Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark

Root bark extract of Calliandra portoricensis (Jacq. ) Benth. chemoprevents N-methyl-N-nitrosourea-induced mammary gland toxicity in rats

Ethnopharmacological relevance:

Calliandra portoricensis (CP) is a herb widely used in Nigeria for the treatment of breast engorgement. However, the scientific evidence of this use and its mechanisms of action is not clearly understood.

Aim of the study:

We assessed the chemopreventive effects of methanol extract of CP on N-methyl-N-nitrosourea (NMU)-induced mammary gland toxicity in rats.

Materials and methods:

Fingerprinting of methanol extract of CP by Gas Chromatography-Mass Spectrometry (GC-MS) was done. Female Wistar rats were assigned into eight groups: Group 1 (control), group 2 received NMU only, groups 3, 4 and 5 received NMU and treated with CP at doses of 100, 200 and 300 mg/kg, respectively. Group 6 received CP (300 mg/kg), group 7 received NMU and vincristine, while group 8 received vincristine.

Conclusion:

CP ameliorated NMU-induced toxicity by modulating different cellular targets.

Keywords:

Inflammation; Mammary gland; N-methyl-N-nitrosourea; Oxidative stress.

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Epub 2020 Feb 6.

A comparative study on root and bark extracts of Eleutherococcus senticosus and their effects on human macrophages

Background:

Eleutherococcus senticosus or Siberian ginseng is a medicinal plant containing adaptogenic substances believed to regulate immune responses. Both, the root and stem bark are commonly used in traditional medicines.

Purpose:

The purpose of the present study is to chemically characterize E. senticosus root and bark extracts and to compare their effects on functions of human primary macrophages.

Study design and methods:

HPLC-DAD-MS analysis was used to characterize chemical constituents of alcoholic extracts from E. senticosus root and bark. The data obtained and available databases were combined for network pharmacology analysis. Involvement of predicted pathways was further functionally confirmed by using monocyte-derived human macrophages and endotoxin-free E. senticosus root and bark extracts.

Results:

Chemical analysis showed that the root extract contained more syringin, caffeic acid, and isofraxidin than the bark extract. At variance, bark extract contained more sesamin and oleanolic acid. Coniferyl aldehyde and afzelin were below the limit of quantification in both extracts. Network pharmacology analysis indicated that constituents of E. senticosus might affect the immune cell phenotype and signaling pathways involved in cell metabolism and cytoskeleton regulation. Indeed, both extracts promoted actin polymerization, migration, and phagocytosis of E. coli by macrophages pointing to macrophage polarization towards the M2 phenotype. In addition, treatment with E. senticosus root and bark extracts decreased phosphorylation of Akt on Ser473 and significantly reduced expression of the hemoglobin scavenger receptor CD163 by macrophages. Neither extract affected expression of CD11b, CD80, or CD64 by macrophages. In addition, macrophages treated with the bark extract, but not with the root extract, exhibited activated p38 MAPK and NF-κB and released increased, but still moderate, amounts of proinflammatory TNF-α and IL-6, anti-inflammatory IL-10, and chemotactic CCL1, which all together point to a M2b-like macrophage polarization. Differently, the root extract increased the IL-4-induced expression of anti-inflammatory CD200R. These changes in monocytes are in agreement with an increased M2a macrophage polarization.

Conclusion:

The ability of E. senticosus root and bark extracts to promote polarization of human macrophages towards anti-inflammatory M2a and M2b phenotypes, respectively, might underlay the immunoregulatory activities and point to potential wound healing promoting effects of this medicinal plant.

Keywords:

Adaptogen; Alternatively activated macrophages; Cytoskeleton; Eleutherococcus senticosus; Network pharmacology; Wound healing.

Conflict of interest statement

Declaration of Competing Interest The authors have stated that there is no conflict of interest associated with the publication and no financial support, which could have influenced the outcome.

Publication types

Jing-Xuan Wan # 
1
Shilei Wang 
2
Xiaolu Wang 
1
Wenqian Guo 
1
Han Ma 
1
Yuqi Wu 
1
Chong-Zhi Wang 
3
Lian-Wen Qi 
2
Ping Li 
2
Haiqiang Yao 
1
Chun-Su Yuan 
3

Free PMC article

Chemical profiling of root bark extract from Oplopanax elatus and its in vitro biotransformation by human intestinal microbiota

Oplopanax elatus (Nakai) Nakai, in the Araliaceae family, has been used in traditional Chinese medicine (TCM) to treat diseases as an adaptogen for thousands of years. This study established an ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) method to identify chemical components and biotransformation metabolites of root bark extract from O. elatus. A total of 18 compounds were characterized in O. elatus extract, and 62 metabolites by human intestinal microbiota were detected. Two polyynes, falcarindiol and oplopandiol were recognized as the main components of O. elatus, whose metabolites are further illustrated. Several metabolic pathways were proposed to generate the detected metabolites, including methylation, hydrogenation, demethylation, dehydroxylation, and hydroxylation. These findings indicated that intestinal microbiota might play an essential role in mediating the bioactivity of O. elatus.

Keywords:

Biotransformation; Intestinal microbiota; Metabolic profiles; Oplopanax elatus; UPLC-Q-TOF/MS.

The authors declare that they have no competing interests.

Figures

Figure 1. The chemical structures of bioactive compounds detected in Oplopanax elatus extract.

(A) Polyynes; (B) Lignans; (C) Phenylpropanoid; (D) Sesquiterpenes; (E) Triterpenoid; (F) Fatty acids.

Figure 2. UPLC-TOF/MS profiles of O. elatus extract in the positive ion mode.

(A) Total ion chromatogram (TIC) of O. elatus extract. (B) TIC of blank sample including dilution medium and human fecal microflora. (C) TIC of biotransformed O. elatus sample by intestinal bacteria.

Figure 3. Metabolites of falcarindiol using UPLC-TOF/MS in the positive ion mode.

(A) Extracted ion chromatograms (EICs); (B) MS/MS spectra and structural elucidation.

Figure 4. Metabolites of oplopandiol using UPLC-TOF/MS in the positive ion mode.

(A) EICs; (B) MS/MS spectra and structural elucidation.

Figure 5. The proposed metabolic pathways of O. elatus extract by human intestinal microflora, including (A) Polyynes; (B) Lignans; (C) Phenylpropanoid; (D) Sesquiterpenes; (E) Triterpenoid; (F) Fatty acids. Methylation (1), demethylation (2), hydrogenation (3), hydroxylation (4), dehydroxylation (5), acetylization (6) and demethoxylation (7) were observed in this biotransformation.

References

This work was supported by grants from the National Natural Science Foundation of China (81803970), NIH/NCCAM Grants AT004418 and AT005362, Young Talent Promotion Project of China Association of Chinese Medicine (CACM-2019-QNRC2-A01), Young Scientists Development Program of Beijing University of Chinese Medicine, Fundamental Research Funds for the Central Universities (2020-JYB-XJSJJ-003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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BMC Complementary and Alternative Medicine

 12, Article number:  ()
Cite this article

Despite the widespread use of roots of Cassia sieberiana in managing several health conditions including gastric ulcer disease, there is little scientific data to support the rational phytotherapeutics as an anti-ulcer agent. This paper reports an evaluation of the in vivo anti-oxidant properties of an aqueous root bark extract of C. sieberiana in experimental gastric ulcer rats in a bid to elucidate its mechanism of action.

Methods

The gastro-cytoprotective effect, inhibition of decrease in activity of gastric anti-oxidant enzymes and MPO as well as the inhibition of gastric LPO level suggests that one of the anti-ulcer mechanisms of C. sieberiana is the anti-oxidant property.

Peer Review reports

Background

Fisher 344 (F344) rats weighing 230.3 ± 14.5 g (mean ± S.D) of both sexes were used for the studies. They were raised on standard laboratory diet (GAFCO, Tema, Ghana). The animal experimentation described in this study was approved by the Scientific and Technical Committee of the Noguchi Memorial Institute for Medical Research (NMIMR) of the University of Ghana and was conducted in accordance with internationally accepted principles for laboratory animal use and care.

Chemicals and reagents

The enzyme immunoassay kits for total anti-oxidants (Item # 709001), lipid hydroperoxide-LPO (Item # 705002), superoxide dismutase-SOD (Item # 706002), catalase-CAT (Item # 707002) and glutathione peroxidase-GPx (Item # 703102), were obtained from Cayman Chemical Company (Ann Arbor, U.S.A). Protein assay kit (Item # 704002) was also obtained from Cayman Chemical Company (Ann Arbor, U.S.A). All other chemicals and reagents were obtained from Sigma-Aldrich Chemical Company, St. Louis, MO, U.S.A.

Administration of plant extract and induction of gastric ulcers

MUI
=

Total

ulcer

score

Number

of

rats

ulcerated

%

Inhibition
=

MUI

of

ethanol

treated
—
MUI

of

extract

pretreated

UI

of

ethanol

treated

×
100

Total antioxidant capacity

The capacity of total anti-oxidants in the serum was assayed as per instructions from Cayman Chemical Company (Ann Arbor, U.S.A). Briefly the blood was allowed to clot for 30 min and then centrifuged at 2,000 g for 15 min at 4°C. The resulting supernatant was assayed for total anti-oxidant capacity using Cayman’s anti-oxidant assay kit (Item # 709001).

Determination of LPO levels and anti-oxidant enzymes activities

In other set of experiments, after euthanasia the stomachs were quickly removed, rinsed in phosphate buffered saline (PBS, pH 7.4) and weighed. The stomachs were then homogenized and centrifuged according to the standard procedures using ELISA kits from Cayman Chemical Company (Ann Arbor, U.S.A) and the supernatant assayed as per instructions for determination of LPO (Item # 705002) and the activity of the anti-oxidant enzymes SOD (Item # 706002), CAT (Item # 707002) and GPx (Item # 703102). Protein levels were estimated by protein assay kits (Item # 704002) from Cayman Chemical Company (Ann Arbor, U.S.A).

Determination of myeloperoxidase (MPO) activity

Table 1
Effect of
C. sieberiana
(CS) root bark extract on ulcer index in ethanol-induced gastric ulcer rats

Full size table

Effect of
C. sieberiana
(CS) root bark extract on gastric ulcer inhibition in F

344

rats.

Full size image

Lipid peroxidation index

Table 2
Effect of
C. sieberiana
(CS) root bark extract on lipid peroxidation indices in ethanol-induced gastric ulcer rats

MPO activity

The results in Figure 2 show the effect of treatments on total anti-oxidant capacity in serum. Analysis of the results indicates that untreated animals induced with gastric ulcer showed a significant decrease (p< 0.001) in serum total anti-oxidants (1.93 ± 0.07 μmol/mg protein) when compared with the intact gastric mucosa (4.76 ± 0.04 μmol/mg protein). Animals pre-treated with ranitidine and extract at the three tested doses showed a significant dose-dependent increase in total serum anti-oxidants. This improvement was most pronounced (4.77 ± 0.07 μmol/mg protein) in the group that received the high dose of C. sieberiana root bark extract (CS-1000). Pre-treatment of animals with ranitidine, the standard anti-ulcer drug showed significantly lower total serum anti-oxidant capacity compared with CS-1000 (p< 0.01).

SOD activity

The results in Figure 3 represent the effect of various treatments on SOD activity. Analysis of the figure shows that the activity of SOD was significantly lowered (p< 0.001) in the gastric mucosa of animals exposed to ethanol (Ulcer control) (2.56 ± 0.11 U/mg protein), when compared with the respective value in intact gastric mucosa (baseline) (5.80 ± 0.06 U/mg protein). Pre-treatment of the rats with C. sieberiana for 7 days showed a dose-dependent significant (p< 0.001) inhibition of the effect of ethanol in decreasing SOD. Pre-treatment with ranitidine also showed a significant (p< 0.001) inhibition of the effect of ethanol on gastric mucosal SOD but this was lower (p< 0.01) than the effect of CS-1000.

CAT activity

In conclusion, the present results suggest that the gastro-cytoprotective effect of C. sieberiana root bark extract against mucosal injury induced by ethanol might be mediated at least partially by its ability to stimulate and thereby maintain baseline activity level of mucosal anti-oxidant enzymes such as SOD, CAT and GPx which constitute endogenous scavengers of ROS. This effect is supported by the elevation of serum total anti-oxidant level and the inhibition of increase in LPO in addition to the inhibition of MPO enzyme activity to maintain them at near basal level.

Abbreviations

Mean ulcer index

Reactive oxygen species

Corresponding author

Correspondence to
Edmund T Nartey.

Additional information

The authors declare they have no competing interests.

Authors’ contributions

ETN and MO worked in the conception, methods, analysing results and revising it critically. CMA worked on the methods, data analysis and revising it critically. All three authors read and approved the final manuscript.

Authors’ original submitted files for images

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About this article

Nartey, E.T., Ofosuhene, M. & Agbale, C.M. Anti-ulcerogenic activity of the root bark extract of the African laburnum “Cassia sieberiana” and its effect on the anti-oxidant defence system in rats.
BMC Complement Altern Med 12, 247 (2012). https://doi.org/10.1186/1472-6882-12-247

Keywords

Oplopanax elatus (Nakai) Nakai is the plant of genus Oplopanax, which belongs to the Araliaceae family. It is mainly distributed in northeast China, Korea and far east of Russia (Dou et al., 2009; Yang et al., 2010). As a traditional medicinal plant, O. elatus is being utilized as a ginseng-like herbal medicine and has been long used as an adaptogen to treat arthritis, diabetes mellitus, rheumatism, neurasthenia, and cardiovascular diseases (Dai et al., 2016; Eom et al., 2017; Knispel et al., 2013; Moon et al., 2013; Panossian et al., 2021). Previous studies have identified several components derived from O. elatus, such as the lignans, saponins, phenolic glycosides, and polyynes (Huang et al., 2010; Shao et al., 2016). To date, polyynes have been chiefly reported with high contents in the root of O. elatus (Huang et al., 2014a). Increasing attention has been paid to two main polyynes facarindiol (FAD) and oplopandiol (OPD), because of their significant anti-tumor activities (Purup, Larsen & Christensen, 2009; Qiao et al., 2017; Sun et al., 2016). However, most studies remain focused on the pharmacological and chemical constituents of O. elatus, while its metabolic profiles are rather obscured.

Recently, various analytical platforms are typically applied to identify metabolic profiles in the complex extracts of TCMs. Most notably, ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is one of the powerful analytical tools (Jin et al., 2018; Wu et al., 2019; Yang et al., 2016). With the newly developed chromatographic technique, the UPLC system allows significant improvements in the resolution, analysis speed, and reduction of solvent waste (Chekmeneva et al., 2018; Du et al., 2017; Wang et al., 2008). Meanwhile, high-resolution Q-TOF/MS can give more specific and accurate mass information on characteristic molecular ions and fragment ions, providing a reliable basis for the qualitative analysis of complex samples (Li et al., 2019; Lou et al., 2015; Wewer et al., 2011). Based on these characteristics, UPLC-Q-TOF/MS was ultimately selected for fast identification of constituents in O. elatus.

In the present study, we focused on the metabolic behavior of human intestinal microflora on O. elatus. A highly selective and sensitive UPLC-Q-TOF/MS method was established to characterize the chemical and metabolic profiles of O. elatus. Furthermore, the proposed metabolic pathways were also summarized. This work will provide a better understanding for exploring the bioactivities of O. elatus in vivo.

Materials & methods

The general anaerobic medium for bacteria culture was obtained from Shanghai Kayon Biological Technology Co. Ltd. (Shanghai, China). Formic acid and HPLC-grade acetonitrile were purchased from Merck (Darmstadt, Germany). Deionized water (18 MΩ·cm) was supplied with a Millipore Milli-Q water system (Milford, MA, USA). All other reagents were from standard commercial sources and of analytical purity.

Preparation of Oplopanax elatus extract

Root bark of O. elatus was obtained from Benxi city (Liaoning, China). The voucher samples were deposited at the Tang Center for Herbal Medicine Research at the University of Chicago (Chicago, IL, USA). The air-dried root bark of O. elatus was pulverized into powder and sieved through an 80-mesh screen. Eight g of the powder were extracted twice by heat-reflux with 70% ethanol for 2 h. The combined extract was evaporated under vacuum and lyophilized with a yield of 28%. The samples were stored at 4 °C until use.

Preparation of human intestinal microflora

Two microliters of the fecal supernatant were added with eight ml anaerobic dilution medium containing five mg of O. elatus extract, which were then anaerobically incubated at 37 °C for 24 h in an anaerobic workstation (Electrotek, UK). The reaction mixtures were extracted three times with water-saturated n-butanol. All the n-butanol layers were mixed and dried under a nitrogen stream and then dissolved in one ml methanol. The solutions were centrifuged at 13,000 rpm for 10 min for analysis.

UPLC-Q-TOF/MS analysis

Mass data were analyzed by the Agilent MassHunter Workstation software (Version B.06.01), based on the accurate measurements of m/z values with online databases (MassBank, etc.), to screen probable compounds. The empirical molecular formula was deduced by comparing the theoretical mass of molecular ions at the mass accuracy of less than five ppm.

Results

To obtain the chromatograms with better resolution and higher baseline stability of O. elatus extract and its primary metabolites, multiple mobile phases such as acetonitrile-water and methanol-water were detected. Acetonitrile-water was applied as the solvent, for its stronger separation ability, shorter retention time, and lower column pressure. Additionally, 0.1% formic acid added in the water as mobile phase adducts may help to achieve higher response and better peak sensitivity (Tao et al., 2016). Therefore, the optimal solvent system consisting of acetonitrile-water (0.1% formic acid), which remarkably enhanced the efficiency of ionization and satisfactory sensitivity, was ultimately selected as mobile phase with a gradient elution.

In addition, the factors related to MS performance, including ionization mode and collision energy, were further improved. The positive ion mode was ultimately employed to gain comprehensive data for structural characterization and metabolite assignment with much lower background noise. The collision energy was optimized to obtain the higher ionization efficiency and relative abundance of precursor and product ions.

Chemical profiling of O. elatus extract

In total, 18 ingredients of O. elatus were detected in this study, and their chemical structures are shown in Fig. 1. There are six types of compounds, including nine polyynes, three lignans, one phenylpropanoid, two sesquiterpenes, one triterpenoid, and two fatty acids. The total ion chromatogram (TIC) of O. elatus extract is shown in Fig. 2A in the positive ion mode by UPLC-Q-TOF-MS. Table 1 shows the detailed information, including retention time, signal intensity, molecular formula, calculated and experimental mass m/z, ppm error, and fragment ions of these 18 components (Schymanski et al., 2014; Wang et al., 2020).

The chemical structures of bioactive compounds detected in Oplopanax elatus extract.

UPLC-TOF/MS profiles of O. elatus extract in the positive ion mode.

Polyynes have been found as the main constituents in the root of O. elatus (Yang et al., 2014). Among them, falcarindiol and oplopandiol were determined to have very high contents in the air-dried root bark. As shown in Table 1, polyynes exhibit the same elemental composition and similar MS/MS behaviors, with the characteristic fragment ions at m/z 79.05 in the positive ion mode.

Detection and identification of metabolites of O. elatus extract

The control sample was prepared in parallel, which used in the dilution medium and human fecal microflora, as shown in Fig. 2B. The biotransformed O. elatus sample by intestinal bacteria is shown in Fig. 2C. Samples were incubated, pretreated, and analyzed under the same conditions as mentioned in “Incubation of sample in intestinal bacteria”. The potential metabolites were detected from the TIC of the transformed O. elatus sample compared to the control group. All the metabolites were further confirmed by the extracted ion chromatograms (EICs) and their MS/MS corresponding fragments. A total of 62 metabolites were identified by UPLC-Q-TOF-MS in the positive mode. Table 2 shows the retention time, signal intensity, experimental and calculated mass m/z, difference between m/z and calculated m/z in ppm, and fragment ions in the MS/MS stage of these 62 metabolites (M1-M62). All these metabolites could not be observed or only in trace amounts in control samples (Schymanski et al., 2014).

UPLC-Q-TOF/MS data of metabolites detected from the biotransformed O. elatus sample in the positive ion mode.

Polyynes

A total of 46 metabolites of nine polyynes generated by the transformation of human intestinal microflora were detected and identified. For each polyyne, at least four types of metabolites were identified. Due to the high biological activities, FAD and OPD selected as the representative compounds of polyynes were stated in detail.

Metabolites of falcarindiol using UPLC-TOF/MS in the positive ion mode.

Metabolites of oplopandiol using UPLC-TOF/MS in the positive ion mode.

M3–M5 were the deglycosylation products of three lignans via the loss of glycose moieties. For example, the parent compound of M3 was determined to be C32H44O16 while M3 was C20H24O6, indicating M3 was the deglycosylation product via the loss of two glucose moieties. M4 and M5 were assigned as the products by losing a glucose moiety from their corresponding parent lignan compounds.

Phenylpropanoids

For two sesquiterpenes, M9, M38, and M58 were identified as the hydroxylation, demethylation, and hydrogenation products of curcumene, while M20 and M45 were the hydroxylation and demethylation product of muurolene, respectively. M37 and M62 were identified as the demethylation and acetylization products of oleanolic acid. In addition, for 2 fatty acids, M12 was the dehydroxylation product of 2-decenoic acid, while M24 and M59-61 were the products of 6,9-octadedicenoic acid.

Proposed metabolic pathways of O. elatus extract

The proposed metabolic pathways of O. elatus extract by human intestinal microflora are presented in Fig. 5. Multiple major metabolite pathways can be observed in this study. The common pathways involved in the biotransformation of O. elatus extract include methylation, demethylation, hydroxylation, dehydroxylation, acetylation, hydrogenation, demethoxylation, and deglycosylation. Among them, polyynes were undoubtedly the most important compounds, as 46 out of 62 metabolites originated from polyynes. By comparing the signal intensity of metabolites, we could find that methylation, dehydroxylation and hydroxylation are major metabolic pathways of polyynes. Moreover, four metabolites of lignans and phenylpropanoid were produced by the loss of glucose. The other metabolites were generated from one triterpenoid and two fatty acids. This indicated that polyynes of O. elatus generated comprehensive biotransformation and were more readily metabolized than other compounds under the same conditions.

The proposed metabolic pathways of O. elatus extract by human intestinal microflora, including (A) Polyynes; (B) Lignans; (C) Phenylpropanoid; (D) Sesquiterpenes; (E) Triterpenoid; (F) Fatty acids. Methylation (1), demethylation (2), hydrogenation (3), hydroxylation (4), dehydroxylation (5), acetylization (6) and demethoxylation (7) were observed in this biotransformation.

In summary, the main metabolic pathways of O. elatus refer to hydrolytic and reductive reactions by gut microorganisms. Because of the complexity of active ingredients or constituent concentrations, in vivo exposure, and individual differences, the metabolic profiles of O. elatus might be affected by several factors.

In this study, a UPLC-Q-TOF-MS/MS method was developed to screen and identify the chemical composition and metabolites from a traditional Chinese herb, the air-dried root bark of O. elatus. A total of 18 ingredients and 62 metabolites biotransformed by human intestinal microflora were characterized from O. elatus in UPLC-Q-TOF/MS positive ion mode. Two polyynes, falcarindiol and oplopandiol, as the main components of O. elatus and their metabolites by human intestinal microflora are mainly illustrated. It could be noted that the major metabolic pathways of O. elatus refer to methylation, dehydroxylation, and hydroxylation. Studies on the chemical and metabolic profiling of O. elatus by human intestinal microflora will be helpful for the understanding of mechanism research on the active components and further in vivo investigation.

Supplemental Information

The mass spectrum of Oplopanax elatus (the herbal medication we studied) and its metabolites by human intestinal microbiota, which were detected by an Agilent 6545 Q-TOF-MS system. Mass data including MS and MS/MS information could be analyzed by Agilent MassHunter Workstation software. The software was applied in the screening and identification of the probable compounds, based on the accurate measurements of m/z values with databases.

BMC Complementary and Alternative Medicine

 14, Article number:  ()
Cite this article

Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity.

In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot.

These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.

In light of the therapeutic potential of mulberry root bark in inflammation-induced colorectal cancer, this study was performed to elucidate the biological mechanism by which mulberry root bark inhibits an inflammatory response in LPS-stimulated RAW264.7 cells and induces the inhibition of cell growth and apoptosis in human colorectal cancer cells. Here, for the first time, we reported that mulberry root bark extracts attenuated NO production by suppressing iNOS expression via regulating the activations of NF-κB and ERK1/2. In addition, it induced cell growth arrest and apoptosis by activating ATF3 expression and cyclin D1 proteasomal degradation in colorectal cancer cells, SW480.

Cell culture media, Dulbecco’s Modified Eagle medium (DMEM) was purchased from Gibco Inc. (NY, USA). LPS (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). SB203580, PD98059 were purchased from Calbiochem (San Diego, CA). SB216763 and N-Acetyl Cysteine (NAC) were purchased from Sigma–Aldrich. Antibodies against iNOS, ATF3 and cyclin D1 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). Other antibodies against IκB-a, p65, ERK1/2, phospho-ERK1/2 (Thr202/Tyr204) and b-actin were purchased from Cell Signaling (Bervely, MA, USA). All chemicals were purchased from Sigma-Aldrich, unless otherwise specified.

Sample preparation

The plant sample, Mulberry (Morus alba L. voucher number: PARK1002(ANH)) root bark, was kindly provided by the Bonghwa Alpine Medicinal Plant Experiment Station, Korea. One kilogram of mulberry root bark was extracted with 1000 ml of 80% methanol with shaking for 24 h. After 24 h, the methanol-soluble fraction was filtered and concentrated to approximately 20 ml volume using a vacuum evaporator and then fractioned with petroleum ether and ethyl acetate in a separating funnel. The ethyl acetate fraction was separated from the mixture, evaporated by a vacuum evaporator, and prepared aseptically and kept in a refrigerator until use.

Cell culture and treatment

Mouse macrophage cell line, RAW264.7 and human colorectal cancer cell line, SW480 were purchased from Korean Cell Line Bank (Seoul, Korea) and grown in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. These cells were maintained at 37°C under a humidified atmosphere of 5% CO2. Mulberry root bark extracts (MRBE) were dissolved in dimethyl sulfoxide (DMSO) and then treated to cells. DMSO was used as a vehicle and the final DMSO concentration was not exceeded 0.1% (v/v).

Measurement of nitric oxide (NO) production

The 3-(4,5-dimethylthizaol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure cell proliferation. Briefly, SW480 cells were seeded onto 96-well culture plate at a density of 50,000 cells per well. The cells were treated with MRBE for 24 h. Then, 50 μl of MTT solution (1 mg/ml) was added to each well. The resulting crystals were dissolved in DMSO. The formation of formazan was measured by reading absorbance at a wavelength of 570 nm.

SDS-PAGE and Western blot

Statistical analysis was performed with the Student’s unpaired t-test, with statistical significance set at *, P < 0.05.

Effect of MRBE on NO production (A) and iNOS (B) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with LPS (1 μg/ml) for the additional 18 h. After treatment, NO production was measured using the media and Griess reagent and cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with iNOS antibody for Western blot. iNOS protein was visualized using ECL detection. Actin was used as internal control. DMSO was used as a vehicle. Values given are the mean ± SD (n = 3). *p < 0.05 compared to LPS treatment without MRBE.

Inhibitory effect of MRBE on LPS-induced NF-κB and ERK1/2 activation in RAW264. 7 cells

Effect of MRBE on IκB-α degradation (A), p65 nuclear translocation (B) and ERK1/2 phosphorylation (C) in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pre-treated with MRBE at the indicated concentrations for 2 h and then co-treated with (1 μg/ml) for 15 min (for Western blot of IκB-α and ERK1/2 phosphorylation) or 30 min (for Western blot of p65). DMSO was used as a vehicle. Cell lysate were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies against IκB-α, p-ERK1/2, total ERK1/2 and p65. The proteins were then visualized using ECL detection. Actin was used as an internal control.

Effect of MRBE on cell viability and apoptosis in human colorectal cancer cell line, SW480

Effect of MRBE on cell growth (A) and apoptosis (B) in SW480 cells. SW480 cells were treated with MRBE at the indicated concentration for 24 h. Cell growth was measured sung MTT solution and expressed as absorbance (A570). *P < 0.05 compared to cell without MRBE treatment. Apoptosis by MRBE was evaluated with Western blot against cleaved PARP. Actin was used as an internal control. DMSO was used as a vehicle.

Effect of MRBE on the levels of ATF3 and cyclin D1 in protein and mRNA in SW480 cells

There is growing evidence that activating transcription factor 3 (ATF3) is linked to cell growth arrest and apoptosis in colorectal cancer. To investigate whether MRBE activates ATF3 expression in human colorectal cancer cells, SW480 cells were treated with MRBE at the indicated concentrations for 24 h. As shown in Figure 4A and4C, MRBE induced ATF3 expression in the levels of both protein and mRNA. Time-course experiment showed that induction of ATF3 by MRBE occurred after 1 h stimulation (Figure 4D).We also evaluated whether MRBE regulates cyclin D1 level in SW480 cells since cyclin D1 is associated with cell growth arrest and apoptosis. As shown in Figure 4B, MRBE decreased the protein level of cyclin D1 in a dose-dependent manner. However, decrease in mRNA level of cyclin D1 by MRBE treatment was not observed (Figure 4C). In time-course experiment for cyclin D1 (Figure 4D), MRBE significantly reduced cyclin D1 protein level after 1 h stimulation.

Effects of MRBE on ATF3 and cyclin D1 expression in mRNA and protein level in SW480 cells. (A, B) SW480 cells were treated with MRBE at the indicated concentrations for 24 h. Cell lysates were subjected to SDS–PAGE and the Western blot was performed using antibodies against cyclin D1, ATF3 and actin. For RT-PCR analysis of ATF3 and cyclin D1 gene expression (C), total RNA was prepared after MRBE treatment for 24 h. (D) SW480 cells were treated with MRBE (25 μg/ml) for the indicated times. Cell lysates were subjected to SDS–PAGE and the Western blot was performed using antibodies against cyclin D1, ATF3 and actin. Actin and GAPDH were used as internal control for Western blot and RT-PCR, respectively. DMSO was used as a vehicle.

GSK3β and ROS-dependent ATF3 activation of MRBE in SW480 cells

To elucidate the molecular mechanism for MRBE-induced ATF3 expression, we evaluated several signaling pathways affected by MRBE. SW480 cells were pretreated with kinase inhibitors such as PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor) and SB216763 (GSK-3β inhibitor), and NAC (ROS scavenger) for 2 h prior to incubation with 25 μg/ml of MRBE. As shown in Figure 5A, MRBE-induced ATF3 expression was observed in the cells pretreated with PD98059 and SB203580. However, pretreatments of SB216763 and NAC diminished MRBE-induced ATF3 expression (Figure 5B). Collectively, these results suggest the pathways of GSK-3β and ROS may be involved in MRBE-induced ATF3 expression.

ROS/GSK3β-dependent ATF3 expression and ROS-dependent cyclin D1 proteasomal degradation by MRBE. (A, B) SW480 cells were pre-treated with PD98059 (20 μM), SB203580 (20 μM), SB216763 (20 μM) or NAC (20 mM) for 2 h and then co-treated with MRBE (25 μg/ml) for 1 h. Cell lysates were subjected to SDS–PAGE and the Western blot was performed using antibodies against ATF3 and actin. (C, D, E) SW480 cells were pre-treated with MG132 (5 and 10 μM), PD98059 (20 μM), SB203580 (20 μM), SB216763 (20 μM) or NAC (20 mM) for 2 h and then co-treated with MRBE (25 μg/ml) for 1 h. Cell lysates were subjected to SDS–PAGE and the Western blot was performed using antibodies against cyclin D1 and actin. Actin was used as an internal control and DMSO was used as a vehicle.

ROS-dependent cyclin D1 proteasomal degradation by MRBE in SW480 cells

Taken together, our report is the first to show that MRBE exerts anti-inflammatory and anti-cancer activity. Anti-inflammatory effect of MRBE is mediated from inhibiting NF-κB and ERK1/2 activation. Anti-cancer activity of MRBE is associated with ROS-dependent cyclin D1 proteasomal degradation and ROS/ GSK3β-dependent ATF3 expression.

Inducible nitric oxide synthease

Extracellular signal-related kinase 1/2

Reactive oxygen species.

This study was supported by the BK21 PLUS program of Ministry of Education and by Bio-industry Technology Development Program (112144-02-2-SB010), Ministry of Agriculture, Food and Rural Affairs.

Author information

Correspondence to
Jin Boo Jeong.

The authors declare that they have no conflict interest.

JBJ directed and HJE designed the study. HJE, JHP, GHP, MHL, JRL and JSK performed the experiments. HJE and JHP drafted manuscript. JHP, GHP, MHL, JRL, JSK and JBJ corrected the manuscript. All authors read and approved the final manuscript.

Hyun Ji Eo, Jae Ho Park contributed equally to this work.

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Cite this article

Eo, H.J., Park, J.H., Park, G.H. et al. Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark.
BMC Complement Altern Med 14, 200 (2014). https://doi.org/10.1186/1472-6882-14-200

Additional Information and Declarations

Jin-Yi Wan performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Jing-Xuan Wan performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Shilei Wang performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Xiaolu Wang performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Wenqian Guo analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Han Ma analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Yuqi Wu analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Chong-Zhi Wang conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Lian-Wen Qi performed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Ping Li analyzed the data, prepared figures and/or tables, and approved the final draft.

Haiqiang Yao conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Chun-Su Yuan conceived and designed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Human Ethics

The raw measurements are available in the Supplementary Files.