based on the NIST Wiley, the confirmed bioactive ability and anti-oxidant activity of the phytochemical derivatives were screened from 35 peaks, and confirmed peak was 11 only, as per previous report of Ukwubilea et al. (2019); Casuga et al. (2016), the peaks related with phytochemicals were found based on the retention time and occupied percentages including coumarins, 3-hydroxy-4-phenyl, di-tert-butyl-2-hydroxybenzylidene-amino-2-indanol, tritriacontane, triterpenoid saponins, oxalates, nonyl, 1,4-diaza-2,5-dioxo-3-isobutyl bicyclo[4.3.0] nonane cycloicosane, tetracosahexaene, 2,3-propanetriol,diacetate, benzene, 1,1′-[oxybis(methylene)]bis, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpr, 1,2,3-trimethyl, hexadecanoic acid, pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpr sitosterol and 12,15-octadecatrienoicacid. All those resulted compounds, derivatives and essential oils were conveyed the information about available properties of R. mucronata extract has excellent reservoir to biological properties. Also, it suggested that the R. mucronata is the best choice to discovery of some novel compounds which act as an inhibition role against pathogenic bacteria. Previously, Rajivagndhi et al. (2018) reported that the R. mucronata is the essential plant to synthesize biological properties. It exhibited some excellent bioactive compounds based on the different environmental parameters and fluctuated atmospheric nature. The undesirable environment may influence the plant stimulants and it produces some excellent biological molecules (Han et al., 2007). Previously, Jamal et al. (2021), reported that the extract of R. mucronata inhibit the quorum sensing molecules and interfere the group formed bacteria. LC-MS based phytochemical detection was very important method to find the bioactive metabolites and it act as an effective identification method.

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Fig. 1

Detection of phytochemical and bioactive compounds of mangrove plant of R. mucronata extract by LC-MS analysis.

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3.2 Measurement of phenolic and flavonoids contents of crude extract

The enormous amount of phenols and flavonoid contents available in the R. mucronata extract were confirmed after reading with UV-spectrometer. In addition, the result was also interpreted with controls result and confirmed that the R. mucronata extract has abundant phenols and flavonoid contents. Concisely, the R² = 0.986 percentage of phenolic content yield was found from the R. mucronata extract as well as the respective values of R² = 0.977 was found for gallic acid, their linearity of the calibration curve was started ranging 25–250 µg/mL concentration for both the samples. In addition, the values of R² = 0.994 was obtained from R. mucronata extract for flavonoid content. Whereas the exhibited standard value curve of rutin was R² = 0.969, it was compromised to the extract due to the lowest value yield of concentration. Based on the interpretation with control results, the available phenol and flavonoid contents were higher in R. mucronata extract instead of protein and sugar content. Based on the reported evidence, the R. mucronata extract was used for further study of this current research. The depicted photogram results of standard gallic acid (Fig. 2a), rutin (Fig. 2c) and available for phenols (Fig. 2b), flavonoid (Fig. 2d) contents were effectively available in Fig. 2. Similarly, marine mangrove plant of R. mucronata extract as an excellent reservoir for producing high level of phytochemical compounds including phenol and flavonoid derivatives (Santini et al., 2015). In addition, it also exhibited very high compared with terrestrial plant extract. It may be happened by influence of marine environmental parameter and nutrients. Because, marine has unpredictable environmental condition and also often fluctuated of intertidal capacity (MacFarlane and Burchett, 2000; Varaprasadham et al., 2010). Further, the protein and sugars percentages of this study were also clearly confirmed with lowest level, and it confirmed that the R. mucronata can be used as a future drug discovery target. Previously, Leen et al., 2020 and Aseer et al. (2015) reported that the R. mucronata is the excellent mangrove plant, and used for discovery of novel compound due to the unfavourable atmospheric nature including nutrients, minerals, temperature, carbon, pH and various stress conditions.

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Fig. 2

Measurement of the phenolic and flavonoid contents of the standard control values of gallic acid (a) and rutin, available phenol (c) and flavonoids (d) of the mangrove plant of R. mucronata extract by UV-spectroscopy.

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3.3 Anti-oxidant and DPPH radical scavenging activities

As like as phenol and flavonoids, the anti-oxidant and formed free radical activities were shown excellent levels in R. mucronata extract, and the results were confirmed that the extract has more anti-oxidant and hydroxyl scavenging activity. Usually, the abundant level of carbohydrate present in the plant extract was usually produced more biomedical and pharmaceutical properties (Ramachandran et al., 2020). As like as, the present result was expected with excellent bioactive compounds synthesizers and it has rich anti-microbial anti-oxidant, larvicidal, immunomodulatory effect, anti-cancer, and other biological activities. In this study, the test sample was closely correlated with controls of gallic acid and rutin at 300 µg/mL. Recently, Suganthy and Pandima Devi (2016) reported, the high content of phenol and flavonoids rich plant extract of R. mucronata may produce excellent anti-oxidant activity (Fig. 3). As followed the previous report, the current study of marine mangrove plant R. mucronata extract was utilized to perform their total antioxidant and DPPH assay using invitro experiments. After comparison, the plant extract was shown very high in anti-oxidant activity and DPPH scavenging assay than the result of control ascorbic acid. The result was more favor to the inter relationship between the sample concentration and antioxidant values. Evidently, 86% of the antioxidant activity and 75% of DPPH scavenging activity were observed for plant extract as same as 76% and 68% were observed against ascorbic acid respectively at 350 and 300 µg/mL concentration (Fig. 4). All the results were proved, the concentration based anti-oxidant activity of control and test samples were more correlated. There were no significant differences for both the samples at tested concentration. Similar report of excellent anti-oxidant and DPPH assay results were reported by Gurudeeban et al. (2015) based on the tested concentration in control and tested samples. The result was agreed by Youssef et al. (2023), the reactive oxygen species and reactive nitrogen species rich plant bioactive compounds or chemical derivatives have played a role in increased anti-oxidant activities in the DPPH assay process, and also decreased the presence of active molecules, carbohydrates, proteins, lipids and protein contents. Therefore, the current results were more useful to the plant extract of R. mucronata for drug discovery research against various biomedical applications. Based on the all results, this study was proved that the mangrove plant Rhizophora mucronata has excellent phytochemical and anti-oxidant producer. It suggested that the research was more helped to future drug discovery research. The supportive result evidence was reported by Jairaman et al., (2019) and DPPH assay of R. mucronata was observed. Also, Vasanthakumar et al., (2019) documented that the unpredictable environmental factors were also influenced the phenols, flavonoids, anti-oxidant and DPPH radical scavenging activity. This statement was also supported by Shekhar Das et al. (2020), and mangrove region was very difficult to understand due to the fluctuated atmospheric nature.

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Fig. 3

Detection of total antioxidant activity of R. mucronata extract by invitro analysis.

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Fig. 4

Detection of DPPH scavenging activity of R. mucronata extract by invitro analysis.

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3.4 Anti-bacterial inactivation

The inactivation of bacterial growth using R. mucronata was exhibited 24 mm and 26 zones of inhibition for K. pneumonia and A. baumannii by agar well diffusion method (Fig. 5a, b). Initially, there are no any zones for below 25 µg/mL concentration. Based on the used concentration, the bacterial growth was arrested significantly in increasing concentration. The zone of inhibition against both the pathogens results were effectively presented in Fig. 5c. In addition, the 250 µg/ml concentration was more efficient to interact with bacterial surface through surface properties. As both the bacteria were gram negative bacteria, the thick theichoic acid layer and lipid contents were digested by R. mucronata extract through the charged particles. Previously, Zhou et al. (2022) reported that the R. mucronata has more phenol, flavonoid and bioactive compounds, all the compounds were efficiently bind with negative charges containing surfaces of the K. pneumonia and A. baumannii. So, the extract was entering into the bacteria and arrests the growth of the bacteria due to the influence of cytoplasmic granules, polysaccharide degradation and nucleolus deformation. Based on the previous report, the excellent permissible ability of the R. mucronata extract arrested the bacterial growth completely (Hardoko et al., 2016). Therefore, the initial screening of the anti-microbial activity result was more favour to R. mucronata extract, and it was excellent anti-bacterial agent against K. pneumonia and A. baumannii.

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Fig. 5

Anti-microbial activity of R. mucronata extract against K. pneumoniae (a) and A. baumannii (b). The various concentration of R. mucronata extract and their zone of inhibition level against K. pneumoniae and A. baumannii (c).

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3.5 Microbroth dilution method

The 96-well plate method of minimum inhibition concentration detection was very efficient method to detect the inhibition concentration of bacteria (Rinto et al., 2022). In our result, the inhibition 90% and 92% of inhibition against K. pneumoniae at 200 µg/mL and 250 µg/mL (Fig. 6) were observed. The bacterial inhibition was confirmed as concentration-dependent due to the visible observation in the 96-well plate. The plates between before and after treatment, the more turbidity was shown at 200 µg/mL and 250 µg/mL treated wells for K. pneumoniae and A. baumannii. In addition, the turbidity was significantly increased in increasing concentration when compared with initial lowest concentration. So, weather the inhibition was based on the concentration dependent or not was confirmed by the result of O.D values. The calculated spectrophotometric O.D values were more favour to the R. mucronata extract, and shown the highest inhibition percentages were observed in higher concentration. The initial death cells were started at 25 µg/mL concentration for K. pneumoniae, 50 µg/mL concentrations for A. baumannii. The half inhibition of 56% and 52% were found at 100 µg/mL concentration for both the bacteria. Therefore, the result was confirmed that the 200 µg/mL and 250 µg/mL concentration for K. pneumoniae and A. baumannii were fixed as a minimum inhibition concentration. Also, these minimum inhibition concentrations were used to damage or cell cycle arrest or intracellular and extracellular destruction in gram negative bacteria. Recent reports of Luksamee et al. (2022); Yunos et al. (2021), the concentration dependent inhibition of R. mucronata extract as an excellent plant source to eradicate the multi-drug resistant bacteria. Similarly, Gurram et al. (2022) also reported that the R. mucronata has potential anti-bacterial components and it was confirmed by GC–MS analysis. Based on the supportive evidence of antimicrobial property results of agar well diffusion and minimum inhibition concentration experiments, the selected R. mucronata is an effective anti-bacterial agent.

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Fig. 6

Detection of concentration dependent and lowest inhibition range effect of R. mucronata extract against K. pneumoniae and A. baumannii by microbroth dilution method.

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3.6 Outer morphology damages by SEM

The anti-bacterial effect of R. mucronata was further evaluated against outer morphology destruction against both the K. pneumonia and A. baumannii. As per altered outer morphology result, the plant extract containing bioactive compounds were influenced the bacterial growth through surface charges. The positive charges of the active chemical components were effectively bound with negative charges on bacterial surface through electrostatic interaction. After, the outer cell membrane was degraded and it lost their original size and shape. If both the bacteria as gram negative, the outer lipid layer was compromised and thioctic acid was digested. When compared with control (Fig. 7a, c), the compromised cell membrane structure with changed morphology, size and shapes were found (Fig. 7b, d). As both bacteria are gram negative, rod shape, the control cells were clearly exhibited with original size and shape of the morphology after 24 h, whereas the belbing formation, crashed cells, destruction morphology, leakages of cytoplasmic materials with absence of rod shape was shown in R. mucronata treated K. pneumoniae and A. baumannii were clearly observed. The morphological changes of gram negative bacteria due to the plant bioactive compounds, chemical constituents were reported by Fenella et al. (2017). Recently, the unfavourable marine environment supplied the additional nutrients to R. mucronata to produce potential bioactive metabolites against various pathogenic infections (Zhou et al., 2022). The outer morphology damages due to the effect of the plant materials were agreed by Famuyide et al. (2020). Altogether, all the invitro anti-bacterial inhibition and morphological damages study results were clearly stated, the marine mangrove plant R. mucronata as an efficient plant materials which can be used for inhibition of various bacterial infections through drug discovery process.

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Fig. 7

Outer cellular morphological differentiation of control and R. mucronata extract treated K. pneumoniae (a) and A. baumannii (b).

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