Photocatalytic degradation and anti-cancer activity of biologically synthesized Ag NPs for inhibit the MCF-7 breast cancer cells

2.1 Preparation of plant extract

Well grinned plant leaves were taken and mixed into the sterile 100 millipore water and continually heated at 80 ℃ for 1 h. After, the process was cool and the fine liquid format of the sample was filtered using whatman No. 5 filter paper. Then, the sample was mixed in methanol of soxhlet experiment procedure and run 3 h with room temperature. Then, methanol was removed gradually and collected filtrate was maintained under reduced pressure at 45 ℃ for 1 day with the help of rotary evaporator. Finally the color changes of the sample turn to darak green was noted and used for synthesis of nanoparticle process.

2.2 GC–MS analysis

The active compounds present in the Rhizophora mucronata extract was spectroscopically analyzed by GC–MS (GKR international, GmbH, and Darmstadt, Germany). In GC–MS, varian chrompack CP-3800 GC/MS/MS-200 equipped with split-splitless inhector, 20 m × o.25 mm, I.D of 0.25 µm thickness of film was used. In addition, the non-polar column was used for fractionation. The following conditions were set in the analyzed GC–MS: The detector temperature − 260–280 ℃; injector temperature – 250 ℃; carrier gas He (1 mL/min), split ratio – 30, injection volume 0.2 µL, Mass range m/z = 20–450. The oven temperature was maintained at 120 ℃ for 2 min; programmed to 280 ℃ at rate of 5℃/min. the consent temperature of 280 ℃ for 15 min was used. The film was coated with non-polar column and also the oven temperature was maintained at 30–380 ℃ time intervals. Additionally, 70 eV ionization voltage of ultra-helium was acted as a gas carrier with flow rate of 1.0 mL/min (Ramachandran et al., 2020).

2.3 Measurement of phenol and flavonoid properties

The Folin–Ciocalteu’s mediated phenolic properties in the Rhizophora mucronata extract was measured by UV-spectrometer analysis using Gallic acid as standard (Rajivgandhi et al., 2020). The 0.5 mL of Rhizophora mucronata extract was diluted by Folin-ciocalteu’s solution with required concentration and followed by 2ML of Na2CO3. The diluted solution was shaken vigorously for 5 min and maintained in dark room at least 30 min. Finally, read on spectrophotometer at 510 nm. Consecutively, the final concentration (10–100 µg/mL) of gallic acid was used as a positive control with same procedure. The result was interpreted with 1 µg of and it equal to 1 g of the powder. Further, the amount of flavonoid content in the Rhizophora mucronata extract was estimated by colorimetric assay using rutin as a standard. The short method of flavonoid detection was followed by Rajivgandhi et al. (2020), and 150 µL sodium nitrate and 400 µL of extract solution taken together with with 150 µL of sodium nitrate was taken initially in a fresh tube. 10% w/v aluminium chloride and KI also added into the side wall of the tube. Then the result was followed by incubated at room temperature in dark condition for 30 min as same as for control also. After incubation, both the samples were measured at 540 nm O.D. 1 mg of flavonoid result was considered when it compared with 1 µg gallic acid.

2.4 Anti-oxidant activity

Phosphor molybdenum method was performed to detect their anti-oxidant activity (Ramesh et al., 2018). Initially, sodium phosphate, ammonium molybdate and sulphuric acid were mixed thoroughly at 97 ℃ for 45 min in water bath. 10 µL of purified extract was diluted into the 1 mL of prepared solution and allowed to stands for 30 min. After incubation, the mixed solution produced anti-oxidant rate was calculated by biophotometer (Sigma, India) at the wave length of 590 nm O.D. Finally, the anti-oxidant activity of tested material was compared with ascorbic acid control result for proper interpretation and the anti-oxidant activity was considered to microgram of ascorbic acid as equivalents to purified fraction.

2.5 DPPH free radical scavenging assay

The DPPH radical scavenging activity of purified Rhizophora mucronata extract was carried out according to the previous method of Rajivgandhi et al. (Rajivgandhi et al., 2020). Various concentration 50, 100, 150, 200, 250, 300, 350 of purified plant extract was aliquot and 3 µL of butylated hydroxyl toluene, which acted as a standard control were taken together in the 200 µL DPPH mixed ethanol tube. The treated and untreated sample tubes were shaken rapidly and maintained at dark condition in room for 30 min. the solution of ethanol containing tube served as a blank. After incubation, the anti-oxidant activity of sample tubes was measured using spectrophotometer. Finally, the test sample result was compared with standard control and calculated the differentiation, $$\mathit{DPPH}scavenged\left(\%\right)=\left[\left(\mathit{Control}OD-TestOD\right)/\left(\mathit{Control}OD\right)\times 100\right]$$

2.6 Synthesis and characterization of Ag NPs

Aqueous solution of 1: 10 ratios of plant material (1 mL) and Ag NO₃ (10 mL) were used to silver nanoparticle synthesis in 50 mL with the help of reported article (Saravanakumar et al., 2019; Kumar et al., 2019). The sample was put it in water bath with one hour and the pH was adjusted properly 1 N H₃PO₄ between the gaps. After one hour, the changed yellow to orange or brown color and it suggested the mixture of the materials were silver nanoparticle. Consecutively, the synthesized Ag NPs was first checked by UV–vis spectroscopy. Synthesized Ag NPs and their electrical vibration connectivity was detected by XRD at the wave ranges of 10°-80°. After, surface morphology was scanned by scanning electron microscope

2.7 Anti-cancer activity analysis

2.8 Photocatalytic activity

The efficiency of degradation of Rhodamine B (Rho B) and Crystal violet (CV) by Ag NPs was evaluated using the method described by Khataee and Kasiri (S.K., et al., 2017). 1 mg/L of Rho B and CV solution was prepared using deionized water. At the same different concentration of Ag NPs (5, 10 and 15 mg/L) was added into the conical flask containing different concentration of Rho B and CV solutions. This experimental setup was maintained by exposing continuous white light fewer than 160 rpm for 3hrs. The aliquots of 5 mL from experimental setup was collected every 30mins to analysis the concentrations of Rho B and CV. The concentration of Rho B was analyzed using UV–visible spectrophotometry. The lambda max values of Rho B and CV was determined as 553 and 585 nm respectively. The percentage of dye degradation was calculated by following formula (1)

3.1 Identification of chemical composition

The exhibited peaks of the GC–MS were shown with 35 different peaks, and all the peaks were exhibited with different pytocompounds. All the compounds were clearly confirmed after interpretation of Sun Yat-Sen University Wiley library, and the compounds have potential biological properties. Among the various peaks, the alkaloids, flavonoids and phenolic compounds were screened separately and followed by bioactive compounds screened separately. Compared with previous reports, the phenol and flavonoid rich compounds have significant anti-oxidant activity (Majeed et al., 2019; Yang et al., 2020). This statement was good agreed by recent studies of Gomathi et al. (Gomathi et al., 2020); pytocompounds have more antioxidant activities. Previously, Rhizophora mucronata extract has rich phenol and flavonoid contents and it possesses excellent anti-oxidant activity with significant biological properties (Rajivgandhi et al., 2020; Rajivgandhi et al., 2020). They anti-cancer ability of the compound names are 7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, Pyrrolo[1,2–a]pyrazine-1,4-dione, hexahydro-3-(2-methylpr, 1-Octadecene, Chirysin 1, 2, 3, Phathalate and Phenol, 2,4-bis(1,1-dimethylethyl). The retention times, occupation area and occupation percentages were shown with 30,42, 28.34, 20, 15, 15, 67, 12, 32 and 24, 78 and 40,6758, 32,4765, 60,7098, 28, 6754, 24, 5674, and 23, 45,342 respectively (Fig. 1). Further, the identified compounds might be potential anti-cancer compounds compared with other sources unfavorable conditions of nutrients, and salt concentration, and other contents.

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Fig. 1

GC–MS analysis reports of available chemical compounds in the Rhizophora mucronata extract.

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3.2 Measurement of phenol and flavonoid properties

After careful comparison with positive controls of gallic acid (R2 = 0. 9661) for phenolic and rutin (R2 = 0.9690) for flavonoid, the linearity calibration curves of phenolic and flavonoid contents were very high at 10–100 μg/mL (Fig. 2a, b). It was comparatively very higher than other plants reported previously. Therefore, when the results were compared with sugar and protein levels, the phenol and flavonoid properties were very high. The agreed result was reported by Yang et al. (Aji Jovitha and Deivasigamani, 2020) and the marine mangrove plant Rhizophora mucronata has more phenolic and flavonoid derivatives compared with other terrestrial plants. It may be influenced by unpredictable mangrove environment including high temperature, pH, NaCl, different ratio of carbon–nitrogen sources and other stress conditions. This extreme condition plant has the ability to produce excellent bioactivity compounds, particularly anti-cancer compounds (Khorrami et al., 2019). So, the Rhizophora mucronata mediated synthesis is an important source for improved bioactivity of nanoparticles synthesis.

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Fig. 2

Measurement of phenolic and flavonoid contents of the Rhizophora mucronata extract.

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3.3 Measurement of anti-oxidant activity

The increasing level of anti-oxidant activity of plant extract was suggested that the plant possess high phenolic and flavonoid content. As well as, the increased content of phenolic and flavonoid derivatives led to stimulate the increased anti-oxidant activity (Majeed et al., 2019; Ramachandran et al., 2020). Based on this statement, our result was indicated that the Rhizophora mucronata extract has increased anti-oxidant activity than synthesized Ag NPs. In addition, the positive control of ascorbic acid was also showed increased anti-oxidant activity than Ag NPs, and closely related value to Rhizophora mucronata extract. The anti-oxidant activities of Rhizophora mucronata extract, Ag NPs and ascorbic acid were exhibited the O.D value of 0.877, 0.490, 0.896 at 150 μg/mL respectively. The total anti-oxidant activity result was concluded that the Ag NPs has decreased activity than extract and control at increased concentration, as well as no significant differences between extract and control was recorded.

The variations of performed samples result was presented in Fig. 3a. Interestingly, the increased free radical activity of DPPH assay result was shown to Ag NPs compared with extract and positive control of ascorbic acid. At increasing concentration, the Ag NPs was exhibited excellent free radical scavenging activity with 72%. When compared with extract 64%, it was high. Instead, it showed low activity compared with positive control 87%. All the activities were also exhibited at 200 μg/mL concentration and shown in Fig. 3b. Previously Aji Jovitha et al. (Abdi et al., 2019) reported that the marine Rhizophora mucronata plant mediated Ag NPs has higher radical scavenging activity than extract and it may influence by Ag NPs. Similarly, Abdi (Zhou et al., 2013) reported that the plant mediated Ag NPs exhibited more anti-oxidant activity and DPPH radical scavenging activity at lowest concentration. Supported evidence of Umashankari (Vijaya Sankar and Abideen, 2019) reported, highest content of phenolic and flavonoid rich sources of plant has excellent anti-oxidant activity and also various biological properties like anti-microbial, anti-cancer, larvicidal and etc.. Sometimes, the increased biological activity of Ag NPs was stimulated by selected plant due to the absorption of nutrients, hormones and other stress responding genes (Yang et al., 2020).

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Fig. 3

Total anti-oxidant (a) and DPPH scavenging activity (b) of Rhizophora mucronata extract by invitro studies.

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3.4 Characterization of Ag NPs

Synthesized Ag NPs surface plasmon resonance was indicated at the place of 415 nm, and it shown in Fig. 4a. The colored solution was changed to no color after addition of Rhizophora mucronata extract at 1 h. The greenish yellow color formation was indicated that the Ag was converted to Ag NPs in the presence of Rhizophora mucronata extract and it suggested with SPR vibrations. The result was good agreement with (Uzma et al., 2020; Titkov et al., 2017) and the range between 410 and 440 is SPR of Ag NPs. In addition, intensity of XRD binding energy was suggest, the three major and two minor diffraction peaks at the position of 30.12, 39.30, 42.34, 51.04, and the respective planes were shown at 61.22 111, 200, 202, 221, 224 (Fig. 4b). The result was clearly revealed that the Ag + ion was transferred in the plant extract and produced the Ag NPs without any impurities. The supportive evidence of Mi theory was significantly correlated with our result (Mortazavi-Derazkola et al., 2020; Al-kawmani et al., 2020).

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Fig. 4

UV-spectrometer (a) and XRD pattern reports of Rhizophora mucronata extract mediated Ag NPs.

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Further, the surface plosman resonance of synthesized Ag NPs was shown with extremely agglomerated spherical shape morphology (Fig. 5) by SEM view (Titkov et al., 2017; Dinparvar et al., 2020).

3.5 Anti-cancer studies

3.5.2

3.5.2 Phase contrast microscopy

Fig. 6b, c was suggested the death and live cells of treated and control wells. Before intracellular inhibition of Ag NPs against MCF-7 cell lines, the shrinked and rough surface of MCF-7 cells was shown by phase contrast microscope after 24 h inhibition. Compared with control, the treated cells were shown irregular, detachment, rough and confused colonies (Fig. 6b). In addition, the treated wells containing solutions were low turbidity and confluence colonies were shown on cover slip after viewed by phase contrast microscope. The differentiation was identified easily by control and treated images due to the respective tightly clumped colonies and loosely arranged colonies of MCF-7 lung cancer. The initial morphological differentiation of phase contrast images were agreed by previous report of Venugopal et al. (Ramesh et al., 2018). Detection of intense damaged cells after treatment with the help of phase contrast microscope. (Ghosal et al., 2020). Similar result was also reported by Solairaj et al. (Mortazavi-Derazkola et al., 2020), and the resulted evidences were more useful to compare treatment effect of silver nanoparticle.

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Fig. 5

Scanning electron microscope of Rhizophora mucronata extract mediated Ag NPs.

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3.5.3

3.5.3 Fluorescence microscopic confirmation

After phase contrast microscope analysis, the confirmation of morphological damages in the Ag NPs treated Ag NPs were viewed by fluorescence microscope using two different fluorescence dyes named as AO/EB (Fig. 6e, d). The live and death cells differentiation was easily identified in AO/EB mediated fluorescence staining assay due to the light intensity observation (Al-kawmani et al., 2020). AO is a green dye having the ability to bind in inside of the cancer cells. Whereas, EB is another fluorescence dye having the binding nature of damaged cells (Saravanakumar et al., 2019). After treatment with biosynthesized Ag NPs of our study result was indicated that the Ag Nps was effectively inhibited the MCF-7 cells effectively. The damage cells were absorbed the EB dye and emitted red color fluorescence images in fluorescence microscope. Compared to control, the treated cells were shown with irregular morphology and more condensed colonies. More places were shown with orange color, indicating the more apoptosis was processed in inside of the cells. The usual shapes were completely modified due to the effect of Ag NPs, instead the regular and normal shapes were confirmed.

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Fig. 6

Percentage of cell viability (a) and IC50 concentration of Ag NPs treated MCF-7 breast cells by phase contrast microscope (b, c) and live/dead cells differentiation (d, e) by fluorescence microscope.

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This differentiation was indicated that the damages were appeared in the Ag NPs treated cells. After inhibition of growth cells, the Ag Nps will not allow the cells to grow next stage or cell cycle process due to the block the nutrient and responsible factors (Parthasarathy et al., 2020; Ramesh et al., 2018). More ever, Ag NPs stimulated the cancer cells to maintain in decline phase and it leads to complete death. Previously, Kumar et al. (Mortazavi-Derazkola et al., 2020); Vijaya Anand et al. (Kumar et al., 2018) reported that the Ag NPs induce the nucleus membrane and produce more condensed cells and it continuously failed to reproduce further. This statement was agreed by Khorrami et al. (Vijaya Anand et al., 2019), reported that the biosynthesized Ag NPs has increased anti-cancer properties.

3.5.4

3.5.4 Photocatalytic studies

The concentration of Rho B was gradually reduced after addition of Ag NPs in time dependent manner (Fig. 7a). As compared to all the tested concentration 15 mg/L seems more efficiency remove the dye as compared to remaining concentrations (5 and 10 mg/L).

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Fig. 7

Photocatalytic degradation of Rhodamine B dye (a) and crystal violet dye (b).

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After 150mins of solar light irradiation reduces the amount Rho B from 85.29 to 10.67% this was relatively higher than the degradation of percentage of CV. The percentage was CV showed insignificant reduction even after exposure to higher concentration of Ag₂O NPs (Fig. 7b). Only 34.04% of CV was reduced after 150mins of Ag₂O NPs exposure. The photocatalytic activity of Ag NPs synthesized via floral extract showed significant reduction of Rho B as compared to other studies. Around 40% of degradation was observed when Rho B was treated with Ag NPs prepared by thermal route (Rajivgandhi et al., 2019). On the other hand, Alpinia nigra fruit extract mediated Ag NPs produced 85.9% of Rho B degradation (Uzma et al., 2020), it was relatively equal to the catalytic efficiency of Ag NPs reported in this study. The present data implicated that the potential of Ag NPs towards the degradation of dye. When discussing with the molecular mechanics, faster degradation of Rho B was directly affected by free radicals generated during the light irradiation. Free radicles such as super oxide and hydroxyl radicles are directly related the dye degradation, however hydroxyl radicles are consider being the more powerful breaker. This result was also agreed by previous report of Khataee and Kasiri, (Tammina et al., 2017); Manal et al. (2021).