Minor Non-Cytotoxic Enantiomeric Constituents from the Leaves of Morus alba

Abstract

Objectives

The current work aimed to isolate and identify new interesting small-molecule natural products from the leaves of a well-known medicinal plant Morus alba.

Methods

The ethanol extract of M. alba leaves was partitioned between EtOAc and water, and the EtOAc soluble part was fractionated by macroporous resin column chromatograph (CC) eluted with gradient EtOH. The 80% EtOH eluate was subject to silica gel, reversed phase C₁₈, MCI gel and Sephadex LH-20 CC and finally purified by HPLC to afford four minor compounds 1–4, and the racemic 1 and 2 were further separated by chiral columns. Structural elucidation of these isolates was achieved via spectroscopic means especially HR-ESIMS and NMR, with the absolute configurations of new compounds assigned by ECD analyses. The cytotoxic screening was performed by MTT assay.

Results

A pair of new quinone enantiomers (1a/1b), a pair of new stilbenoid enantiomers (2a/2b), a known prenyl stilbenoid (3) and a known dihydrochalcone (4) were isolated and structurally characterized from the leaves of M. alba. They did not show significant cytotoxicity against MDA-MB231, A549, Hela and 4T1 tumor cell lines.

Conclusions

The M. alba leaves represent a rich source of new natural products, and the currently obtained constituents did not exert obvious growth inhibitory activity against the tested tumor cell lines. The bioactivities of the new isolates deserve further exploration in the future.

Introduction

The genus Morus is the most famous plant tribe of family Moraceae in that most Morus species possess significant economic values because of their nature as both food and natural medication in human's long history. ¹ M. alba (mulberry) as the type species of genus Morus is an indigenous plant of China but now has spread out to most Asian and European countries. ¹ In East Asian countries especially China, M. alba has been used for over a thousand years in different aspects of daily life. ¹ Of particular note, all parts of mulberry tree are with various usages/values: the fruit is a popular food in early summer, and the leaf (Mori folium), tender twig (Mori ramulus) and root bark (Mori cortex) are catalogued in Chinse National Pharmacopeia as herbal medicines. ² Modern researches including natural product investigations have demonstrated that the great values of mulberry tree are closely related to its bioactive chemical constituents, which has been well summarized in several comprehensive reviews.^3,4 Among the different plant parts of M. alba, the leaf represents an interesting and specialized one owing to the fact that it is the only part that has been recognized by Chinese National Health Commission to be a homology of both food and medicine. Previous phytochemical studies and biological evaluations into mulberry leaf revealed that it is rich in small-molecule natural compounds including alkaloids, benzofurans, catechins, coumarins, flavonoids, etc, with flavonoids being the predominant components, and more interestingly, these metabolites and the leaf extract were reported to show a variety of pharmacological properties, making mulberry leaf an attractive research topic for years till now.^5,6

Recently, several reports on mulberry root barks collected in the authors’ hometown have drawn our attention due to the large number of new diversified structures and promising therapeutical potential for diabetes and renal fibrosis.^7–9 As is known to all, natural products vary according to the body part, habitat, collecting time and even local seasonal climate of the source species. Therefore, we wonder if mulberry leaf from the same province also contains such rich new chemical constituents. An intensive phytochemical investigation was thus conducted on mulberry leaf collected in Xiajin County of Shandong province, a place famous for its ancient mulberry trees. As a result, new quinone (1a/1b) and stilbenoid (2a/2b) enantiomers, as well as known stilbenoid (3) and chalcone (4) (Figure 1), were separated and structurally characterized as minor constituents. It is noteworthy that the mostly reported flavonoid and stilbenoid constituents from mulberry leaf usually exist as flavones and benzofurans, respectively, while we have obtained their likely biosynthetic precursors (3 and 4) in the present study. More interestingly, compounds 1a/1b represent the first examples of quinone type of metabolites from M. alba. Thus, details of the isolation, structural identification and a preliminary biological assessment of these interesting compounds will be presented below.

Figure 1.

Chemical Structures of Compounds 1a/1b−4 from M. alba.

Materials and Methods

General

Optical rotations: Rudolph VI polarimeter (Rudolph Research Analytical, Hackettstown, USA); UV and ECD data: Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, Surrey, UK); NMR experiments: in CD₃OD (δ_H 3.30, δ_C 49.15), Bruker Avance DRX600 spectrometer (Bruker BioSpin AG, Fallanden, Switzerland); HR-ESIMS spectra: Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies Inc., Waldbronn, Germany); HPLC analyses and separations: SilGreen-C₁₈ column (5 μm, 10 × 250 mm, Greenherbs Science and Technology, Beijing, China), Daicel CHIRALPAK AD-H and IC columns (both 5 μm, 4.6 × 250 mm, Daicel Chiral Technologies Co. Ltd, Shanghai, China) on Shimadzu LC instrument (Shimadzu, Kyoto, Japan). Conventional column chromatography (CC): D101-macroporous absorption resin (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China), reversed-phase (RP) C₁₈ silica gel (Merck KGaA, Darmstadt, Germany), MCI gel (CHP20/P120, Mitsubishi Chemical Co. Ltd, Tokyo, Japan), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and silica gel (300-400 mesh or H; Qingdao Marine Chemical Co. Ltd, Qingdao, China).

Plant Materials

The leaves of Morus alba L. (family Moraceae) were collected at the end of October 2024 in Xiajin County, Shandong Province (China), and the species authentication was accomplished by Prof. Xiushan Yin from Shandong First Medical University (Accession No.: TMUCIH-2024-006).

Extraction and Isolation

The air-dried leaves (5.0 kg) of M. alba were milled and the powder was percolated with 15 L EtOH at room temperature for four times (once a week). The EtOH extraction was solvent-removed under reduced pressure and the obtained residue (600 g) was partitioned between water (1.0 L) and EtOAc (0.5 L × 4). The organic phases were combined and condensed in vacuum to afford the EtOAc soluble part (256 g), which was then fractionated on D101 macroporous resin CC with 30%, 50%, 80% and 95% (all 5.0 L) EtOH in H₂O successively. Each eluate was concentrated to remove the solvent to yield the respective fractions.

The 80% EtOH-H₂O eluate (89 g) was first separated by silica gel CC (petroleum ether-EtOAc, 30:1 to 1:1) to give 12 fractions (Fr-A to Fr-L). Fr-J (702 mg) was then fractionated by a RP-C₁₈ column (50%−100% MeOH-H₂O) to obtain six further subfractions (Fr-J1 to Fr-J6), among which Fr-J2 was further processed with Sephadex LH-20 CC (CH₂Cl₂-MeOH 1:1) to yield subfractions Fr-J2-1 to Fr-J2-4. Fr-J2-3 was finally purified by HPLC (50% MeCN-H₂O) to give compound 2 (t_R = 9.8 min, 0.8 mg, 3.00 mL/min). Fr-K (2.11 g) was separated on an MCI column (50%−100% MeOH-H₂O) to return seven subfractions (Fr-K1 to Fr-K7), and Fr-K3 was then processed with Sephadex LH-20 CC (MeOH) to yield another seven subfractions Fr-K3-1 to Fr-K3-7. Fr-K3-7 was finally purified by HPLC (55% MeCN-H₂O, 3.00 mL/min) to give compound 3 (t_R = 13.3 min, 0.5 mg). Fr-L (5.05 g) was first separated on an MCI column (50%−100% MeOH-H₂O) to return eight subfractions (Fr-L1 to Fr-L8), and Fr-L3 was then processed with Sephadex LH-20 CC (MeOH) to give another four subfractions Fr-L3-1 to Fr-L3-4. Fr-L3-2 was finally purified by HPLC (40% MeCN-H₂O, 3.00 mL/min) to afford compound 4 (t_R = 11.2 min, 0.5 mg). Fr-L4 was processed using the same procedure as Fr-L3 (Sephadex LH-20: MeOH; HPLC: 50% MeCN-H₂O) to yield compound 1 (t_R = 14.0 min, 0.4 mg).

Compound 1 was further separated on a Daicel IC chiral column (28% isopropanol in hexane, 1.00 mL/min) to yield 1b (t_R = 8.4 min, 0.1 mg) and 1a (t_R = 10.5 min, 0.1 mg), and compound 2 was further separated on a Daicel AD-H chiral column (25% isopropanol in hexane, 1.00 mL/min) to yield 2b (t_R = 13.8 min, 0.2 mg) and 2a (t_R = 16.2 min, 0.2 mg).

Compounds 1a/1b: pale-yellow solid; [α]²⁷_D −100/+90 (c 0.01, MeOH); UV (MeOH) λ_max (log ε) 249 (3.80) nm; ECD (c 0.1 mg/mL, MeOH) λ (Δε) 233 (+7.41), 262 (–5.42), 347 (–1.20) nm (1a), λ (Δε) 233 (–6.50), 261 (+4.37), 349 (+0.75) (1b); ¹H and ¹³C NMR (in CD₃OD) see Table 1; (+)-HR-ESIMS m/z 293.1392 [M + H]⁺ (calcd C₁₆H₂₁O₅⁺, 293.1384).

Table 1.

¹H (600 MHz) and ¹³C (150 MHz) NMR Data for Compounds 1a/1b and 2a/2b in CD₃OD.

No.	1		No.	2
	δC	δH (J in Hz)		δC	δH (J in Hz)
1	188.3		1	130.1	7.48, d (16.2)
2	141.9 a		2	124.9	6.89, d (16.2)
3	141.8 a		1′	117.0
4	188.9		2′	158.5
5	142.1		3′	103.7	6.31, d (2.4)
6	145.0		4′	160.7
7	12.5 a	2.00, s	5′	108.8	6.31, dd (8.0, 2.4)
8	12.4 a	2.00, s	6′	129.5	7.37, d (8.0)
9	12.3	2.04, s	1″	151.1
10	22.3	2.66, m	2″	106.5	6.51, d (1.4)
		2.60, m	3″	161.0
11	34.4	1.84, ddd (13.6, 11.5, 5.0	4″	106.4
		1.78, ddd (13.6, 11.5, 5.3)	5″	162.9
12	91.3		6″	106.7	6.56, d (1.4)
13	74.2	4.20, dd (6.0, 2.3)	1‴	199.2
14	39.4	3.08, dd (18.1, 6.0)	2‴	77.5	4.33, s
		2.40, dd (18.1, 2.3)	3‴	83.7
15	177.8		4‴	26.8a	1.55, s
16	23.1	1.42, s	5‴	18.8a	1.29, s

a

Assignments with the same superscript might be interchangeable.

Compounds 2a/2b: pale-yellow solid; [α]²⁷_D +15/–15 (c 0.02, MeOH); λ_max (log ε) 375 (3.40) nm; ECD (c 0.2 mg/mL, MeOH) λ (Δε) 223 (+1.50), 310 (–0.70) nm (2a), λ (Δε) 224 (–2.12), 312 (+1.00) nm (2b); ¹H and ¹³C NMR (in CD₃OD) see Table 1; (+)-HR-ESIMS m/z 343.1174 [M + H]⁺ (calcd C₁₉H₁₉O₆⁺, 343.1176).

ECD Calculations

Compounds 1a/1b and 2a/2b were first submitted to random conformational analysis with the MMFFs force field module in Maestro 10.2 software. The acquired conformers were further optimized using ground state method at WB97XD/6-311G(d) level in the gas phase. Frequency analyses of the optimized conformers were conducted at the same level of theory. Then the obtained conformers were subject to subsequent TD-DFT ECD calculations with the PCM model. The ECD spectrum of each conformer was simulated by SpecDis 1.71 with a half-bandwidth of 0.26 eV, and their final ECD spectra were acquired according to the Boltzmann contribution of each conformer.

Cytotoxic Assay

Tumor cell lines were obtained from the cell bank of Chinese Academy of Sciences, Beijing Branch (Beijing, China) and cultured according to the supplier's instructions. Briefly, MDA-MB231, A549 and Hela cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% (v/v) fetal bovine serum (FBS), and 4T1 cells were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% (v/v) FBS. All cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

In brief, tumor cells were seeded in 96-well plates at the density of 6 × 10³ cells/well. After 24 h, different concentrations of tested compounds were added and co-incubated for another 24 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide solution (MTT, 5 mg/mL, 10 μL) was added to each well and the plate was cultured for another 2 h. Following removal of the culture suspension, 100 μL dimethyl sulfoxide was added to each well. Finally, the optical density values at 490 nm were measured using a microplate reader (Tecan, Austria). Adriamycin (Aladdin, Beijing, China) was used as a reference compound.

Statistical Analysis

Statistical analysis (if applicable) was performed using GraphPad Prism 8.02 (GraphPad Software, San Diego, CA, USA) and a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.

Results

Compound 1 was obtained as a pale-yellow solid and exhibited a protonated molecular ion peak at m/z 293.1392 (calcd 293.1384) in HR-ESIMS analysis, corresponding to the molecular formula C₁₆H₂₀O₅ with seven indices of hydrogen deficiency (IHD). According to its ¹H and ¹³C NMR data shown in Table 1, three carbonyls (δ_C 188.7, 188.2, 177.7), four sp² quaternary carbons (δ_C 145.0, 142.1, 141.9, 141.8), a sp³ quaternary carbon (δ_C 91.3), an oxymethine (δ_H 4.20 (dd, J = 6.0, 2.3 Hz), δ_C 74.2], three methylenes (δ_H 3.08 (dd, J = 18.1, 6.0 Hz), 2.66 (m), 2.60 (m), 2.40 (dd, J = 18.1, 2.3 Hz), 1.84 (ddd, J = 13.6, 11.5, 5.0 Hz), 1.78 (ddd, J = 13.6, 11.5, 5.3 Hz); δ_C 39.4, 34.4, 22.3), and four methyls (δ_H 2.04 (s, 3H), 2.00 (s, 6H), 1.42 (s, 3H); δ_C 23.1, 12.5, 12.4, 12.3), were clearly resolved. These NMR features (three carbonyls and two double bonds) accounted for five IHD, and the remaining two IHD suggested the presence of a bicyclic backbone for 1. Further analyses of 2D NMR data (Figure 2) revealed two ¹H-¹H COSY fragments of CH₂-10/CH₂-11 and CH-13/CH₂-14, and the critical HMBC correlations from H₃-7 to C-1, C-2, and C-3, from H₃-8 to C-2, C-3 and C-4, from H₃-9 to C-4, C-5 and C-6, and from H₂-10 to C-1 and C-5, established the peri-substituted 1,4-quinone unit as shown. Furthermore, the HMBC signals from H₃-16 to C-11, C-12 and C-13, and from H-14 to C-12 and C-15, as wells as considering the chemical shifts for C-12 and C-15 and the IHD numbers, the right-hand part with a γ-lactone ring in the structure of 1 was thus constructed. In the NOESY spectrum, H-13 showed remarkable correlation with the Me-16 protons, indicating their coplanar relationship in the lactone ring (Figure 2). Thus, the constitution structure with relative configuration of 1 was unambiguously assigned.

Figure 2.

Illustration of key 2D NMR Correlations of Compounds 1 and 2.

It was interesting to note that the specific optical rotation of 1 was close to zero and there was also no obvious Cotton effect observed in its ECD spectrum, which alerted us of its possibly racemic nature according to recent years’ increasing reports of natural enantiomers. ¹⁰ Excitingly, chromatographic analysis of 1 on chiral column indeed resolved two distinct peaks that were further separated and identified to be a pair of enantiomers. By employing the time-dependent density functional theory (TD-DFT) method, the ECD spectra of the two enantiomers of 1 were acquired at WB97XD/6-311G(d) level and further compared with the experimental curves (Figure 3), which established the absolute configurations of 1a and 1b as (12R,13R) and (12S,13S), respectively.

Figure 3.

Experimental and Calculated ECD spectra for Compounds 1a/1b and 2a/2b.

Compound 2 was assigned the molecular formula C₁₉H₁₈O₆ based on the protonated HR-ESIMS molecular ion peak at m/z 343.1174 (calcd 343.1176), incorporating 11 IHD. Analyses of its ¹H and ¹³C NMR data (Table 1) revealed the presence of a conjugated ketone (δ_C 199.2), an 1,2,4-trisubstituted benzene (ABX spin system: δ_H 7.37 (d, J = 8.0 Hz), 6.31 (dd, J = 8.0, 2.4 Hz), 6.31 (d, J = 2.4 Hz); δ_C 160.7, 158.5, 129.5, 117.0, 108.8, 103.7), an 1,3,4,5-tetrasubstituted benzene (δ_H 6.56, 6.51 (both d, J = 1.4 Hz); δ_C 162.9, 161.0, 151.1, 106.7, 106.5, 106.4), an E-configured double bond (7.48, 6.89 (both d, J = 16.2 Hz); δ_C 130.1, 124.9), a sp³ oxygenated quaternary carbon (δ_C 83.7), an oxymethine (δ_H 4.33 (s); δ_C 77.5), and two singlet methyl groups (δ_H 1.55, 1.29; δ_C 26.8, 18.8). These functionalities occupied 10 IHD, and the last IHD indicated an extra ring in the structure of 2. The NMR data of 2 were close to those reported for the known stilbenoid 4-prenyloxyresveratrol (3), ¹¹ implying an analogue of the latter. Further examination of 2D NMR (Figure 2) confirmed this deduction, revealing diagnostic ¹H-¹H COSY correlations of H-1/H-2 and H-5′/H-6′, as well as key HMBC signals from H-1 to C-2′ and C-6′, from H-3′ to C-1′, C-2′ and C-4′, from H-5′ to C-4′, from H-2″ to C-2, C-3″ and C-4″, and from H-6″ to C-2, C-4″ and C-5″, which established the stilbenoid core structure as shown. The main structural differences between the two co-isolates were attributed to that the prenyl unit in 2 underwent oxidation, hydroxylation and cyclization compared with the free one in 3, which was corroborated by the HMBC correlations from H-2″ and H-2‴ to C-1‴, and from H-4‴ to C-3″, C-2‴, C-3‴ and C-5‴ (Figure 2). The constitution structure of 2 was thereby characterized to bear only one chiral carbon. As with the case of 1, the [α]_D value and ECD spectrum of 2 also suggested a nearly racemic mixture, and subsequent chiral separation afforded a pair of enantiomers 2a/2b. The absolute configurations of 2a/2b were assigned as S/R as drawn by comparing their ECD curves with the calculated ones (Figure 3).

Two known minor constituents, namely 4-prenyloxyresveratrol (3) ¹¹ and 2,4,2′,4′-tetrahydroxy-dihydrochalcone (4), ¹² were also isolated in the present work, and they were identified by spectroscopic analyses and comparison with literature data.

As the novel quinone (1) incorporates α,β-conjugated ketone functionality that can act as the receptor of Michael addition reaction to some key target proteins, and also due to the limited natural supply of these isolates, compounds 1a/1b−4 were only assessed for their growth inhibitory activity against three human tumor cell lines (MDA-MB231 (breast), Hela (cervical) and A549 (lung)) and a murine tumor cell line 4T1 (breast), as a routine biological screening in our laboratory. However, none of them exerted significant cytotoxicity toward all tumor cells (<50% inhibitory rate at 30 μM).

Discussion

In the current work, two pairs of new minor enantiomers (1a/1b and 2a/2b) and two known minor compounds (3 and 4) were isolated and identified from the leaves of the famous medicinal plant M. alba. Subsequent cytotoxic evaluation of these isolates in four tumor cell lines demonstrated their non-cytotoxic nature. Unfortunately, assessments in other biological models were not further carried out due to the scarce sample amount. As recorded in the literature, the title species contains rich phenolic constituents, and future work should focus more on anti-oxidant and anti-inflammatory evaluations. It is well known that the congenital abundance and limited supply of natural products always present a huge challenge for the biological evaluation and further development. Therefore, other strategies, such as total synthesis, could be applied to these interesting molecules to afford enough samples for future in-depth studies.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251385042 - Supplemental material for Minor Non-Cytotoxic Enantiomeric Constituents from the Leaves of Morus alba

Supplemental material, sj-docx-1-npx-10.1177_1934578X251385042 for Minor Non-Cytotoxic Enantiomeric Constituents from the Leaves of Morus alba by Jie Xu, Yanfang Yang, Qianqian Li, Ziyi Dong, Yaoyang Guo and Zhansheng Jiang in Natural Product Communications