Elsevier

Journal of Ethnopharmacology

Volume 248, 10 February 2020, 112296
Journal of Ethnopharmacology

Multiple In vitro biological effects of phenolic compounds from Morus alba root bark

https://doi.org/10.1016/j.jep.2019.112296Get rights and content

Abstract

Ethnopharmacological relevance

Morus alba L. is used in traditional Chinese medicine for the treatment of various diseases, including bacterial infections and inflammation. As a rich source of phenolic compounds, the plant is an object of many phytochemical and pharmacological studies.

Aim of the study

The aim of the study was to isolate and evaluate possible parallel antiviral, antibacterial, and anti-inflammatory activities of phenolic mulberry compounds.

Materials and methods

Extensive chromatographic separation of mulberry root bark extract and in vitro biological screening of 26 constituents identified promising candidates for further pharmacological research. Selected compounds were screened for anti-infective and anti-inflammatory activities. Antiviral activity was determined by the plaque number reduction assay and by the titer reduction assay, antibacterial using broth microdilution method, and anti-inflammatory activity using COX Colorimetric inhibitor screening assay kit. One compound was evaluated in vivo in carrageenan-induced paw-edema in mice.

Results

Five prenylated compounds 1, 2, 8, 9, and 11, together with a simple phenolic ester 13, exhibited inhibitory activity against the replication of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), with IC50 values ranging from 0.64 to 1.93 μg/mL, and EC50 values 0.93 and 1.61 μg/mL. Molecular docking studies demonstrated the effects of the active compounds by targeting HSV-1 DNA polymerase and HSV-2 protease. In antibacterial assay, compounds 1, 4, 11, and 17 diminished the growth of all of the Gram-positive strains tested, with MIC values of 1–16 μg/mL. The anti-inflammatory ability of several compounds to inhibit cyclooxygenase 2 (COX-2) was tested in vitro, and compound 16 displayed greater activity than the indomethacin, positive control. Mulberrofuran B (11) showed anti-inflammatory activity in vivo against carrageenan-induced paw-edema in mice.

Conclusions

Experimental investigation showed promising antiviral, antibacterial, and/or anti-inflammatory activities of the phenolic mulberry constituents, often with multiple inhibitory effects that might be used as a potential source of new medicine.

Introduction

Morus alba L. (Moraceae), commonly known as mulberry, is a medium-size deciduous tree. Originally from China, it is now cultivated for various purposes throughout Asia, Europe, North and South America, and Africa, and is economically important for sericulture in East, Central, and South Asia (Yang et al., 2011). In traditional Chinese medicine, mulberry has been used for centuries and the Chinese pharmacopoeia mentions its different parts as ingredients for medicine preparations for prevention and therapy of throat infections, inflammation, fever, asthma, hypertension or diabetes (Asano et al., 2001; Chen et al., 2016; Kumar and Chauhan, 2008; Tang and Eisenbrand, 1992; Xia et al., 2019). In Japan and Korea, mulberry also finds use in folk medicine (Yuan and Zhao, 2017). Mulberry root bark (Sang-Bai-Pi) is a rich source of prenylated flavonoids and other prenylated compounds for which anti-inflammatory (Vochyanova et al., 2017), antibacterial (Sohn et al., 2004), antiviral (Du et al., 2003), antioxidant (Lan et al., 2013), neuroprotective (Kim et al., 2011), and cytotoxic activities (Lee et al., 2008) have been reported in vivo and in vitro. As an example, a methanol extract of the root bark exerted anti-inflammatory activity by inhibition of NF-κB mediated by an inhibition of phosphorylation of ERK1/2 (Eo et al., 2014). Another study identified arylbenzofurans and prenylated flavonoids that inhibited the secretion of NO in LPS-stimulated RAW 264.7 cells and thus contributed to the anti-inflammatory effect of mulberry (Yang et al., 2011). Strong antibacterial activity against various bacterial strains has been reported for some prenylated flavonoids (Sohn et al., 2004).

These promising activities warrant further investigation of the multiple inhibitory activities of these natural compounds that may provide effective therapeutic approaches to the treatment of diseases such as infections of herpes simplex virus (HSV). HSV, a double-stranded DNA virus belonging to the Herpesviridae family, involves two human antigen types, HSV-1 and HSV-2 (Birkmann and Zimmermann, 2016). Clinical manifestations of this viral infection range from benign to severe and life-threating syndromes in immunocompromised patients. Most clinical symptoms, such as pain, redness, and swelling, are induced by the inflammatory response of the immune system to viral infection (Harmenberg et al., 2010). Medications that combine antiherpetic and anti-inflammatory agents have proven more effective in preventing ulcerative viral lesions than antiherpetics alone (El Hayderi et al., 2017; Harmenberg et al., 2010). Moreover, ulcerations caused by HSV can easily lead to secondary infections caused by bacteria, including Staphylococci, and antibacterial activity is therefore desirable in the therapeutic preparation administered.

This work was focused on the screening of mulberry compounds for their parallel anti-infective and anti-inflammatory effects. Thirteen known mulberry constituents were evaluated for inhibitory activity against the replication of HSV-1 and HSV-2. In an effort to understand the mechanism of the effect, in silico molecular docking studies were performed to assess the affinity of the active compounds for key viral enzymes. The antimicrobial activity against seven Gram-positive bacterial strains and the potential anti-inflammatory effect caused by inhibiting COX-2 were also tested on 13 other, previously isolated mulberry compounds. The anti-inflammatory activity of mulberrofuran B (11) was further tested in vivo using carageenan-induced paw edema in mice.

Section snippets

Plant material

The root bark of the mulberry tree (Morus alba L., Moraceae) was collected on the campus of the University of Veterinary and Pharmaceutical Sciences Brno (UVPS Brno; N 49°13′52″, E 16°35′32″), Brno, Czech Republic, in April 2011. The plant material was identified by Associate professor K. Šmejkal and voucher specimen (No. MA-11A) has been deposited in the herbarium of the Department of Natural Drugs, Faculty of Pharmacy, UVPS Brno.

General experimental procedures

UV spectra were acquired with Lambda 25 UV–Vis spectrometer

Isolation of compounds from Morus alba

Extensive chromatographic separation of Mori Cortex radicis led to the isolation of 26 compounds from the chloroform and the ethyl acetate extracts. Among these compounds were the flavonoids kuwanon C (1), kuwanon T (2), sanggenon H (3), morusin (4), morusinol (5), cyclomorusin (6), kuwanon S (7), kuwanon E (8), and kuwanon U (9); the 2-arylbenzofurans mulberrofuran A (10), mulberrofuran B (11), moracin M (14), moracin C (15), mulberrofuran L (22), moracin G (23), mulberrofuran Y (24), and

Conclusion

Multiple bioassays were used to identify several compounds obtained from the root bark of M. alba with promising antiviral, antibacterial, or anti-inflammatory effects. The pleiotropic effects of these compounds are desirable for the treatment of herpetic infections, which typically cause local inflammation, and possibly also for other, secondary bacterial infections. The discovery that the effectiveness of mulberrofuran B (11), the most promising compound, could be confirmed in vivo at a dose

Funding information

This work was supported by the Grant Agency of the Czech Republic (project no. 16-07193S to K. Š.; J. H.; P. S.); and by the Internal Grant agency of the University of Veterinary and Pharmaceutical Sciences Brno (project no. 310/2018/FaF to A. S.).

Notes

We dedicate this paper to prof. Suchý in the occasion of his 83rd birthday.

Declaration of competing interest

The authors declare there is no conflict of interest.

Acknowledgment

The authors thank Dr. F. T. Campbell for proofreading the manuscript.

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