Phenolic profiles and antioxidant activity of Morus alba L. infusions prepared from commercially available products and naturally collected leaves

Abstract

The aim of this study was to compare the phenolic compound content and antioxidant activity of infusions made from commercially available Morus alba L. leaves with those of infusions made from a naturally collected source. The phenolic profile was determined using RP-HPLC–DAD and LC-Q-TOF–MS/MS. The total phenolic content (TPC) was determined using Folin-Ciocâlteu reagent. To assess the antioxidant and reducing properties, Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) were used. Our analysis revealed the presence of two phenolic acids and six flavonoids. The most abundant compound was chlorogenic acid. The TPC, TEAC, and FRAP results indicated that infusions prepared from naturally collected samples exhibited greater phenolic content (19.7—52.6 vs 18.1—35.2 mg/100 ml) and stronger antioxidant (0.0605—0.1842 vs 0.0453—0.0822 mmol Trolox/100 ml) and reducing (0.244—0.597 vs 0.202—0.371 mmol Fe²⁺/100 ml) activities than those of commercially available products in the Polish market. Samples L1-L3 from the natural collection were those with the highest values. These results were further supported by principal component analysis (PCA), which categorized observations into three distinct clusters.

Introduction

Morus alba L., commonly known as white mulberry, is a deciduous woody tree belonging to the Moraceae family that was originally harvested from Asia. Its leaves have been integral to Traditional Chinese Medicine for over three millennia and are utilized to address various ailments, including diabetes, cancer, and cold¹. They are commercially available as tea or beverages in many countries, while in certain regions of China, young leaves are consumed as vegetables. The adaptability of white mulberry to diverse climatic and soil conditions, its resilience to air pollutants, and its resistance to diseases and pests have led to its introduction into various temperature zones in Europe, North and South America, and Africa².

In recent years, there has been an escalating demand for plant-derived constituents and products, prized for their applications in functional foods, nutraceuticals, drug development, and discovery^3–7. Consequently, materials such as white mulberry leaves have garnered significant interest due to their status as valuable nutrient sources for extraction and incorporation into food formulations^8–11.

The bioactive compounds present in white mulberry leaves are known for their potential as effective α-glucosidase inhibitors with the capacity to induce hypoglycaemic effects^12,13. The literature also suggests the feasibility of using white mulberry leaf extracts to address other conditions, such as hypercholesterolemia¹⁴. Due to these properties, functional tea crafted from white mulberry leaves has gained popularity as a beverage¹⁵.

Moreover, the bioactive molecules found within white mulberry leaves may play a crucial role in mitigating myocardial infarction and hypertension through the regulation of antioxidant defensive mechanisms¹⁶. In diabetic animals, the consumption of mulberry leaf extract significantly enhanced the activities of enzymes involved in the antioxidative defence system¹³. Both mulberry leaf extracts and isolated compounds exhibit cytotoxicity against various human cancer cell lines, including HeLa cervical carcinoma cells, MCF- 7 breast carcinoma cells, Hep3B hepatocarcinoma cells, and HepG2 hepatoma cells^17,18. Previous reports have discussed the antiobesity, anti-inflammatory, and antiatherosclerotic potential of mulberry leaves¹³. These multifaceted properties are attributed to the high concentrations of bioactive compounds, such as phenolic acids, flavonoids, proteins, organic acids, and macronutrients. Notably, these compounds include chlorogenic acid, rutin, isoquercitrin, astragalin, and 1-deoxynojirimycin^9,13,19–23.

Due to their valuable nutritional composition and versatile pharmacological properties, mulberry leaves have extensive applications in the food industry, dietary supplements, pharmaceuticals, and cosmetic production.

In Poland, dietary supplements incorporating white mulberry leaves have been very popular for many years mainly among diabetes and prediabetes patients to maintain a normal blood glucose homeostasis. They are easy accessible and affordable nutritional products, available mainly in a wide range of ready-to-drink tea beverages rather than solid forms (capsules and tablets). A regular practice among patients with impaired glucose management system is used concomitantly white mulberry leaves dietary products with conventional hypoglycemic agents to increase the effectiveness of therapy and help to achieve therapeutic goals and reduce adverse effects. According to manufacturers, an infusion of 2.0 g of dried mulberry leaves, consumed 2–3 times daily, can have hypoglycaemic effects. However, there remains a knowledge gap regarding the composition and quantities of the active constituents in mulberry leaf supplements. Additionally, the antioxidant potential of mulberry teas available on the Polish market remain unclear.

Unfortunately, in Poland, the reliable control of each food supplement is a big challenge. There are lack of detailed studies and requirements on food supplements sold in Polish market, confirming the composition in line with the label and safety for the consumer. Moreover, there is also no obligation to test the content of active substances or to standardize the raw material for specific active substances in these products²⁴. Those information are crucial with respect to both safety and efficacy of dietary supplements in the treatment of diabetes. Laboratory tests commissioned by The Supreme Audit Office (state institution in Poland) revealed that many supplements do not possess the qualities declared by the manufacturers, while some of them are even harmful to health. The checks showed the presence of supplements containing i.a pathogenic bacteria or banned substances from the list of psychoactive drugs²⁵. In addition, much lower requirements in the manufacturing, controlling and marketing of dietary supplements compared to synthetic drugs may eventually lead to changes to quality and quantity of ingredients within products. Worth of attention is also complications of combined pharmacotherapy due to possible pharmacokinetic and pharmacodynamic interactions between conventional drugs and plant derived products metabolites.

Therefore, based on the aforementioned considerations, the primary goal of our research was to carry out analysis of the composition and antioxidant potential of food supplements that incorporate dried white mulberry leaves available on the Polish market and compare them with with those derived from natural collections. To the best of our knowledge, there is no clear information in recent literature about quality assessment and antioxidant properties of products sold as dietary supplements with Morus alba leaves in Poland. Additionally, some reports about available products seem to be insufficient in terms of phenolic compounds profile, when compared with reports from other regions^26,27 This study aimed to provide crucial insights into the antioxidant potential and phenolic composition of these supplements, with a particular emphasis on elucidating any variations that might exist between commercially available products and those obtained from natural sources. We believe that consumers need and deserve to have access to detailed and necessary product information to help them use products safely, as well as avoid interaction between food-food supplements, drugs-food supplements, gene-food supplements, and minimize the risk of their harmful and allergic consequences. In our opinion, in the case of herbal substances or herbal preparations with constituents of known therapeutic activity, clear and accurate product labelling including identification and quantitation of active substances, as well as identification of unauthorized compounds empowers consumers to make informed, wise and first of all health-promoting choices. Moreover, by addressing this knowledge gap, our research sought to contribute valuable data that could not only inform both consumers and the industry about the nutritional and pharmacological attributes of mulberry leaf-based supplements, provide suggestions on the consumption and regulations of supplements in order to reduce the adverse effect cases caused by them and facilitating more informed choices and product development strategies as well further research with mulberry-based products.

Materials and methods

Chemicals

All reagents used in the study were of the highest analytical grade, and the standard solutions were prepared with deionized water. Quercetin, quercetin- 3-O-rutinoside (rutin), quercetin- 3-O-glucoside (isoquercitrin), kaempferol- 3-O-glucoside (astragalin), chlorogenic acid, caffeic acid, Folin–Ciocâlteu’s phenol reagent, formic acid, sodium persulfate, phosphate-buffered saline, 2,2’-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS), 6-hydroxy- 2,5,7,8-tetramethyl-chroman- 2-carboxylic acid (Trolox), (+)-catechin, sodium carbonate, ferrous chloride, acetate buffer, and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) were obtained from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile, methanol and trifluoroacetic acid were acquired from Avantor Performance Materials (Gliwice, Poland). Water was purchased from Merck (Darmstadt, Germany).

Plant material and preparation of samples

The research material consisted of 15 herbal medicines containing white mulberry leaves, as shown in Table 1. Among them, 9 were purchased locally in pharmacies and herbal stores in Kuyavian-Pomeranian Voivodeship (M1-M9). They were stored in their original packaging until analysis and are reffered as ‘comercial products’. The other 3 herbage samples of mulberry leaves, L1-L3, were harvested from trees located near the complex of Antoni Jurasz University Hospital No. 1 in Bydgoszcz (Kuyavian-Pomeranian Voivodeship, Poland). Plant verification and identification were performed by Assoc. Prof. Anna Kurzyńska-Kokorniak, PhD, DSc (Department of Ribonucleoprotein Biochemistry, Institute of Bioorganic Chemistry Polish Academy of Sciences Poznań, Poland) using the intersimple sequence repeat polymerase chain reaction technique (ISSR-PCR). The leaves were collected at the end of May in the afternoon (1–2 p.m.) on sunny days, preceded by a rainless period lasting at least a few days. The harvested mulberry leaves were gently cleaned to remove dirty materials and immediately dried under natural conditions (on plain paper in a ventilated place without direct sunlight). The dried samples were transferred to paper bags and kept at room temperature in the dark until use to prevent any physical or microbial damage. The remaining 3 products (L4-L6) were donated by a Polish pharmaceutical company (Natur-Vit, Pińczów, Poland). Samples were collected from Pakistan, China and Poland. Samples L1-L6 are referred as ‘raw plant material’. All the samples (n = 15) were grouped into two collections: commercial products (M1-M9, n = 9) and raw plant material (L1-L6, n = 6).

Table 1.

The characteristics of the 15 analysed samples containing white mulberry leaves.

Sample	Origin	Form
Commercial products
M1	Poland, China	Tea bags
M2	Poland	Tea bags
M3	Poland	Tea bags
M4	Poland	Tea bags
M5	Bulgaria	Tea bags
M6	Poland	Tea bags
M7	Bulgaria	Tea bags
M8	China	Tea bags
M9	Turkey, Bulgaria	Tea bags
Raw plant material
L1	Poland	Loose leaf
L2	Poland	Loose leaf
L3	Poland	Loose leaf
L4	China	Loose leaf
L5	Pakistan	Loose leaf
L6	Poland	Loose leaf

Extraction

Two grams of leaves were infused with 100 mL of freshly boiled deionized water for 5 min. Next, the extract was decanted, filtered through Chromafil PES 45/25 single-use syringe filters (0.45 µm, Macherey–Nagel, Düren, Germany) and adjusted to ambient temperature.

Identification and quantification of the phenolic compounds of Morus alba

Liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-Q-TOF–MS/MS)

Phenolic compounds were identified²⁸ using an Eksigent microLC 200 system coupled with a TripleTOF 5600 + mass spectrometer (AB Sciex, Framingham, MA, USA). Chromatographic separation was carried out on an Eksigent Halo C18 column (0.5 × 50 mm, 2.7 µm; AB Sciex). The mobile phase consisted of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B), using a linear gradient system that increased the proportion of solvent B from 1 to 90% over 3 min. The electrospray ionization source (ESI) was operated in negative ion mode, with an ion spray voltage of 4.5 kV. The nebulizer gas (GS1) and curtain gas flow rates were set to 30 L/min, while the heater gas (GS2) flow rate was 35 L/min. The turbo spray temperature was maintained at 350 °C. For full-scan MS, the declustering potential (DP) was set to − 90 or 90 V, and the collision energy (CE) was − 20 or 20 eV. For MS2 mode, the DP and CE values were adjusted to − 80 or 80 V and − 30, − 10 or 10 eV, respectively. The time-of-flight (TOF) mass spectrometer was operated in the 100–1200 m/z mass range.

Reversed phase high performance liquid chromatography with diode-array detector (RP-HPLC–DAD) analysis

The separation of phenolic compounds^11,29 was carried out by using the RP-HPLC–DAD Shimadzu Nexera X2 system (Shimadzu, Kyoto, Japan), which was equipped with a CBM- 20 A system controller, a CTO- 20 AC column oven, a DGU- 20 A5R degassing unit, two LC- 30 AD pumps, a SIL- 30 AC autosampler, and an SPD-M30 A photodiode array detector. Phytochemical separation was accomplished using a Luna Omega C18(2) column (4.6 × 150 mm, 3 µm; Phenomenex, Torrance, CA, USA). The analyte was separated at 25 °C in binary gradient mode using the following mobile phases to determine the phenolic content: solvent A, acetonitrile–water-trifluoroacetic acid (5:95:0.1; v/v/v); solvent B, acetonitrile-trifluoroacetic acid (100:0.1; v/v) for 0–10 min, 0–25% B; 10–15 min, 60% B; 15–17 min, 0% B; and 17–21 min, 0% B. The flow rate was 1 mL/min. The samples were filtered (0.22 µm, polyethersulfone membrane filter, TPP Techno Plastic Products AG, Trasadingen, Switzerland) and injected (10 µl). The detection of phenolics by UV–Vis spectra means of the photodiode detector was monitored over a wavelength range of 200–400 nm. Phenolic quantification by the external standard method was carried out using caffeic acid, chlorogenic acid, quercetin- 3-O-rutinoside (rutin), quercetin- 3-O-glucoside (isoquercitrin), kaempferol- 3-O-glucoside (astragalin), kaempferol and quercetin at a wavelength of 360 nm. The content of the studied compound was expressed as mg/100 ml of infusion (mg/100 mL). All measurements were performed in triplicate.

Determination of total phenolic content (TPC)

A colorimetric method with Folin-Ciocâlteu’s reagent (FCR) was used to determine the total phenolic content³⁰. First, 0.25 ml of sample, 0.25 ml of FCR, 0.5 ml of saturated sodium carbonate solution and 4 ml of water were mixed. The reaction mixture was centrifuged (5 min, 5000 × g, MPW- 350R, MPW Med. Instruments, Warsaw, Poland) after 25 min of the reacting time. Supernatants were subjected under absorbance measurement at 750 nm (DU- 7500 spectrophotometer, Beckman Instruments, Fullerton, CA, USA). The concentrations of TPC were expressed as mg of catechin equivalent (CAE) per 100 ml of infusion (mg CAE/100 ml). Measurements were performed in triplicate.

Determination of 2,2’-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) radical cation scavenging capacity—Trolox equivalent antioxidant capacity (TEAC)

The ability of white mulberry infusions to quench the synthetic radical cation ABTS was measured according to the procedure proposed by Re et al.³¹. Following original protocol, ABTS• + was generated and diluted. 2 ml of ABTS• +, 20 µl of sample were mixed and incubated (TH- 24 block heater, Meditherm, Warsaw, Poland) in 30 °C for 6 min. Absorbance was measured at 734 nm. The results are expressed as Trolox equivalents and are presented as mmol Trolox per 100 mL of infusion (mmol Trolox/100 mL).

Determination of ferric reducing antioxidant power (FRAP)

A method described previously by Benzie and Strain³² was used to measure the ability of white mulberry infusions to reduce iron Fe³⁺ to Fe²⁺. FRAP reagent was prepared before the analysis. Next, 2.25 ml of the reagent was mixed with 225 µl of water and 75 µl of sample. Reacting mixture was incubated in 37 °C for 30 min and after absorbance was measured at 593 nm. The results are expressed as mmol Fe²⁺/100 ml of infusion (mmol Fe²⁺/100 ml).

Statistical analysis

Analysis of the results was performed using statistical inference methods^33–35. The basic statistical descriptors included the mean value x and standard deviation (± SD). The normality of the distribution was tested with the Kolmogorov–Smirnov test, while the equality of variance in different samples was tested with the Levene test. Analysis of variance was used to find significant differences between the means, and in the case of significant differences, Tukey’s post hoc test was employed. Correlations among the samples were evaluated using Pearson’s correlation analysis and principal component analysis³⁶. The level of significance for all the statistical tests was set at α = 0.05. The statistical calculations mentioned above were carried out with MS Excel 2019 software (Microsoft, Redmond, WA, USA, 2019) and Statistica 13.3 (Dell, Round Rock, TX, USA, 2022) and PAST 3.2 (Hammer UiO, Norway, 2018) software.

Results and discussion

The analysis of individual phenolic compounds in Morus alba (white mulberry) leaf extract was conducted using RP-HPLC–DAD and LC-Q-TOF–MS/MS. A total of eight compounds were identified. Typical chromatogram of the extract of Morus alba is presented in the Fig. 1. Their UV–Vis absorbance maxima (λ_max), molecular ions, and fragment ions used for characterization are presented in Table 2. For the identification of phenolic acids we used negative ionisation mode and for flavonoids both negative as well as positive ionisation modes in case of LC-Q-TOF–MS/MS.

Fig. 1.

Typical chromatogram of the extract of Morus alba using RP-HPLC–DAD. 1–3-caffeoylquinic acid (neochlorogenic acid); 2 – quercetin- 3,7-O-di-hexoside; 3–5-caffeoylquinic acid (chlorogenic acid); 4 – quercetin- 3-O-rutinoside (rutin); 5 – quercetin- 3-O-glucoside (isoquercitrin); 6 – quercetin- 3-O-(6’’-O-malonyl)-glucoside; 7 – kaempferol- 3-O-glucoside (astragalin); 8 – kaempferol 3-O-(6’’-O-malonyl)-glucoside.

Table 2.

UV–Vis absorption maxima (λ_max) and fragmentation patterns of phenolic compounds identified in Morus alba leafs extracts.

Peak	λmax (nm)	Collision energy (eV)	Molecular ion (m/z)	Fragment ions (m/z)	Compound
1	325	− 10	353	191, 179, 173, 135	3-Caffeoylquinic acid (neochlorogenic acid)
2	257, 359	− 10	625	463, 301	Quercetin- 3,7-O-di-hexoside
3	325	− 10	353	191, 179	5-Caffeoylquinic acid (chlorogenic acid)
4	257, 356	− 10	609	301	Quercetin- 3-O-rutinoside (rutin)
		10	611	303
5	257, 354	− 10	463	301	Quercetin- 3-O-glucoside (isoquercitrin)
		10	465	303
6	257, 355	− 30	549	505, 300	Quercetin- 3-O-(6’’-O-malonyl)-glucoside
		10	551	303
7	265, 348	− 30	447	327, 284	Kaempferol- 3-O-glucoside (astragalin)
		10	449	287
8	268, 348	− 30	533	489, 285	Kaempferol 3-O-(6’’-O-malonyl)-glucoside
		10	535	287

Peaks 1, 2, 6, and 8 were tentatively identified. Compounds 3, 4, 5, and 7 were identified using reference standards during RP-HPLC–DAD analysis. We observed two caffeoylquinic acid derivatives: 3-caffeoylquinic acid (neochlorogenic acid) (peak 1) and 5-caffeoylquinic acid (chlorogenic acid) (peak 3) characterized by their fragment ions at m/z 191 and 179. The presence of a fragment ion at m/z 135 in the spectrum of peak 1 suggests that it corresponds to neochlorogenic acid. In contrast, the absence of this fragment in peak 3 supports its identification as chlorogenic acid^28,37. Six compounds attributed to flavonoids were identified, including four quercetin derivatives (peaks 2, 4–6) and two kaempferol derivatives (peaks 7 and 8). Observed UV spectra, with λ_max at 257 and 354–359 nm for quercetin derivatives and 265–268 and 348 nm for kaempferol derivatives, are characteristic of these two flavonoid classes. The quercetin derivatives exhibited fragment ions at m/z 300, 301, or 303, corresponding to the quercetin aglycone, while kaempferol derivatives showed diagnostic fragment ions at m/z 284, 285, or 287. Among the glycosylated flavonoids, quercetin- 3-O-rutinoside (rutin), quercetin- 3-O-glucoside (isoquercitrin, peak 5), and kaempferol- 3-O-glucoside (astragalin) fragmentation resulted in ions characteristic of these compounds, derived from the loss of sugar moieties. For rutin, the loss of rhamnose-glucose (− 308 Da) was observed, whereas isoquercitrin and astragalin exhibited a loss of glucose (− 162 Da). Peak 2, with a signal at m/z 625, was identified as quercetin- 3,7-O-di-hexoside. This identification is based on the progressive cleavage of glycosidic bonds between the quercetin core and the two sugar moieties, with each cleavage resulting in characteristic fragment ions at m/z 463 (− 162 Da) and 301 (− 162 Da and − 162 Da). Peaks 6 and 8 were attributed to quercetin- 3-O-(6’’-O-malonyl)-glucoside and kaempferol- 3-O-(6’’-O-malonyl)-glucoside, respectively. These compounds displayed fragments resulting from the decarboxylation of the malonyl group (− 44 Da) and the loss of malonyl-glucose (− 284 Da). These findings align with previous research conducted by other authors, including Jeong et al.⁸, Sánchez-Salcedo et al.^21,38, Hu et al.²², Dugo et al.²⁶, Ju et al.³⁹, Li et al.⁴⁰, Pothinuch et al.⁴¹ providing a substantial basis for the thorough examination of phenolic compounds within white mulberry infusion.

The chromatographic separation of phenolic compounds from mulberry leaves is exemplified by typical chromatograms, as presented in studies conducted by Sánchez-Salcedo et al.²¹, Dugo et al.²⁶, Polumackanycz et al.^9,27, Ju et al.³⁹ and Pothinuch et al.⁴¹. The outcomes reported by the^21,26,39, closely resemble our own findings, with a visual inspection revealing comparable elution patterns. Notably, the primary focus of³⁹was the analysis of flavonoids; however, an unassigned, distinct peak with a relatively short retention time is discernible at the outset of the chromatogram. In our interpretation, this peak likely corresponds to a phenolic acid compound, which elutes earlier than the monoglycosides of flavonoids, and in our study, this compound was identified as chlorogenic acid, distinguished by a strong signal in the chromatogram. Similar findings are presented in²¹and⁴¹. Conversely, the second research group, as delineated by Polumackanycz et al.^9,27, presented chromatograms for both hydromethanolic and aqueous extracts that exhibited significant differences from our findings. Latter study was based on samples of Morus alba obtained from Polish market. In both cases, the dominant compounds were phenolic acids. Rutin and aglycones were the only flavonoids identified in those samples. These variations are attributed to differences in their extraction methodologies. In the hydromethanolic extraction performed by the abovementioned team, a solid‒liquid ratio of 1:4 was employed, while for the aqueous extraction, a ratio of 1:200 was utilized. In contrast³⁹, opted for a 1:10 ratio for hydromethanolic extraction, whereas our study utilized a ratio of 1:50, which closely resembles the preparation of a home infusion. Differences between the methodologies of the abovementioned teams manifest in noticeable differences in the obtained outcomes. Moreover, it is worth noting that Polumackanycz et al.^9,27 did not identify any glycosides apart from rutin. However, aglycones apigenin and quercetin were identified in Morus. alba leaves from the Polish market²⁷, along with myricetin and naringenin in Morus. alba leaves collected in Italy⁹. Ju et al.³⁹ suggested that not all flavonoids, particularly acylated compounds, may be present in all Morus alba varieties. In our study, we observed two peaks that were identified as acylated compounds.

Chan et al.⁴² comprehensively summarized the phenolic profile of Morus alba in their review, which included naringenin but did not mention apigenin or myricetin. Nevertheless, they listed 6-geranylapigenin and 8-geranylapigenin, suggesting that mulberry may have the capacity to biosynthesize these molecules. This inference is supported by the work of Hu et al.²², whose research team provided compelling evidence for the accumulation of apigenin and naringenin glycosides in white mulberry leaves.

However, in our study, the absence of those compounds could be attributed to the use of an aqueous extraction method and, potentially, their low concentrations. Notably, in general, apigenin and naringenin derivatives were not reported by other researchers^21,26,41. These observations underscore the need for further investigations into supplements based on Morus alba leaves available on the Polish market.

The quantification of all analysed compounds is presented in Table 3. Predominantly, chlorogenic acid emerged as the dominant compound, displaying a content range of 2.07 to 25.0 mg/100 ml (M4 and L2, respectively), with the highest levels observed in samples derived from a natural collection. The neochlorogenic acid exhibited a content within the range of 0.157–0.611 mg/100 ml, a trend consistent with findings reported by Sánchez-Salcedo et al.³⁸, reaffirming the predominance of phenolic acids over flavonoids, with chlorogenic acid as the primary compound.

Table 3.

Content of individual phenolic compounds in Morus alba L. infusions (mg/100 ml).

Sample	3-Caffeoylquinic acid (neochlorogenic acid)	Quercetin- 3,7-O-di-hexoside	Chlorogenic acid	Quercetin- 3-O-rutinoside (rutin)	Quercetin- 3-O- glucoside (isoquercitrin)	Quercetin- 3-O-(6’’-O-malonyl)-glucoside	Kaempferol- 3-O-glucoside (astragalin)	Kaempferol 3-O-(6’’-O-malonyl)-glucoside
Commercial products
M1	0.157 ± 0.014f	0.122 ± 0.020gh	2.07 ± 0.234d	0.307 ± 0.052e	0.292 ± 0.061 h	0.135 ± 0.025e	0.162 ± 0.028f.	0.099 ± 0.014f.
M2	0.445 ± 0.052b–d	0.211 ± 0.021cd	5.13± 0.715cd	1.02 ± 0.145e	0.956 ± 0.130e–g	0.615 ± 0.101c–e	0.443 ± 0.046c–f	0.416 ± 0.047c–e
M3	0.223 ± 0.025ef	0.123 ± 0.004gh	3.22 ± 0.430cd	0.432 ± 0.050e	0.512 ± 0.078gh	0.263 ± 0.038de	0.230 ± 0.036d–f	0.176 ± 0.020ef
M4	0.305 ± 0.052b–f	0.132± 0.021f–h	3.47 ± 0.589cd	0.600 ± 0.163e	0.478 ± 0.123gh	0.351 ± 0.102de	0.291 ± 0.038d–f	0.296 ± 0.054c–f
M5	0.455 ± 0.042a–c	0.176 ± 0.005d–f	5.19 ± 0.312cd	1.18 ± 0.079ed	1.09 ± 0.067d–f	0.701 ± 0.059c–e	0.509 ± 0.036b–d	0.485 ± 0.044 cd
M6	0.246 ± 0.013ef	0.133 ± 0.012e–h	3.17 ± 0.314	0.484 ± 0.039e	0.589 ± 0.033f–h	0.143 ± 0.014e	0.287 ± 0.012d–f	0.114 ± 0.006f.
M7	0.359 ± 0.024b–e	0.155 ± 0.006e–g	4.54 ± 0.331cd	0.651 ± 0.050e	1.32 ± 0.034c–e	0.469 ± 0.041c–e	0.477 ± 0.021b–e	0.286 ± 0.026c–f
M8	0.429 ± 0.010b–d	0.105 ± 0.011h	3.64 ± 0.145cd	0.713 ± 0.033e	0.787 ± 0.074e–h	0.275 ± 0.007de	0.364 ± 0.026c–f	0.211 ± 0.006d–f
M9	0.433 ± 0.037b–d	0.129 ± 0.017gh	3.51 ± 0.281cd	0.447 ± 0.009e	0.692 ± 0.029f–h	0.164 ± 0.010e	0.447 ± 0.004c–f	0.180 ± 0.005ef
Raw plant material
L1	0.611 ± 0.122a	0.247 ± 0.021bc	16.68 ± 5.835b	5.06 ± 1.032a	2.89 ± 0.538b	1.23 ± 0.978bc	0.632 ± 0.272bc	0.476 ± 0.294 cd
L2	0.289 ± 0.073d–f	0.274 ± 0.019b	25.0 ± 2.024a	3.84 ± 0.326b	1.71 ± 0.208c	5.40 ± 0.425a	0.282 ± 0.037d–f	2.084 ± 0.182a
L3	0.293 ± 0.059c–f	0.162 ± 0.016e–g	13.4 ± 0.445	2.19 ± 0.076c	0.878 ± 0.028e–g	1.548 ± 0.053b	0.170 ± 0.020ef	0.560 ± 0.017c
L4	0.377 ± 0.028b–e	0.154 ± 0.007e–g	3.72 ± 0.288cd	0.446 ± 0.039e	0.747 ± 0.081e–h	0.147 ± 0.018e	0.352 ± 0.034c–f	0.116 ± 0.012f.
L5	0.459 ± 0.092ab	0.179 ± 0.018de	4.68 ± 0.639cd	0.900 ± 0.122e	1.61 ± 0.226cd	0.476 ± 0.066c–e	0.764 ± 0.115b	0.339 ± 0.050c–f
L6	0.430 ± 0.034b–d	0.330 ± 0.016a	7.14 ± 0.139c	1.91 ± 0.149c	3.69 ± 0.311a	1.01 ± 0.008b–d	2.20 ± 0.251a	0.870 ± 0.058b

Values in the same row marked with different letters differ significantly (p < 0.05). Content of compound 1 is expressed as caffeic acid equivalent, compounds 2 and 6 as quercetin equivalents and compound 8 as kaempferol equivalent.

Among the glycosides of flavonoids, rutin had the highest content, ranging from 0.307 to 5.06 mg/100 ml, with sample L1 from the natural collection exhibiting the highest concentration. The contents of isoquercitrin and two other quercetin derivatives (quercetin- 3,7-O-di-hexoside and quercetin- 3-O-(6’’-O-malonyl)-glucoside), along with astragalin and a kaempferol 3-O-(6’’-O-malonyl)-glucoside, exhibited diversity within the following ranges: 0.292–3.692, 0.105–0.330, 0.135–5.398, 0.162–2.198, and 0.099–2.084 mg/100 ml, respectively. Notably, glycosides of quercetin were more abundant than those of kaempferol, aligning with the scientific discourse outlined in Sánchez-Salcedo et al.³⁸ and Ju et al.³⁹.

Spectrophotometric techniques were used to assess the total phenolic content and antioxidant activity of white mulberry infusions through two different assays. The results of the TPC, Trolox equivalent antioxidant capacity, and ferric reducing antioxidant power analyses are summarized in Table 4.

Table 4.

Total phenolics content (TPC), Trolox equivalent antioxidant capacity (TEAC) and ferric-reducing antioxidant power (FRAP).

Sample	TPC	TEAC	FRAP
	(mg/100 ml)	(mmol Trolox/100 ml)	(mmol Fe2+/100 ml)
Commercial products
M1	19.0 ± 1.307e	0.0453 ± 0.000fg	0.242 ± 0.010ef
M2	27.2 ± 0.724b–e	0.0822 ± 0.006c–e	0.319 ± 0.014b–f
M3	23.0 ± 1.138de	0.0472 ± 0.00fg	0.251 ± 0.010d–f
M4	18.1 ± 1.137e	0.0626 ± 0.002ef	0.291 ± 0.016c–f
M5	18.1 ± 1.137e	0.0742 ± 0.001de	0.310 ± 0.011c–f
M6	20.9 ± 1.568e	0.0354 ± 0.026g	0.202 ± 0.150f
M7	35.2 ± 0.779b–d	0.0649 ± 0.005ef	0.371 ± 0.009b–d
M8	27.1 ± 0.582b–e	0.0580 ± 0.002e–g	0.327 ± 0.008b–e
M9	35.2 ± 1.704b–d	0.0700 ± 0.003ef	0.305 ± 0.005c–f
Raw plant material
L1	52.6 ± 1.931a	0.1470 ± 0.008b	0.597 ± 0.023a
L2	49.9 ± 1.481a	0.1842 ± 0.006a	0.572 ± 0.019a
L3	36.8 ± 0.734b	0.0962 ± 0.012 cd	0.400 ± 0.012bc
L4	19.7 ± 1.483e	0.0605 ± 0.001ef	0.244 ± 0.004ef
L5	23.8 ± 1.818c–e	0.0801 ± 0.007de	0.336 ± 0.022b–e
L6	36.2 ± 2.614bc	0.1071 ± 0.002c	0.436 ± 0.026b

The TPC ranged from 18.1 to 52.6 mg (CAE)/100 ml. These findings are comparable to the literature^9,38. Those studies express the results as gallic acid equivalent (GAE). It should be noted that CAE delivers slightly higher values when compared with GAE⁹. shows in case of plants cultivated in Italy that TPC values were higher in case of infusions when compared with hydromethanolic extract. In contrary³⁸, focused on hydromethanolic extraction of the leaves collected from plants originating from Spain and obtained higher results than⁹. What is more³⁸, shows also that significant differences in the TPC were observed in case of clones of the same specimen. Moreover, the antioxidant activities, as determined by TEAC and FRAP assays, differed across the samples, ranging from 0.0354 to 0.184 mmol TE/100 ml and from 0.202 to 0.597 mmol Fe²⁺/100 ml, respectively.

Notably, the TEAC and FRAP values obtained in our investigation were relatively lower than those reported in the literature^9,38. These discrepancies are likely attributed to following factors. First are differences in the methodological modifications employed in our study compared to the referenced research. Another one are differences between plants used in the study. Not only place of the origin, climatic and soil conditions are essential. Phenotypic factor of the plants cultivated in the same area influences significantly the variability within a chemical profile of a plant.

The majority of the recorded values were notably higher for infusions prepared using leaves sourced from the natural collection, specifically L1, L2, L3, and L6. Conversely, samples L4-L5, which also originated from a natural collection, exhibited lower values that closely resembled those of commercially available products. For instance, the content of rutin (p < 0.001), a recognized potent antioxidant, is an example. Furthermore, the phenolic composition of mulberry leaves can be influenced by various factors, including food processing methods⁴³, such as the drying technique and growth period²². Employing diverse processing methods may result in the accumulation or loss of specific groups of compounds, such as flavonoids, along with alterations in their antioxidant activity. Among the available drying methods, air drying at room temperature has emerged as the most efficient approach for retaining flavonoids in white mulberry leaves, both in terms of preservation and economic feasibility, compared to freeze drying.

Notably, the growth period assumes significance when examining samples from a natural collection, as their growth cycles lack standardization, similar to those from commercially managed orchards. This factor can also influence the phenolic content within infusions prepared from commercially available products or those sourced from a natural collection. Nonetheless, our study conclusively demonstrated that samples derived from natural collections are characterized by a notably high phenolic compound content.

To explore the potential influence of individual compound contents on both the TPC and antioxidant activity of the infusions, we calculated Pearson correlation coefficients (p ≤ 0.05), as presented in Table 5. The rutin content exhibited strong positive correlations with the TPC (R = 0.86), TEAC (R = 0.91), and FRAP (R = 0.94). Similarly, the chlorogenic acid content displayed notably high positive correlation coefficients with the TPC, and the results of the antioxidant assays (R = 0.83, 0.95, and 0.88, respectively) were comparable to those observed for rutin. Conversely, the contents of neochlorogenic acid and astragalin showed non-significant correlations (p≤ 0.05) with the results obtained from spectrophotometric methods. The contents of the remaining compounds exhibited correlations with the TPC, TEAC, and FRAP at similar levels. Additionally, we observed significant correlations between the contents of various phenolic compounds within the samples. However, neochlorogenic acid exhibited a correlation exclusively with the content of isoquercitrin. These strong positive correlations between the results obtained from spectrophotometric methods and the contents of phenolic compounds in mulberry leaves align with the findings of other researchers⁹. In the present study, similar findings were observed, including a notable correlation between the flavonoid content and phenolic acid content. However, it is worth highlighting that the FRAP assay results were not significantly correlated with the phenolic content. This difference from the present study may be attributed to the reported variance in phenolic fingerprints, as discussed earlier, which likely influenced the outcomes of the referenced study. It is essential to emphasize that the total phenolic content, antioxidant activity, and phenolic compound contents exhibited remarkable similarities among infusions prepared from commercially available samples. In contrast, the outcomes varied significantly for infusions derived from a natural collection.

Table 5.

Correlations between the compounds, total phenolics, TEAC and FRAP. Values of the correlation coefficient (shaded fields) that are statistically significant are shown in italic.

R/p	3-Caffeoylquinic acid (neochlorogenic acid)	Quercetin- 3,7-O-di-hexoside	Chlorogenic acid	Quercetin- 3-O-rutinoside (rutin)	Quercetin- 3-O-glucoside (isoquercitrin)	Quercetin- 3-O-(6’’-O-malonyl)-glucoside	Kaempferol- 3-O-glucoside (astragalin)	Kaempferol 3-O-(6’’-O-malonyl)-glucoside	Total phenolics content	TEAC	FRAP
3-Caffeoylquinic acid (neochlorogenic acid)		0.129	0.542	0.099	0.021	0.867	0.122	0.944	0.144	0.158	0.067
Quercetin- 3,7-O-di-hexoside	0.41		0.012	0.004	< 0.001	0.022	0.003	0.003	0.012	0.001	0.002
Chlorogenic acid	0.17	0.63		< 0.001	0.077	< 0.001	0.976	< 0.001	< 0.001	< 0.001	< 0.001
Quercetin- 3-O-rutinoside (rutin)	0.44	0.69	0.90		0.007	0.004	0.519	0.008	< 0.001	< 0.001	< 0,001
Quercetin- 3-O-glucoside (isoquercitrin)	0.59	0.89	0.47	0.67		0.231	< 0.001	0.083	0.011	0.008	0.002
Quercetin- 3-O-(6’’-O-malonyl)-glucoside	0.05	0.59	0.92	0.69	0.33		0.967	< 0.001	0.007	< 0.001	0.002
Kaempferol- 3-O-glucoside (astragalin)	0.42	0.72	0.01	0.18	0.84	− 0.01		0.474	0.432	0.385	0.276
Kaempferol 3-O-(6’’-O-malonyl)-glucoside	0.02	0.71	0.86	0.66	0.46	0.97	0.20		0.011	< 0.001	0.002
Total phenolics content	0.40	0.63	0.83	0.86	0.64	0.67	0.22	0.64		< 0.001	< 0.001
TEAC	0.38	0.79	0.95	0.91	0.65	0.86	0.24	0.87	0.86		< 0.001
FRAP	0.48	0.74	0.88	0.94	0.74	0.73	0.30	0.73	0.92	0.95

To obtain a better understanding of the arrangement of the observations within the dataset, we performed principal component analysis. The results of this analysis are presented in Fig. 2. Principal components 1 and 2 (PC1 and PC2) accounted for the majority of the variance (99.24%). In particular, PC1 explained the vast majority of the data structure, representing 92.83% of the total variation. A strong positive correlation emerged between the TPC, chlorogenic acid content, and PC1. Consequently, this correlation delineated the formation of three distinct clusters of observations. In the preceding discussion section, we reported strong, positive Pearson correlation coefficients between the TPC and chlorogenic acid, the dominant compound within white mulberry leaves. This correlation pattern manifested in the arrangement of the observations. The first cluster encompasses two observations, namely, L1 and L2, exhibiting the highest positive values for PC1. Both of these samples are infusions prepared from leaves sourced from a natural collection. The second cluster comprises four samples, namely, M7, lL6, M9, and L3, which display intermediate values for PC1. This cluster groups infusions prepared from both a natural collection and commercially available products. Finally, the third cluster comprises the remaining observations characterized by negative values for PC1. This cluster contains two samples from a natural collection.

Fig. 2.

Principal component analysis plot of the distribution of variables.

Other researchers have also employed PCA to gain a deeper understanding of the interrelationships and patterns among variables connected to Morus alba^2,21,38. In the visual distribution depicted by Sánchez-Salcedo et al.³⁸, certain similarities were noted concerning the influence of specific variables, although without a pronounced impact of any single variable. In our study, the TPC and chlorogenic acid content were the variables that exerted the most substantial influence on the distribution of observations, although the number of variables considered differed.

Gai et al.²⁹demonstrated that individual compounds may contribute to the total variance to different degrees when assessing the activity of the entire mixture (infusion, extract, etc.) under polar conditions (TPC, TEAC, FRAP). Discrepancies with the PCA performed in other studies^2,21 present a complex challenge for interpretation and rely upon the type and quantity of variables investigated in each study.

It is crucial to recognize that when spectrophotometric methods are employed to measure factors such as antioxidant activity, the occurrence of synergistic, additive, and antagonistic effects among specific molecules during the reaction period must be acknowledged. These effects depend on the ratio of individual antioxidants present in the reaction mixture. Furthermore, when more than two antioxidants are present, the nature of these effects becomes even more complex⁴⁴, such as in a natural matrix (Morusalba infusion, extract).

Conclusions

Phenolic acids were found to be the most abundant compounds in white mulberry leaf infusions, with two phenolic acids identified, of which chlorogenic acid was the most abundant. In contrast, among the flavonoids, rutin was the most abundant. While phenolic acids displayed greater overall abundance, flavonoids exhibited greater diversity, including four quercetin derivatives and two kaempferol derivatives, within the infusions. Notably, the former four flavonols were present in greater quantities than the latter two.

A trend revealed that samples sourced from a natural collection exhibited the highest phenolic compound contents. This trend was consistently reflected in the results of the TPC, TEAC, and FRAP analyses, which is consistent with a more robust phenolic fingerprint.

Furthermore, the spectroscopic results correlated significantly with the contents of individual compounds, as indicated by strong positive Pearson correlation coefficients. For instance, the TPC exhibited a noteworthy correlation with the dominant compound, which was further confirmed by PCA. This analysis grouped the observations into three distinct clusters, primarily guided by this pair of variables. The first cluster contained samples from a natural collection, the third predominantly included commercially available samples, and the second cluster exhibited a mixed composition.

If the quality of white mulberry leaf infusion is at least partially defined as the levels of dominant antioxidant compounds present in the beverage, our approach successfully segregated the samples with the highest quality. Moreover, our findings underscore the comparability of commercially available products, signalling the need for novel production methods or alternative raw materials. Our findings suggest the possibility of further investigations into white mulberry leaf products available on the Polish market.

Abbreviations

ABTS

2,2’-Azinobis(3-ethylbenzothiazoline- 6-sulfonic acid)

CAE

Catechin equivalent

FCR

Folin-Ciocâlteu’s reagent

FRAP

Ferric reducing antioxidant power

ISSR-PCR

Intersimple sequence repeat polymerase chain reaction technique

LC-Q-TOF–MS/MS

Liquid chromatography quadrupole time-of-flight tandem mass spectrometry

PCA

Principal component analysis

SD

Standard deviation

TEAC

Trolox equivalent antioxidant capacity

TPC

Total phenolic content

TPTZ

2,4,6-Tri(2-pyridyl)-s-triazine

Trolox

6-Hydroxy- 2,5,7,8-tetramethyl-chroman- 2-carboxylic acid

RP-HPLC–DAD

Reversed phase high performance liquid chromatography with diode-array detector

Author contributions

Michał Adam Janiak: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Anna Gryn-Rynko: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Katarzyna Sulewska: Investigation, Data curation, Formal analysis, Ryszard Amarowicz: Conceptualization, Methodology, Data curation, Formal analysis, Visualization, Supervision. Kamila Penkacik: Investigation, Data curation, Formal analysis. Radomir Graczyk: Methodology, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. Dorota Olszewska-Słonina: Conceptualization, Visualization, Supervision. Michał Stanisław Majewski: Visualization, Supervision. All authors have read and agreed to the published version of the manuscript.

Data availability

The data that support the findings of this study will be available on request from the authors (Michał Adam Janiak, Anna Gryn-Rynko).

Declarations

Competing interests

The authors declare no competing interests.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-97223-9.