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The essential list of guidances for software medical devices
This page gathers the guidances and other documents about CE mark and FDA 510k for software medical devices. I limited the list to documents, which have an impact on design.
Go to the list of FDA guidances
CE Mark Guidances
I picked the documents in websites of the following organisms:
- European Commission,
- Notified Body Operations Group (NBOG),
- European Association for Medical devices of Notified Bodies (Team-NB),
- International Medical Device Regulators Forum (IMDRF). FYI: the International Medical Device Regulators Forum (IMDRF) replaced the Global Harmonization Task Force (GHTF) late 2012. Not a big deal for our subject. All GHTF documents are available on IMDRF website.
If you're looking for documents about other steps of the software lifecycle (clinical evaluation & investigation, post-market surveillance and vigilance), you'll find what you're looking for in those websites.
Is your software a medical device?
The MEDDEV 2.1.6 Qualification and Classification of stand alone software contains information to let you determine if your software is a medical device. I hope for you that your software falls out of the scope of medical devices. You will save money! But I'll be sad, because you won't need to read my blog any more :-)
Has it a measuring function?
The MEDDEV 2.1/5 of June 1998 Medical Devices with a measuring function contains contains criteria which indicate that a device has a measuring function. Note that usually standalone software doesn't have a measuring function. If valid measurement data are given by another medical device, and are processed by the standalone software to compute new data, then it shouldn't have a measuring function.
What is its classification?
The MEDDEV 2.4.1 rev9 Classification of medical devices contains a comprehensive interpretation of the classification rules of the 93/42 directive.
Not yet sure of its classification?
Have a look at the last version of the Manual on borderline and classification of medical devices. It has, amongst other things, a section about Picture Archiving and Communication Systems (PACS) and a chapter dedicated to mobile applications.
How should I proceed to CE mark?
The NB-MED 2.2/rec4 revision 5 Software and Medical Devices contains indications about how applying the annexes II to VII of the 93/42 CE directive.
This is explained with more details on the page How to qualify, classify, and CE mark Software.
How should I design it?
Official documents on CE mark are not talkative about software design. You have to rely on IEC 62304 standard (see posts in the "standards" category of this blog).
The FAQ on IEC 62304 of TEAM-NB gives a good bunch of recommendations to apply this standard for CE mark compliance. It also gives a lot of additional hints related to questions that are asked in this web page.
Outside Europe, the FDA published more that 10 years ago the "General Principles of Software Validation" guidance. The link to this very useful guidance is in the section about FDA guidances below on this page.
How to do clinical evaluation?
I put the clinical evaluation in the list hence it may have an impact on design. The MEDDEV 2.7.1 rev 4 Clinical evaluation - Guide for manufacturers and notified bodies contains recommendations about clinical evaluation.
The NBOG CL 2010-1 Checklist for audit of Notified Body's review of Clinical Data-Clinical Evaluation is also interesting to see what is expected from the notified body during clinical data evaluation.
What information should I put in the technical file?
The NB-MED/2.5.1/Rec5 Technical Documentation contains the structure of the design dossier submitted (or not, if you're in class I) to the notified body. It's a very generic document and doesn't contain a lot about software.
The GHTF SG1 N11 Summary Technical Documentation for Demonstrating Conformity to the Essential Principles of Safety and Performance of Medical Devices (STED) has some more information about software.
See also my templates repository page on design documents you should provide with your design dossier.
Furthermore, you may have a look at the NBOG_BPG_2009_1 Guidance on Design-Dossier Examination and Report to see what data notified bodies expect to find in your design dossier.
What do I put in the declaration of conformity?
If you do it by yourself (you're in class I), the MDEG – 2009–12-01 Guidance notes for manufacturers of class I medical devices contains all information about CE mark of class I devices.
Have a look also at the NB-MED 2.5.1 Rec4 Content of mandatory certificates contains interesting information on what notified bodies want to see in the declarations of conformity of manufacturers.
How do I handle design changes?
This is a big issue with software, which are always perfectible and need to be continuously improved. The NB-MED/2.5.2/Rec2 Reporting of design changes and changes of the quality system contains information on when and how to report changes to design.
Have a look also at the NB-MED/2.5.1/Rec6 Renewal of EC Design-Examination and Type-Examination and the NBOG BPG 2014-1 Renewal of EC Design-Examination and Type-Examination Certificates. They both contain recommendations about when to decide to renew the design examination by a notified body. The first one is somewhat replaced by the second one, which is newer. However the NBOG BPG 2014-1 doesn't say that it supersedes the NB-MED/2.5.1/Rec6.
But the most interesting document on design changes was published in 2014: the NBOG BPG 2014-3 Guidance for manufacturers and Notified Bodies on reporting of Design Changes and Changes of the Quality System. It contains the section 5.1 with lots of examples related to software changes.
Do I have to translate it?
Another really big issue for software, where translation may lead to design changes. The MDEG 2008-12- II-6.3 Mandatory Languages Requirements for Medical Devices is made for you.
Warning! It's a bit old. You should have a look at the most recent regulations of member states. The advantage is if you find that a language is mandatory in this document, you won't have to do further investigations.
Do I have to print the instructions for use?
The easiest way to deliver the IFU is to give it in an electronic format. The Council Decision on electronic instructions for use of medical devices gives information about when printing instructions is mandatory.
See also the NB-MED position about e-IFU.
Here's my list so far for CE mark.
Let's see now the FDA 510k guidances for software.
FDA 510k Guidances
I picked all the documents on FDA's website: medical devices guidances search page
This is my unique source of information. Compared to what is found about CE mark, the FDA has the immense virtue to gather all the precious information! I think that FDA guidances are a little verbose. But their content is always of good level and may be used to find answers on subjects that EU guidances don't tackle.
What is the classification of my device?
Classification of devices is most of times straightforward, with the help of the FDA database on classification of medical devices. However, things are changing quickly for software with the rush of medical devices manufacturers on mobile applications. We have the :
- Medical Device Data Systems, Medical Image Storage Devices, and Medical Image Communications Devices guidance,
- General Wellness: Policy for Low Risk Devices Guidance,
- Medical Device Accessories: Defining Accessories and Classification Pathway for New Accessory Types Draft Guidance, and the
- Mobile Medical Applications guidance.
That's a lot of information. There's an article dedicated to these guidances.
How do I design it ?
Surely the most important document of the FDA about software design (after the CFR, of course!), the General Principles of Software Validation; Final Guidance for Industry and FDA Staff is one of the most comprehensive documents about software design, verification and validation in the medical devices industry. It covers all steps of software design and more. Read it carefully!
What about COTS and SOUP?
COTS (Components Off The Shelf) are software not developed internally. COTS include software found at runtime and software used for design. For example: COTS include java runtime and java SDK.
IEC 62304 standard defines the concept SOUP (Software of Unknown Provenance) as software used in the medical device, but not developed with the level of scrutiny required by regulations. The SOUP definition doesn't include software used for design. For example: COTS include java runtime only, not java SDK.
The guidance on Off-The-Shelf Software Use in Medical Devices deals with problems about hazards brought by this kind of software.
What about human factors engineering?
Human factors in a recent subject of concern of regulation agencies. FDA is not the last one in the race for new guidance about it with the Guidance on Applying Human Factors and Usability Engineering to Optimize Medical Device Design.
This guidance integrates the latest developments on ergonomics found in IEC 62366 and AAMI HE75 standards.
There is also a long page on FDA website dedicated to human factors.
What about cyber security?
Software security is a non-functional requirement, which is probably the most difficult to implement if software wasn't initially designed the right way. There are two documents on cybersecurity while designing software medical device.
The first one is the guidance on Content of Premarket Submissions for Management of Cybersecurity in Medical Devices. It is not easy to apply as it defines a cybersecurity management process.
The second document is the guidance about Cybersecurity for Networked Medical Devices Containing Off-the-Shelf (OTS) Software. Its scope is narrower as it focuses on problems about updating COTS software (like installing a patch delivered by the COTS editor), which have impact on security.
The last guidance on cybersecurity deals with phases after design, when software is placed on the market: the draft Guidance on Postmarket Management of Cybersecurity in Medical Devices. It is still a draft guidance but is the only one you'll find on cybersecurity management when the device is placed on the market.
There is also a page on FDA website dedicated to cybersecurity.
Do I have to print the instructions for use?
The guidance on Acceptable Media for Electronic – Product User Manuals contains new information about cases where delivering user manuals in printed format is mandatory. If you fall out of the scope of mandatory print copies of instructions for use, enjoy.
What do I put in my 510k submission ?
FDA gives instructions about software in medical device in the Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices. Another document more specific on medical Imaging software is the Guidance for the Submission Of Premarket Notifications for Medical Image Management Devices.
My device changes, should I submit a new 510k ?
The guidance on 510(k) Device Modifications: Deciding When to Submit a 510(k) for a Change to an Existing Device contains a very well done decision tree and a lot of explanations with new information about software. This guidance was issued in 1997 and a new draft guidance Deciding When to Submit a 510(k) for a Change to an Existing Device was published by the FDA.
The two guidances above are for all types of medical devices, a new draft guidance was also published by the FDA: Deciding When to Submit a 510(k) for a Software Change to an Existing Device. Rely on this last guidance when changes are only made in software. Even if it is in draft state, this is the only one on this subject.
First version on February 22nd 2012
Second version on October 29th 2015.
Updated on August 2016
Reason: periodic review of articles
Fixed dead links and updated guidances info.
Published on Wednesday 22 February 2012 by Mitch


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I haven't wrote about Formula 1 on my blog in a while but let's be honest, there hasn't been much to talk about. At time of writing, Mercedes' have just clinched the 2015 Constructors' Championship and in two weeks time, Hamilton could win the Championship, the question is, what does he need to do in order to clinch his third world title?
From October 2015
OS X El Capitan – Review
Let me say this right away, everyone running OS X Yosemite should upgrade to OS X El Capitan. Apple have focused on performance, stability and security enhancements so although there aren't many new features El Capitan is a brilliant update and I'm going to say it's nearly on par with OS X Snow Leopard and OS X Mountain Lion, arguably two of Apple's best OS X updates.
iOS 9 – Review
I'm going to get straight to the point, iOS 9 is better than iOS 8. Siri is a lot smarter, there's a new Low Power mode, new Multitasking experiences for iPad and much more. One of the best things about iOS 9 is that every device that was compatible with iOS 8 last year is compatible with this update.
watchOS 2 – Review
I'll be honest, watchOS 2 is what should of shipped when Apple Watch was originally released. It includes much-needed features and optimisation enhancements. If you haven't already got an Apple Watch or you're still on the fence, watchOS 2 might make you change your mind. The software that shipped with Apple Watch (which I will simply refer to as watchOS) was sluggish and the biggest feature that was missing was native apps. watchOS 2 was due for release on the 16th of September 2015 alongside iOS 9 but due to a critical bug, watchOS 2 was delayed. It was released on the 21st of September 2015, five days later than originally planned.


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- Research article
- Open Access
- Open Peer Review
Immunomodulatory activity of polysaccharides isolated from Clerodendrum splendens: Beneficial effects in experimental autoimmune encephalomyelitis
- Koffi Kouakou†1, 2,
- Igor A Schepetkin†1,
- SangMu Jun3,
- Liliya N Kirpotina1,
- Ahoua Yapi4,
- Daria S Khramova5,
- David W Pascual3,
- Yury S Ovodov5,
- Mark A Jutila1 and
- Mark T Quinn1Email author
© Kouakou et al.; licensee BioMed Central Ltd. 2013
- Received: 22 February 2013
- Accepted: 20 June 2013
- Published: 28 June 2013
Extracts of leaves from Clerodendrum have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts.
Polysaccharides from the leaves of Clerodendrum splendens were extracted and fractionated by ion exchange and size-exclusion chromatography. Molecular weight determination, sugar analysis, degree of methyl esterification, and other chemical characterization of the fractions were performed. Immunomodulatory activity of the fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO), cytokine production, and mitogen-activated protein kinase (MAPK) phosphorylation. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice, and severity of EAE was monitored in mice treated with intraperitoneal (i.p.) injections of the most active polysaccharide fraction. Lymph nodes (LN) and spleen were harvested, and levels of cytokines in supernatants from LN cells and splenocytes challenged with myelin oligodendrocyte glycoprotein peptide were determined.
Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high-molecular weight sub-fraction CSP-AU1 (average of 38.5 kDa) induced NO and cytokine [interleukin (IL)-1α, -1β, -6, -10, tumor necrosis factor (TNF; designated previously as TNF-α), and granulocyte macrophage-colony stimulating factor (GM-CSF)] production by human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages. CSP-AU1- induced secretion of TNF was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS, indicating a role for TLR4 signaling. Treatment with CSP-AU1 also induced phosphorylation of a number of MAPKs in human PBMC and activated AP-1/NF-κB. In vivo treatment of mice with CSP-AU1 and CSP-NU1 resulted in increased serum IL-6, IL-10, TNF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α/CCL3, and MIP-1β/CCL4. CSP-AU1 treatment of mice with EAE (50 mg/kg, i.p., daily, 13 days) resulted in significantly reduced disease severity in this experimental model of multiple sclerosis. Levels of IL-13, TNF, interferon (IFN)-γ, IL-17, and GM-CSF were also significantly decreased, whereas transforming growth factor (TGF)-β was increased in LN cells from CSP-AU1-treated EAE mice.
Polysaccharide CSP-AU1 is a potent natural innate immunomodulator with a broad spectrum of agonist activity in vitro and immunosupressive properties after chronic administration in vivo.
- Clerodendrum splendens
- Polysaccharide
- Macrophage
- Cytokine
- Immunomodulation
Natural products comprise one of the most popular sources of complementary and alternative medicines for treating inflammatory and immune disorders [1]. While the mechanisms of action of many plant products remain to be elucidated, further study in this area is essential for the identification of novel therapeutics with immunomodulatory activity [2]. Clerodendrum splendens belongs to the family of Lamiaceae and the genus Clerodendrum, consisting of about 400 herbs, vines, shrubs, and trees of the tropics, many of which are grown as garden plants [3–5]. The plant is a climbing evergreen bush with attractive red flowers produced during the dry seasons of the year and it is very widely distributed in tropical and subtropical regions of the world [3]. Extracts of roots, leaves, and bark from C. splendens are used in traditional medicine to treat malaria, coughs, buboes, venereal infections, including gonorrhea and syphilis, skin diseases, ulcers, rheumatism, asthma, and uterine fibroid [3, 6, 7]. Leaves of this plant have also been used to treat various skin diseases and as wound-healing medicine [4]. For example, the leaves of C. splendens have been formulated into lotion and applied to bruises and sores, and dry powdered leaves have also been applied to blisters caused by burns [4].
Antimicrobial and wound healing properties of chloroform and methanol extracts from C. splendens have been confirmed in recent pharmacological studies [8, 9]. The volatile oil of C. splendens, prepared by extraction of fresh flowers with n-hexane, was found to be active against Staphylococcus aureus and Candida albicans[10]. Antipyretic and anti-inflammatory effects of C. splendens extracts have also been described [11]. C. splendens has been extensively investigated, and a number of chemical constituents, including steroids, terpenoids, flavonoids, volatile constituents, cyanogenic glycoside, alkaloids, tannins, saponins, and phenolic compounds have been isolated [3, 7, 12–14].
Although there are many publications describing the chemical structure and bioactive effects of small molecules isolated from C. splendens, little is known about the immunomodulatory effects of polysaccharides extracted from the leaves of this plant. Since decoction in water is the most common mode of preparation, and the most common modes of administration are oral and local application, we hypothesized that polysaccharides extracted from C. splendens could have immunomodulatory properties and contribute to the therapeutic effects of extracts from this plant. To address this question, we fractionated water-soluble polysaccharides from leaves of C. splendens and evaluated their immunomodulatory activities. We found that the most active Clerodendrum polysaccharide fractions contained type II arabinogalactan and had potent immunomodulatory activity in vitro and in vivo. To investigate the molecular mechanism responsible for the polysaccharide-induced immunomodulatory responses, the roles of signaling pathways including mitogen-activated protein kinase (MAPK) phosphorylation and transcription factors activating protein-1 (AP-1) and nuclear factor κB (NF-κB) were evaluated.
Plant material
Leaves of C. splendens were collected in the Bingerville areas of Cote d'Ivoire. This plant was identified and authenticated by Dr. Aké-Assi, Emeritus Professor of Botany, using voucher specimens deposited at various periods, including voucher specimen #16877, deposited at the National Herbarium of the National Centre of Floristique of the University of Cocody-Abidjan. Parts of plants tested were air-dried for 7–10 days at room temperature away from direct sunlight and powdered under laboratory conditions.
Polysaccharide fractionation
Ground leaves (500 g) were extracted with 3 L boiling distilled H2O for 1 hr, and the aqueous extracts were centrifuged at 2,500 × g for 15 min. A four-fold volume of ethanol was added to each supernatant to precipitate the polysaccharides overnight at 4°C. The precipitates were pelleted by centrifugation, dissolved in distilled H2O, centrifuged at 8,000 × g for 1 hr, and re-precipitated with a four-fold volume of ethanol. The supernatants were re-dissolved in distilled H2O and filtered through a 0.22 μm filter and concentrated in an Amicon concentrator with a 1 kDa PLAC membrane (Millipore, Biillerica, MA) to obtain crude extracts. The crude polysaccharide extracts were further purified using ion-exchange chromatography on a DEAE-cellulose column equilibrated with 0.05 M Tris–HCl buffer (pH 8.0). For each fractionation, the column was eluted with equilibration buffer to obtain the crude neutral polysaccharide fraction. The bound material was eluted with equilibration buffer containing 2 M NaCl. The eluates were concentrated in an Amicon concentrator with a 1 kDa PLAC membrane to obtain the crude neutral (CSP-N) and acidic (CSP-A) polysaccharide fractions. These fractions were further fractionated on Diaion HP-20 absorbent resin column (2.5 × 20 cm). For each fractionation, the column was eluted with distilled H2O, and the eluates were lyophilized to obtain unbound fractions (designated as CSP-NU and CSP-AU). Diaion-bound polysaccharides were eluted with methanol and dried to obtain fractions CSP-NB and CSP-AB.
Fractions CSP-AU and CSP-NU were further sub-fractionated by size-exclusion chromatography on a Sepharose-6B column (2.5×95 cm) eluted with distilled H2O at a flow rate of 21 ml/hr. The carbohydrate elution profile was determined by the phenol-H2SO4 method, modified to a microplate format [15], and absorbance was measured at 488 nm using a SpectraMax Plus microplate reader (Molecular Devices, Palo Alto, CA). The polyphenol elution profile was determined by the Folin-Ciocalteu assay (see below). The relevant fractions were pooled and concentrated. Buffer salts were removed from the polysaccharide samples by repeated (6×) concentration in an Amicon concentrator (1 kDa cut-off PLAC membrane) and dilution with a 10-fold volume of distilled H2O. For analysis of biological activity, the fractions were diluted in Hanks balanced-salt solution without Ca2+ and Mg2+ (HBSS-) to a concentration of 5 mg/ml and filtered through sterile 0.22 μm filters.
Characterization of polysaccharide fractions
Average molecular weights of the polysaccharide fractions were determined by high performance size-exclusion chromatography (HP-SEC) using a Shimadzu Class VP HPC and Shodex OHpak SB-804 HQ column (8 mm × 300 mm) eluted with 50 mM sodium citrate buffer, pH 7.5, containing 0.15 M NaCl and 0.01% NaN3 at a flow rate of 0.3 ml/min. Peaks were detected using a refractive index detector (RID-10A; Shimadzu, Torrance, CA). Average molecular weights of the polysaccharide fractions were estimated by comparison with retention times of pullulan standards P-100, 50, 20, 10, and 5 (Phenomenex, Torrance, CA), which have molecular weights of 112, 47.3, 22.8, 11.8, and 5.9 kDa, respectively. Reproducibility of the retention times was typically >98%.
The presence of arabinogalactan in the samples was detected by single radial gel diffusion in 1% agarose gels containing 100 μg/ml β-glucosyl Yariv reagent, which selectively interacts with and precipitates compounds containing type II arabinogalactan structures [16]. Four μl of polysaccharide samples (10 mg/ml; w/v) were loaded into the wells, and the samples were incubated at 25°C for 24 hr in a humid atmosphere. A positive reaction was indicated by a reddish circle (halo) around the well, and arabic gum (4 mg/ml) (Fluka Chemie GmbH, Germany) served as a positive control.
The total amount of polyphenols in the polysaccharide fractions was determined by the Folin-Ciocalteu assay [17]. Briefly, 250 μL of Folin's phenol reagent was added to the samples dissolved in 500 μL distilled water. After 3 min at room temperature, 1.25 mL of 20% sodium carbonate was added, mixed, and the mixture was allowed to stand for 40 min. The absorbance was measured at 750 nm in a SpectraMax Plus microplate reader. Tannic acid from Rhus semialata (Sigma-Aldrich, St. Louis, MO) was used to generate a standard curve.
For monosaccharide analysis, the polysaccharide fractions were lyophilized and submitted for analysis to the Oklahoma Center for Glycobiology Analytical Core Lab (Oklahoma City, OK). The polysaccharide samples or background blanks were subjected to methanolysis (methanolic 2 M HCl, 16 hr, 80°C), followed by acid hydrolysis (2 M trifluoroacetic acid, 4 hr, 100°C), and the resulting monosaccharide mixtures were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) on a Dionex DX-600 HPAEC system equipped with an ED50 detector (Dionex Corporation, Sunnyvale, CA). The samples were separated on a Dionex CarboPac PA20 column eluted isocratically with 12 mM NaOH at a flow rate of 1 ml/min at 22°C. For analysis of uronic acids, the column was eluted with 10 mM NaOH for 20 min, followed by a gradient of 100 mM NaOH/150 mM sodium acetate (0–100% in 45 min). Background signals were subtracted from all samples, and individual components were quantified based on electrochemical detection relative to known standards.
The content of methoxyl groups was determined using the calibration plot for methanol, as previously described [18]. Photocolorimetry was carried out at 412 nm. The degree of methyl esterification was calculated as the ratio of the molar amount of methanol to the molar amount of uronic acids (galacturonic and glucuronic acids) and is expressed as a molar percentage. M.W., molecular weight.
Determination of endotoxin contamination
Analyses of endotoxin concentration were performed via the kinetic method with Limulus Amebocyte Lysate (LAL) kit (GenScript, Piscataway, NJ), and samples were monitored using a SpectraMax Plus microplate reader.
Cell culture
Murine macrophage J774.A1 cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated, endotoxin-free fetal bovine serum (FBS; Atlanta Biologicals, Atlanta, GA), 100 μg/ml streptomycin, and 100 U/ml penicillin. Cells were grown to confluence in sterile tissue culture flasks and gently detached by scraping. Human monocyte-macrophage MonoMac-6 cells (DSMZ, Germany) were grown in RPMI 1640 supplemented with 10% (v/v) endotoxin-free FBS, 10 μg/ml bovine insulin, 100 μg/ml streptomycin, and 100 U/ml penicillin. Human monocytic THP-1Blue cells obtained from InvivoGen (San Diego, CA) were cultured in RPMI 1640 medium supplemented with 10% (v/v) endotoxin-free FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 100 μg/ml zeocin, and 10 μg/ml blasticidin S. These cells are stably transfected with a secreted embryonic alkaline phosphatase gene that is under the control of a promoter inducible by AP-1/NF-κB.
PBMCs were purified from human blood using dextran sedimentation, followed by Histopaque 1077 gradient separation and hypotonic lysis of erythrocytes. Blood was collected from healthy donors in accordance with a protocol approved by the Institutional Review Board at Montana State University, and informed consent was obtained from all donors for the use of their blood in this study. PBMCs were cultured at 37°C in a humidified atmosphere containing 5% CO2. Cell number and viability were assessed microscopically using trypan blue exclusion.
Analysis of nitric oxide (NO) production
J774.A1 cells were plated at a density of 1.5 × 105 cells/well in a final volume of 200 μl in 96-well flat-bottom tissue culture plates and incubated in medium alone or medium containing various concentrations of polysaccharide fractions or lipopolysaccharide (LPS) from Escherichia coli K-235 (Sigma Chemical Co., St. Louis, MO) as a positive control. Cells were incubated at 37°C in the presence of 5% CO2 for 24 hr, and 100 μl of the cell culture supernatants were removed and analyzed for nitrite using a colorimetric method with NaNO2 as the standard. Briefly, supernatants were mixed with an equal volume of Griess reagent, which was prepared by mixing one part of 0.1% (w/v) N-(1-naphthyl)ethylenediamine with one part of 1% (w/v) sulfanilamide in 5% phosphoric acid. After 20 min, absorbance was measured at 540 nm using a SpectraMax Plus microplate reader.
Determination of cytokine production
J774.A1 cells, human PBMCs, or human MonoMac-6 monocytic cells were incubated for 24 hr in culture medium supplemented with 3% (v/v) endotoxin-free FBS, with or without polysaccharide fractions or LPS as a positive control. Concanavalin A (Con A) from Canavalia ensiformis (Calbiochem, San Diego, CA) was also used as positive control in some experiments. Cells were plated in 96-well plates at a density of 2×105 cells in 100 μL per well and incubated for 24 hr. Enzyme-linked immunosorbent assay (ELISA) kits for human tumor necrosis factor (TNF) and granulocyte macrophage-colony stimulating factor (GM-CSF), or human and mouse interleukin (IL)-6 (all from Biolegend) were used to test cytokine protein levels in the cell supernatants. Where indicated, PBMCs were pretreated with or without TLR4 antagonist LPS-RS (LPS from Rhodobacter sphaeroides; Invivogen, San Diego, CA) for 30 min, followed by treatment with or without polysaccharide sub-fraction CSP-AU1, LPS, or 200 nM of phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, St. Louis, MO) for 24 hr, and TNF levels were evaluated in cell supernatants using an ELISA kit. A human cytokine Multi-Analyte ELISArray™ Kit (SABiosciences Corporation; Frederick, MD) was also utilized to evaluate various cytokines (IL-4, IL-6, IL-8, IL-10, IL-12, IL-17A, IFN-γ, TNF, and GM-CSF) in supernatants of PBMCs.
Analysis of AP-1/NF-κB activation
Activation of AP-1/NF-κB transcription factors was measured using an alkaline phosphatase reporter gene assay in THP-1Blue cells. Polysaccharide fractions or LPS (100 ng/ml) were added, and the cells (2 × 105 cells/well) were incubated for 24 hr. Alkaline phosphatase activity was measured in cell supernatants using QUANTI-Blue mix (InvivoGen). Activation of AP-1/NF-κB is reported as absorbance at 655 nm and compared with samples treated with positive control LPS.
Cytotoxicity assay
Cytotoxicity was analyzed with a CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI), according to the manufacturer's protocol. Following treatment, the cells were allowed to equilibrate to room temperature for 30 min, substrate was added, and the samples were analyzed with a Fluoroscan Ascent FL microplate reader.
Mitogen-activated protein kinase (MAPK) profile
Analysis of the phosphorylation states of all MAPKs was performed using a human phospho-MAPK array kit Proteome Profiler™, which is similar to immunoprecipitation and Western blot analysis (R&D Systems, Minneapolis, MN). Human PBMCs were incubated for 60 min with the selected polysaccharide fractions or negative control (HBSS). The cells were rinsed with HBSS and lysed with the buffer provided. Arrays were incubated overnight at 4°C with lysates obtained from 3 × 106 cells for each sample. The arrays were washed three times with 20 ml of the wash buffer provided and incubated for 2 hr with the provided detection antibody cocktail containing phospho-site-specific MAPK biotinylated antibodies. The wash steps were repeated, after which the arrays were exposed to chemiluminescent reagents, and the signal was captured with an Alpha Innotech FluorChem FC2 imaging system.
EAE induction and treatment
Female 6–8 week old C57BL/6 mice (Frederick Cancer Research Facility, National Cancer Institute, Frederick, MD) were used throughout the study of experimental autoimmune encephalomyelitis (EAE). Mice were maintained under pathogen-free conditions in individual ventilated cages and were fed sterile food and water ad libitum. All animal experiments were performed in accordance with National Institutes of Health guidelines and approved by the Montana State University Institutional Animal Care and Use Committee.
For EAE induction, mice were challenged subcutaneously in the flank with 200 μg of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (Biosynthesis, Inc., Lewisville, TX) in 100 μl of incomplete Freund's adjuvant (IFA) (Sigma–Aldrich, St. Louis, MO) containing 400 μg killed Mycobacterium tuberculosis (Difco Laboratories, Detroit, MI) on day 0, as described previously [19]. On days 0 and 2, mice were treated by intraperitoneal (i.p.) injection with 200 ng of Bordetella pertussis toxin (List Biological Laboratories, Campbell, CA). Starting on day 4, mice were treated daily i.p. with 50 mg/kg of CSP-AU1 in 100 μl of phosphate buffer saline (PBS) or with 100 μl of PBS (control). Mice were monitored and scored daily by a technician blinded to treatments for clinical signs of EAE using the following scoring system: 0, normal; 1, a limp tail; 2, hind limb weakness; 3, hind limb paresis; 4, quadriplegia; 5, moribund state.
Cytokine analysis in cultures of lymph node (LN) and splenic cells
To assess the cytokines induced, spleen and head and neck LNs were harvested at day 17 of the disease, and total mononuclear cells (5 × 106 cells/ml) were cultured with 10 μg/ml of MOG35-55 peptide for 4 days in a complete medium consisting of RPMI 1640 medium with the following supplements (Invitrogen-Life Technologies): 1 mM sodium pyruvate, 1 mM nonessential amino acids, penicillin/streptomycin (10 U/ml), and 10% FBS. TNF, GM-CSF, IL-13, IL-17, IFN-γ, and TGF-β were measured from culture supernatants, as described previously [19].
Statistical analysis
Mann–Whitney U test was used for statistical analysis of clinical scores in the EAE model. One-way ANOVA and Student t test were performed to analyze NO and alkaline phosphatase production, MAP kinase phosphorylation, and cytokine-specific ELISA data. Results were considered statistically significant if the p value was <0.05. The statistical analysis was performed using Prism 5 (GraphPad Software, San Diego, CA).
Ethnopharmacological information
Traditional Uses of Clerodendrum splendens in Côte d'Ivoire a
Specific parts of the plant
Preparation and administration
The leaves were boiled in water, and the decoction was consumed; part of the decoction was mixed in shea butter and applied locally to hemorrhoids
Sinus disease
A vapor bath was inhaled once a day for 3 days
Menstrual troubles
The leaves were boiled in water, and the decoction was consumed 2 times a day for 2 months
The leaves were boiled in water, and the decoction was consumed 3 times a day for 3 days
Healing scars
The pounded fresh leaves were applied as an ointment or rubbed on the scars
Febrifuge agent
The leaves were boiled in water, and the decoction was consumed 2 times a day for 1 week
Vermifuge agent
The leaves were boiled in water, and the decoction was consumed 2 times a day for 1 week
Scrofulous infection
Leaves and bark
The pounded or crashed fresh leaves and barks were applied or rubbed on skin infections until cured.
Preparation and partial characterization of Clerodendrum polysaccharide fractions
Biochemical properties of C. splendens polysaccharide fractions
Fractionation step & Fraction name
M.W. (kDa)
Type II Arabino-galactan
Polyphenol content (%)
Methoxyl content (Mol %)
DM (%)a
DEAE cellulose
Diaion HP-20
Sepharose 6B
9.3 ± 0.29
1.0 ± 0.04
8.8 ± 0.25
0.9 ± 0.04
7.6 ± 0.22
0.9 ± 0.04
8.1 ± 0.14
1.3 ± 0.03
0.9 ± 0.03
1.1 ± 0.01
0.5 ± 0.01
1.0 ± 0.02
0.2 ± 0.02
6.5 ± 0.14
Analytical HP-SEC of the individual fractions showed that each fraction was represented by a single and generally symmetrical peak on the chromatogram. Based on calibration curves derived from analysis of pullulan standards, we determined average molecular weights of the polysaccharide fractions. Sequential fractionation of C. splendens extract by DEAE-cellulose and Diaion HP-20 gave parent fractions with relatively low molecular weights ranging from 12.0 kDa (fraction CSP-NB) to 24.5 kDa (fraction CSP-AU). Polysaccharides that bound to the Diaion resin had lower molecular weights in comparison with unbound polysaccharides (13.2 vs. 24.5 kDa, and 12.0 vs. 21.7 kDa, respectively). Sub-fractions CSP-AU1 and CSP-NU1, which were obtained by size-exclusion chromatography on Sepharose 6B, had relatively high molecular weights of 38.5 and 60.6 kDa, respectively (Table 2). The samples were also tested for possible endotoxin contamination using the LAL assay. All fractions contained less than 0.2 ng endotoxin/mg of polysaccharide, which is considered to be insignificant for various bioactive products [20, 21].
Analysis of polyphenols showed that polyphenol content was minimal (<1%) in neutral polysacchride fractions from C. splendens (CSP-NB, CSP-NU, and CSP-NU1), whereas all acidic fractions and the low-molecular weight neutral sub-fraction CSP-NU2 contained a relatively high polyphenol content (from 6.5 to 9.3%). Thus, the polyphenol component of CSP-NU is enriched in the low-molecular weight sub-fraction CSP-NU2, as compared to the high-molecular weight sub-fraction CSP-NU1 (Table 2).
Analysis of the polysaccharide fractions using the Yariv test showed that Diaion-unbound fraction CSP-NU and sub-fractions CSP-AU1 and CSP-NU1 contained type II arabinogalactan, whereas fractions that bound to this resin (CSP-AB and CSP-NB), unbound fraction CSP-AU, and low molecular weight sub-fractions CSP-AU2 and CSP-NU2 all tested negative for the presence of type II arabinogalactan (Table 2). Thus, it appears that the relative amount of type II arabinogalactan in parent fraction CSP-AU was too dilute to be detected by the Yariv reagent and that it became concentrated enough to be detected in the high-molecular weight sub-fraction CSP-AU1 after chromatography.
Sugar composition of polysaccharide fractions isolated from C. splendens a
Mol %
Galacturonic Acid
Glucuronic Acid
The acidic fractions were enriched with polyphenols (up 9.5%), whereas the neutral, relatively high molecular weight fractions (CSP-NB, CSP-NU, and CSP-NU1) had minimal amounts of polyphenols (<1%).
Effects of the Clerodendrum polysaccharide fractions on macrophage NO production
Effects of Clerodendrum polysaccharide fractions on cytokine production in cell cultures
To determine if polysaccharides from different plant sources could stimulate similar levels of GM-CSF production, we evaluated the effect of polysaccharide fractions from several different plants. These previously characterized polysaccharide fractions included fraction A-I from Artemisia tripartite[23], fraction T-I from Tanacetum vulgarie[24], and fraction C-I from Opuntia polyacanta[25]. Interestingly, none of these polysaccharide fractions induced GM-CSF production by human PBMCs (data not shown). On the other hand, our previous studies showed that these polysaccharides could potently activate production of various other cytokines, such as TNF and IL-6, in phagocytes [23–25].
Effect of the Clerodendrum polysaccharide fractions on AP-1/NF-κB activity
Effect of Clerodendrum polysaccharide fractions on cell viability
Although our functional assays suggested that the polysaccharides extracted from C. splendens were relatively non-toxic, we evaluated their potential cytotoxic effects to determine if the results might be influenced by background toxicity. Using a cytotoxicity assay, we determined that none of the polysaccharides significantly affected viability of human PBMCs, THP-1Blue, or MonoMac-6 cells over the concentration range of 10–250 μg/ml polysaccharides, verifying that these polysaccharides were not toxic to these cells (data not shown).
Effect of CSP-AU1 on TLR4-dependent pathway and phosphorylation of MAPK family
Effect of CSP-AU1 on cytokine concentration in serum
Because CSP-AU1 was the most abundant fraction among the isolated Clerodendrum polysaccharide fractions (its yield was ~5 times larger than yield of CSP-NU1; data not shown), we used CSP-AU1 for subsequent in vivo studies. After i.p. administration of CSP-AU1 (single injection in dose of 250 mg/kg), a rapid rise in serum TNF concentration was observed, peaking at ~2 hr (Figure 10, Inset A). When mice were treated with different doses of CSP-AU1, a dose–dependent increase in serum TNF concentration was found at 2 hr (Figure 10, Inset B).
Effect of CSP-AU1 on EAE
Historically, C. splendens has been used in African folk medicine for treating a number of diseases and medical conditions. However, despite the widespread traditional use of C. splendens in tropical countries, little is known regarding the active components responsible for its therapeutic properties. Previously, antipyretic and anti-inflammatory effects of methanolic extracts from C. splendens have been described [11], whereas the immunomodulatory activities of high-molecular weight polysaccharide fractions from this plant are unknown. In the present work, we provide evidence demonstrating that C. splendens polysaccharides have potent immunomodulatory properties in vitro and in vivo and suggest that this may contribute, at least in part, to the therapeutic potential of C. splendens-derived medicines.
Our ethnobotanical survey and literature review indicate that a decoction in water of the leaves is the mode of extraction most commonly used to prepare remedies from C. splendens in African folk medicine. Based on this observation, we isolated eight polysaccharide fractions from the hot-water extract of C. splendens leaves and provided initial structural and pharmacological characterization. Average molecular weights of the fractions were in the range of 5.5 to 60.6 kDa. We found that the high-molecular weight sub-fractions CSP-AU1 (mol. weight 38.5 kDa) and CSP-NU1 (mol. weight 60.6 kDa) were quite potent in biological tests, whereas the low-molecular weight sub-fraction CSP-AU2 was inactive or minimally active in the same assays. Indeed, sub-fractions CSP-AU1 and CSP-NU1 contained potent immunomodulatory activity, as demonstrated by their ability to induce NO production in macrophage J774.A1 cells and cytokines by monocyte/macrophages and human PBMCs. These cytokines included of IL-1α, IL-1β, IL-6, TNF, IL-10, and GM-CSF. In addition, we found that CSP-AU1 and CSP-NU1 induced increased levels of certain serum cytokines in vivo, including the anti-inflammatory cytokine IL-10 and pro-inflammatory cytokines IL-6, TNF, MCP-1, MIP-1α, and MIP-1β. In contrast, the low-molecular weight sub-fraction CSP-AU2 was minimally active in vivo (Figure 10).
A common feature of plant polysaccharides that modulate macrophage functions may be higher molecular weight, as we found previously that immunomodulatory activity positively correlated with increased molecular weight of various plant polysaccharides [23–25, 36]. Although the immunologic effects of Clerodendrum polysaccharides following oral administration were not studied in the present work, numerous dietary polysaccharides do appear to elicit diverse immunomodulatory effects in various animal tissues, including the blood, gastrointestinal tract and spleen [37–42]. Note, however, that our recent studies suggest that nasal application of some plant polysaccharides may be even more effective than oral administration for stimulation of innate immunity [43]. In any case, further studies are now in progress to determine the therapeutic potential of Clerodendrum polysaccharide sub-fraction CSP-AU1 and its structural features conferring biological activity.
Active and inactive polysaccharide fractions contained polyphenols, which were present over a broad range. Polyphenols have been previously noted in polysaccharide fractions of various plants (e.g. [42, 44]), and it has been reported that polysaccharides are able to bind to polyphenols by intermolecular interactions [45], changing the molecular conformation of carbohydrates. Therefore, the presence of polyphenols in polysaccharide fractions must be considered. However, activities of the fractions did not correlate with polyphenol content in our biological studies. Indeed, one of the most active fractions (CSP-AU1) contained a relatively high level of polyphenols (7.6%), whereas other polyphenol-enriched fractions (CSP-AB, 9.3% polyphenols; CSP-AU2, 8.8% polyphenols) were inactive or had low activity in most biological assays. Finally, the highly active sub-fraction CSP-NU1 had the lowest polyphenol content (0.2%).
We found that both of the Diaion-bound fractions, CSP-AB and CSP-NB, generally were inactive or had low biological activity. Sugar analysis showed that these fractions contained much less galacturonic acid but more neutral sugars (glucose and mannose) than the Diaion-unbound fractions CSP-AU and CSP-AU. Moreover, analysis of the fractions using the Yariv reagent demonstrated that both CSP-AB and CSP-NB tested negative for the presence of type II arabinogalactan. Conversely, the most potent immunomodulatory properties were associated with the fractions that exhibited a positive reaction for type II arabinogalactan. Thus, native type II arabinogalactans could be the main structures responsible for immunomodulatory properties of the C. splendens- derived polysaccharides, although presence of type I arabinogalactan in the fractions could not be excluded. In any case, it appears that Diaion HP-20 resin could be used to enrich bioactive C. splendens polysaccharides by removing inactive material that bound to the resin [46]. Note, we also evaluated if the inactive Diaion-bound material might possibly have inhibitory activity by assessing its ability to inhibit LPS-induced cytokine production but found that this material was inactive, having no effect on LPS induced cytokine production (data not shown). Future chemical and physical analysis of these fractions will be necessary for characterization of Diaion-bound and unbound polysaccharide fractions and definition of the polysaccharide structures responsible for immunomodulatory activities.
Previously it was proposed that selective immunostimulatory and anti-inflammatory properties of citrus polysaccharides could be attributed to different degrees of methyl esterification [47–49]. In the present study, we found that the polysaccharide fraction with the most potent immunomodulatory activity (CSP-AU1) had a low degree of methyl esterification of uronic acid residues. Thus, free carboxylic groups and associated electrical charge may enhance the bioavailability and binding potential of CSP-AU1 polysaccharides to different cell receptors.
Clerodendrum polysaccharides and recently characterized polysaccharides from Alchornea cordofolia[50] seem to be unique, as compared to LPS and many other plant polysaccharides, in their ability to induce GM-CSF production by MonoMac-6 cells and PBMCs. Indeed, previously characterized polysaccharide fractions A-I from A. tripartite[23], T-I from T. vulgare[24], C-I from O. polyacanta[25], and even high concentrations of LPS (up to 10 μg/ml) were unable to stimulate GM-CSF production by human PBMCs, whereas CSP-AU, CSP-AU1, CSP-NU1, and CSP-NU2 all significantly induced production of this cytokine by MonoMac-6 cells and PBMCs.
Because Con A stimulated GM-CSF production by human PBMCs (Figure 5), we considered whether the GM-CSF-inducing activity of Clerodendrum polysaccharide fractions might correlate with hemagglutinating activity but found that the Clerodendrum polysaccharides, as well as polysaccharides isolated from A. cordofolia[50], had no hemagglutinating activity (up to 1 mg/ml). On the other hand, we found that polysaccharide CP-AU isolated from Combretum racemosum had relatively high hemagglutinating activity but minimally stimulated GM-CSF production by human PBMCs and only at the highest tested concentration (0.5 mg/ml) [51]. Thus, plant polysaccharide structures responsible for GM-CSF stimulation and hemagglutination appear to be distinct from each other.
Differences in intracellular signaling pathways were observed between Clerodendrum polysaccharides and previously reported immunomodulatory botanical polysaccharides. For example, polysaccharides from algae Capsosiphon fulvescens induced ERK1/2, but not p38 [52], polysaccharide SP1 from Caulerpa lentillifera increased the phosphorylation of p38 [53], whereas polysaccharide AP-1 from Platycodon grandiflorum activated phosphorylation of all three MAPKs [46]. Here, we demonstrated that CSP-AU1 induces phosphorylation of Akt2, Akt3, GSK-3α/β, p38β/γ/δ, p70S6K1, RSK2, and mTOR, but not ERK1/2 in human peripheral blood mononuclear cells. Thus, it appears that various immunomodulatory polysaccharides exert their biological activities through different modes of action.
Plant polysaccharides can modulate leukocyte activity via different receptors, including complement receptor 3 (CR3), scavenger receptor, dectin-1, mannose receptor, galectin 3, and TLR4 (reviewed in [22]). For example, polysaccharides ASP and G1-4A, which were isolated from Acanthopanax senticosus and Tinospora cordifolia, respectively, activated macrophages and B cells by interacting with TLR4 [54, 55]. TLR4 was also found to be one of the cellular receptors mediating TNF secretion induced by ZPF1, polysaccharide isolated from Dioscorea batatas[56]. Likewise, polysaccharide CWSP from Chlorella pyrenoidosa induced cytokine secretion in macrophages via TLR4-mediated signaling pathways [57]. In the present work, we found that the Clerodendrum polysaccharide sub-fraction CSP-AU1 induced secretion of TNF in human PBMCs mainly via TLR4, but binding affinity for this TLR agonist was much lower than that of E.coli-derived LPS. Indeed, a 10-fold higher concentration of LPS-RS over LPS was necessary to completely inhibit LPS-induced TNF production in human PBMC. In comparison, a much lower relative concentration of LPS-RS was needed to inhibit CSP-AU1 activity. Thus, the pharmacological properties of such low-affinity TLR4/MD-2 ligands may be different than those of bacterial LPS. Indeed, although the profiles of pro-inflammatory mediators (NO and most of the cytokines studied here) for in vitro stimulatory activity by bacterial LPS and CSP-AU were fairly similar (Figures 1, 2, 3, and 6), the profile was quite different for production of GM-CSF (Figure 4). Because several plant polysaccharides were recently found having therapeutic effects in different models of autoimmune diseases, such as arthritis [31, 34], lupus erythematosus-like syndrome [30, 33], autoallergic mouse model of Sjogren's syndrome [58], asthma [59], and EAE [32], we suggest that such natural low-affinity TLR4 agonists may have clinically beneficial effects on autoimmune diseases by their induction of immunosuppression during chronic administration [29].
Although medical application of hot-water extracts from C. splendens for the treatment of autoimmune diseases has not been reported to date, in the present work we studied therapeutic effects of CSP-AU1 in a mouse model of EAE. We found that chronic i.p. administration of CSP-AU1 induced clinically beneficial effects in EAE, supporting the immunomodulatory properties of Clerodendrum polysaccharides in vivo. Our data showed that CSP-AU1 treatment of mice with EAE delayed disease onset and reduced the clinical severity of EAE. Only one plant polysaccharide, ginsan from Panax ginseng, has been tested to date in this animal model of multiple sclerosis [32]. Similar to our results, these authors observed reduced EAE severity. The polysaccharide ginsan also has been reported to exhibit an anti-allergic reaction in an ovalbumin-induced murine asthma model [60]. Although various plant polysaccharides have been reported to have therapeutic activity in some other models of autoimmune diseases [30, 31, 33, 34, 58], mechanisms of immune suppression by these high-molecular weight molecules in autoimmune disease remain unresolved. Because different plant polysaccharides (e.g., [61]) and CSP-AU1 activate cells, presumably via TLR4, it is possible that chronic stimulation of the innate immune system via TLR4 could lead to the induction of immunosuppression [29]. Indeed, Vaknin et al.[29] demonstrated that repetitive administration of LPS was sufficient to induce bystander T-cell immunosuppression. Moreover, LPS-stimulated dendritic cells induced tolerance against established collagen-induced arthritis [62].
In attempts to determine possible immunomodulatory mechanisms of EAE delay by CSP-AU1, we evaluated cytokine production by MOG-peptide reactivated cells in culture, after isolation from CSP-AU1- and PBS-treated EAE mice, and found that the beneficial effect of CSP-AU1 administration was accompanied by reduced IFN-γ, IL-13, IL-17, TNF, GM-CSF and increased TGF-β production. Indeed, a review of the literature regarding immune mechanisms of multiple sclerosis and EAE showed that these cytokines play an important role in progression of EAE disease. In EAE, increased number of IL-17- and IFN-γ-producing cells in the spinal cord resulted from peripheral expansion of these cells after immunization with myelin-derived antigen (reviewed in [63]). During the EAE disease effector phase, GM-CSF was reported to sustain neuroinflammation via myeloid cells that infiltrated the central nervous system [64]. TNF produced by myeloid cells accelerated the onset of EAE disease by regulation of chemokine expression in the CNS, driving the recruitment of inflammatory cells into the target organ [65]. On the other hand, we recently found that regulatory T cells confer protection against EAE via TGF-β [19]. Thus, the higher level of TGF-β production by splenic and lymph node lymphocytes from CSP-AU1-treated EAE mice could be related to the beneficial therapeutic effects of this polysaccharide fraction.
In this study, we elucidate the molecular mechanism of monocyte/macrophage activation by polysaccharides isolated from leaves of C. splendens. We found that Diaion HP-20-unbound polysaccharides from C. splendens were not cytotoxic and activated PBMCs and monocyte/macrophages, resulting in stimulation of NO and cytokine production. Our data demonstrated that Clerodendrum polysaccharide sub-fraction CSP-AU1, which was one of the most potent activators of cytokine production, activated PBMCs and monocyte/macrophages via TLR4 and signaling cascades involving Akt2/3, GSK-3α/β, p38β, p38δ, p38γ, p70S6K1, RSK2, mTOR, and AP-1/NK-κB. We also found that chronic i.p. administration of CSP-AU1 induced clinically beneficial effects in EAE, an experimental model of multiple sclerosis, supporting the immunomodulatory properties of Clerodendrum polysaccharides in vivo. Thus, our data provide a molecular basis to explain at least part of the beneficial therapeutic effects for hot-water extracts of C. splendens, and suggest that the polysaccharides from C. splendens have potential for immune modulation.
We would like to thank Drs. Christopher West and Christa L. Feasley, Oklahoma Center for Medical Glycobiology Core Laboratory, Oklahoma City, OK for expert monosaccharide analysis. This work was supported in part by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number GM103500, National Institutes of Health grant AT04986, United States Department of State Fulbright Visiting Scholar Program (Grant ID 68110029), an equipment grant from the M. J. Murdock Charitable Trust, and the Montana State University Agricultural Experimental Station.
Authors' Affiliations
- Venkatesha SH, Rajaiah R, Berman BM, Moudgil KD: Immunomodulation of Autoimmune Arthritis by Herbal CAM. Evid Based Complement Alternat Med. 2011, 2011: 986797-View ArticlePubMedGoogle Scholar
- Huang CF, Lin SS, Liao PH, Young SC, Yang CC: The immunopharmaceutical effects and mechanisms of herb medicine. Cell Mol Immunol. 2008, 5 (1): 23-31. 10.1038/cmi.2008.3.View ArticlePubMedPubMed CentralGoogle Scholar
- Shrivastava N, Patel T: Clerodendrum and healthcare: an overview. Medicinal Aromatic Plant Sci Biotech. 2007, 1: 140-150.Google Scholar
- Irvine FR: Woody Plants of Ghana. 1961, London: Oxford University Press, 750-754. 1Google Scholar
- Steane DA, Scotland RW, Mabberley DJ, Olmstead RG: Molecular systematics of Clerodendrum (Lamiaceae): its sequences and total evidence. Am J Bot. 1999, 86 (1): 98-107. 10.2307/2656958.View ArticlePubMedGoogle Scholar
- Okwu DE, Iroabuchi F: Isolation of an antioxidant flavanone diglycoside from the Nigeria medicinal plant Clerodendron splendens, a. Cheval. Int J Chem Sci. 2008, 6: 631-636.Google Scholar
- Okwu DE, Iroabuchi F: Phytochemical composition and biological activities of Uvaria chamae and Clerodendoron splendens. E-Journal Chem. 2009, 6: 553-560. 10.1155/2009/190346.View ArticleGoogle Scholar
- Gbedema SY, Emelia K, Francis A, Kofi A, Eric W: Wound healing properties and kill kinetics of Clerodendron splendens G. Don, a Ghanaian wound healing plant. Pharmacognosy Res. 2010, 2 (2): 63-68. 10.4103/0974-8490.62948.View ArticlePubMedPubMed CentralGoogle Scholar
- Abouzid SF, Wahba HM, Elshamy A, Cos P, Maes L, Apers S, Pieters L, Shahat AA: Antimicrobial activity of some Clerodendrum species from Egypt. Nat Prod Res. 2013, 27 (11): 1032-1036. 10.1080/14786419.2012.686905.View ArticlePubMedGoogle Scholar
- el-Deeb KS: The volatile constituents in the absolute of Clerodendron splendens G. Don oil. Bull Fac Pharm Cairo Univ. 2003, 41: 259-263.Google Scholar
- Shehata AH, Yousif MF, Soliman GA: Phytochemical and pharmacological investigations of Clerodendron splendens G. Don growing in Egypt. Egypt J Biomed Sci. 2001, 7: 145-163.Google Scholar
- Pinto WJ, Nes WR: 24β-Ethylsterols, n-alkanes and n-alkanols of Clerodendron splendens. Phytochem Rev. 1985, 24: 1095-1097. 10.1016/S0031-9422(00)83192-1.View ArticleGoogle Scholar
- Yang H, Jiang B, Hou AJ, Lin ZW, Sun HD: Colebroside A, a new diglucoside of fatty acid ester of glycerin from Clerodendrum colebrookianum. J Asian Nat Prod Res. 2000, 2 (3): 177-185. 10.1080/10286020008039909.View ArticlePubMedGoogle Scholar
- Okwu DE, Uchenna NF: Exotic multifaceted medicinal plants of drugs and pharmaceutical industries. Afr J Biotechnol. 2009, 8: 7271-7282.Google Scholar
- Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S, Lee YC: Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal Biochem. 2005, 339 (1): 69-72. 10.1016/j.ab.2004.12.001.View ArticlePubMedGoogle Scholar
- Yariv J, Rapport MM, Graf L: The interaction of glycosides and saccharides with antibody to the corresponding phenylazo glycosides. Biochem J. 1962, 85: 383-388.View ArticlePubMedPubMed CentralGoogle Scholar
- Singleton VL, Rossi JA: Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Amer J Enol Viticult. 1965, 16: 144-158.Google Scholar
- Wood PJ, Siddiqui IR: Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity. Anal Biochem. 1971, 39 (2): 418-428. 10.1016/0003-2697(71)90432-5.View ArticlePubMedGoogle Scholar
- Jun S, Ochoa-Reparaz J, Zlotkowska D, Hoyt T, Pascual DW: Bystander-mediated stimulation of proteolipid protein-specific regulatory T (Treg) cells confers protection against experimental autoimmune encephalomyelitis (EAE) via TGF-β. J Neuroimmunol. 2012, 245 (1–2): 39-47.View ArticlePubMedPubMed CentralGoogle Scholar
- Catchpole B, Hamblin AS, Staines NA: Autologous mixed lymphocyte responses in experimentally-induced arthritis of the Lewis rat. Autoimmunity. 2002, 35 (2): 111-117. 10.1080/08916930290016619.View ArticlePubMedGoogle Scholar
- Brito LA, Singh M: Acceptable levels of endotoxin in vaccine formulations during preclinical research. J Pharm Sci. 2011, 100 (1): 34-37. 10.1002/jps.22267.View ArticlePubMedGoogle Scholar
- Schepetkin IA, Quinn MT: Botanical polysaccharides: macrophage immunomodulation and therapeutic potential. Int Immunopharmacol. 2006, 6 (3): 317-333. 10.1016/j.intimp.2005.10.005.View ArticlePubMedGoogle Scholar
- Xie G, Schepetkin IA, Siemsen DW, Kirpotina LN, Wiley JA, Quinn MT: Fractionation and characterization of biologically-active polysaccharides from Artemisia tripartita. Phytochemistry. 2008, 69 (6): 1359-1371. 10.1016/j.phytochem.2008.01.009.View ArticlePubMedPubMed CentralGoogle Scholar
- Xie G, Schepetkin IA, Quinn MT: Immunomodulatory activity of acidic polysaccharides isolated from Tanacetum vulgare L. Int Immunopharmacol. 2007, 7 (13): 1639-1650. 10.1016/j.intimp.2007.08.013.View ArticlePubMedPubMed CentralGoogle Scholar
- Schepetkin IA, Xie G, Kirpotina LN, Klein RA, Jutila MA, Quinn MT: Macrophage immunomodulatory activity of polysaccharides isolated from Opuntia polyacantha. Int Immunopharmacol. 2008, 8 (10): 1455-1466. 10.1016/j.intimp.2008.06.003.View ArticlePubMedPubMed CentralGoogle Scholar
- Kikkert R, de Groot ER, Aarden LA: Cytokine induction by pyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants. J Immunol Methods. 2008, 336 (1): 45-55. 10.1016/j.jim.2008.03.010.View ArticlePubMedGoogle Scholar
- Coats SR, Pham TT, Bainbridge BW, Reife RA, Darveau RP: MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex. J Immunol. 2005, 175 (7): 4490-4498.View ArticlePubMedGoogle Scholar
- Guha M, Mackman N: LPS induction of gene expression in human monocytes. Cell Signal. 2001, 13 (2): 85-94. 10.1016/S0898-6568(00)00149-2.View ArticlePubMedGoogle Scholar
- Vaknin I, Blinder L, Wang L, Gazit R, Shapira E, Genina O, Pines M, Pikarsky E, Baniyash M: A common pathway mediated through Toll-like receptors leads to T- and natural killer-cell immunosuppression. Blood. 2008, 111 (3): 1437-1447.View ArticlePubMedGoogle Scholar
- Wang Z, Li H, Xu H, Yue XL, Cheng XQ, Hou WJ, Zhang YY, Chen DF: Beneficial effect of Bupleurum polysaccharides on autoimmune disease induced by Campylobacter jejuni in BALB/c mice. J Ethnopharmacol. 2009, 124 (3): 481-487. 10.1016/j.jep.2009.05.013.View ArticlePubMedGoogle Scholar
- Jiang JB, Qiu JD, Yang LH, He JP, Smith GW, Li HQ: Therapeutic effects of astragalus polysaccharides on inflammation and synovial apoptosis in rats with adjuvant-induced arthritis. Int J Rheum Dis. 2010, 13 (4): 396-405. 10.1111/j.1756-185X.2010.01555.x.View ArticlePubMedGoogle Scholar
- Hwang I, Ahn G, Park E, Ha D, Song JY, Jee Y: An acidic polysaccharide of Panax ginseng ameliorates experimental autoimmune encephalomyelitis and induces regulatory T cells. Immunol Lett. 2011, 138 (2): 169-178. 10.1016/j.imlet.2011.04.005.View ArticlePubMedGoogle Scholar
- Jiang YW, Li H, Zhang YY, Li W, Jiang YF, Ou YY, Chen DF: Beneficial effect of Bupleurum polysaccharides on autoimmune-prone MRL-lpr mice. Clin Dev Immunol. 2012, 2012: 842928-View ArticlePubMedPubMed CentralGoogle Scholar
- Suszko A, Obminska-Mrukowicz B: Influence of polysaccharide fractions isolated from Caltha palustris L. on the cellular immune response in collagen-induced arthritis (CIA) in mice. A comparison with methotrexate. J Ethnopharmacol. 2013, 145 (1): 109-117. 10.1016/j.jep.2012.10.038.View ArticlePubMedGoogle Scholar
- Batoulis H, Addicks K, Kuerten S: Emerging concepts in autoimmune encephalomyelitis beyond the CD4/T(H)1 paradigm. Ann Anat. 2010, 192 (4): 179-193. 10.1016/j.aanat.2010.06.006.View ArticlePubMedGoogle Scholar
- Schepetkin IA, Faulkner CL, Nelson-Overton LK, Wiley JA, Quinn MT: Macrophage immunomodulatory activity of polysaccharides isolated from Juniperus scopolorum. Int Immunopharmacol. 2005, 5 (13–14): 1783-1799.View ArticlePubMedGoogle Scholar
- Popov SV, Golovchenko VV, Ovodova RG, Smirnov VV, Khramova DS, Popova GY, Ovodov YS: Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L. Vaccine. 2006, 24 (26): 5413-5419. 10.1016/j.vaccine.2006.03.076.View ArticlePubMedGoogle Scholar
- Popov SV, Vinter VG, Patova OA, Markov PA, Nikitina IR, Ovodova RG, Popova GY, Shashkov AS, Ovodov YS: Chemical characterization and anti-inflammatory effect of rauvolfian, a pectic polysaccharide of Rauvolfia callus. Biochemistry (Mosc). 2007, 72 (7): 778-784. 10.1134/S0006297907070139.View ArticleGoogle Scholar
- Takeuchi A, Kamiryou Y, Yamada H, Eto M, Shibata K, Haruna K, Naito S, Yoshikai Y: Oral administration of xanthan gum enhances antitumor activity through Toll-like receptor 4. Int Immunopharmacol. 2009, 9 (13–14): 1562-1567.View ArticlePubMedGoogle Scholar
- Khramova DS, Popov SV, Golovchenko VV, Vityazev FV, Paderin NM, Ovodov YS: Abrogation of the oral tolerance to ovalbumin in mice by citrus pectin. Nutrition. 2009, 25 (2): 226-232. 10.1016/j.nut.2008.08.004.View ArticlePubMedGoogle Scholar
- Ramberg JE, Nelson ED, Sinnott RA: Immunomodulatory dietary polysaccharides: a systematic review of the literature. Nutrition. 2010, 9: 54-10.1186/1475-2891-9-54.View ArticleGoogle Scholar
- Smiderle FR, Ruthes AC, Van AJ, Chanput W, Iacomini M, Wichers HJ, Van Griensven LJ: Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells. BMC Complement Altern Med. 2011, 11: 58-10.1186/1472-6882-11-58.View ArticlePubMedPubMed CentralGoogle Scholar
- Skyberg JA, Rollins MF, Holderness JS, Marlenee NL, Schepetkin IA, Goodyear A, Dow SW, Jutila MA, Pascual DW: Nasal Acai polysaccharides potentiate innate immunity to protect against pulmonary Francisella tularensis and Burkholderia pseudomallei Infections. PLoS Pathog. 2012, 8 (3): e1002587-10.1371/journal.ppat.1002587.View ArticlePubMedPubMed CentralGoogle Scholar
- Holderness J, Schepetkin IA, Freedman B, Kirpotina LN, Quinn MT, Hedges JF, Jutila MA: Polysaccharides isolated from Acai fruit induce innate immune responses. PLoS One. 2011, 6 (2): e17301-10.1371/journal.pone.0017301.View ArticlePubMedPubMed CentralGoogle Scholar
- Renard CM, Baron A, Guyot S, Drilleau JF: Interactions between apple cell walls and native apple polyphenols: quantification and some consequences. Int JBiol Macromol. 2001, 29 (2): 115-125. 10.1016/S0141-8130(01)00155-6.View ArticleGoogle Scholar
- Yoon YD, Kang JS, Han SB, Park SK, Lee HS, Kang JS, Kim HM: Activation of mitogen-activated protein kinases and AP-1 by polysaccharide isolated from the radix of Platycodon grandiflorum in RAW 264.7 cells. Int Immunopharmacol. 2004, 4 (12): 1477-1487. 10.1016/j.intimp.2004.06.012.View ArticlePubMedGoogle Scholar
- Salman H, Bergman M, Djaldetti M, Orlin J, Bessler H: Citrus pectin affects cytokine production by human peripheral blood mononuclear cells. Biomed Pharmacother. 2008, 62 (9): 579-582. 10.1016/j.biopha.2008.07.058.View ArticlePubMedGoogle Scholar
- Ramachandran C, Wilk BJ, Hotchkiss A, Chau H, Eliaz I, Melnick SJ: Activation of human T-helper/inducer cell, T-cytotoxic cell, B-cell, and natural killer (NK)-cells and induction of natural killer cell activity against K562 chronic myeloid leukemia cells with modified citrus pectin. BMC Complement Altern Med. 2011, 11: 59-10.1186/1472-6882-11-59.View ArticlePubMedPubMed CentralGoogle Scholar
- Popov SV, Markov PA, Popova GY, Nikitina IR, Efimova L, Ovodov YS: Anti-inflammatory activity of low and high methoxylated citrus pectins. Biomed Prevent Nutr. 2013, 3: 59-63. 10.1016/j.bionut.2012.10.008.View ArticleGoogle Scholar
- Kouakou K, Schepetkin IA, Yapi A, Kirpotina LN, Jutila MA, Quinn MT: Immunomodulatory activity of polysaccharides isolated from Alchornea cordifolia. J Ethnopharmacol. 2013, 146 (1): 232-242. 10.1016/j.jep.2012.12.037.View ArticlePubMedPubMed CentralGoogle Scholar
- Schepetkin IA, Kouakou K, Yapi A, Kirpotina LN, Jutila MA, Quinn MT: Immunomodulatory and hemagglutinating activities of acidic polysaccharides isolated from Combretum racemosum. Int Immunopharmacol. 2013, 15: 628-637. 10.1016/j.intimp.2013.01.015.View ArticlePubMedPubMed CentralGoogle Scholar
- Go H, Hwang HJ, Nam TJ: Polysaccharides from Capsosiphon fulvescens stimulate the growth of IEC-6 Cells by activating the MAPK signaling pathway. Mar Biotechnol. 2011, 13 (3): 433-440. 10.1007/s10126-010-9314-y.View ArticlePubMedGoogle Scholar
- Maeda R, Ida T, Ihara H, Sakamoto T: Immunostimulatory activity of polysaccharides isolated from Caulerpa lentillifera on macrophage cells. Biosci Biotechnol Biochem. 2012, 76 (3): 501-505. 10.1271/bbb.110813.View ArticlePubMedGoogle Scholar
- Han SB, Yoon YD, Ahn HJ, Lee HS, Lee CW, Yoon WK, Park SK, Kim HM: Toll-like receptor-mediated activation of B cells and macrophages by polysaccharide isolated from cell culture of Acanthopanax senticosus. Int Immunopharmacol. 2003, 3 (9): 1301-1312. 10.1016/S1567-5769(03)00118-8.View ArticlePubMedGoogle Scholar
- Raghu R, Sharma D, Ramakrishnan R, Khanam S, Chintalwar GJ, Sainis KB: Molecular events in the activation of B cells and macrophages by a non-microbial TLR4 agonist, G1-4A from Tinospora cordifolia. Immunol Lett. 2009, 123 (1): 60-71. 10.1016/j.imlet.2009.02.005.View ArticlePubMedGoogle Scholar
- Liu JY, Yang FL, Lu CP, Yang YL, Wen CL, Hua KF, Wu SH: Polysaccharides from Dioscorea batatas induce tumor necrosis factor-alpha secretion via Toll-like receptor 4-mediated protein kinase signaling pathways. J Agric Food Chem. 2008, 56 (21): 9892-9898. 10.1021/jf8018722.View ArticlePubMedGoogle Scholar
- Hsu HY, Jeyashoke N, Yeh CH, Song YJ, Hua KF, Chao LK: Immunostimulatory bioactivity of algal polysaccharides from Chlorella pyrenoidosa activates macrophages via Toll-like receptor 4. J Agric Food Chem. 2010, 58 (2): 927-936. 10.1021/jf902952z.View ArticlePubMedGoogle Scholar
- Wang Y, Yan T, Shen J, Guo H, Xiang X: Preventive effect of Ophiopogon japonicus polysaccharides on an autoallergic mouse model for Sjogren's syndrome by regulating the Th1/Th2 cytokine imbalance. J Ethnopharmacol. 2007, 114 (2): 246-253. 10.1016/j.jep.2007.08.014.View ArticlePubMedGoogle Scholar
- Jung ID, Kim HY, Park JW, Lee CM, Noh KT, Kang HK, Heo DR, Lee SJ, Son KH, Park HJ: RG-II from Panax ginseng C.A. Meyer suppresses asthmatic reaction. BMB Rep. 2012, 45 (2): 79-84. 10.5483/BMBRep.2012.45.2.79.View ArticlePubMedGoogle Scholar
- Lim YJ, Na HS, Yun YS, Choi IS, Oh JS, Rhee JH, Cho BH, Lee HC: Suppressive effects of ginsan on the development of allergic reaction in murine asthmatic model. Int Arch Allergy Immunol. 2009, 150 (1): 32-42. 10.1159/000210378.View ArticlePubMedGoogle Scholar
- Schepetkin IA, Quinn MT: Immunomodulatory effects of botanical polysaccharides. Polysaccharides in Medicinal and Pharmaceutical Applications. Edited by: Popa V. 2011, Shawbury, UK: Smithers Rapra, 988-994. 35Google Scholar
- Salazar L, Aravena O, Abello P, Escobar A, Contreras-Levicoy J, Rojas-Colonelli N, Catalan D, Aguirre A, Zuniga R, Pesce B: Modulation of established murine collagen-induced arthritis by a single inoculation of short-term lipopolysaccharide-stimulated dendritic cells. Ann Rheum Dis. 2008, 67 (9): 1235-1241.View ArticlePubMedGoogle Scholar
- Petermann F, Korn T: Cytokines and effector T cell subsets causing autoimmune CNS disease. FEBS Lett. 2011, 585 (23): 3747-3757. 10.1016/j.febslet.2011.03.064.View ArticlePubMedGoogle Scholar
- Codarri L, Gyulveszi G, Tosevski V, Hesske L, Fontana A, Magnenat L, Suter T, Becher B: RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation. Nat Immunol. 2011, 12 (6): 560-567. 10.1038/ni.2027.View ArticlePubMedGoogle Scholar
- Kruglov AA, Lampropoulou V, Fillatreau S, Nedospasov SA: Pathogenic and protective functions of TNF in neuroinflammation are defined by its expression in T lymphocytes and myeloid cells. J Immunol. 2011, 187 (11): 5660-5670. 10.4049/jimmunol.1100663.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/149/prepub
Pre-publication history
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Open Access is a wonderful opportunity for researchers, innovators, teachers, students, media professionals and the general public. It encourages the circulation of knowledge on a planetary scale and thus contributes to academic and scientific discoveries, innovation and socio-economic development (UNESCO Declaration, 2012).
Open Access for researchers and universities
It has now been proven that publications deposited on Open Access are cited much more than those which are less accessible because they have to be paid for. At the level of ORBi, an article deposited on Open Access is downloaded on average 2 times more than those on restricted access.
The advantage of OA in terms of impact for researchers is thus undeniable and testified (see the studies carried out by Open Citation Project), but there are many other benefits as well.
Open Access for the citizens
See also
To follow tomorrow…Open Access concerns all of us! Part 2 – What is the situation for publishers?


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557 fans witnessed a real battle the last time these 2 sides met at Jalan Besar and it took a last minute goal from Yasuhiro Yamakoshi, who thanked the heavens after finding the ball falling to his feet with the goal at his mercy, following a defensive mix-up between Fabien Lewis and Ahmadulhaq Che Omar.
Four months have passed since that game and while the White Swans have maintained their position at the top half of the S.League table, the Rams seemed to have lost their footing right after that match, falling from 3rd position into the cellar. The Rams, which had won two consecutive matches before that fateful game, never really recovered from that loss to Albirex.
Although a win is a win, the result in March only serves to flatter the White Swans as they laboured to find a way past a stubborn Woodlands defence. Japanese hitman Toshikazu Soya, who had scored 3 goals before meeting Woodlands, was kept quiet throughout the match and cut a frustrated figure up front.
In order to grind out a result at Jurong East, our defence must aim to keep the potent Japanese outfit out like they did at Jalan Besar.
|Expect a real battle in midfield between Armanizam Dolah and Musashi Okuyama.
Photo Credit: Andrew Him (The Black Sheep)
We will also probably see the White Swans marking Moon Soon-Ho tightly, as the Korean is generally perceived as the greatest threat to the Japanese. With an average of a goal scored in every two matches, Moon is equally dangerous as a creator and has 3 assists to his name so far, the highest among a list of assistants that include Daniel Hammond, Armanizam Dolah and Goh Swee Swee.
Moon also seems to be Albirex's bogeyman, scoring in the first minute when he played against the White Swans last season after joining Woodlands during the mid-season transfer window. The former Cheonan City star could have made it 2 goals in 2 matches against Albirex but Albirex custodian Takuma Ito managed to somehow defy gravity and palm away Moon's audacious lob in the first leg of this fixture in 2012. Moon's partnership with Hussein Akil, who scored his third goal in our last match against Geylang, will also be something to look out for. Moon has assisted Akil in two of those goals, and Akil could very use make use of the tight marking on the Korean to peel away with the ball with his blistering pace.
|Moon will be tightly marked by the Japanese.
Photo Credit: Andrew Him (The Black Sheep)
Albirex's recent Singapore Cup match against Tampines shows that the Japanese are suspect to breaches in concentration, having given away a first half lead to concede two goals to the Stags in the second half. The White Swans went on to lose the game and the Rams must exploit these lapses.
The Albirex man to watch out for would be the talismatic Yasuhiro Yamakoshi, who always seems to be in the right place at the right time. Yamakoshi has 5 goals to his name so far, as many as first choice striker Soya, and our defence needs to keep a close eye on him. At 172cm, he may not pose much of an aerial threat but our twin towers need to be wary of his quick feet.
Albirex's midfield fulcrum, Atsushi Shimono, may not be the most graceful player on the pitch, nor the making the headlines by getting on the scoresheet for the White Swans, but the midfielder with the luscious locks is one of the most hardworking players on the pitch. A quiet but solid performer, he puts in the tackles where they matter, and his crisp passing would cause problems for any team. Armanizam Dolah will need to be at his best to match Shimono in this one.
|The work of the tireless Atsushi Shimono must be nipped at the bid.
Photo Credit: Andrew Him (The Black Sheep)
Albirex's "smiling assassin", Sho Kamimura, would also be a handful for the Rams. The 23 year old may only have 3 goals at the moment, but he is always on the prowl for more goals.
Against an organized Albirex Niigata (S), Woodlands will need to maintain their composure and churn out a result. It will be a match where there's plenty of passing and running involved, and the Rams will need to adopt a patient approach.
The last time we lost to Albirex, we gave them too much respect and only started attacking after a good 15 minutes had gone by. Yes, let's respect the opponent, but not to the point where we are simply being eaten like a bunch of sacrificial lambs.
Come On You Rams! We shall Never Surrender!
Match Information
Date: Wednesday, 11th June, 2012 (7.45pm kick-off)
Venue: Jurong East Stadium


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We are located in Rohnert Park, California
Spice Sensuality is located in Rohnert Park, California where there is ample parking.
Spice Sensuality Boutique
6597 Commerce Blvd
Rohnert Park, California 94928
Telephone: (707) 588-0525
Store Hours:
Monday-Saturday 10:00am-11:00pm
Sunday 12:00pm-9:00pm
Email Us:
or Contact Us online
Meet Chuck
Chuck is thrilled to open his doors to all who have a sense of fun, and a willingness to explore and expand their physical relationships. His choice to open Spice left some surprised, but for Chuck, the rewards have been amazing, and he is not talking just financial rewards, but the difference he feels Spice has made for many couples. Chuck has been running retail shops for over two decades. He enjoys working with people to help them find products that will add a new dimension to their lives. His first chain of stores was so enriching, he decided to take his retail knowledge and spiral it off into a new direction, one which is full of the best kind of Spice!
Chuck and his staff provide a source of products and information designed to help residents of Marin and Sonoma counties learn how to develop and build strong, intimate relationships. Education is an important part of Chuck's vision for Spice. He and his staff have conducted many sexual education classes at local colleges and other public venues. The staff at Spice is highly trained to understand the benefit each product can provide. Chuck believes his staff's most important role is to help customers find products and materials that will enrich their relationships and lives. Chuck strives to create a welcoming, clean shop where men and women of all sizes and inclinations feel welcomed and supported as they embrace their sensual side. Chuck envisions a store where his customers feel comfortable to ask questions, knowing that they will get advice that will help them understand which products are a good fit for them and/or their partner.
Meet Courtney
Courtney came to Spice from the jewelry business, and she loves to sell, especially in specialized markets! Her vibrant, warm personality helps remove any inhibitions as soon as you walk through the door. As the business grows, she has been delighted to realize that more and more people feel at ease exploring their sexuality. If you are shy or uncomfortable with the topic of sex it takes but a few minutes with Courtney to put you at ease and you will always walk out with a smile.
Imagine the most open childhood possible, and you will understand the environment in which Courtney learned to live and love. Her parents embraced the hippy lifestyle, even though her father was a police officer. She grew up learning not to judge but to appreciate the best in everyone. Courtney's husband is an IT Specialist in the US Coastguard, stationed at CAMSPAC, Point Reyes. When the couple moved from Michigan, Courtney found herself exploring a small boutique selling intimate products. Before long, she was employed by the owner, and has never looked back! "This is my calling, I enjoy helping people feel relaxed in an environment where they naturally feel they should not be comfortable."
"We should not feel ashamed of our sexuality, we should embrace it and enjoy it. "Courtney believes that we naturally have "hang-ups" about experimenting. She admits that she was initially intimidated by products involving bondage, and was reticent to sell them, in her words, "how do you hurt someone safely?" She realizes now that although there is a fine line between pleasure and pain, education is key, and there is much pleasure to be derived from all aspects of sexual play. Spice carries an excellent variety of floggers, paddles and whips.
Courtney loves the lingerie Spice sells, and as the lingerie buyer spends many hours picking different styles to fit every size. She believes that size is not an issue when picking intimate outfits and enjoys helping all her customers find just the right garment to make them feel sexy! If you think you can't look sexy in a corset, you have yet to meet Courtney!
Meet Jessica
Jessica majored in math, and always thought working at Spice would be a side venture. However she loves helping her customers find a strong intimate connection with themselves and their partners. She is always delighted to hear how sexual experiences have improved as a result of education, and with the help of great products, obviously!
Jessica believes that finding a comfort level with your own body is essential before you can share experiences with your partner. "You would be surprised at how many people are not comfortable sexually in their own skin. Finding and evaluating the correct products is vital, it is not just about titillation it is so much more than that".
"Masturbation should not be a taboo subject." It is a natural part of sexual experience and should be treated as such, and talked about, instead of hidden behind closed doors. We carefully evaluate our products for quality and satisfaction, and welcome feedback from our customers.
When Jessica started working at Spice she realized that all assumptions should be cast aside when it comes to sex! As an example, she initially felt sure that most harnesses were designed for women to use with other women, but has since realized many straight couples enjoy using harnesses too! Spice sells harnesses of exceptional quality, habd selected from Spareparts Hardwear.
There are many items on the market, and everyone has different needs. What is right for one person or couple may not be right for another. Jessica can provide valuable insight and ideas on all of the products in the boutique.
Jessica lives by the belief that "wondering will always hurt more than knowing the answer." This is true of all aspects of life. Being open and liberal has helped her explore all sexual avenues to find what it is she truly desires.


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Having the chance to relax is very rare nowadays. Everyone's busy with their job and this leaves less time to just click clack sit back. Here are a few ways to make yourself time so you can enjoy yourself.
1. Clear It All Got a heavy burden that you need finished. Why not do it after you read this blog? Get all your work up to date and you'll feel great. The feeling you get when everything's off your chest is amazing and then you will finally pay all your attention to relaxing. Or you could also do your work but by bit then there should be that special day off.
2. Sit Down And Nap A great way to relax is sleep. Just sit on your couch and then while you slowly doze off BAM! Your asleep. And then you wake up a few hours later feeling fresh and ready to finish off the day. It all starts on the couch or bed and then your asleep.
3. Or Just Relax Sometimes the best solution when you've got a pile of work in front of your face is too just sit back and relax. But the only problem with this one is that if you didn't do the work when you were relaxing then you probably won't do the work when you wake up. Just sit on your cocah or bed and give yourself time to refresh and life should look a lot better after that nap. But if you get fired from work because you didn't do any work don't blame me since you can do it after you wake up :).
Sorry for the short articles lately just been busy anyways that's the article. If you liked it please add me to circles and share. Thanks for reading