|Year : 2021 | Volume
| Issue : 4 | Page : 405-411
Comparison of anti-inflammatory activities of biogenic gymnema sylvestre- and panicum sumatrense-mediated titanium dioxide nanoparticles
Moses Stella Bharathy, Gnanasekar Dayana Jeyaleela, Joseph Devaraj Rosaline Vimala, Aranganathan Agila, Sagaya Adaikalaraj Margrat Sheela
Department of Chemistry, Holy Cross College (Autonomous), Affiliated to Bharathidasan University, Tiruchirappalli, Tamil Nadu, India
|Date of Submission||05-Aug-2021|
|Date of Acceptance||16-Oct-2021|
|Date of Web Publication||14-Dec-2021|
Moses Stella Bharathy
Department of Chemistry, Holy Cross College (Autonomous), Affiliated to Bharathidasan University, Tiruchirappalli - 620 002, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Studying the anti-inflammatory effect of the wound-healing property in immune responsive compounds such as interleukins and cytokinins plays a vital role in targeting various inflammatory diseases such as asthma, rheumatoid arthritis, tuberculosis, and periodontitis. The goal of the present work is to compare the anti-inflammatory activity of Gymnema sylvestre (GS)- and Panicum sumatrense (PS)-mediated titanium dioxide (TiO2) nanoparticles (NPs) by in vitro studies. Methods: G. sylvestre- and P. sumatrense-mediated TiO2 NPs were synthesized by Greener method. The synthesized TiO2 NPs were spectroscopically characterized such as Ultraviolet-visible, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscope (SEM), and Energy Dispersive X-Ray Analysis (EDAX). The in vitro anti-inflammatory activities of GS-TiO2 and PS-TiO2 NPs were carried out by albumin denaturation assay. Results: The size of G. sylvestre-mediated TiO2 (GS-TiO2) NPs was found to be 16–22 nm and that of P. sumatrense (PS-TiO2)-mediated NPs was in the range of 26–32 nm. Rectangle, hexagonal, and square types of NPs were recorded in the SEM analysis of both the GS-TiO2 and PS-TiO2. When comparing the X-ray powder diffraction and EDAX results of GS-TiO2 and PS-TiO2, G. sylvestre-mediated TiO2 showed less impurity and along with that, it revealed pure titanium in 50.50% and oxygen in 35.54%. Similarly, G. sylvestre-mediated TiO2 nanoparticles exhibited 91.52% of inhibitory effect on protein denature (in vitro anti-inflammatory activity), whereas P. sumatrense (PS-TiO2)-mediated NPs showed only 84.80%. Conclusion: The overall study concludes that G. sylvestre-mediated TiO2 nanoparticle can have a scope in alternative treatment/remedy for inflammation diseases.
Keywords: Anti-inflammatory activities of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide, green synthesis of titanium dioxide, Gymnema sylvestre-titanium dioxide, Panicum sumatrense-titanium dioxide
|How to cite this article:|
Stella Bharathy M, Dayana Jeyaleela G, Rosaline Vimala JD, Agila A, Margrat Sheela SA. Comparison of anti-inflammatory activities of biogenic gymnema sylvestre- and panicum sumatrense-mediated titanium dioxide nanoparticles. Biomed Biotechnol Res J 2021;5:405-11
|How to cite this URL:|
Stella Bharathy M, Dayana Jeyaleela G, Rosaline Vimala JD, Agila A, Margrat Sheela SA. Comparison of anti-inflammatory activities of biogenic gymnema sylvestre- and panicum sumatrense-mediated titanium dioxide nanoparticles. Biomed Biotechnol Res J [serial online] 2021 [cited 2022 Jan 20];5:405-11. Available from: https://www.bmbtrj.org/text.asp?2021/5/4/405/332452
| Introduction|| |
Nanotechnology/nanoscience comprises a synthesis and application of nano-sized particles with an average size of 1–100 nm and is widely applied in the field of energy, chemical, health care, and cosmetics. In scientific research, the most fastest-growing field is nanotechnology which was first proposed by the scientist Richard Feynman in the year 1959. Among the metal oxide nanoparticles (CuO, ZnO, CeO2, Co3O4, etc.,) titanium dioxide (TiO2) nanoparticles possess high stability, insoluble in water, and exhibit unique magnetic, thermal, optical, and electrical properties. Numerous metal oxide nanoparticles were reported through chemical, physical, and greener methods. Among all, a chemical reduction method is regularly practiced in large-scale productions. Chemical and physical methods need toxic chemicals and also require high temperature and pressure which can pollute the environment and also it limits the mass production of NPs, respectively.,, Greener reducing method is an eco-friendly alternative method in which the reducing agents are derived from natural resources such as microorganisms, plant extracts, and marine sources. Recently, agricultural waste, algae, biomass, and animal wastes are utilized for NP synthesis, which can reduce the usage of expensive chemicals and toxicity of NPs as compared to other methods. TiO2 is a naturally occurring mineral which can be derived from brookite, rutile, and anatase. It is categorized as a human carcinogenic chemical (2B) by the IARC. Because of its unique optical properties, nontoxicity, high chemical stability, and good photocatalytic and corrosion resistance behavior, TiO2 NPs have huge demand in various industrial sectors. It is diversely applied in pharmaceuticals, food preparations, sunscreens, drug delivery, and cosmetics, which is due to its high refractive index and ultraviolet (UV) light absorption characteristics. Micronized titanium dioxide nanoparticles used in sunscreens protect the skin from the UV rays and enhances skin whiteness also is applied in paper, paints, plastic, toothpaste, ink production industries.,,,
Plant extracts contain medicinally active organic compounds such as phenolics, alkaloids, flavonoids, anthocyanins, proteins, and carbohydrates, which can reduce the size of TiO2 NPs and stabilize the formed NPs in the synthesis. These phyto-organic compounds are present in different levels and types, which control the shape, size, and distribution of the TiO2 NPs. Various plant extract-mediated TiO2NPs have been reported, and most of the results confirmed the round or spherical shapes with clustered form. Few green TiO2 NPs have been reported with porous structure forms, with comparatively large crystals showing the interesting and unique surface morphology. Owing to the porous structures, TiO2 has revolutionary applications in the environmental industry.,,
Medicinal plant Gymnema sylvestre (G. sylvestre) is an indigenous herb belonging to the Asclepiadaceae family. Its extract is majorly used in the Ayurvedic system of medicine to treat malaria, snakebites, and diabetes. G. sylvestre is a good source of bioactive phytomolecules such as gymnemic acids, gurmarin, gymnemasaponins, stigmasterol, gymnemanol, d-quercitol, anthraquinones, hydroxycinnamic acids, β-amyrin-related glycosides, lupeol, and coumarols. G. sylvestre extract possesses pharmacological activity such as anti-diabetic, anti-arthritic, anti-microbial, anti-cancer, anti-inflammatory, immunostimulatory, anti-hyperlipidemic, hepatoprotective, and wound-healing activities, and also it is used in the treatment of dental caries. Panicum sumatrense (P. sumatrense) is a species of millet and is well known as little millet which belongs to the Poaceae family. It is a good source of nutraceuticals such as lignans, starch, phenolics, sterols, gama-aminobutyric acid, and phytates. P. sumatrense extract possesses enzymic inhibitory, hypolipidemic, anti-carcinogenic, hypoglycemic, anti-glycation, hypocholesterolemic, anti-inflammatory, and vasodilatory activities. Owing to antioxidants, it reduces degenerative diseases such as osteoporosis, cardiovascular diseases, and cancer., The present work is to compare the anti-inflammatory activity of G. sylvestre-mediated TiO2 NPs and P. sumatrense-mediated TiO2 NPs. Both G. sylvestre- and P. sumatrense-mediated TiO2 NPs were characterized by UV-visible (Vis), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction, scanning electron microscope (SEM), and EDAX.
| Methods|| |
Collection of plant materials and preparation of plant extracts
The millet P. sumatrense was purchased from a local country drug store and the herbal plant G. sylvestre was collected from Kollimalai, Tamil Nadu, India. Both the plant materials were washed under running tap water and spread out in a plane paper and then dried at room temperature for about 2–5 days in the shade. After that, both the plant materials were made into fine powder. 100 g of each plant powder was transferred into two separate 500-mL beakers and then 250 mL of 70% ethanol was poured into the respective beakers and then left for 1 week. After that, the whole content was filtered through a normal filter paper and the filtrate was again filtered through Whatman No. 1 filter paper. Double-filtered filtrates were used for further qualitative analysis and the TiO2 NP synthesis. The taxonomy of the plant was identified by the Rapinat Herbarium, St. Joseph's College (Autonomous) Trichy with authenticate number of MSB001.
Preliminary phytochemical screening of 70% ethanolic extracts of Gymnema sylvestre and Panicum sumatrense
Phytochemical screening tests which are chemical qualitative tests were carried out on P. sumatrense and G. sylvestre extracts using the standard procedures to identify the presence of phytochemicals in them. Xofowara (1993), Treaxe and Evans (1989), and Harborne (1973) methods were used in the screening process.,,
Synthesis of Gymnema sylvestre-mediated titanium dioxide and Panicum sumatrense-mediated titanium dioxide nanoparticles
One hundred milliliter of 1 mM TiO2 was taken in a 500-mL beaker and to this, 20 mL of 70% ethanolic extract of G. sylvestre was added and kept on a magnetic stirrer. After 30-min interval, again 20 mL of the extract was added at continuous stirring condition. Similarly, for every 30-min time interval, 20 mL of the extract was added, and after the complete addition of 100-mL extract, the reaction mixture was stirred well for 4 h. Afterward, the formed TiO2 NPs were collected by centrifuge method at 3000 rpm. Then, the particles were washed with water followed by ethanol to remove the impurities in the TiO2 NPs. Similarly, P. sumatrense-mediated TiO2 NPs was synthesized.
Characterization of synthesized Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide nanoparticles
The reduction of Ti ions was preliminarily confirmed by UV-Vis spectroscopy. Plant metabolites and their functional groups involved in the reduction process were studied by FT-IR analysis. The phase evolution of calcined GS-TiO2 and PS-TiO2 NPs was analyzed by X-ray powder diffraction (XRD) in the 2theta ranges of 15° to 70° with Cu Ka radiation and a scan rate of 0.04/s. The size and morphology of GS-TiO2 and PS-TiO2 were determined by SEM using JEOLJsm-6480 LV.,,
In vitro anti-inflammatory activity of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide nanoparticles by albumin denaturation method
The in vitro anti-inflammatory activities of GS-TiO2 and PS-TiO2 NPs were carried out by albumin denaturation assay. Five milliliters of the mixture solution was taken in reaction tubes which contain 2.8 mLof phosphate-buffered saline, pH – 6.4, 0.2 mL of egg albumin, and 2 mL of different concentrations of synthesized TiO2 NPs (20, 40, 60, 80, and 100 μg/mL). Instead of TiO2 NPs, 2 mL of double distilled water served as a control. After additions, the reaction mixtures were incubated at room temperature for 15 min followed by heating at 70°C for 5 min. After cooling the mixture, the absorbance of each solution was measured with the aid of spectrophotometry at 660 nm. The same procedures were performed for standard diclofenac sodium from 20 to 100 μg/mL concentrations which were used as reference drugs. The protein denaturation percentage by Nps and standard was calculated by using the following formula.,,
% Inhibition = 100× (Vt/Vc − 1)
Vt = absorbance of a test sample,
Vc = absorbance of control.
| Results|| |
Phytochemical screening results of 70% ethanolic extracts of Gymnema sylvestre and Panicum sumatrense
The screening results of G. sylvestre and P. sumatrense extracts are shown in [Table 1] and [Supplementary Figure 1] and [Supplementary Figure 2]. Both the ethanolic extracts revealed the presence of medicinally important metabolites in them, which can reduce the metal ions and form the NPs. G. sylvestre extract showed the highest percentage of phytochemicals than the P. sumatrense extract.
|Table 1: Phytochemical screening results of Gymnema sylvestre and Panicum sumatrense extracts|
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Characterization of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide nanoparticles
The formation of TiO2NPs was confirmed by visual color changes before and after the addition of the extracts. The precursor titanium solution is colorless, G. sylvestre extracts are of yellowish-brown, and P. sumatrence extracts are of light yellow. In GS-TiO2 NPs synthesis, after adding the extract, the titanium solution turned into dark brown with NPs. Similarly, after adding the extract, PS-TiO2 NPs changed into dark yellow with NP precipitate. The color changes in GS-TiO2 and PS-TiO2 preliminarily confirmed the formation of TiO2 NPs, which is shown in [Supplementary Figure 3].
Ultraviolet/visible spectroscopy results of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
UV-Vis is a reliable and simpler method to examine the stability of NPs which can examine whether the NP solution has destabilized or not. The UV-Vis results of GS-TiO2 and PS-TiO2 revealed that G. sylvestre-mediated titanium NPs showed absorbance at 398 nm and P. sumatrence-mediated titanium NPs showed 367 nm [Figure 1], which confirms the formation of TiO2NPs.
|Figure 1: Ultraviolet-visible spectral analysis of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide|
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Fourier transform infrared spectroscopy spectroscopic analysis results of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
FT-IR is an essential tool to identify the functional groups in biomolecules which are involved in NP synthesis. In both the GS-TiO2 and PS-TiO2 NPs [Figure 2], OH stretching of alcohols (phenolic compounds) appeared at 3278 cm−1 and 3426 cm−1; similarly, C–C and C = C stretching of aromatic rings appeared at 2925 cm−1, 2426 cm−1, and 2099 cm−1. At 1641 cm−1 and 1601 cm1, peaks were observed that could be the multiple C = O groups in the biomolecules. More biomolecules are bounded in PS-TiO2 than that in the GS-TiO2, which can reduce the purity of the NPs. Metal-oxygen bonds (Ti-O) were highly found in GS-TiO2 and it is less bounded with plant biomolecules.
|Figure 2: Fourier transform infrared spectroscopy spectral analysis of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide|
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Scanning electron microscope analysis results of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
SEM analysis was carried out on GS-TiO2 and PS-TiO2 to understand the topological and morphological changes in its surface. Polydispersed shapes of TiO2NPs were found in both GS-TiO2 and PS-TiO2 with various sizes. Rectangle, hexagonal, and square types of TiO2 NPs were observed in both the G. sylvestre- and P. sumatrense-mediated TiO2 NPs. The average size of GS-TiO2 was found to be 16–22 nm and PS-TiO2NPs was in the range of 26–32 nm [Figure 3]. Compared to PS-TiO2, GS-TiO2 revealed the lower average size of NPs with less aggregation observed under SEM.
|Figure 3: Scanning electron microscope images of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide|
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X-ray powder diffraction results of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
XRD is used to determine the chemical compositions and crystal patterns in the sample. The crystalline nature of GS-TiO2 and PS-TiO2 NPs was studied from X-ray diffraction analysis, and their results are shown in [Figure 4]. The characteristic numbers of Bragg reflections were observed in GS-TiO2 with a 2 θ range of 38.18°, 48.62°, 63.62°, and 84.22°, which is responsible for the (111), (210), (220), and (320) planes, respectively. In PS-TiO2 XRD, patterns were observed at 28.34°, 36.22°, 42.16°, 45.02°, 54.58°, and 69.22°, which indicate the (101), (111), (210), (220), (211), and (320) planes. GS-TiO2 is more crystalline with low impurities, whereas in PS-TiO2 NPs, little amorphous nature is found. Furthermore, both NPs are well correlated with the TiO2 standard pattern of JCPDS No. 04-0783.
|Figure 4: X-ray powder diffraction patterns of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide|
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Energy-dispersive spectroscopy results of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
Energy-dispersive spectroscopy (EDX) was used to find the elemental composition in the formed TiO2NPs. EDX of GS-TiO2 and PS-TiO2 is given in [Figure 5]. GS-TiO2 NPs revealed the presence of pure titanium in 73.23% and oxygen in 26.77%. Similarly, PS-TiO2 showed 67% of titanium and 31% of oxygen and then, the remaining percentage of chlorine and carbon elements are present in it.
|Figure 5: Energy-dispersive spectroscopy patterns of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide|
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In vitro anti-inflammatory activity of Gymnema sylvestre-titanium dioxide and Panicum sumatrense-titanium dioxide
P. sumatrense- and G. sylvestre-mediated TiO2 NPs were studied against the anti-inflammatory activity by protein denature model (albumin denaturation), and their results are summarized in [Table 2] and [Figure 6]. The anti-inflammatory activity images of GS-TiO2, PS-TiO2, and the standard are depicted in [Supplementary Figure 4], [Supplementary Figure 5], [Supplementary Figure 6]. The results reveal that the percentage of inhibition of protein denature was increased by increasing the concentration of GS-TiO2, PS-TiO2, and standard diclofenac sodium. At 100-μg/mL concentration, GS-TiO2 showed 93.52% of inhibitory effect on protein denaturation, PS-TiO2 showed 82.80%, and the standard exhibited 96.25%. These results suggest that GS-TiO2 reveals higher inhibitory effect than PS-TiO2 and more or less equivalent to standard.
|Table 2: Summarized anti-inflammatory activity results of Gymnema sylvestre-titanium dioxide, Panicum sumatrense-titanium dioxide, and diclofenac sodium (standard)|
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|Figure 6: In vitro anti-inflammatory activity results of Gymnema sylvestre-titanium dioxide, Panicum sumatrense-titanium dioxide, and diclofenac sodium (standard)|
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| Discussion|| |
According to a thorough review of the literature, nanotechnology provides the ideal platform for pharmacology by allowing drug delivery systems to target cells affected with bacteria that cause inflammatory disorders.,, Hence, we considered TiO2 NPs mediated by G. sylvestre in the present study as an alternate treatment/remedy for treating inflammatory disorders. The different kinds of phytochemicals in the extract and their proportion/compositions play the predominant role in the reduction and stabilization process during the NP synthesis. Because G. sylvestre extract possesses the highest percentage of phytochemicals, it can reduce more silver ions (Ag+) and stabilize the formed NPs than the P. sumatrense. UV absorbance of NPs is completely based on the size, composition, shapes, agglomeration, and aggregation states of the formed particles. Usually, the optical properties of TiO2 NPs are changed by the aggregation's effect. The aggregation effect influences the UV-visible absorption spectrum of synthesized nanoparticles (wavelength). PS-TiO2 are more aggregated/agglomeration and their particle size is much larger than that of GS-TiO2, which destabilizes the NPs. Unassigned peaks were observed at GS-TiO2 and PS-TiO2 in XRD and are known as impurities that may come from the plant extracts (biomolecules) and are adsorbed on the NPs or bounded with the TiO2. From XRD, it is evidenced that the formed GS-TiO2 NPs were obtained with purity (less impurity) than PS-TiO2.
Denature of protein and membrane lysis leads to the formation of autoantigen in the human body. That auto-antigen is the main reason for the inflammation and inflammatory diseases such as asthma, rheumatoid arthritis, chronic peptic ulcer, tuberculosis, periodontitis, and Crohn's disease. To stop/reduce the production of autoantigen, the protein denaturation and membrane lysis percentage should control. The egg albumin protein denaturation method is the cost-effective alternative method of testing the anti-inflammatory activity of any samples. The anti-inflammatory activity of plant extract-mediated TiO2 NPs has not been much reported earlier. Because the size of GS-TiO2 NPs is smaller and its purity is higher, it can easily penetrate, which results in a significant inhibitory effect than PS-TiO2.
| Conclusion|| |
G. sylvestre- and P. sumatrense-mediated TiO2 NPs were first time reported in this article. The anti-inflammatory activity on TiO2 NPs is not much reported in earlier studies. Because G. sylvestre-mediated TiO2 NPs are smaller in size with high purity than P. sumatrense-mediated TiO2 NPs, the anti-inflammatory effect showed by GS-TiO2 is significantly higher. The definite inhibitory effect of protein denaturation by NPs is an unpredictable mechanism that can include hydrogen, electrostatic hydrogen, disulfide, or hydrophilic bonding between the NPs and the protein. In general, the inflammatory activity (anti-inflammatory activity) can be inhibited/tested by inhibiting the protein denaturation. In this work, diclofenac sodium is used as a reference drug, however it causes side effects such as gastric irritations, ulceration, and hemorrhage. These side effects can be rectified by green synthesis NPs and nowadays, TiO2 NPs are being applied in various pharmacological effects. GS-TiO2 NPs can be used as the alternative therapeutic agent against inflammatory diseases, which can be utilized as wound-healing pastes or gels in future.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]