Phytochemical Composition and Antioxidant Activity of Mulberry (Morus spp.) Fruit Extracts: A Comparative Analysis

Introduction

Oxidative stress, a condition resulting from the accumulation of reactive oxygen species (ROS) beyond the body’s natural antioxidant defense, plays a central role in the pathogenesis of numerous chronic and degenerative diseases such as cancer, cardiovascular disorders, neurodegenerative ailments, diabetes, and accelerated aging [1-2]. In recent decades, scientific attention has shifted toward exploring natural antioxidants from fruits and medicinal plants as safer and more sustainable alternatives to synthetic antioxidants, which may pose potential health risks when consumed over time [3-5].Mulberry (Morus spp.), belonging to the Moraceae family, is a fast-growing, deciduous plant widely cultivated in Asia, Europe, Africa, and North America [6]. Traditionally known for its use in sericulture, the plant also holds a prominent place in traditional medicine systems due to its therapeutic potential. Among its various parts, mulberry fruits have gained significant interest for their rich composition of bioactive compounds, including polyphenols, flavonoids, anthocyanins, vitamin C, and resveratrol—all of which are known for their potent antioxidant properties [7].Recent studies have revealed that mulberry fruit extracts not only scavenge free radicals but also modulate cellular antioxidant enzyme systems, contributing to overall redox homeostasis. The phytochemical content and antioxidant efficacy of mulberry can vary depending on species (e.g., Morus alba, Morus nigra, Morus rubra), geographical origin, climatic conditions, harvesting time, and extraction techniques [8-10], mulberry fruit extracts have consistently shown high potential in in vitro antioxidant assays, such as DPPH, ABTS, FRAP, and ORAC.Given the increasing demand for functional foods and nutraceuticals, investigating the antioxidant capacity of mulberry fruits has become more relevant than ever. This study aims to systematically evaluate the antioxidant activity of mulberry fruit extracts using different extraction solvents, and to correlate the results with total phenolic and flavonoid contents [11-14]. The findings may provide insights into optimizing extraction conditions for maximum antioxidant yield and could pave the way for the development of mulberry-based dietary supplements, beverages, and pharmaceuticals.

Materials and Methods

1. Collection of Plant Material

Fresh and ripe mulberry (Morus spp.) fruits were collected from Bihar district of Supaul, Indiaduring the peak harvesting season. The fruits were thoroughly washed under running water to remove dust and debris, shade-dried at room temperature for 4–5 days, and then ground into a fine powder using a mechanical grinder. The powder was stored in an airtight container at 4°C until further use.

2. Preparation of Extracts
Ten grams of powdered fruit material was subjected to extraction using 100 mL of solvents with varying polarity—methanol, ethanol, and distilled water—via the maceration method for 48 hours with intermittent shaking. The resulting mixtures were filtered through Whatman No. 1 filter paper, and the filtrates were concentrated under reduced pressure using a rotary evaporator at 40 °C. The crude extracts were then stored at 4 °C in amber glass bottles until further analysis.

3. Determination of Total Phenolic Content (TPC)
Total phenolic content was determined using the Folin–Ciocalteu reagent method. Briefly, 0.5 mL of the extract was mixed with 2.5 mL of 10% Folin–Ciocalteu reagent, followed by the addition of 2.0 mL of 7.5% sodium carbonate solution. The mixture was incubated at room temperature for 30 minutes. Absorbance was then recorded at 765 nm using a UV-Visible spectrophotometer. Gallic acid was used as the standard, and results were expressed as milligrams of gallic acid equivalents (mg GAE) per gram of dry weight.

4. Determination of Total Flavonoid Content (TFC)
Total flavonoid content was estimated using the aluminum chloride colorimetric method. In brief, 0.5 mL of the extract was mixed with 0.5 mL of 2% aluminum chloride solution and 3.0 mL of methanol. After incubation at room temperature for 30 minutes, the absorbance was measured at 415 nm. Quercetin was used as the standard, and results were expressed as milligrams of quercetin equivalents (mg QE) per gram of dry weight.

5. Antioxidant Activity Assays

a. DPPH Radical Scavenging Assay

The antioxidant activity of the extracts was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. A 1 mL aliquot of 0.1 mM DPPH solution in methanol was mixed with 1 mL of extract at various concentrations (25–200 µg/mL). The mixture was incubated in the dark for 30 minutes, and absorbance was measured at 517 nm. The percentage of DPPH inhibition was calculated, and IC₅₀ values were determined.

b. ABTS Radical Cation Scavenging Assay
The ABTS assay was conducted by generating ABTS•⁺ radicals through the reaction of ABTS stock solution with potassium persulfate, followed by incubation in the dark at room temperature for 12–16 hours. The resulting ABTS•⁺ solution was then diluted with ethanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm. For the assay, 1 mL of the diluted ABTS•⁺ solution was mixed with 100 µL of the sample extract. After a 10-minute incubation at room temperature, the absorbance was measured at 734 nm. The percentage inhibition was calculated, and the IC₅₀ values were determined to assess antioxidant activity.

6. Statistical Analysis
All experiments were conducted in triplicate, and the results are presented as mean ± standard deviation (SD). The IC₅₀ values were determined through linear regression analysis. Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test to assess differences between groups. A p-value of less than 0.05 was considered statistically significant.

Lower IC₅₀ values indicate higher antioxidant activity.

Notes:

• r indicates Pearson correlation coefficient.
• Negative values indicate an inverse correlation (i.e., higher phenolic or flavonoid content is associated with lower IC₅₀ values, implying better antioxidant activity).
• p < 0.01, highly significant correlation.

Results and Discussion

Phytochemical Analysis

The phytochemical analysis of the mulberry fruit extracts indicated ahigh concentration of polyphenols and flavonoids, which are well-known for their antioxidant potential. The Total Phenolic Content (TPC)ranged between 112.5 ± 2.3 to 138.7 ± 1.9 mg gallic acid equivalents (GAE)/g, while the Total Flavonoid Content (TFC)ranged between67.2 ± 1.1 to 85.6 ± 1.5 mg quercetin equivalents (QE)/g of extract (Table 1). These findings are in agreement with earlier studies, which have shown that mulberries (especially Morus alba and Morus nigra) are rich in secondary metabolites such as flavonoids, anthocyanins, and tannins (Li et al., 2020).

Antioxidant Activity Assays

Two in vitro antioxidant assays — DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) — were employed to assess the radical scavenging potential of the mulberry extracts. The extracts demonstrated concentration-dependent scavenging activity, with IC₅₀ values ranging from:

• DPPH assay: 42.6 ± 0.8 µg/mL to 61.4 ± 1.2 µg/mL
• ABTS assay: 35.7 ± 0.9 µg/mL to 49.3 ± 1.0 µg/mL

These values are indicative of strong antioxidant potential, particularly in darker-colored mulberry varieties, which often possess higher anthocyanin content. These findings are consistent with those reported by Kim et al. (2013), who noted that darker mulberries contain greater bioactive capacity due to pigment-associated polyphenols.

Correlation Between Phytochemical Content and Antioxidant Activity

The Pearson correlation analysis (Table 2) revealed a significant negative correlation between the TPC and IC₅₀ values of the DPPH (r = –0.986, p < 0.01) and ABTS assays (r = –0.981, p < 0.01). Similarly, TFC was negatively correlated with DPPH (r = –0.972) and ABTS (r = –0.968). These findings suggest that polyphenols and flavonoids are the primary contributors to the observed antioxidant activity, as lower IC₅₀ values indicate higher radical scavenging potential.

Comparison with Other Natural Antioxidants

The antioxidant capacity of mulberry fruit extract was found to be comparable or superior to many other fruits such as blueberries, strawberries, and grapes. The presence of bioactive compoundssuch asresveratrol, rutin, quercetin, chlorogenic acid, and anthocyaninsin mulberry enhances its therapeutic relevance in oxidative stress-related diseases (Sánchez-Salcedo et al., 2015). These compounds not only act as free radical scavengers but also help in modulating enzyme activity, inhibiting lipid peroxidation, andreducing oxidative DNA damage.

Health Implications and Future Perspectives

The antioxidant-rich nature of mulberry fruits makes them ideal candidates for nutraceutical formulation, functional food development, andphytopharmaceutical applications. Antioxidants from natural sources play a critical role in preventing chronic degenerativediseases, including cardiovascular disorders, neurodegeneration, and cancer. Given the safety profile and sustainability of plant-derived antioxidants, mulberries offer a viable alternativeto synthetic antioxidants [15-21].

Future research should aim to:

• Isolate and characterize the individual phenolic compounds responsible for antioxidant activity.
• Conduct in vivo studies and clinical trials to validate the therapeutic efficacy.
• Explore novel delivery mechanisms (e.g., nanoformulations) to improve bioavailability of mulberry phytochemicals.

Conclusion

The present study highlights the significant antioxidant potential of mulberry fruit extracts, attributed primarily to their rich content of phenolic and flavonoid compounds. The extracts exhibited strong radical scavenging activity in both DPPH and ABTS assays, with a clear correlation between phytochemical content and antioxidant efficacy. These findings reinforce the role of mulberry as a potent natural source of antioxidants with promising applications in the fields of functional foods, nutraceuticals, and preventive healthcare. Further studies, particularly in vivo and clinical evaluations, are recommended to establish the therapeutic relevance and commercial viability of mulberry-derived antioxidant formulations.

References

1. Mehmood Abbasi, A., Shah, M. H., Guo, X., & Khan, N. (2016). Comparison of nutritional value, antioxidant potential, and risk assessment of the Mulberry (Morus) fruits. International Journal of Fruit Science, 16(2), 113-134.
2. Iqbal, S., Younas, U., Sirajuddin, Chan, K. W., Sarfraz, R. A., & Uddin, K. (2012). Proximate composition and antioxidant potential of leaves from three varieties of Mulberry (Morus sp.): a comparative study. International journal of molecular sciences, 13(6), 6651-6664.
3. Song, W., Wang, H. J., Bucheli, P., Zhang, P. F., Wei, D. Z., & Lu, Y. H. (2009). Phytochemical profiles of different mulberry (Morus sp.) species from China. Journal of agricultural and food chemistry, 57(19), 9133-9140.
4. Natić, M. M., Dabić, D. Č., Papetti, A., Akšić, M. M. F., Ognjanov, V., Ljubojević, M., &Tešić, Ž. L. (2015). Analysis and characterisation of phytochemicals in mulberry (Morus alba L.) fruits grown in Vojvodina, North Serbia. Food chemistry, 171, 128-136.
5. Hussain, F., Rana, Z., Shafique, H., Malik, A., & Hussain, Z. (2017). Phytopharmacological potential of different species of Morus alba and their bioactive phytochemicals: A review. Asian Pacific journal of tropical biomedicine, 7(10), 950-956.
6. Saensouk, S., Senavongse, R., Papayrata, C., &Chumroenphat, T. (2022). Evaluation of color, phytochemical compounds and antioxidant activities of mulberry fruit (Morus alba L.) during ripening. Horticulturae, 8(12), 1146.
7. Polumackanycz, M., Sledzinski, T., Goyke, E., Wesolowski, M., &Viapiana, A. (2019). A comparative study on the phenolic composition and biological activities of Morus alba L. commercial samples. Molecules, 24(17), 3082.
8. Saeed, A., Kauser, S., Hussain, A., & Nadeem, A. A. (2023). Comparative variability of nutrients, minerals, phenolics and anthocyanins with antioxidant potentials during fruit development stages in five Mulberry (Morus) cultivars. Journal of Berry Research, 13(4), 355-377.
9. REBAI, O., BELKHIR, M., FATTOUCH, S., & AMRI, M. (2017). Phytochemicals from mulberry extract (Morus sp.): Antioxidant and neuroprotective potentials. Journal of Applied Pharmaceutical Science, 7(1), 217-222.
10. Sun, Z., Zhou, Y., Zhu, W., & Yin, Y. (2023). Assessment of the fruit chemical characteristics and antioxidant activity of different mulberry cultivars (Morus spp.) in semi-arid, sandy regions of China. Foods, 12(18), 3495.
11. Memete, A. R., Timar, A. V., Vuscan, A. N., Miere, F., Venter, A. C., &Vicas, S. I. (2022). Phytochemical composition of different botanical parts of Morus species, health benefits and application in food industry. Plants, 11(2), 152.
12. Altaf, L., Wani, S. A., Hussain, P. R., Suradkar, P., Baqual, M. F., & Bhat, A. A. (2024). Bioactive compounds and antioxidant activity in various parts of Morus alba L. Cv. Ichinose: a comparative analysis. Discover Life, 54(1), 7.
13. Bao, T., Xu, Y., Gowd, V., Zhao, J., Xie, J., Liang, W., & Chen, W. (2016). Systematic study on phytochemicals and antioxidant activity of some new and common mulberry cultivars in China. Journal of Functional Foods, 25, 537-547.
14. Shreelakshmi, S. V., Nazareth, M. S., Kumar, S. S., Giridhar, P., Prashanth, K. H., & Shetty, N. P. (2021). Physicochemical composition and characterization of bioactive compounds of mulberry (morusindica L.) fruit during ontogeny. Plant Foods for Human Nutrition, 76, 304-310.
15. Serio, G., Asteggiano, A., Gatti, N., La Rosa, L., Bertea, C. M., Farina, V., … & Gentile, C. (2024). Comparative profiling of secondary metabolites and antioxidant properties of twelve Morus varieties: Insights into the diversity of M. alba and M. nigra grown in Sicily. Food Bioscience, 61, 104782.
16. Ramappa, V. K., Srivastava, D., Singh, P., Kumar, U., Kumar, D., Gosipatala, S. B., … & Raj, R. (2020). Mulberries: a promising fruit for phytochemicals, nutraceuticals, and biological activities. International Journal of Fruit Science, 20(sup3), S1254-S1279.
17. Fauzi, A., Kifli, N., Noor, M. H. M., Hamzah, H., &Azlan, A. (2024). Bioactivity, phytochemistry studies and subacute in vivo toxicity of ethanolic leaf extract of white mulberry (Morus alba Linn.) in female mice. Journal of Ethnopharmacology, 325, 117914.
18. Cao, X., Yang, L., Xue, Q., Yao, F., Sun, J., Yang, F., & Liu, Y. (2020). Antioxidant evaluation-guided chemical profiling and structure-activity analysis of leaf extracts from five trees in Broussonetia and Morus (Moraceae). Scientific reports, 10(1), 4808.
19. Sánchez-Salcedo, E. M., Mena, P., García-Viguera, C., Martínez, J. J., & Hernández, F. (2015). Phytochemical evaluation of white (Morus alba L.) and black (Morus nigra L.) mulberry fruits, a starting point for the assessment of their beneficial properties. Journal of functional foods, 12, 399-408.
20. Gungor, N., &Sengul, M. (2008). Antioxidant activity, total phenolic content and selected physicochemical properties of white mulberry (Morus alba L.) fruits. International Journal of Food Properties, 11(1), 44-52.
21. Wang, Y., Xiang, L., Wang, C., Tang, C., & He, X. (2013). Antidiabetic and antioxidant effects and phytochemicals of mulberry fruit (Morus alba L.) polyphenol enhanced extract. PloS one, 8(7), e71144.