Pigment extraction from mulberry: A review

Introduction

The various colors seen in plants, animals, and microbes are caused by pigments, which are found throughout the biological world (Siddharthan et al., 2020) [17] . Because of their potential uses in a variety of industries, such as food, cosmetics, textiles, and pharmaceuticals, the extraction of these pigments from natural sources has attracted a lot of attention (Juric et al., 2020) [10] . A promising source of natural pigments, especially anthocyanins, which give its fruits their vivid colors, is the genus of flowering plants known as mulberries, which belong to the Moraceae family (Suhaimi et al., 2021) [18] . As primary or secondary metabolites, pigment production in microorganisms is essential to their survival and plays a key role in photoreactive mechanisms that protect against oxidative stress and reduce DNA damage from UV radiation (Ghosh, 2025) [8] . The process of pigment extraction from mulberry plants involves a multifaceted approach, encompassing various techniques aimed at isolating and purifying the desired compounds while preserving their structural integrity and bioactivity. The recovery of anthocyanins and phenolic compounds from plant materials is commonly achieved through solid-liquid extraction utilizing polar solvents such as methanol, ethanol, and acetone under acidic conditions (Suhaimi et al., 2021) [18] . Natural sources, chemical characteristics, stabilization procedures, and applications in actual food systems are some of the major concerns surrounding the use of these natural colorants in foods. They cover a variety of pigment groups, many of which have strong biological potential. Major regulatory bodies have approved the use of natural pigments in foods, which provide a variety of colors and intensities. However, their higher cost and less stability in comparison to synthetic alternatives can occasionally impede their adoption (Juric et al., 2020) [10] . The choice of extraction method is contingent upon several factors, including the type of pigment being extracted, the plant matrix, and the desired purity and yield. Solvent extraction, a conventional technique widely employed at the laboratory scale, involves the use of organic solvents to selectively dissolve and separate target compounds from insoluble solids through absorption and release of bioactive components (Kattil et al., 2024) [12] . The efficiency of solvent extraction is influenced by several parameters, including solvent type, temperature, extraction time, and solid-to-solvent ratio. The selection of a suitable nontoxic extraction solvent is critical for the safe recovery of pigments intended for use as food colorants (Daud et al., 2021) [7] . Optimization of anthocyanin extraction involves careful control of factors like solvent type to maximize extraction rates (Tan et al., 2022) [19] . Mulberry plants, which are members of the Morus genus, are a valuable but underutilized resource because of their varied nutritional makeup, phytochemical richness, and possible pharmacological uses (Kattil et al., 2024) [12] . Like other berries, mulberry fruits are syncarps made up of clusters of tiny fruits called drupes that undergo color, texture, and biochemical changes as they ripen following pollination (Kattil et al., 2024) [12] . The fact that these plants are found in temperate and subtropical climates in Asia, Africa, Europe, and the Americas highlights their accessibility potential for pigment extraction projects (Chetia et al., 2023) [6] .The shift towards natural pigments has accelerated, driven by heightened awareness of the safety and potential toxicity associated with synthetic counterparts, thereby creating a compelling impetus for exploration of plant-based alternatives such as mulberry (Bakhsh al., 2023) [3] . The extraction of pigments from mulberry plants is a multifaceted process influenced by several critical parameters, including the choice of extraction solvent, the optimization of extraction methodologies, and precise control over environmental conditions such as temperature, pH, and light exposure. Solvent extraction is a common and conventional technique for extracting pigments from plant materials that relies on dissolving the desired compounds in a suitable solvent (Kalra et al., 2020) [11] . Polar solvents, including ethanol and acetone, have demonstrated efficacy in extracting anthocyanins from mulberry fruits, underscoring their role in solubilizing these pigments (Suhaimi et al., 2021) [18] . Novel technologies, including ultrasound-assisted extraction and microwave-assisted extraction, have emerged as promising alternatives to conventional solvent extraction methods, as they offer the advantages of reduced extraction time, lower solvent consumption, and enhanced extraction efficiency (Masyita et al., 2025) [15] . These advanced techniques leverage the principles of sonication and microwave irradiation, respectively, to disrupt plant cell walls and facilitate the release of pigments into the extraction solvent. Furthermore, the optimization of extraction parameters, such as solvent composition, extraction time, temperature, and pH, is crucial for maximizing pigment yield and purity. Different cultivars of mulberry exhibit variations in anthocyanin profiles and antioxidant activity, suggesting the importance of cultivar selection in pigment extraction strategies (Kim & Lee, 2020) [13] .

Pigment Extraction from Mulberry

From sample preparation to the ultimate pigment isolation and purification, the process of extracting pigments from mulberry plants usually entails multiple steps. The particular process can change based on the desired pigment type and whether the leaves or fruits of the mulberry plant are being used. Gather fresh mulberry leaves, usually from certain spots (such as third and fourth from the top). Use distilled water to thoroughly wash the gathered leaves in order to get rid of any dirt or contaminants. To maximize the surface area for extraction, chop them into smaller pieces, such as those that are about 1 cm in size (Chetia et al., 2023) [6] . Fresh mulberry fruits can be processed into juice (Liu et al., 2004) [14] or consumed raw (Qin et al., 2010) [16] . Mulberry pomace, a byproduct of juice and wine making, can also be utilized (Zhang et al., 2011) [21] . Various methods are employed for extracting pigments, often involving different solvents and conditions.

Conventional Solvent Extraction method involves soaking the prepared leaf material in a solvent. Water is a common solvent for natural dye extraction (Chetia et al., 2023) [6] . Organic solvents like ethanol are also used for extracting phenolic compounds and flavonoids (Kattil et al., 2024) [12] . Optimal ratio of Material to Liquor (M:L) can range (e.g., 1:10, 1:20, 1:30). Elevated temperatures are often used to enhance extraction efficiency (e.g., 60 °C, 70 °C, 80 °C). Extraction time can vary (e.g., 60 minutes, 70 minutes, 80 minutes, 90 minutes). For optimal dye yield from leaves, a material-to-liquor ratio of 1:30, temperature of 80 °C, and extraction time of 80 minutes have been reported. After extraction, the mixture is typically filtered to separate the solid residue from the pigment extract. The liquid extract can then be concentrated (e.g., by drying in a hot water bath to evaporate water) (Chetia et al., 2023) [6] .

Anthocyanin extraction from Mulberry Fruits

Solvent Extraction is a common laboratory-scale method. Polar solvents are preferred, such as ethanol, methanol, or acetone, often in aqueous solutions (e.g., 70-80% in water) and under acidic conditions (e.g., with 0.1% HCl or trifluoroacetic acid) to enhance recovery of anthocyanins and phenolic compounds (Kattil et [12,16,18] . Soaking the sample in a large volume of solvent at ambient temperature with occasional shaking. For example, 50% ethanol for 4 hours at 25°C. Ultrasonic-Assisted Extraction method uses ultrasonic waves to improve extraction efficiency. Examples include 63.8% methanol with 1% trifluoroacetic acid at 43.2°C for 40 minutes, or 80% ethanol at 20°C for 12 minutes. Microwave-Assisted Extraction uses microwave energy to heat the solvent and sample, such as 59.6% methanol at 425 W power for 132 seconds (Kattil et al., 2024) [12] . In alcoholic extraction, fresh mulberry fruits can be extracted with 95% alcohol/0.1% HCl (1:1 ratio) at room temperature for 4 hours in the dark (Qin et al., 2010) [16] .

Purification and Concentration

After initial extraction, purification steps are often necessary to increase pigment purity and concentration, especially for anthocyanins. Extracts are typically filtered to remove solid particles (Chetia et al., 2023) [6] . Macroporous resins (e.g., X-5, AB-8) are effective for adsorbing anthocyanins, removing impurities like mono-, di-, and polysaccharides, minerals, proteins, and organic acids. The adsorbed anthocyanins can then be eluted using acidified ethanol (e.g., 0.5% (v/v) hydrochloric acid in ethanol) (Liu et al., 2004; Zhang et al., 2011) [14,21] . This process can significantly increase the color value (Liu et al., 2004) [14] . The eluted pigment solution can be concentrated using a rotary evaporator under reduced pressure, followed by lyophilization (freeze-drying) to obtain pigment powder (Liu et al., 2004) 14[] . For high-purity isolation of individual anthocyanin monomers, methods like high-speed counter current chromatography combined with column chromatography can be used (Chen et al., 2017) [5] . Sephadex LH-20 columns can be used for separation, eluting with solvents like 10% ethanol containing 1% acetic acid (Zhang et al., 2011) [21] .

Analysis and Characterization

After extraction and purification, the pigments are typically analyzed to identify and quantify their components. Techniques like UV spectrophotometry can be used to determine total anthocyanin content using methods like the pH differential method (Liu et al., 2004) [14] . HPLC used for identification and quantification of individual pigments and their relative concentrations (Liu et al., 2004; Qin et al., 2010) [14,16] . LC-MS and NMR are advanced techniques for detailed identification of pigment structures (Qin et al., 2010) [16] . FT-IR used to confirm the presence of specific functional groups like alcohols, halogens, flavonoids, phenols, and tannins in the extract (Chetia et al., 2023) [6] . This systematic approach allows for the efficient and targeted extraction of various pigments from different parts of the mulberry plant, catering to diverse applications. The optimization of pigment extraction from mulberry plants necessitates a detailed evaluation of various methodologies to maximize yield and purity. Pigment extraction from mulberry plants yields a variety of natural coloring agents, primarily from both leaves and fruits. The results of these extractions depend on the specific plant part used, the mulberry cultivar, and the extraction methodology.

Identification of Pigments

Mulberry plants are rich in various pigments and bioactive compounds. From Mulberry Leaves lutein (30.86%) and βcarotene (26.30%) are reported as primary coloring agents (Chetia et al., 2023) [6] . Other compounds include quercetin and kaempferol (Chetia et al., 2023) [6] , as well as flavonoids, phenolic compounds, carbohydrates, halogen group, and tannins (Chetia et al., 2023) [6] . Anthocyanins are abundant in mulberry fruits with cyanidin 3-O-rutinoside (60%) and cyanidin 3-O-glucoside (38%) being the most prevalent. Minor anthocyanins like pelargonidin are also present (Qin et al., 2010) [16] . Cyanidin-3-glucoside and cyanidin-3-rutinoside are identified as major anthocyanins (Go et al., 2022) [9] . The total anthocyanin content varies significantly among cultivars, ranging from 147.68 to 2725.46 mg/L juice, and is influenced by environmental factors such as climate and production area (Liu et al., 2004) [14] . Mulberry fruits also contain flavonoids and phenolic compounds (Centhyea et al., 2021; Kattil et al., 2024) [3,12] .

From Mulberry Leaves (for dye extraction)

Optimal conditions for dye extraction were found to be a material-to-liquor ratio (M:L) of 1:30, a temperature of 80 °C, and an extraction time of 80 minutes. Under these conditions, a dye yield of 13.38±0.59a was achieved. The extracted dye from mulberry leaves produced colors ranging from brown to yellowbrown (Chetia et al., 2023) [6] .

From Mulberry Fruits (for anthocyanin extraction)

Purification processes, such as using macroporous resins, can significantly enhance the color value of extracted pigments, often increasing it from less than 4.0 to over 100, while also removing impurities and improving stability (Liu et al., 2004) [14] . Mulberry pomace, a byproduct of juice production, has shown potential as a source of anthocyanins, with contents exceeding 250 mg/100 g in some varieties, representing 74%-79% of the anthocyanin content in the whole fruit (Zhang et al., 2011) [21] . Various extraction methods and their reported yields for anthocyanins from different mulberry fruit types include: [12] Dried mulberry fruit Ultrasonic-Assisted Extraction 63.8% methanol + 1% trifluoroacetic acid, 43.2 °C, 40 min 64.70 mg/100 g (Kattil et al., 2024) [12] Black mulberry fruit juice Ultrasonic-Assisted Enzymatic Extraction 80% ethanol, 20 °C, 12 min 55.32 mg/100 g (Kattil et al., 2024) [12] Fresh mulberry fruits Solvent Extraction 95% alcohol/0.1% HCl (1:1 ratio), room temperature, 4 h Not specified, but yields abundant anthocyanins (Qin et al., 2010) [16] (Qin et al., 2010) [16] Pigment Characteristics and Extraction Efficiency Mulberry leaves are a source of natural dyes, primarily containing lutein (30.86%) and β-carotene (26.30%) as main coloring agents. Other compounds such as flavonoids, phenols, and tannins are also present (Chetia et al., 2023) [6] . Optimized extraction parameters, including material-to-liquor ratio, temperature, and extraction time, are crucial for maximizing the dye yield from leaves. For instance, a yield of 13.38±0.59% was achieved under specific conditions (1:30 material-to-liquor ratio, 80°C, 80 minutes) (Chetia et al., 2023) [6] . Mulberry fruits, conversely, are rich in anthocyanins, which are responsible for their natural color (Kattil et al., 2024) [12] . Key anthocyanins identified include cyanidin-3-o-glucoside (c3g) and cyanidin-3-o-rutinoside (c3r), along with pelargonidin-3glucoside (Kattil et al., 2024) [12] . While raw mulberry juice has a low color value due to non-pigment components, purification with resins significantly enhances the color value (to over 100) and improves stability by removing impurities (Liu et al., 2004) [14] . This purification process is critical for producing highquality pigment for commercial use (Liu et al., 2004) [14] . The total anthocyanin content in fruits can vary greatly among cultivars, with ranges observed between 147.68 and 2725.46 mg/L juice, and is also influenced by climate and production area (Liu et al., 2004) [14] .

Applications and Bioactivity

The extracted pigments from mulberry plants offer a wide array of applications due to their coloring properties and bioactive compounds:

• Natural Dyes: Mulberry leaves are a promising natural source for dyeing fabrics, offering a potential reduction in the cost of producing natural dyes and promoting the utilization of agricultural byproducts (Chetia et al., 2023) [6] . • Food Colorants: Mulberry fruit anthocyanins are attractive as natural food colorants. They are water-soluble and provide appealing colors like orange, red, and blue (Liu et al., 2004) [14] . The utilization of these anthocyanins as natural food colorants can enhance the overall profitability of the mulberry plant (Liu et al., 2004) [14] . • Health and Therapeutic Benefits: Beyond their coloring attributes, mulberry pigments, particularly anthocyanins, possess significant health benefits. They are potent antioxidants and have been reported to improve visual acuity. They also exhibit antineoplastic, radiationprotective, vasotonic, vasoprotective, anti-inflammatory, chemo-protective, and hepato-protective activities (Liu et al., 2004) [14] Mulberry is known to protect from liver damage, strengthen joints, facilitate urine release, and decrease blood stress (Kattil et al., 2024) [12] . Additionally, other phytochemicals like flavonoids and phenolics in mulberry contribute to properties such as laxative, hypoglycemic, expectorant, anthelmintic, odantalgic, and emetic effects (Kattil et al., 2024) [12] .

• Emerging Technologies: Mulberry pigments also show potential in advanced applications, such as their use as dye sensitizers in Dye-Sensitized Solar Cells (Suhaimi et al., 2021) [18] .

Conclusion

Mulberry plants, both leaves and fruits, present a valuable and diverse source of natural pigments with broad applications. The successful extraction and purification of these pigments are crucial for their effective utilization in industries ranging from textiles and food to pharmaceuticals and emerging technologies. The increasing demand for natural and sustainable products positions mulberry pigments as a significant area of research and commercial interest, driven by their aesthetic qualities and compelling health benefits. Plant species with varying colors and properties can be harnessed in the textile, printing, cosmetics, and food industries (Alegbe & Uthman, 2024) [1] . The increasing attention toward natural pigments reflects a growing awareness of the ecological impacts and health hazards linked to synthetic colorants (Bakhsh et al., 2023; Venil et al., 2020) [3,20] . Natural pigments are expected to have increased use in the pharmaceutical, medical, and food sectors because of their nontoxic qualities, biodegradability, and biocompatibility (Chetia et al., 2023; Masyita et al., 2025) [6,15] . The food industry is turning to natural pigments as consumer awareness grows regarding the possible health risks of artificial food coloring. The use of bacterial pigments shows promise for a wide array of new biotechnological uses, including functional food production Mulberry's rich pigments, particularly anthocyanins, not only serve as natural colorants but also offer various health benefits, including antioxidant, antiinflammatory, and anti-cancer properties. and the creation of cutting-edge medications and biomedical therapies ( [4,12,9]

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