Open access peer-reviewed chapter - ONLINE FIRST
Abstract
Rutin, a quercetin-3-O-rhamnoglucoside, is a naturally occurring flavonol ubiquitous in plants, especially Morus species. Rutin, with its antioxidant, antibacterial, and radical scavenging properties, is a promising anti-inflammatory and anticancer agent, potentially improving cardiovascular well-being by reducing inflammation and enhancing blood vessel functionality. Mulberry leaves, rich in nutrients and bioactive components, are used in medicine, human consumption, and animal rearing for their ability to reduce inflammation and act as potent antioxidants. Flavonoids, particularly rutin, possess strong therapeutic and antioxidant properties but have not been fully characterized, necessitating further research to understand their components and pharmacological characteristics. High-performance thin-layer chromatography, high-performance liquid chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, and crystal isolation can be used to isolate and characterize Rutin, a prevalent flavonoid in Morus species. These methods in combination allow for quantitative assessment of rutin content in Morus species, uncovering significant variations and highlighting the need for comprehensive phytochemical studies.
Keywords
- rutin
- HPTLC
- HPLC
- NMR
- FTIR
- characterization
- isolation of rutin crystals
- Morus species
1. Introduction
Flavonoids are an important group of bioactive phytochemicals that are widely acknowledged for their varied biological effects and widespread occurrence in the plant world. The compounds possess a phenolic structure and are ubiquitous in a variety of sources, such as fruits, vegetables, cereals, bark, roots, stems, flowers, tea, and wine. Flavonoids play a crucial role in the pigmentation and pollination processes of plants, contributing to the vibrant colors observed in various fruits and flowers. In addition to their visual attractiveness, flavonoids serve vital functions in plant defense systems against UV radiation, pests, and diseases, underscoring their importance in ecological interactions and plant adaptation processes [1, 2].
Scientific interest in flavonoids extends beyond their roles in plants, as they exhibit potential health benefits for humans. Research suggests that flavonoids possess antioxidant, anti-inflammatory, and immune-modulating properties, which may contribute to their protective effects against chronic diseases such as cardiovascular disorders and certain cancers. The therapeutic potential of flavonoids has spurred investigations into their dietary intake and bioavailability, aiming to understand how these compounds might promote human health and disease prevention through dietary interventions and pharmaceutical applications [3, 4, 5].
Flavonoids are synthesized within plants through the phenylpropanoid pathway, a complex metabolic process involving multiple enzymatic steps and regulatory mechanisms. Flavonoids are synthesized through the conversion of phenylalanine into cinnamic acid, by the action of the enzyme phenylalanine ammonia-lyase, and cinnamic acid undergoes transformations using enzymes such as cinnamate 4-hydroxylase and 4-coumarate CoA ligase. This leads to the formation of chalcone, an intermediary in the synthesis of flavonoids. The enzyme chalcone isomerase converts chalcone into naringenin, which is the precursor for various flavonoids, including flavones, flavonols, flavanones, anthocyanins, and isoflavonoids [6]. The pathway diverges in several directions based on the particular enzymes involved and external variables, which impact the synthesis of distinct kinds and concentrations of flavonoids in various plant tissues and under different circumstances. The knowledge of flavonoid biosynthesis becomes essential for comprehending their functions in plant physiology and adaptations, as well as for implementing their potential advantages in agriculture, nutrition, and medicine [7].
Flavonoids are a diverse group of plant secondary metabolites characterized by their common structure consisting of 15 carbon atoms arranged into two aromatic rings joined by a heterocyclic ring. This basic structure is known as the flavonoid skeleton, which can undergo various modifications such as hydroxylation, methylation, glycosylation, and prenylation. These modifications lead to the extensive structural diversity observed among flavonoids, giving rise to numerous subclasses including flavones, flavonols, flavanones, flavan-3-ols (catechins), anthocyanidins, and iso-flavonoids [8].
Flavonoids obtained from plant species of the genus
Rutin, also known as vitamin P, rutoside, quercetin-3-O-rutinoside, and sophorin, is a flavonol glycoside found in
1.1 Structure and biosynthesis
Rutin’s chemical structure consists of two main components: a flavonol backbone and a rutinose sugar moiety. The flavonol backbone, quercetin, is a flavonoid compound with a characteristic structure of a diphenyl propane skeleton, hydroxyl groups at positions 3, 5, 7, 3′, 4′, and 5′, and a double bond between carbons 2 and 3 in the C ring. The rutinose sugar, attached
Figure 1.
Chemical structure of rutin [15].
Synonyms: Rutoside, Quercetin 3-rutinoside, Vitamin P, etc.
Chemical formula: C27H30O16;
Molecular weight: 610.518;
Chemical class: Flavonol.
Rutin biosynthesis in plants involves the conversion of the precursor compound quercetin to rutin through the addition of a rutinose sugar group catalyzed by specific glycosyltransferases. This process occurs in various plant tissues, particularly in the leaves, flowers, and seeds, where rutin serves protective roles against UV radiation and oxidative stress [17].
1.2 Sources and dietary intake
Rutin is abundantly found in dietary sources such as buckwheat (
1.3 Pharmacological effects and health benefits
The pharmacological effects of rutin are extensive. Rutin is a significant ingredient found in apples that serves multiple biological functions, thereby supporting the well-known adage “an apple a day keeps the doctor away.” The extensive investigation of rutin, a vital phytochemical, is necessary to establish a safe profile for its medicinal use in humans.
The antitumor activity of the combination of rutin and cisplatin against murine ascites Dalton’s lymphoma (DL) has been discovered. This combination enhances cytotoxicity and induces apoptosis in DL cells. The described intervention enhances the ability of the host to withstand stress and decreases the levels of glutathione in cancerous cells [18].
A study by Calzada et al. has discovered that rutin derived from
Patil et al. have determined in their research that rutin and quercetin have the ability to safeguard human lymphocytes from DNA damage caused by radiation. This protective effect is attributed to rutin’s antioxidant properties, which enables it to effectively neutralize free radicals and inhibit oxidative stress [20].
A study by Hunyadi et al. reported that rutin, which is the primary constituent of
Figure 2.
Health benefits of rutin [21].
Rutin exhibits a range of pharmacological effects that contribute to its health benefits: [23].
Antioxidant activity: Rutin acts as a potent antioxidant, scavenging free radicals and protecting cells from oxidative damage. This property is attributed to the hydroxyl groups on the quercetin backbone, which stabilizes free radicals and inhibits lipid peroxidation.
Anti-inflammatory properties: Studies have shown that rutin can suppress inflammatory mediators such as cytokines and prostaglandins, thereby reducing inflammation in various experimental models.
Vasoprotective effects: Rutin is known for its beneficial effects on vascular health. It strengthens capillaries and blood vessels, improves microcirculation, and reduces the risk of vascular permeability and edema.
Anticancer potential: Research suggests that rutin may have potential anticancer effects, attributed to its antioxidant properties and ability to induce apoptosis in cancer cells.
2. Isolation of rutin
The extraction and isolation of phytochemicals, which contribute to biologic activity, is a crucial step in identifying and characterizing plant drugs. Various methods have been used for years, depending on the nature of the phytoconstituent to be isolated and the best isolation technique for the plant drug [24]. Rutin, a phenolic flavonoid, is the rhamno-glucoside of quercetin and precisely follows phenolic compound isolation protocols; after extraction, isolation is typically conducted using chromatographic techniques for further characterization [25]. Different techniques used for the extraction of rutin from various bioresources have been enumerated in the previous studies given below.
One of the main methods that have been used for the isolation entails the exhaustive soxhlation of the powdered/ground plant material with alcohol [26, 27, 28]. Thereafter, filtration and evaporation and then subsequent mixing with distilled water (mostly hot) and extraction with petroleum ether and/or chloroform. The aqueous layer is let to stand for up to 72 hours, and the yellow precipitate obtained is separated from solution. After filtering, the precipitate is cleaned using a suitable solution (like chloroform: ethyl acetate: ethanol-50:25:25), and the undissolved portion is dissolved in methanol and filtered; the filtrate is then dried by evaporating to get yellow powder, which is a flavonoid solution containing rutin [29, 30, 31]. This flavonoid-rich solution can then be subjected to chromatographic techniques like HPLC [32, 33, 34] for further concentration (Figure 3).
Figure 3.
Diagrammatic representation of method commonly used for isolation of rutin [30].
An alternate method involves taking the impure solution after alcoholic soxhlation to be further refined through modest amounts of cold alcohol extraction, repeated crystallization from aqueous pyridine and/or diluted acetic acid (1%), and/or hot water to give rutin crystals [35].
Another method involves obtaining the hexane extract, and the dried sample is first extracted with hexane at room temperature (25 ± 2°C) for 10 days. The ethanolic extract is then produced by macerating the vegetable material that had been extracted using hexane with ethanol. Additionally, rota-evaporation and filtration are used to concentrate the ethanolic extract, producing a yellow-green material yield. It can then be examined using infrared, 1H, and 13C nuclear magnetic resonance spectroscopy. Rutin (99% purity) can be obtained by recrystallization of this material with methanol [36].
Yet in another alternate method the raw plant material is repeatedly extracted using methanol and 0.1% concentrated hydrochloric acid; then, the filtered extract is concentrated under low pressure and refined by partitioning against ethyl acetate [37].
3. Characterization of rutin using various analytical techniques
The characterization of flavonoid structures is closely linked to the elucidation of their spectroscopic spectra, which are obtained using techniques such as nuclear magnetic resonance (NMR), mass spectrometry (MS), spectrophotometric ultraviolet (UV), and infrared (IR). These techniques offer valuable insights into the chemical bonds, functional groups, and overall structure of flavonoids [38].
The characterization of rutin from the leaves of
3.1 Characterization of rutin by ultraviolet-visible (UV-vis) spectrophotometry
Ultraviolet-Vis spectrophotometry is a widely recognized technique for identifying and characterizing phenolic compounds, particularly flavonoids. It provides rapid and easily interpretable results, making it advantageous in natural product chemistry. It expedites the evaluation of bioactive phytochemicals in plant extracts and provides valuable information about the purity and quantity of phenolic chemicals, making it an essential tool for researchers in various scientific disciplines [40].
UV spectroscopy reveals two prominent absorption bands in the presence of flavonoids in methanol spectra. Band I, typically 300–380 nm, is associated with the B-ring cinnamoyl system, while Band II, 240–280 nm, is associated with the A-ring benzoyl system. These bands aid in identifying and quantifying flavonoids in plant extracts. Researchers can compare UV spectra of unknown samples to recognized standards to assess flavonoids’ classes and quantities. Flavonoids are classified based on the presence of hydroxyl groups in the A and B rings of the flavonoid structure [41].
The ultraviolet spectrum of rutin in methanolic solution reveals three main absorption bands at 236, 257, and 358.5 nm, indicating a flavonol structure [29]. Savale et al. reported that the rutin solution in methanol exhibited maximum absorption at 257 nm, which was reported as λmax [42]. Hunyadi et al. found that the antidiabetic activity of
Pitchaya et al. analyzed the UV spectra of mulberry leaves, revealing rutin as the main flavonoid compound with λmax values at 256 and 353–354. This finding aligns with previous studies, which also identified rutin as a significant flavonoid in mulberry leaves. Rutin is known for its antioxidant and anti-inflammatory properties, suggesting mulberry leaves could be a promising health benefit source [44].
Zhang et al.‘s study analyzed rutin’s UV spectra to identify its characteristic absorption wavelength for HSCCC detection and HPLC analysis. The chosen wavelength was 257 nm. The results showed rutin had a significant absorption peak at 257 nm, making it suitable for both techniques. This could improve the precision and effectiveness of rutin analysis in diverse samples (Figure 4) [45].
Figure 4.
UV spectra of rutin [4].
Taraba et al. studied UV-visible spectra of rutin in EtOH, revealing two absorption bands at 356 nm (band I) and 258 nm (band II), which is similar to the earlier reported studies. Band I correspond to cinnamoyl system between two rings of the flavonoid structure, while band II to the benzoyl moiety. The addition of Triton X-114 (TX114) to the Ru-EtOH combination reduced the intensity of both absorption bands, suggesting interactions between the components [46].
UV absorption is a powerful analytical tool for identifying and characterizing phenolic compounds, particularly flavonoids, in industries such as pharmaceuticals, food and beverage, and cosmetics, providing prompt, interpretable results and quantifying flavonoid concentrations in samples.
3.2 Characterization of rutin by Fourier transform infrared spectroscopy (FTIR)
Flavonoids can be identified using chromatographic techniques and mass spectrometry, but Fourier Transform Infrared (FT-IR) spectroscopy is a cost-effective and user-friendly alternative. It eliminates the need for complicated sample preparation and additional reagents [47]. However, interpreting the spectra can be challenging, so a spectrum library or expert consultation may be necessary. These methods can differentiate functional groups, analyze bond structure, and examine conformation, making them valuable for identification and quantification [47]. Understanding the structure of flavonoids is important for determining their antioxidant and antibacterial/antifungal properties, and FT-IR spectroscopy can help analyze their chemical composition and detect substituents that affect functionality [48]. The extracts of medicinal plants are combined with KBr salt using a mortar and pestle, and then compressed into a thin pellet. The pellet is subsequently subjected to infrared spectroscopy for the purpose of identifying the functional groups that are present in the plant extracts. The infrared spectra and peak values are generally recorded using a Fourier transform infrared (FTIR) spectrometer, within the range of 4000 to 400 cm−1. The fingerprint region is characterized by prominent absorption bands in the 1500–500 cm−1 range, and the FTIR bands within the 4000–1500 cm−1 range indicate the presence of a functional group. The observed peaks in the spectra correspond to the vibrations of distinct functional groups, enabling the identification of specific compounds present in the plant extracts.
Fourier transform infrared (FT-IR) spectroscopy is a widely employed analytical technique that enables the examination of structural variations among different samples. However, the interpretation of FT-IR spectra can be challenging when the samples exhibit similar structural characteristics. An example of this is that all spectra include bands that are specific to the stretching vibrations of the C〓C ring. However, these bands are found at slightly varying wavenumbers. The vibrational frequency of flavonoids is consistently observed at approximately 1650–1560 cm−1. Additionally, all flavonoid groups, except flavanols, exhibit a C‒C ring vibration band within the range of 1560–1465 cm−1. The spectral peak observed at the wavenumber range of 1475–1400 cm−1 is attributed to the presence of the C‒C bond in flavonols and anthocyanins compounds. The vibrational (C‒C) band of the ring is detected within the range of 1310–1200 cm−1 for all flavonoid groups, with the exception of anthocyanins. The presence of the band at 1225–1180 cm−1 is exclusive to the spectra of isoflavones, flavonols, and flavanols. The presence of a band at 1046–970 cm−1 is exclusively detected in the spectra of isoflavones, flavonols, and anthocyanins. The spectral bands below 970 cm−1 display distinct characteristics for each flavonoid group, primarily determined by their ascription rather than their position [49].
The rutin molecule is distinguished by the replacement of the C3 hydroxyl group of quercetin with the disaccharide rutinose (α-L-rhamnopyranosyl-(1 → 6)-β-D-glucopyranose). The primary differences in IR spectra are attributed to the vibration of the C ring and the pyranosyl rings. The presence of a carboxylic acid in rutin can be identified by a distinct band observed between 3550 and 3400 cm−1, which corresponds to the O‒H bond. This bond is known to contribute to the formation of strong intermolecular hydrogen bonding. The band observed in the range of 1700–1600 cm−1 is associated with the stretching vibration of the C〓O bond. The presence of a distinct peak in the wavenumber range of 1500–1400 cm−1 indicates the presence of C‒O and O‒H bonds. The rocking vibration frequencies of the C‒H bonds in rhamnopyranosyl and glucopyranosyl rings are observed at 1121 and 1168 cm−1, respectively. The infrared (IR) spectra exhibit peaks at 1041 and 1093 cm−1, indicating the oscillation of the C‒O bond between rhamnopyranosyl and glucopyranosyl rings [50]. The infrared (IR) band observed at 999 cm−1 can be attributed to the combined wagging vibrations of the C‒H bonds in the rhamnopyranosyl ring and the linkage between the rhamno- and glucopyranosyl rings. Additionally, the presence of substitutions in the Ar-H group is indicated by a band observed in the range of 800–600 cm−1. The infrared spectrum of rutin offers significant insights into its functional groups and molecular structure.
In their study, Tatke et al. conducted research focused on the isolation of rutin from
Figure 5.
FTIR spectra of rutin [49].
The spectral peaks of rutin (728, 1062, 1599, 1654, 2878, and 3344 cm−1) were analyzed in
A study on
3.3 Characterization of rutin by high-performance thin layer chromatography (HPTLC) analysis
High-performance thin layer chromatography (HPTLC) is a valuable analytical tool for evaluating plant quality and formulations containing flavonoids as biomarkers. It helps identify and quantify specific flavonoids like rutin by comparing chromatographic fingerprints from samples and ensuring the quality and standardization of medicinal products. The literature review suggests that a mobile phase of ethyl acetate, formic acid, glacial acetic acid, and water in different ratios is commonly used for high-performance thin-layer chromatography (HPTLC) analysis of rutin, especially in
Based on the findings of Sánchez-Salcedo et al. as well as Polumackanycz et al., rutin, a biomarker found in the leaves of various
A study by Jan et al. reported the presence of rutin and chlorogenic acid in
Figure 6.
HPTLC chromatogram of rutin in
The study by Kathia et al. evaluated the phenolic content, rutin, and antioxidant and antibacterial activity of methanolic extracts from
The high-performance thin-layer chromatography (HPTLC) technique is widely recognized as a valuable method for the qualitative analysis of flavonoids in plant extracts. This method is highly sensitive and specific, allowing for the detection and precise identification and quantification of flavonoids, thereby offering valuable data for subsequent research on the potential health advantages associated with these plant extracts [61].
Mulberry leaves are a rich source of flavonoids, providing antioxidant, anti-tumor, anticancer, and anti-inflammatory benefits. HPTLC analysis can assess the quality of
3.4 Characterization of rutin by high-performance liquid chromatography (HPLC) analysis
The high-performance liquid chromatography (HPLC) method is a preferred chromatographic technique for analyzing flavonoids due to its efficient time-saving nature, elimination of derivatization, and its ability to be conducted at room temperature, ensuring the preservation of delicate compounds such as flavonoids without the risk of decomposition at higher temperatures [62]. It involves isolating phenolic chemicals from the samples and using HPLC with a gradient mobile phase. Target flavonoids are identified and quantified by comparing them to reference standards using a polar organic solvent and mild acid [63].
Researchers have widely studied flavonoid components in mulberry species, detecting phenolic acids and flavonol glycosides in leaves using HPLC analysis. Thabti et al. identified compounds such as rutin, kaempferol-7-O-glucoside, and quercetin-3-O-rhamnoside-7-O-glucoside in mulberry leaves. Furthermore, it was determined that the leaves of
A study by Ju et al. used high-performance liquid chromatography (HPLC) to analyze the flavonoid content in Korean mulberry leaves. The results showed that the leaves had high levels of quercetin and rutin, known for their antioxidant properties. These flavonoids, which are abundant in Korean mulberry leaves, could potentially contribute to the health benefits associated with flavonoid-rich foods. The findings suggest that these compounds could significantly influence the consumption of these foods [65].
A study by Sarita et al. reported that rutin and quercetin were the most abundant flavonoids in mulberry leaf extract, with flavonoids such as quercetin, isoquercetin, and luteolin also present. The RP-HPLC methodology was used to identify these components, using HPLC with a UV-visible detector and a C18 column for chromatographic separation. Isocratic elution was performed using mobile phase of acetonitrile and 0.1 percent v/v solution of glacial acetic acid [66].
HPLC is a vital analytical technique in the pharmaceutical industry, ensuring accurate data for drug discovery, development, formulation, and quality control. A validated method helps pharmaceutical analysts deliver better-quality products and meet required standards.
3.5 Characterization of rutin by nuclear magnetic resonance spectroscopy (NMR)
NMR spectroscopy is a powerful tool for analyzing flavonoid molecule structures in complex materials. It helps identify specific chemical groups within flavonoids and determine their spatial arrangement, providing crucial information for understanding their biological activity and potential health benefits [67].
The study by Selvaraj et al. analyzed flavonoid compounds using TLC, PTLC, and HPLC. UV-vis, FTIR, and NMR were used to examine their structures and chemical interactions. Rutin and quercetin were reported, showing significant antioxidant radical scavenging action. Nuclear magnetic resonance (1H and 13C) spectra were acquired in CDCl3 using a Bruker NMR Avance and TCI cyroprobe at 600 MHz [68].
Ghareeb et al. conducted a study to examine several extracts of
The study by Shuang et al. explored the antioxidant properties of
Fatima et al. conducted a study on the characterization and quantitation of flavonoids and phenolic components extracted from lyophilized mulberry leaves from two different cultivars. They identified the major flavonoid, rutin, using a hybrid IT-TOF MS system coupled to HPLC, and performed NMR analysis [71].
NMR spectroscopy is a valuable tool for understanding the structure of complex molecules, particularly flavonoids such as rutin and other phenolic components found in mulberry leaves.
3.6 Characterization of rutin by liquid chromatography-mass spectroscopy (LC: MS)
LC-MS is a widely used technique for identifying flavonoids and flavonoid glycosides, combining chromatography’s separation capabilities with precise detection of compound-specific precursors and fragments. It is particularly useful for examining complex plant extract combinations, where conventional techniques may lack sensitivity to measure low flavonoids levels, making it a valuable tool in phytochemical analysis and drug discovery [72].
HPLC can be integrated using multiple detectors like MS/MS and is a widely used method for identifying species and ensuring the quality of herbal medicines, specifically applied to ginkgo and mulberry leaves, including
The study by Xiaoyun et al. developed a precise and cost-effective chromatographic fingerprinting profile for evaluating mulberry leaves’ quality using HPLC, DAD, and MS methods. The study used an Agilent Technologies 1200 analytical HPLC system with a quaternary pump autosampler and triple quadrupole mass spectrometer for elution at 30°C using a Poroshell 120 EC-C18 column, with a mobile phase of 0.1% formic acid and acetonitrile. The method was validated and effectively used to identify rutin in mulberry leaves [75].
Rutin was reported in the leaves of two mulberry cultivars, Yai-Burirum (YB) and Khunphai (KP), which were processed into green tea (GT) and black tea (BT). The LCMS analysis showed that the rutin content in GT and BT extracts was significantly higher than the FL extract. The highest rutin content was found in the YB GT extract [76].
Research conducted by Ling Wan et al. using LCMS has shown that mulberry leaf ethanolic extract includes many flavonoids, including rutin, isoquercetin, and astragalin, that have the potential to inhibit xanthine oxidase enzyme, leading to extract’s potent antioxidant and anti-inflammatory capabilities [77].
A study by Yu Meng et al. found that mulberry leaves contain numerous polyphenols, including protocatechuic acid, chlorogenic acid, nicotiflorin, rutin, and astragalin. The most abundant bioactive flavonoid in mulberry leaf polyphenol extract (MLPE) was rutin, which regulates autophagy in HepG2 cells by influencing p53 signaling and also has antioxidative and anticancer properties [78].
Wang et al. performed a research on mulberry leaves using qualitative LC-ESI-QTOF analysis, which revealed a strong correlation between mulberry leaves’ antioxidant activities and the levels of isoquercitrin, chlorogenic acid, and rutin in free phenolic extracts. Additionally, syringic acid and rutin were revealed to be the primary contributors to the antioxidant activities of bound phenolic fractions [79].
Liquid chromatography-mass spectrometry (LC-MS) could be a useful analytical technique for quantifying rutin concentration in mulberry leaf extracts, with further research exploring their potential physiological benefits.
4. Conclusion
Rutin, a flavonoid-derived compound from quercetin glycosides, has various biological activities such as antioxidant, anti-radical, estrogenic, anti-inflammatory, antiviral, anticancer, cytotoxic, and organ-protective effects. This chapter highlights the importance of natural product chemistry in identifying bioactive compounds like rutin with potential health benefits, emphasizing the significance of these methods in the field of natural product chemistry. Rutin can be identified and characterized using techniques like UV-VIS spectrophotometry, FTIR, HPTLC, HPLC, NMR, and LCMS, providing valuable data for future research in the pharmaceutical and nutraceutical sectors.
The identification, isolation, and characterization of flavonoid rutin from
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