ANTI-INFLAMMATORY AND ANTIFUNGAL ACTIVITY OF ZINC OXIDE NANOPARTICLE USING RED SANDALWOOD EXTRACT

Ashna Yuvaraj¹, R Priyadharshini¹*, Rajesh Kumar², Palati Sinduja¹

ABSTRACT

Pterocarpus santalinus is valued for its strong, dark-purple, bitter heartwood. The gentle aroma of P. santalinus heartwood is caused by the presence of terpenoids, which gives it its color and fragrance. P. santalinus heartwood dye is used as a light microscopy stain, and as a coloring agent in pharmaceutical preparations. The Antibacterial, antimicrobial, and UV-blocking capabilities of ZnO NPs are outstanding compared with other metal oxides. The study aims to analyze the antiinflammatory and antifungal activity of zinc oxide nanoparticles using red sandalwood extract. A plant extract sample was taken and 100ml of distilled water was added. Heated at 50°C for 5-10 minutes. The heated solution was filtered. .0.861g of zinc oxide was measured and dissolved in 70 ml of distilled water. The mixture was transferred to extract the sample. The solution was heated and labeled. The heating mantle was used with a temperature of 50-60°C and the time took was 6-8 minutes. The results obtained were statistically analyzed using spearman correlation analysis and using SPSS software. The sample shows an increasing absorbance rate and increases in the zone of inhibition with the increase in the concentration of the extract. It also showed an effective antifungal activity against C.albicans. The correlation analysis is statistically significant with p value<0.05. From the present study, it is concluded that red sandal-mediated zinc oxide nanoparticles possess anti-inflammatory and antifungal activity. 

Keywords: Anti-inflammatory, Anti-fungal, Innovative technique, Pterocarpus santalinus, Red sandalwood, Zinc oxide nanoparticle

Introduction

Natural herbal extracts of red sandalwood, botanical name Pterocarpus santalinus, have been used in therapeutics for centuries and are considered to have anti-diabetic, anti-inflammatory, antioxidant, and analgesic effects [1, 2]. In English, Pterocarpus santalinus L. f. (Fabaceae) is known as red sandalwood, but it also goes by other names in other languages. Pterocarpus is derived from the Greek words pteron (wing) and karpos (fruit), referring to the winged pod, while santalinus is derived from the Latin words sandal and inus (meaning similar to), referring to a plant that has characteristics similar to Indian sandalwood, Santalum album L [3, 4].

P. santalinus (also known as African or Nepalese sandalwood and Indian sandalwood) is valued for its strong, dark-purple, bitter heartwood. The active components of Red Sandalwood, such as anthocyanins, saponins, tannins, isoflavonoids, terpenoids, and associated phenolic compounds, beta-sitosterol, and lupeol, are considered to be multifunctional in the treatment of various diseases [5-7]. The gentle aroma of P. santalinus heartwood is caused by the presence of terpenoids, which gives it its color and fragrance. P. santalinus heartwood dye is used as a light microscopy stain, and a coloring agent in pharmaceutical preparations, and the food, leather, and textile industries [7-10]. Many studies have taken place using red sandalwood-mediated zinc oxide nanoparticles. Various activities such as anti-diabetic, antioxidant, cytotoxicity, and anti-cancer activities have been done by many authors. The previous studies have faced challenges mainly in the method that has been adopted to synthesize the nanoparticles. The chemical method of synthesis has its disadvantages such as the distribution of the size and control of the deposit parameters but it's also a widely used technique as it is very cost-efficient, requires no chemical purification, and large-scale production of the nanoparticle can be achieved through this method [11, 12].

Due to their unique physical and chemical properties, zinc oxide nanoparticles (ZnO NPS), one of the most common metal oxide nanoparticles, are widely used in a variety of fields [13-16]. ZnO NPs were first used in the rubber industry to provide wear resistance to rubber composites, increase the durability and strength of high polymers, and provide anti-aging properties, among other things. ZnO is increasingly used in personal care products, such as cosmetics and sunscreen, due to its good UV absorption properties [17]. ZnO NPs also have outstanding antibacterial, antimicrobial, and UV-blocking properties. Zinc is commonly recognized as an important trace element that can be found in all body tissues, including the brain, muscle, bone, and skin [18-20].

Zinc participates in the body's metabolism and plays important roles in protein and nucleic acid synthesis, hematopoiesis, and neurogenesis as the key component of various enzyme systems. Because of the small particle size of Nano-ZnO, zinc is more readily absorbed by the body [21]. As a result, nano-ZnO is widely used in food. Furthermore, the US Food and Drug Administration has listed ZnO as a “GRAS’ (generally accepted as safe) substance (FDA) [22, 23]. ZnO NPs have gotten a lot of attention in biomedical applications because of these properties. ZnO NPs, which are relatively inexpensive and less toxic than other metal oxide NPs, have a wide variety of biomedical uses, including anticancer, drug delivery, antibacterial, and diabetes treatment; anti-inflammation; wound healing; and bioimaging [24-27].

This study aims to determine the anti-inflammatory and antifungal activity of red sandalwood-mediated zinc oxide nanoparticles.

Materials and Methods

Preparation of Plant Extract 

Plant samples such as Pterocarpus santalinus were extracted from fresh or dried plant material. There was no bias and a random sampling method was adopted. Since the plant extract was already available in powdered form, the extract was directly prepared. 100ml of distilled water was taken in a measuring cylinder 1.008g of Pterocarpus santalinus was measured and taken using a weighing machine. (Figure 1) Then, the weighted powder is mixed with the distilled water that is taken in a conical flask. The solution is labeled and heated by using a machine called the heating mantle. The temperature of the heating mantle was set to about 50°C and the time is taken to heat is about 5-10 minutes. The heating solution is taken out of the heating mantle when there is an appearance of small bubbles. After the heating process, the heated solution is filtered using filter paper (Figure 2).

Preparation of Zinc Oxide Nanoparticle

0.861g of zinc oxide was measured and dissolved in 70 ml of distilled water (Figure 3). Now the calculated amount of zinc oxide and distilled water has been applied to the previously prepared plant extract. The solution was to be red. The measured powder which was mixed with distilled water was taken in a conical flask. The solution was heated and labeled. The heating mantle was used with a temperature of 50-60 degrees Celsius and the time taken was 6-8 minutes. The annealed powder was used as a sample for analysis.

Anti-Inflammatory Activity

Bovine serum albumin was used for the assay. 2 ml of bovine albumin was mixed with 400 microliters of zinc oxide nanoparticles in different concentrations was used as standard and then incubated at 55°C and then the results were analyzed spectrometrically.

Antifungal Activity

Candida albicans were used as a tested pathogen by agar well diffusion assay (Figure 4). Sabouraud’s Dextrose Agar is used to prepare the medium. The prepared and sterilized medium was swabbed with test organisms and nanoparticles with different concentrations were added to the well. The plates were incubated at 28° C for 48-72hours. After the incubation time, the zone of inhibition was measured.

The results obtained were tabulated accordingly and their graphs were plotted using correlation analysis in the statistical software “SPSS version 23”. The graphs thus collected were studied and analyzed. The data collected and studied were validated by the guide and the nanoparticle researcher.

Results and Discussion

Anti-Inflammatory

Table 1. The table depicts the concentration and the absorbance rate obtained for the activity.

Anti Fungal (C albicans)

Table 2. The table depicts the concentration and the zone of inhibition obtained for the activity.

Anti-Inflammatory

The test for anti-inflammatory activity was assessed using bovine serum albumin. 2 ml of the bovine albumin was added to the red sandalwood ZnO nanoparticle extract with the extract concentration ranging from 1 microlitre to 5 microlitres and the result was seen after incubation. The study showed a positive increase in the absorbance rate with the increase in the concentration of the nanoparticle extract (Figure 5). The test for antifungal activity was assessed using the fungal pathogen, Candida albicans (Figure 6). The test was done using Sabouraud’s dextrose agar in a sterile medium and the plates were incubated for 48-72 hours at 28 degrees Celsius and the zone of inhibition was obtained. The study showed positive results as the zone of inhibition of Candida Albicans showed a positive increase with an increase in the concentration of the extracted sample. From the results obtained from our study, it has been observed that the red sandalwood-mediated zinc oxide nanoparticle has a positive effect on anti-inflammatory and antifungal activity.

Nanomaterials are particles with nanoscale dimensions, are extremely small particles with increased catalytic reactivity, thermal conductivity, nonlinear optical efficiency, and chemical stability due to their wide surface area to volume ratio [28]. Nanoparticles have been incorporated into many consumer industries, including industrial, health, food, feed, space, chemical, and cosmetics, necessitating a green and environmentally friendly approach to their synthesis [29, 30]. Biosynthesis of nanoparticles is a method of synthesizing nanoparticles for biomedical applications using microorganisms and plants. This method is eco-friendly, cost-effective, biocompatible, safe, and environmentally friendly. Plants, bacteria, fungi, algae, and other species are all used in green synthesis [31]. They allow the development of ZnO NPs on a large scale without the addition of impurities. Biomimetic NPs have higher catalytic activity and needless costly and toxic chemicals to manufacture [32].

Similar studies have been done earlier with different fungal pathogens that have shown positive results. In the study by Saquib, because of the large-scale development, easy downstream processing, and economic viability, extracellular synthesis of NPs from the fungus is extremely useful. Fungal strains are favored over bacteria due to their higher resistance and ability to bioaccumulate metals [33-35]. SEM, TEM, and XRD analysis confirmed that NPs synthesized with Candida albicans had a similar size range of 15–25 nm. For the synthesis of ZnO NPs, Aspergillus species have been commonly used, and NPs synthesized from these fungal strains were mostly spherical [36, 37]. In the study by MD Jayappa, the anti-inflammatory activity of the ZnO NPs was assessed using the human red blood cells membrane stabilization method where the blood samples were collected without nonsteroidal and anti-inflammatory drugs (NSAIDs). This study showed positive absorbance of 560 nm for the anti-inflammatory activity [38, 39]. Several other studies have shown that the zinc oxide nanoparticle has been very effective and significant in antioxidant, anti-diabetic, and anti-cancer activities.

Other green sources for the synthesis of nanoparticles include biocompatible chemicals. It is a simple and cost-effective process that removes the production of any side products during the nucleation and synthesis of nanoparticles. With its well-dispersed existence, it results in the creation of nanoparticles of regulated shape and size [40]. The limitations of this study were mainly the sample size and the errors that might’ve been caused in the measurement of the concentrations in the extract. In the last decade, the biosynthesis of nanoparticles using an environmentally friendly approach has been a subject of study. For the synthesis of shape and size-controlled nanoparticles, green sources act as both a stabilizing and a reducing agent. Plant-based nanoparticles have a wide range of applications in the food, pharmaceutical, and cosmetic industries, and have thus become a major research subject [41, 42].

Conclusion

From the present study, it is concluded that red sandal-mediated zinc oxide nanoparticles possess anti-inflammatory and antifungal activity. In summary, this is a simple, effective biosynthetic method that could be used to substitute chemical and physical methods for large-scale ZnO-NP development. The use of such environmentally-friendly ZnO-NPs in a variety of fields, including medicine, catalysis, and drug delivery systems, makes this bioreduction method an excellent choice for large-scale synthesis.

Acknowledgments: We would like to thank Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, and Saveetha University for supporting us to conduct the study.

Conflict of interest: None

Financial support: The present project is supported by

• Saveetha Institute of Medical and Technical Sciences
• Saveetha Dental College and Hospitals, Saveetha University

In Genius technologies, Chennai.

Ethics statement: None

1.        Cheng YL, Lee SC, Lin SZ, Chang WL, Chen YL, Tsai NM, et al. Anti-proliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo. Cancer Lett. 2005;222(2):183-93.

2.        Tan ML, Sulaiman SF, Najimuddin N, Samian MR, Muhammad TT. Methanolic extract of Pereskia bleo (Kunth) DC. (Cactaceae) induces apoptosis in breast carcinoma, T47-D cell line. J Ethnopharmacol. 2005;96(1-2):287-94.

3.        Ramawat KG, Goyal S. The Indian Herbal Drugs Scenario in Global Perspectives. Bioact Mol Med Plants. 2008;325-47.

4.        Aluri JSR. Pollination ecology of the Red Sanders Pterocarpus santalinus (Fabaceae), an endemic and endangered tree species. Curr Sci. 2002;83(9):23-8.

5.        Bulle S, Reddy VD, Padmavathi P, Maturu P, Ch VN. Modulatory role of Pterocarpus santalinus against alcohol-induced liver oxidative/nitrosative damage in rats. Biomed Pharmacother. 2016;83(3):1057-63.

6.        Nandita R, Priyadharshini R, Rajeshkumar S, Sinduja P. Cytotoxic Effect of Pterocarpus santalinus and stevia-based Mouthwash-A Lab-based Analysis. J Pharm Res Int. 2021;33(58B):437-47.

7.        Warakagoda PS, Kumari DLC, Subasinghe S. In vitro shoot tip culture of Red Sandalwood (Pterocarpus santalinus L.). Proc Int Forest Environ Symp. 2013;2(1):28-32.

8.        Nivedha VM, Priyadarshini R, Rajeshkumar S, Sinduja P. Anti-diabetic and Antioxidant Activity of Pterocarpus santalinus and Stevia Herbal Formulation. J Pharm Res Int. 2021;33(62B):124-34.

9.        Brundha MP. A Comparative Study-The Role of Skin and Nerve Biopsy in Hansen’s Disease. Res J Pharm Biol Chem Sci. 2015;7(10):837.

10.     Harsha L, Brundha MP. Prevalence of Dental Developmental Anomalies among Men and Women and its Psychological Effect in a Given Population. J Pharm Sci Res. 2017;9(6):869-73.

11.     Modan EM, University of Piteşti, Romania, Plăiașu AG, University of Piteşti, Romania. Advantages and Disadvantages of Chemical Methods in the Elaboration of Nanomaterials. Ann “Dunarea de Jos” University of Galati Fascicle IX, Metall Mater Sci. 2020;43(1):53-60.

12.     Timothy CN, Samyuktha PS, Brundha MP. Dental pulp Stem Cells in Regenerative Medicine--A Literature Review. Res J Pharm Technol. 2019;12(8):4052-6.

13.     Jiang J, Pi J, Cai J. The Advancing of Zinc Oxide Nanoparticles for Biomedical Applications. Bioinorg Chem Appl. 2018;12(3):1-18.

14.     Wu D, Chen Z, Cai K, Zhuo D, Chen J, Jiang B. Investigation into the antibacterial activity of monodisperse BSA-conjugated zinc oxide nanoparticles. Curr Appl Phys. 2014;14(11):1470-5.

15.     Rajeshkumar S, Sandhiya D. Biomedical Applications of Zinc Oxide Nanoparticles Synthesized Using Eco-friendly Method. Nanopart their Biomed Appl. 2020;5(2):65-93.

16.     Rajeshkumar S, Lakshmi T, Naik P. Recent advances and biomedical applications of zinc oxide nanoparticles. Green Synth, Charact Appl Nanopart. 2019;6(2):445-57.

17.     Mirzaei H, Darroudi M. Zinc oxide nanoparticles: Biological synthesis and biomedical applications. Ceram Int. 2017;43(1):907-14.

18.     Zhang Y, R Nayak T, Hong H, Cai W. Biomedical applications of zinc oxide nanomaterials. Curr Mol Med. 2013;13(10):1633-45.

19.     Agarwal H, Nakara A, Menon S, Shanmugam VK. Microbe-mediated Synthesis of Zinc Oxide Nanoparticles and Its Biomedical Applications. InMicrobial Nanotechnology 2020 May 25 (pp. 162-177). CRC Press.

20.     Barui AK, Kotcherlakota R, Patra CR. Biomedical applications of zinc oxide nanoparticles. InInorganic frameworks as smart nanomedicines 2018 Jan 1 (pp. 239-278). William Andrew Publishing.

21.     Ruszkiewicz JA, Pinkas A, Ferrer B, Peres TV, Tsatsakis A, Aschner M. Neurotoxic effect of active ingredients in sunscreen products, a contemporary review. Toxicol Rep. 2017;4(2):245-59.

22.     Zhang ZY, Xiong HM. Photoluminescent ZnO Nanoparticles and Their Biological Applications. Materials. 2015;8(6):3101-27.

23.     Zhang HJ, Xiong HM. Biological Applications of ZnO Nanoparticles. Curr Mol Imaging. 2013;2(2):177-92.

24.     Kim S, Lee SY, Cho HJ. Doxorubicin-Wrapped Zinc Oxide Nanoclusters for the Therapy of Colorectal Adenocarcinoma. Nanomaterials. 2017;7(11):354.

25.     Kim S, Lee SY, Cho HJ. Berberine and zinc oxide-based nanoparticles for the chemo-photothermal therapy of lung adenocarcinoma. Biochem Biophys Res Commun. 2018;501(3):765-70.

26.     Xiong HM. ZnO Nanoparticles Applied to Bioimaging and Drug Delivery. Adv Mater. 2013;25(37):5329-35.

27.     Xiong HM. ChemInform Abstract: ZnO Nanoparticles Applied to Bioimaging and Drug Delivery. ChemInform. 2013;44(49):82-6.

28.     Khan ST, Musarrat J, Al-Khedhairy AA. Countering drug resistance, infectious diseases, and sepsis using metal and metal oxides nanoparticles: Current status. Colloids Surf B Biointerfaces. 2016;146(3):70-83.

29.     Vidya C, Manjunatha C, Chandraprabha MN, Rajshekar M, Antony Raj MA. Hazard-free green synthesis of ZnO nano-photo-catalyst using Artocarpus Heterophyllus leaf extract for the degradation of Congo red dye in water treatment applications. J Environ Chem Eng. 2017;5(4):3172-80.

30.     Hiremath S, Vidya C, Antonyraj MAL, Chandraprabha MN, Seemashri S, Shetty B, et al. Photocatalytic Degradation Study of Rhodamine-B by Green Synthesized Nano TiO2. Asian J Chem. 2017;29(1):221-5.

31.     Elumalai K, Velmurugan S, Ravi S, Kathiravan V, Ashokkumar S. Retraction notice to: Green synthesis of Zinc oxide nanoparticles using Moringa oleifera leaf extract and evaluation of its antimicrobial activity. Spectrochim Acta A Mol Biomol Spectrosc. 2019;206(3):651.

32.     Elumalai K, Velmurugan S, Ravi S, Kathiravan V, Ashokkumar S. Retracted: Green synthesis of zinc oxide nanoparticles using Moringa oleifera leaf extract and evaluation of its antimicrobial activity. Spectrochim Acta A Mol Biomol Spectrosc. 2015;143(2):158-64.

33.     Pavani KV, Sunil Kumar N, Sangameswaran BB. Synthesis of Lead Nanoparticles by Aspergillus Species. Pol J Microbiol. 2012;61(1):61-3.

34.     Kumar RR, Priyadharsani KP, Thamaraiselvi K. Mycogenic synthesis of silver nanoparticles by the Japanese environmental isolate Aspergillus tamarii. J Nanopart Res. 2012;14(5):1-7.

35.     Saquib Q, Faisal M, Al-Khedhairy AA, Alatar AA, editors. Green Synthesis of Nanoparticles: Applications and Prospects. Springer Singapore, Imprint: Springer; 2020;2(3):40-6.

36.     Hoffmann MR, Martin ST, Choi W, Bahnemann DW. Environmental Applications of Semiconductor Photocatalysis. Chem Rev. 1995;95(1):69-96.

37.     Mashrai A, Khanam H, Aljawfi RN. Biological synthesis of ZnO nanoparticles using C. albicans and studying their catalytic performance in the synthesis of steroidal pyrazolines. Arab J Chem. 2017;10:S1530-6.

38.     Jayappa MD, Ramaiah CK, Kumar MAP, Suresh D, Prabhu A, Devasya RP, et al. Green synthesis of zinc oxide nanoparticles from the leaf, stem and in vitro grown callus of Mussaenda frondosa L.: characterization and their applications. Appl Nanosci. 2020;10(8):3057-74.

39.     Sinduja P, Ramani P, Gheena S, Ramasubramanian A. Expression of metallothionein in oral squamous cell carcinoma: A systematic review. J Oral Maxillofac Pathol. 2020;24(1):143.

40.     Nagarajan S, Kuppusamy KA. Extracellular synthesis of zinc oxide nanoparticle using seaweeds of gulf of Mannar, India. J Nanobiotechnol. 2013;11(1):39.

41.     Anita S, Ramachandran T, Rajendran R, Koushik CV, Mahalakshmi M. A study of the antimicrobial property of encapsulated copper oxide nanoparticles on cotton fabric. Text Res J. 2011;81(10):1081-8.

42.     Gharagozlou M, Baradaran Z, Bayati R. A green chemical method for synthesis of ZnO nanoparticles from solid-state decomposition of Schiff-bases derived from amino acid alanine complexes. Ceram Int. 2015;41(7):8382-7.