Introduction

For centuries, traditionally made grape wine has been enjoyed around the world. However, many other fruits such as bananas, cherries, kiwis, plums, and papayas are also used in winemaking. These fruits are not only nutritious but also gain additional polyphenols and volatile compounds through the process of fermentation¹.

Excessive alcohol intake is linked to the progression of diseases such as chronic liver disease^2,3, liver cancer², hypertension, and cardiovascular diseases⁴ and an increased risk of colorectal malignancies⁵, on the other hand moderate alcohol intake is linked with a lower risk of coronary heart disease, as shown in several studies^6,7. Numerous in vitro and in vivo studies, along with epidemiological surveys, suggest that moderate wine consumption, despite its ethanol content, is related to lower risks of type 2 diabetes⁶, cardiovascular diseases, neurodegenerative disorders⁷, platelet aggregation, and oxidative damage, largely due to the polyphenols present in wine⁸. Polyphenols are regarded as key compounds responsible for wine’s potential health benefits⁹.

Bioactive compounds, particularly polyphenols, form a major component of wine¹⁰. The polyphenolic content of wines depends on the type and variety of fruits selected for wine-making¹¹. Polyphenols comprise a large class of phytochemical compounds that consist of many subclasses, namely flavonoids, phenolic acids, stilbenes, and lignans¹². Flavonoids are the major polyphenols present in wine and can be further subdivided into groups such as flavan-3-ols (catechin and epicatechin), flavonols (quercetin, kaempferol, and myricetin), flavones, isoflavones, and anthocyanins (malvin and petunin)^{8, 9, 12}. In red grape wine, the most abundant phenolic antioxidants include catechin, proanthocyanidins, resveratrol, epicatechin, quercetin, anthocyanins, and rutin¹³. Cherry wine is reported to contain naringenin and apigenin as the major compounds¹⁴. Rhododendron mucronulatum flowers which are rich in myricetin, quercetin, and kaempferol and have been used to make wine in the past.

 Dietary polyphenols, especially those found in wines, play a significant role in shaping the composition and function of the human gut and oral microbiota¹⁰. Wine-derived polyphenols exhibit prebiotic properties that help in the proliferation of beneficial gut bacteria¹⁵. They also exhibit antimicrobial effects against pathogenic bacteria¹⁶. Grape-derived antioxidants have been demonstrated to possess antitumor properties through various in vitro and in vivo models. Studies on red wine indicate that polyphenols, such as quercetin, resveratrol, catechin, and gallic acid, are possible cancer chemopreventive representatives¹⁷. Additionally, polyphenols exhibit anti-inflammatory and antimutagenic activities¹⁸.

Polyphenols in wine have garnered significant attention for their potent antioxidant properties. Studies have shown strong correlations between total phenolic content (TPC) and antioxidant capacity. Phenolic acids, such as hydroxycinnamic and hydroxybenzoic acids, demonstrate effective free radical scavenging, helping to sustain the balance of reactive oxygen intermediates in vivo¹⁹. The flavonoids in wine also exhibit powerful scavenging abilities against reactive oxygen, and nitrogen species²⁰. While the health benefits of polyphenols in grape wines are well-documented, the potential of other fruit and flower wines remains underexplored. The present study pursues to evaluate and compare the polyphenolic content and antioxidant properties of home-brewed wines derived from plum, cherry, rhododendron, and grape.

Materials and Methods

Materials

Fresh fruits such as green grapes (Vitis vinifera), cherries (Prunus avium), and plums (Prunus salicina) were procured from the local market (Silvassa). Rhododendron flowers (dried) purchased from Paraman store through Amazon. Absolute ethanol, Methanol, AlCl3, Folin Ciocalteau Reagent, LPS, MTT reagent, Tannic acid, were purchased from Sigma-Aldrich. DMEM and FBS (South American origin) purchased from MP Biomedicals. Trypsin EDTA solution, Nutrient Agar, Yeast Extract-Peptone-Dextrose (YPD) media were purchased from HiMedia.

Manufacturing of wine 

Wines were prepared according to published protocol^{21, 22} after several modifications. such as green grapes (Vitis vinifera), cherries (Prunus avium), and plums (Prunus salicina) were cleaned with distilled water. The fruits were mashed, and the pulp and skin were used for fermentation. Ten kilograms of fruits were chopped into small pieces and juiced using a mortar and pestle. They were not mashed for a long time to avoid pectin release. Two kilograms of powdered table sugar was added to the fruit pulp/flower juice. Subsequently, 30 g of Saccharomyces cerevisiae was added in 100 mL of warm water with 5 g of glucose and after 15 to 20 minutes, bubbles or foam were observed, indicating the activation of the yeast. This mixture was added to the fruit pulp/flower juice. The volume was adjusted to 10 liters with distilled water, and the mixture was transferred to an amber-colored bottle with adequate headspace, after which the fermentation rate was monitored by counting bubbles per minute. The mixture was kept for 21 days. Two hundred milliliters of diluted egg white (1:10) was prepared using water as the diluent and added to further clarify the wine. After a week, the wine was decanted and filtered through two layers of muslin cloth and stored in a glass bottle in a refrigerator (as mentioned in the flow-chart 1). The method was slightly different for manufacturing flower wine. In the case of rhododendron (Rhododendron arboreum) wine, 100 g of dried flowers were soaked in 500ml of boiling water and the mixture was kept for 24 h at room temperature and then kept at 4 °C for 24 h before being strained. Then 300g of powdered table sugar and 3gms of activated yeast were added to the flower juice. The volume was adjusted to 1L with sterile distilled water. The mixture was kept for 21 days in an air-lock container at 25^oC. The wine was decanted and filtered through two layers of muslin cloth and stored in a glass bottle in a refrigerator (as mentioned in the flow-chart 2).

Flow-Chart 1: Wine manufacturing with Plum, Cherry and Grape fruits       Click here to view Flowchart

Flow-Chart 2: Wine manufacturing with rhododendron flower       Click here to view FlowChart

Determination of physicochemical properties

Estimation of pH

 pH was measured using a pH meter (Thermo Fisher Orion Versa Star Pro).

Estimation of Titratable acidity

Titratable acidity was determined using the alkaline titration method with 0.1 N NaOH solution using phenolphthalein as an indicator. A 3mL sample of wine was placed in a flask, and the volume was then brought up to 25 mL with distilled water. The sample was titrated until a pink color appeared. Titratable acidity was calculated in terms of tartaric acid(g/L) using the following formula²³, where 75= milliequivalent factor for tartaric acid

Estimation of alcohol content

Using hydrometer

The concentration of alcohol in wines was assessed using a hydrometer. The initial fruit/flower juice was filled in a hydrometer tube, and the hydrometer was immersed in the liquid (allowing it to freely float). Subsequently, the initial specific gravity was recorded. After fermentation, the same procedure was repeated, and the final reading was noted. The percent alcohol concentration was estimated using the following equation ²⁴

(Initial specific gravity- Final specific gravity) ×131.25

Using the dichromate method.

The potassium dichromate reagent was used to estimate the alcohol concentration of the wine samples. Absolute ethanol was used as the standard (3-6% v/v) for this assay. One milliliter of the standard or wine sample was added to a 100 mL flask, followed by 10 mL of 0.1N potassium dichromate reagent and 10 mL of 50% v/v sulfuric acid. After incubating the flask at 60°C for 20 minutes and allowing it to cool, the solution was diluted to 50 mL with distilled water. The absorbance was then measured at 587 nm using a spectrophotometer (Epoch II, BioTek). A standard graph was plotted, and alcohol concentrations were calculated using a linear equation obtained from the standard curve²⁵.

Estimation of total polyphenolic content using Folin–Ciocalteau and Folin–Denis methods

The total polyphenolic content of the wine samples were assessed using the Folin–Ciocalteau and Folin–Denis methods with slight modifications²⁶. Twenty microliters of undiluted wine were mixed with 100 µL of Folin–Ciocalteau or Folin–Denis reagent in a 96-well plate, followed by 80 µL of sodium bicarbonate (0.1M) after 10 minutes. Absorbance at 760 nm was measured after 30 minutes of incubation at 25°C using a plate reader (Epoch II, BioTek). Tannic acid (10-100 µg/mL) served as the reference standard. The TPC was calculated using a calibration curve and expressed as µg/mL of tannic acid equivalent.  

Estimation of flavonoid content by AlCl₃ assay

An aluminum chloride (AlCl₃) assay was used to measure the flavonoid content in the wine samples²⁷. In a 96-well plate, 100 µL of 2% aluminum chloride was mixed with 50 µL of the wine sample and kept for 30 minutes at 25°C. Absorbance at 420 nm was recorded using a plate reader (Epoch II, BioTek). Flavonoid content was determined using a quercetin standard curve prepared with water as a solvent (0-200 µg/mL) and expressed as µg/mL of quercetin equivalent.

Estimation of antioxidant activity using ferric ion-reducing antioxidant power method

The ferric ion reducing antioxidant power (FRAP) assay was performed according to the reported protocol²⁸ with some modifications. 150 μL of FRAP solution was mixed with 50 μL of each wine sample in a 96-well plate. A range of standard concentrations of quercetin (0-50 µg/mL) was prepared using water as the solvent. The absorbance was taken at 593 nm using a plate reader (Epoch II, BioTek). The antioxidant properties of wines were calculated based on the linear equation obtained from quercetin standard curve. 

Estimation of antioxidant activity using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ( ABTS) method

The free radical scavenging activity of the wines was assessed using the ABTS method as described earlier²⁹. Aqueous quercetin (50 µg/mL) served as a positive control. Absorbance was measured at 734 nm with a plate reader (Epoch II, BioTek), and the antioxidant activity was calculated based on the percentage inhibition of the ABTS radical.

Estimation of antioxidant activity using 2, 2-diphenyl-β-picrylhydrazyl (DPPH)

The free radical scavenging ability of the wine samples was evaluated using the DPPH assay as outlined by the reference³⁰. Briefly, 50 µL of each wine sample was mixed with 150 µL of 200 µM methanolic DPPH solution and incubated in the dark at room temperature for 30 minutes. Aqueous solution of quercetin (50 µg/mL) was used as a positive control, and methanol served as a negative control. Absorbance was taken at 517 nm using a microplate reader (Epoch II, BioTek).

Cell culture and cell viability assay

RAW 264.7 macrophages were sourced from the National Centre for Cell Science (Pune, India) and cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum under a 5% CO₂ atmosphere. Cell viability was assessed using an MTT assay as described earlier³¹.

Nitric oxide production

RAW 264.7 cells were cultured in 96-well plates at 5 × 10⁵ cells/mL and incubated overnight. After the incubation, the cell supernatant was replaced with fresh medium containing 100 µL of 1 µg/mL LPS (prepared in DMEM medium), with or without 100 µL of wine samples, and incubated for another 24 hours. Nitrite levels in the culture supernatant, indicative of NO production, were measured with the Griess reagent. Equal volumes of the culture supernatant and Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine-HCl) were mixed and incubated for 10 minutes, and absorbance was read at 540 nm. Fresh culture medium served as the blank, and nitrite concentration was determined using a sodium nitrite standard curve.

Measurement of reactive oxygen species production

For measuring reactive oxygen species (ROS), 5 X10⁵ cells/mL of cell suspension was seeded in a black 96-well plate. The experiment was performed as described earlier²⁸. Briefly, the cells were cultured and treated with LPS, as described above. Then, the medium was replaced with 10 µM dichlorodihydrofluorescein diacetate (DCFDA, Sigma-Aldrich, Germany), and the cells were incubated for 30 min at 37 °C and 5% CO2. The medium was discarded, and the cell layer was washed with phosphate-buffered saline. Subsequently, 200 μL of serum-free medium was added to each well, and the fluorescence intensity was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a spectrophotometer (iD3 SpectraMax, Molecular Devices, San Jose, CA, USA).

High-performance liquid chromatography

The wine samples were analyzed using a Eurosphere C-18 reversed-phase cartridge (dimensions: 300 mm in length and 4 mm in diameter, particle size: 5µm; KNAUER HPLC, Germany). Standard stock solutions of catechin, quercetin, and gallic acid were prepared separately in methanol. Then, the final stock solutions of the standards (10-100µg/ml was prepared in the mobile phase which was a mixture of 28% acetonitrile and 2% aqueous acetic acid v/v. The sample injection volume was 10µl. The polyphenols were monitored at 360 nm and identified based on their retention times. ChromGate software was used for data analysis.

Statistical analysis

All the experiments were done at least three times in triplicate. Statistical analyses were executed using Microsoft Excel and GraphPad Prism version 5.0. Data are shown as mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied to identify significant differences between means. A P value less than 0.05 was considered significant. Pearson’s correlation coefficient (r) was determined to explore correlations between different parameters.

Results and Discussion

Determination of physicochemical properties: alcohol content, pH, titratable acidity

The alcohol concentration of wines was estimated using the hydrometer and dichromate method and was found to range between 4% and 8% (Table 1). The highest alcohol concentration was observed in grape wine, while the lowest was found in rhododendron wine. Kashyap and Deepshikha have reported an alcohol level of 6.3% in a mixture of rhododendron and mahua flower wines³². Li and colleagues reported an average alcohol content of 10.9% in cherry wine³², which is higher than the values obtained in this study. This difference may be attributed to the fact that home-brewed wines typically have lower alcohol content. Our findings are consistent with the general range of 5-13% for home-brewed wines²⁹.

Titratable acidity and pH were determined for all wines and were found to be in the range of the standard values normally found in the respective fruit wines in the Indian subcontinent. The pH of the wine samples were in the range of 3.4 to 3.6 and cherry wine had the highest titratable acidity (Table 1). According to a study, the pH of red wine ranges between 3 and 4.5, which is comparable to the pH of home-brewed red wines in the present study³³.

Table 1: Physico-chemical characteristics of fruit and flower wines 

Estimation of total phenolic content and flavonoid content of wine

Folin–Ciocalteau and Folin–Denis both methods were utilized to estimate the polyphenol content in wines (Fig. 1a-b and Table 2). Rhododendron wine contained significantly higher (P<0.05) concentrations of polyphenols than other fruit wines, as estimated using both methods. A positive correlation was observed between the Folin–Ciocalteau and Folin–Denis methods (r=0.9938, P<0.0001), validating both methods for the estimation of TPC. The Folin–Denis method yielded higher TPC values than the Folin–Ciocalteau method. A previous study also reported similar results²⁵. The composition of wine with respect to the compounds present varies, depending on the fruit type, climate, terrain, conditions of winemaking, and reactions that occur during the aging of wine, which could account for the results obtained³⁴. Literature data on rhododendron wine show a polyphenol content of 790 µg/mL, which is higher than that observed in the present study³². TPC in cherry wine was lower than the previously reported value of 1940 µg/mL¹⁴. Previous research has indicated that plum wine contains more polyphenols compared to cherry wine, a result that aligns with the findings of this study³⁵. The polyphenol content of wines may decrease owing to unfavorable biochemical reactions such as oxidation, degradation, formation of complexes with proteins, and precipitation with sugars present in wine³⁶.

The flavonoid content in wines was measured using the aluminum chloride assay (Fig. 1c). Rhododendron wine showed the highest flavonoid content (167.75±9.53 µg/mL quercetin equivalent) compared with other wines. Sometimes, fruit wine contains more flavonoids than the fruit itself because of the fermentation process³⁷. Moreover, the bioavailability of the flavonoids and polyphenols are high in wines because of the presence of alcohol ^{38, 39}.

Figure 1: In-vitro assays for determination of total polyphenolic content and total flavonoid content of Grape, Cherry, Plum, and Rhododendron wine samples by a)Folin–Ciocalteau assay, b) Folin–Denis assay, and c) AlCl3 assay       Click here to view Figure

Table 2: Total polyphenolic content and flavonoid content of the wine samples

 High-performance liquid chromatography

The concentrations of catechin, quercetin, and gallic acid in plum, cherry, grape, and rhododendron wines were estimated using high-performance liquid chromatography. Figure 2 presents the chromatograms of standard catechin, quercetin, and gallic acid, with retention times of 10.43 min, 11.76 min, and 6.93 min, respectively (Fig. 2a–c). Standard curves were generated using known concentrations of each polyphenol and their corresponding area under the curve (AUC) values. Similarly, plum, cherry, rhododendron, and grape wine samples were analyzed, and the concentrations of the selected polyphenols were determined based on the standard curves (Fig. 2d–g, Table 3). The concentrations of catechin and quercetin present in the grape wine samples were within the range reported previously^{40, 41, 42}. However, only a few reports are available on plum, cherry, and rhododendron wine samples. To our knowledge, this study is the first to evaluate the antioxidant potential of rhododendron wine and to quantify its catechin, quercetin, and gallic acid concentrations.

Table 3: Polyphenolic content of fruit and flower wines

Figure 2: High- performance liquid chromatogram of (a) standard catechin, (b) quercetin, (c) gallic acid, (d) Plum wine, (e) Cherry wine, (f) Rhododendron wine, and (g) Grape wine at 360 nm.        Click here to view Figure

Estimation of antioxidant activity using FRAP, ABTS, and DPPH methods

The antioxidant capacity of the wine samples was assessed using the FRAP assay, revealing that Rhododendron wine had the highest antioxidant capacity (1723.8±143.19 µg/mL quercetin equivalent) (Fig. 3a). Both Rhododendron and plum wines exhibited significantly higher antioxidant capacities (P<0.05) compared to grape wine in FRAP assay. Additional evaluations with the ABTS and DPPH assays confirmed that all four wines demonstrated robust free-radical scavenging activity (Fig. 3b and 3c), though no significant differences were found among them. Specifically, plum wine had the highest ABTS activity (77.5±3.64%), while Rhododendron wine showed the greatest DPPH scavenging ability (82.16±7.38%). These variations are likely attributed to differences in phenolic and flavonoid compounds, which significantly impact antioxidant capacity. For example, the antioxidant properties of flavonoids are influenced by factors such as the presence of hydroxyl groups, their hydrophobicity, and molecular planarity^{43, 44}. Previous studies have reported varying antioxidant capacities for different wines. For example, cherry wine was found to have a high antioxidant capacity, and a further increase in total phenolic content (2.73 g gallic acid equivalent/L) and antioxidant activity (22.07 mM Trolox equivalent/L) after adding green tea to it⁴⁴. Kashyap and Deepshikha reported the antioxidant capacities of rhododendron wines³². Similarly, plum wine reported to have a phenolic content of 469 ± 7 mg/L gallic acid equivalents and a total antioxidant activity of 304.36±6.24 µg/L (Trolox equivalents)⁴⁵. Correlations between the FRAP, ABTS, Folin–Ciocalteu, Folin–Denis, and AlCl3 assays were positive in the present study (e.g., FRAP-ABTS: r=0.52; Folin–Ciocalteau-FRAP: r=0.78). The literature reveals diverse results regarding the relationship between antioxidant capacity and phenolic or flavonoid contents of wine. Some studies indicate a linear correlation between antioxidant capacity and total phenolic content⁴⁵, while others suggest that antioxidant capacity is more closely related to specific flavonoid fractions. The antioxidant activity of these compounds relies on their proton-donating capacity and the number of hydroxyl groups, with glycosylation also affecting antioxidant potency⁴⁶.

Figure 3: In-vitro assays for determination of antioxidant activity using a). FRAP assay, b) ABTS assay, and c) DPPH assay (*** P < 0.001; ns, not significant [on comparison with grape wine])       Click here to view Figure

Determination of antioxidant potential using RAW 264.7 cells

The antioxidant potential was assessed by measuring the reactive nitrogen species (RNS) and ROS in mouse macrophage cells (RAW 264.7) by inducing oxidative stress with LPS (1 µg/mL). LPS, which is predominantly found in the outer cell wall of gram-negative bacteria, triggers an inflammatory response in the host, leading to elevated production of ROS/RNS and other pro-inflammatory mediators⁴⁷. Macrophages exposed to 1 µg/mL LPS triggered ROS and RNS without inducing cytotoxicity. The cell viability was determined in the presence and absence of LPS and wine samples using MTT assay in macrophage cells (Fig. 4a). More than 86% cell viability was observed for all wine samples, except for plum wine, where the cell viability was relatively low (76%). The ability of wine samples to prevent RNS/ROS generation is shown in Fig. 4b-c. Wine samples, particularly grape and plum wines, significantly reduced nitrite concentration compared with samples treated with LPS only. None of the four wine samples showed significant RNS production in RAW 264.7 cells compared with the media control. LPS (1 µg/ml) induced a high level of ROS production (1,415,300 ± 147,303 RFU), which was significantly reduced by all four wine samples (ranging from 406,965 to 635,281 RFU) after treating with LPS in RAW 264.7 cells. These results indicate that plum, cherry, and rhododendron wines demonstrate ROS/RNS scavenging potential similar to grape wine, without significant cytotoxic effects on macrophage cells.

Figure 4: In-vitro assays using mouse macrophage cells (RAW 246.7) for the estimation of antioxidant activity       Click here to view Figure

Effects of lipopolysaccharide (LPS) and wine samples on the viability of RAW 246.7 macrophage cells determined using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

Measurement of nitrite concentration using Griess reagent: In this assay, RAW 264.7 macrophage cells were subjected to oxidative stress using 1µg/mL LPS with or without wine samples and its stable conversion product nitrite (NO₂^–) was measured.

Measurement of ROS using DCFDA: Mouse macrophage cells (RAW 246.7) were treated with or without 1µg/mL LPS and wine samples for 24 h, and ROS production of the treated and untreated cells was determined using DCFDA (diacetyldichlorofluorescein) staining. The relative fluorescence unit estimation was performed using a fluorescence plate reader (iD3 spectradrop, Molecular devices).

Conclusion

In the present study, we performed a comparative analysis between traditional grape wine and the conventionally less-explored fruit wines of cherry, plum, and rhododendron wines. Our study showed that rhododendron wine possesses greater antioxidant activity compared to grape wine, using various in vitro assays. However, plum, cherry, and rhododendron wines showed significant antioxidant potential in macrophage cells treated with LPS compared with grape wine. Further investigation is needed to quantify additional individual bioactive compounds in wines and to elucidate the health benefits of wine polyphenols using a mouse model

Acknowledgement

We thank Dr. Sangeetha Chavan for her valuable suggestions during the writing of this manuscript.

Funding Sources

The project was funded by Mumbai University (Mumbai University-project no. 362) and the Department of Biotechnology (DBT-Builder-BT/INF/22/SP41293/2020), Government of India.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Author Contributions

Concept and design of the experiments: Pampi Chakraborty, Avik Chakraborty.

Performed the experiments: Mrunmayee Saraff, Juili Mirgule, Shivani Dharmadhikari, Pratik Nazareth, Sarah Thomas and Clarissa Rodrigue.

 Analyzed the data:  Pampi Chakraborty, Avik Chakraborty, Mrunmayee Saraff and Juili Mirgule.

Preparation of the manuscript: Mrunmayee Saraff, Juili Mirgule, Shivani Dharmadhikari and Pampi Chakraborty.

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