EXPLORATION OF THREE INDIAN SPICES FOR ZINC OXIDE NANOPARTICLES SYNTHESIS AND ASSESSMENT OF THE THERAPEUTIC POTENTIAL OF THESE SYNTHESIZED NANOPARTICLES

EXPLORATION OF THREE INDIAN SPICES FOR ZINC OXIDE NANOPARTICLES SYNTHESIS AND ASSESSMENT OF THE THERAPEUTIC POTENTIAL OF THESE SYNTHESIZED NANOPARTICLES

Snehal Kulkarni, Suneeta Panicker *, Shahin Akbar Ali Siddiqui, Madhuri Lalbahadur Sahani and Arti Kishor Singh

Department of Microbiology, Dr. D.Y. Patil Arts, Commerce and Science College, Sant Tukaram Nagar, Pimpri, Pune, Maharashtra, India.

ABSTRACT: Nanotechnology has revolutionized multiple scientific domains, especially biomedical sciences. Zinc Oxide (ZnO) nanoparticles (NPs) due to their unique properties find a wide application in biomedicine, food, agriculture, cosmetics, pharmaceutical, textile, rubber and electronic industries. Green synthesis of ZnO NPs employing plant-based extracts is an eco-friendly and sustainable alternative to conventional chemical synthesis. Present study explores the potential of Indian Spices viz. Brassica hirta (Yellow Mustard), Piper cubeba (Kabab Chini), and Amomum subulatum (Black Cardamom) as reducing and stabilizing agents in green synthesis of ZnO NPs and its detailed characterization with respect to UV-Visible Spectroscopy, X-Ray Diffraction (XRD), and Field Emission Scanning Electron Microscopy (FE-SEM). The UV-Visible spectra of the synthesized ZnO nanoparticles revealed the absorption maxima in the range between 290-350 nm which confirms the synthesis of ZnO nanoparticles. The XRD pattern confirmed the crystalline nature and hexagonal wurtzite structure of the ZnO NPs. The FESEM analysis revealed the cylindrical shape in case of Brassica hirta ZnO NPs, hexagonal shape in case of Piper cubeba ZnO NPs and irregular shape in case of Amomum subulatum ZnO NPs. These biogenically produced ZnO NPs were then used to evaluate their possible antibacterial, antioxidant and anti-inflammatory properties. ZnO NPs demonstrated significant antimicrobial activity against Staphylococcus aureus, Bacillus, Escherichia coli, Proteus, Aspergillus niger, and Candida albicans, while their antioxidant and anti-inflammatory properties were also comparable to the respective standards use in the assays. This research highlights the potential biomedical applications of Indian Spice-mediated ZnO NPs, demonstrating their efficacy in various therapeutic interventions.

Keywords: Zinc oxide nanoparticles, Green synthesis, Phytochemical analysis, Antibacterial, Antifungal, Antioxidant, Anti-inflammatory

INTRODUCTION: There is tremendous demand of nanoparticles in the field of chemistry, medicine as well as biotechnology ¹. Different metals and metal oxides are used to produce nanoparticles such as silver, copper, gold, zinc, titanium, magnesium etc.

Out of which, ZnO NPs are particularly significant due to their unique properties like high electron mobility, wide band gap, high piezoelectric property, Biocompatibility and environmental sustainability ².

Along with these properties, ZnO NPs are reported for their antimicrobial, anticancer, antidiabetic, antioxidant, antifungal, antiparasitic, anti-inflammatory activity and wound healing properties and thus have potential biomedical applications. ZnO NPs have been listed by US FDA as “Generally Recognized as Safe” (GRAS) due to their non-toxic nature at low concentrations ³. Traditional methods for ZnO NPs synthesis involve physical and chemical processes that require hazardous chemicals and high energy input ⁴. Green synthesis of ZnO NPs utilizes biological agents such as plant extracts, bacteria, fungi, algae etc. for nanoparticle synthesis which has many advantages viz. cost-effective, non-toxic nature, biodegradability, non-hazardous to environment and use of natural resources like plants and microorganisms ⁵.

Indian spices such as Brassica hirta (Yellow Mustard seeds), Piper cubeba (Kabab Chini), and Amomum subulatum (Black cardamom) possess notable antimicrobial, antioxidant properties and anti-inflammatory properties and thus have long been used in traditional Indian medicine. The bioactive compounds like Phenolic acids, flavonoids, terpenes etc. found in the aqueous extract of these plants can act as an oxidizing, reducing, and capping agent for the synthesis of biogenic ZnO NPs. Various researchers have demonstrated the diverse biomedical applications of ZnO NPs, including: Antimicrobial Activity ⁶, Antioxidant Properties ⁷, Anti-inflammatory Effects ⁸.

Although plant-mediated green synthesis of ZnO NPs has been widely explored using extracts from various plant species, including some species of Brassicai ⁹ and Piper ¹⁰ genus, the authors did not identify any scientific reports stating, the use of Brassica hirta or Piper cubeba extracts for ZnO NP synthesis in the databases searched. Accordingly, this study explores these species as novel biogenic agents for ZnO NP synthesis.

Thus, current study is aimed synthesize ZnO NPs using Indian spices like Brassica hirta, Piper cubeba and Amomum subulatum and its detailed characterization with respect to UV-Visible spectroscopy, X- ray Diffraction studies and Field Emission Scanning Electron Microscopy. Furthermore, the antimicrobial, antioxidant and anti-inflammatory properties of ZnO NPs were studied.

MATERIALS AND METHODS:

Sample Collection: Spices such as Brassica hirta (Yellow Mustard seeds), Piper cubeba (Kabab Chini) and Amomum subulatum (Black Cardamom) were procured from local markets in Pimpri, Pune, Maharashtra, and authenticated based on morphological characteristics.

Chemicals and Reagents: All chemicals and reagents used in the present study were of analytical grades, and Reagent-grade (purity ≥ 98%) in particular Zinc acetate dihydrate [Zn (CH₃CO₂).2H₂O], Sodium hydroxide, Nutrient Agar, Mueller-Hinton Agar, Potato dextrose Agar, Bovine serum albumin, Potassium ferricyanide, Ferric chloride, Trichloroacetic acid, Ascorbic acid and Dimethyl Sulfoxide (DMSO) were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Ciprofloxacin (500mg), Ketoconazole and Diclofenac sodium tablet were purchased from Local medical store.

Preparation of Aqueous Extracts of Brassica hirta, Piper cubeba and Amomum subulatum by Decoction Method ¹¹: The decoction method was used for aqueous extraction of three spices viz. Brassica hirta, Piper cubeba and Amomum subulatum.

The seeds of above three spices were washed with distilled water to remove dust particles and completely air-dried to remove the moisture contents and were crushed using mortar and pestle to prepare the fine powder. 10g powder of each spice was soaked in 150 mL distilled water separately and boiled for 20 minutes.

The extracts were filtered through Whatman filter paper No. 1 and the obtained extract of each spice was used for phytochemical analysis and ZnO NPs synthesis.

Phytochemical Analysis of Aqueous Extract of Brassica hirta, Piper cubeba, Amomum subulatum: The aqueous extract of Brassica hirta, Piper cubeba, Amomum subulatum was tested for presence or absence of phytochemicals like flavonoids, saponins, carbohydrates, proteins, alkaloids, tannins, steroids, glycosides, coumarin and amino acids which acts as a reducing and capping agent for the synthesis of ZnO NPs. Qualitative phytochemical analyses for the extracts were performed according to a study ¹².

TABLE 1: PROTOCOLS FOR PHYTOCHEMICAL ANALYSIS OF AQUEOUS EXTRACTS OF ALL THREE SPICES

Green Synthesis of ZnO NPs using aqueous extracts of Brassica hirta, Piper cubeba, Amomum subulatum: For green synthesis of ZnO NPs, 10 mL aqueous extract of all three spices viz. Brassica hirta, Piper cubeba, Amomum subulatum was added separately drop wise to the 50 mL, 0.25 M zinc acetate dihydratesolution under continuous stirring at 60°C for 2 hours on a hot plate magnetic stirrer. While stirring, 0.1M NaOH was added dropwise in the above solution to maintain the pH at 12. There was formation of off-white precipitate in Brassica hirta aqueous extract, brown precipitate in Piper cubeba aqueous extract and light brown precipitate in Amomum subulatum aqueous extract at the bottom of the beaker. All three solutions were centrifuged at 8000 rpm for 20 minutes to obtain the pellet. The white, brown and light brown precipitate of ZnO NPs was washed with distilled water thrice and driedthem overnight at 60°C ¹³.

Characterization of ZnO NPs: Characterization of synthesized ZnO NPs was performed using different techniques like; UV-Visible spectroscopy, X-ray Diffraction analysis and Field emission Scanning Electron microscopy (FESEM). The obtained ZnO NPs were dissolved in 1mL DMSO and the UV-spectra for these samples were scanned between the range of 200-800 nm on SHIMADZU UV-Spectrophotometer (SerialNo-A11455009292) to observe the characteristic peak confirming ZnO NPs formation ¹⁴. The crystallinity of the ZnO NPs was analysed using Single crystal X-Ray Diffractometer (Bruker D8 Venture), using Cu/40k V/40mAas the X-Ray source with K-beta filter ¹⁵. To analyse the surface morphology of the ZnO NPs, FESEM was performed using FEI Nova Nano SEM 450 and EDS: Bruker X Flash 6I30 used SEM grids were prepared by placing a small amount of sample powder on a copper coated grid and drying under lamp ¹⁶.

Antimicrobial Activity of Synthesized ZnO NPs ¹¹: The antibacterial activity of synthesized ZnO NPs was evaluated by agar well diffusion method. Two Gram Positive bacteria viz. Staphylococcus aureus and Bacillus sp. and two Gram Negative bacteria viz. Escherichia coli and Proteus sp. were used for the assay. Overnight grown broth culture (100 μL) of the test bacteria was spread on the sterile Muller-Hinton agar plate. Wells were punched on the spread plate using sterile 8 mm cork borer. 100 μL of each ZnO NPs having concentration of 1mg/ml was added into respective well using sterile micropipette. DMSO was used as negative control. Antibiotic Ciprofloxacin having concentration 500 mg/mL (Leeford) was taken as positive control.

The plates were first incubated at 4°C for 10 minutes for pre-diffusion and then at 37°C for 24 hours. Anti-bacterial activity was determined by measuring the diameter of zone of inhibition (mm).

The antifungal activity of synthesized ZnO NPs was also evaluated by agar well diffusion method using the above protocol with few modifications. Candida albicans and Aspergillus niger, sterile Potato Dextrose Agar plates and Ketoconazole (100 mg/mL) was used for the antifungal assay. The plates with Candida albicans were incubated at 37°C and those with Aspergillus niger were incubated at 30°C.

Anti-Inflammatory Activity of Synthesized ZnO NPs: The in-vitro anti-inflammatory activity of synthesized ZnO NPs was checked Bovine Serum Albumin (BSA) denaturation assay ¹⁷. Briefly, 10, 20, 30, 40 and 50 μg of ZnONPs and diclofenac sodium was taken from a stock of 1 mg/ml solution in tubes labelled respectively.

To each test tube, 2 mL of 1% BSA and 390, 380, 370, 360 and 350 μL of distilled water was added. The tubes were incubated at room temperature for 10 minutes at 50°C in water bath for 10 minutes. Absorbance was measured at 660 nm in UV Spectrophotometer. The percentage of protein denaturation was determined using the following equation:

% inhibition = (Absorbance of control - Absorbance of sample × 100) / Absorbance of control

Antioxidant Activity of Synthesized ZnO NPs ¹⁴: The antioxidant property of ZnO NPs was checked by reducing power assay using Potassium Ferricyanide by measuring their capacity to reduce ferric to ferrous ions. Different concentrations of ZnO NPs and Ascorbic acid (standard) ranged from 2 mg/mL to 8 mg/mL each was added in respectively labelled tubes. 1mL of absolute ethanol was added and mixed with 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (10 g/L); mixture was then incubated at 50°C for 20 min; 2.5 mL of trichloro acetic acid (100 g/L) was added to the mixture, which was then centrifuged at 3000 rpm for 10 minutes. Finally, 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml ferric chloride (1 g/L). Absorbance was measured at 700 nm in UV-Visible Spectrophotometer. Increased absorbance of the reaction mixture indicates stronger reducing power.

RESULTS:

Phytochemical Analysis of Aqueous Extract of Brassica hirta, Piper cubeba and Amomum subulatum: To find out the active components in the aqueous extracts of Brassica hirta, Piper cubeba and Amomum subulatum, phytochemical analysis ofthese extracts was performed.

It was observed that aqueous extract of Brassica hirta contains flavonoids, saponins, reducing sugars, alkaloids, proteins, tannins, steroids, glycosides, coumarins, and amino acids, but it was devoid of quinones, the aqueous extract of Piper cubeba showed the presence of saponins, quinone, alkaloids, proteins, steroids, and amino acids, but was devoid of  flavonoids, reducing sugars, tannins, glycosides, and coumarins while the aqueous extract of Amomum subulatum contained saponins, alkaloids, proteins, steroids, coumarins, and amino acids, but was devoid of flavonoids, quinones, reducing sugars, tannins, and glycosides, the results are depicted in Table 2.

TABLE 2: PHYTOCHEMICAL ANALYSIS FOR BRASSICA HIRTA, PIPER CUBEBA, AMOMUM SUBULATUM

Green Synthesis of ZnO NPs Synthesized using Aqueous Extracts of Brassica hirta, Piper cubeba, Amomum subulatum: As the phytochemical analysis of aqueous extract of Brassica hirta, Piper cubeba, Amomum subulatum revealed that it contains Flavonoid, saponins, reducing sugars, proteins, alkaloids, tannins, steroids etc. which can act as an oxidizing, reducing, and capping agent for green synthesis of ZnO NPs, aqueous extract of Indian spices like Brassica hirta, Piper cubeba, and Amomum subulatum were used for green synthesis of ZnO NPs.

White precipitate of ZnO NPs was observed in the case of Brassica hirta and dark brown and brown precipitate of ZnO NPs was formed in case of Piper cubeba, and Amomum subulatum. The above ZnO NPs were characterized in detail with respect to its morphology, UV-visible spectrum, size, antimicrobial property, anti-inflammatory and antioxidant property.

Characterization of the ZnO NPs: The formed ZnO NPs were characterized with respect to UV-Visible spectroscopy, X-Ray Diffraction, and Scanning Electron Microscopy.

UV-Visible Spectroscopy of ZnO NPs: In order to study the optical absorption property of the synthesized ZnO NPs using, Brassica hirta, Piper Cubeba, Amomum subulatum, the UV–visible absorbance spectrum was monitored at room temperature in the wavelengths of 200 to 700 nm. The spectrum showed a peak at 350 nm in case of Brassica hirta ZnO NPs Fig. 1A, 300 nm in case of Piper cubeba ZnO NPs Fig. 1B and 290 nm in case of Amomum subulatum ZnO NPs which is specific for ZnO NPs Fig. 1C.

FIG. 1A: UV-VISIBLE SPECTRUM OF BRASSICA HIRTA ZNO NPs

FIG. 1B: UV-VISIBLE SPECTRUM OF PIPER CUBEBA ZNO NPs                                         

FIG. 1C: UV-VISIBLE SPECTRUM OF AMOMUM SUBULATUM ZNO NPs

FESEM Analysis of ZnO NPs: The surface morphology of the green synthesized ZnO NPs using Indian spices Brassica hirta, Piper cubeba, and Amomum subulatum was determined through Field Emission Scanning Electron Microscopy. The FESEM images provided insights into the structural characteristics of the synthesized NPs Fig. 2A, B, C. Brassica hirta ZnO NPs exhibited a morphology resembling cylindrical with the average sizes ranging from 18 to 35 nm Fig. 2A. Piper cubeba ZnO NPs exhibited a morphology showing irregular cylindrical shapes with the average sizes ranging from 27 to 48 nm Fig. 2B. Amomum subulatum ZnO NPs exhibited a morphology resembling irregular shape with the average sizes ranging from 30 to 113 nm Fig. 2C.

FIG. 2A: FE-SEM IMAGE OF BRASSICA HIRTA ZNO NPs

FIG. 2B: FE-SEM IMAGE OF PIPER CUBEBA ZNO NPs                                

FIG. 2C: FE-SEM IMAGE OF AMOMUM SUBULATUM ZNO NPs

X-Ray Diffraction Analysis of ZnO NPs: The samples of ZnO NPs formed using aqueous extracts of Brassica hirta, Piper Cubeba, Amomum subulatum were analysed for XRD using Single crystal X-Ray Diffractometer.

XRD Analysis of Brassica hirta ZnO NPs: The X-ray diffraction pattern of ZnO NPs synthesized by using Brassica hirta is shown in Fig. 3A. The XRD pattern showed ten intense peaks in the whole spectrum of 2θ values ranging from 09 to 100 (29.72, 31.78, 34.42, 36.28, 47.56, 56.64, 62.90, 66.40, 67.98, and 69.76), which could be attribute to the 12, 65, 66, 100, 18, 39, 27, 09, 26, and 15 planes for zinc, respectively.

All the diffraction peaks confirmed the hexagonal wurtzite structure of ZnO NPs. The strong and narrow diffraction peaks confirmed the crystalline nature of synthesized ZnO NPs.

FIG. 3A: XRD ANALYSIS OF BRASSICA HIRTA ZNO NPs

XRD Analysis of Piper cubeba ZnO NPs: The X-ray diffraction pattern of ZnO NPs synthesized by using Piper cubeba is shown in Fig. 3B. The XRD pattern showed ten intense peaks in the whole spectrum of 2θ values ranging from 23 to 100 (27.14, 29.94, 32.00, 34.66, 36.48, 47.82, 56.80, 63.00, 68.12, and 69.36), which could be attribute to 28, 42, 72, 70, 100, 29, 45, 31, 33, and 23 planes for zinc, respectively. All the diffraction peaks confirmed the hexagonal wurtzite structure of ZnO NPs. The strong and narrow diffraction peaks confirmed the crystalline nature of synthesized ZnO NPs.

FIG. 3B: XRD ANALYSIS OF BIOSYNTHESIZED PIPER CUBEBA ZNO NPs

XRD Analysis of Amomum subulatum ZnO NPs: The X-ray diffraction pattern of ZnO NPs synthesized by using Amomum subulatum is shown in Fig. 3C. The XRD pattern showed eight intense peaks in the whole spectrum of 2θ values ranging from 13 to 100 (31.84, 34.50, 36.32, 47.62, 56.66, 62.92, 66.52, and 68.00), which could be attribute to 67, 69, 100, 24, 39. 29, 13 and 26 planes for zinc, respectively. All the diffraction peaks confirmed the hexagonal wurtzite structure of ZnO NPs. The strong and narrow diffraction peaks confirmed the crystalline nature of synthesized ZnO NPs.

FIG. 3C: XRD ANALYSIS OF BIOSYNTHESIZED AMOMUM SUBULATUM ZNO NPs

Antibacterial Activity of ZnO NPs: The antibacterial activity of synthesized ZnO NPs (1 mg/ml) using Brassica hirta, Piper cubeba, and Amomum subulatum extract was against two Gram positive (Staphylococcus aureus and Bacillus sp.) and two Gram negative (Escherichia coli and Proteus sp.) bacteria. The ciprofloxacin antibiotic (500 mg/ml) was used as a positive control and DMSO as negative control.

The data from Table 3 illustrates the antibacterial efficacy of green-synthesized ZnO Nanoparticles (ZnO NPs) against four bacterial strains: two Gram-positive (Staphylococcus aureus, Bacillus sp.), and two Gram-negative (Escherichia coli, Proteus sp.). Piper cubeba derived ZnO NPs demonstrated the strongest overall performance among the three variants, particularly against Gram-positive bacteria. However, all ZnO NPs showed moderate activity compared to the standard antibiotic Ciprofloxacin. This trend is consistent with extensive literature on green-synthesized ZnO NPs, which report them as viable, eco-friendly antibacterials.

TABLE 3: ANTIBACTERIAL ACTIVITY OF ZNO NPs

Antifungal Activity of ZnO Nanopaticles: The antifungal activity of ZnO NPs synthesized by using Indian spices like Brassica hirta, Piper cubeba, and Amomum subulatum at a concentration of 1 mg/ml was tested against Aspergillus niger and Candida albicans, with Piper cubeba derived nanoparticles showing the highest efficacy among the three samples.

It was determined that all the ZnO NPs possessed same antifungal capacity at the tested concentration. Antifungal Ketoconazole (100 mg/ml) was used as a positive control and DMSO as a negative control. The measured diameters of zones of inhibition are depicted in Table 4. The synthesized ZnO NPs showed intermediate antifungal activity at 1 mg/ml concentration but at higher concentrations they can exhibit significant antifungal activity many against fungi.  While the ZnO NPs are effective, they currently show lower potency than the standard antifungal Ketoconazole, which produced the largest zones of inhibition. This aligns with existing literature highlighting ZnO NPs as a promising, low-toxicity alternative to traditional azole-based treatments. Aspergillus niger was consistently more sensitive to all tested ZnO NP variants compared to Candida albicans, a trend supported by studies identifying A. niger as highly susceptible to green-synthesized metal oxides.

TABLE 4: ANTIFUNGAL ACTIVITY OF ZNO NPs

Anti-Inflammatory Activity of Synthesized ZnO NPs using Brassica hirta, Piper cubeba, Amomum subulatum: The anti-inflammatory activity of ZnO nanoparticles synthesized using Brassica hirta, Piper cubeba, and Amomum subulatum was evaluated in comparison with diclofenac sodium Fig. 5 and Table 5. All experiments were performed in triplicate, and results are expressed as mean ± SD (n = 3).

Although ZnO nanoparticles exhibited slightly higher mean inhibition values than diclofenac sodium at higher concentrations, the observed differences were modest. Given that diclofenac is a clinically established anti-inflammatory drug, the present findings suggest that ZnO NPs demonstrate comparable efficacy under in-vitro conditions, rather than definitive superiority.

The enhanced activity may be attributed to nanoscale effects and phytochemical capping from the spice extracts. Further in-vivo and mechanistic studies are required to confirm their therapeutic relevance.

FIG. 4: ANTI-INFLAMMATORY ACTIVITY OF SYNTHESIZED NPs

TABLE 5: ANTI-INFLAMMATORY ACTIVITY OF SYNTHESIZED ZNO NPs

Antioxidant Activity of Synthesized ZnO NPs: The ZnO NPs synthesized using Indian spices Brassica hirta, Piper cubeba, and Amomum subulatum was found possess antioxidant property. In this study, the reducing capacity of the ZnO NPs was assessed using the Fe³⁺ to Fe²⁺ reduction assay, where a colour changes from yellow to blue indicating the reduction of Fe. The antioxidant property of ZnO NPs increased with the increase in the concentration of ZnO NPs and the activity was comparable with the standard antioxidant, Ascorbic acid. The results are demonstrated in the Table 6.

TABLE 6: ANTIOXIDANT ACTIVITY OF SYNTHESIZED ZNO NPs

DISCUSSION: The present study supported the earlier findings that aqueous extracts of Brassica hirta, Piper cubeba, and Amomum subulatum contain various phytochemicals, including alkaloids, flavonoids, tannins, saponins, glycosides, coumarins, steroids, and phenols ¹⁸.

The bioactive compounds like phenolic acids, flavonoids, saponins, alkaloids, tannins, terpenes and steroids etc. found in the aqueous extract of Brassica hirta, Piper cubeba and Amomum subulatum might act as an oxidizing, reducing, and capping agent for the synthesis of biogenic ZnO NPs. These compounds are responsible for the reducing properties of the spice extract, which are crucial in the formation of ZnO NPs, as well as for their capping and stabilizing effects. They enable the production by providing electrons to reduce metal ions (Zn²⁺) into stable ZnO NPs and then attach to the surface to avoid aggregation.

There are very few reports of use of Indian spices for synthesis of ZnO NPs, which include Black cardamom and Black Pepper ¹⁹, Curry leaf ²⁰, nutmeg ²¹, Zinger and Garlic Bulb ¹¹, Tejpatta ²², Black cumin or Kalonji ²³. The present study also confirmed the successful synthesis of ZnO NPs using three spice extracts independently, as evidenced by UV-Vis spectroscopy, with absorption peaks at 350 nm, 300 nm, and 290 nm for Brassica hirta, Piper cubeba, and Amomum subulatum, respectively and confirmed by XRD and SEM. Several researchers studied the UV-visible spectroscopic analysis of ZnO NPs and reported that the absorbance peak of ZnO NPs ranges between 310 nm and 360 nm of wavelength ^{14, 1}. Thus, the results of current study correlate with the earlier reports. In the present work, peaks at 2θ values and the corresponding planes confirmed the synthesis of ZnO NPs using Brassica hirta and similar peaks were earlier reported for ZnO NPs synthesized by using other spices ^{24, 15}.

The agglomeration of smaller ZnO NPs particles resulted in the formation of larger particles, which imparts irregular forms to their structures and the same was reported by a similar study ²⁵. The hexagonal form and uneven distribution pattern of the produced ZnO NPs were also demonstrated in prior investigations ²⁶.

The antimicrobial properties of the synthesized ZnO NPs were evaluated against Staphylococcus aureus, Bacillus, Escherichia coli, and Proteus sp., demonstrating significant antibacterial activity at the tested concentration. The ZnO NPs assessed for their antibacterial properties demonstrated better results than obtained by another group with the ZnO NPs synthesized by using garlic bulb and Z. officinale root extract ¹¹. According to a recent study ²⁷, ZnO NPs synthesized utilizing Beta vulgaris, had antifungal activity against Aspergillus niger, but ZnO NPs prepared from Cinnamomum tamala were active against Candida albicans. Furthermore, ZnO NPs synthesized using Brassica oleracea var. italica extract demonstrated efficacy against both strains of fungus. Similarly, ZnO NPs synthesized from three different spice extracts in this present study, possessed significant antifungal activity against both these fungi.

Further, ZnO NPs exhibited potent antioxidant and anti-inflammatory properties, as confirmed by Fe³⁺ to Fe²⁺ reduction assays, indicating their ability to scavenge free radicals. The observed activity is likely attributed to their small size and high surface reactivity ¹⁴. The findings highlight the potential of ZnO NPs synthesized from Indian spices for antimicrobial, antifungal, and therapeutic applications.

CONCLUSION: This study successfully synthesized ZnO NPs using Brassica hirta, Piper cubeba, and Amomum subulatum. To the best of our knowledge, this is the first report of the synthesis of ZnO NPs using Piper cubeba and Brassica hirta. These ZnO NPs were spherical and/ irregular in shape and average particle size was 61.90 nm. The biosynthesized ZnO NPs exhibited significant antimicrobial, anti-inflammatory, and antioxidant properties, highlighting their potential in nanomedicine, pharmaceuticals, and biotechnology. ZnO nanoparticles synthesized using Piper cubeba, exhibited higher medicinal potential in concentration-dependent manner as compared to the other two plants studied here. The antiviral and anti-cancer properties of these NPs should be investigated since they may play a significant role in modern medical care. Green synthesis provides an eco-friendly, cost-effective alternative to chemical methods, promoting sustainable nanotechnology applications.

ACKNOWLEDGEMENT: Authors are thankful to Dr. D. Y. Patil Arts, Commerce & Science College, Pimpri, Pune, India, for providing facilities available at their laboratory.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Kulkarni S, Panicker S, Siddiqui SAA, Sahani ML and Singh AK: Exploration of three Indian spices for zinc oxide nanoparticles synthesis and assessment of the therapeutic potential of these synthesized nanoparticles. Int J Pharm Sci & Res 2026; 17(6): 1900-11. doi: 10.13040/IJPSR.0975-8232.17(6).1900-11.

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