European Journal of Plant Pathology

February 2015

Date: 28 Feb 2015

Alternative control of Chalara paradoxa, causal agent of black rot of pineapple by plant extract of Mormodica charantia

• Wilza Carla Oliveira de Souza,
• Luciana Cordeiro do Nascimento,
• Daniele Lavra Vieira,
• Taciana Silva dos Santos,
• Francisco Miguel de Assis Filho

Abstract

Black rot of pineapple caused by the fungus Chalara paradoxa is considered an aggressive and difficult disease to control. The use of natural products has been studied with the goal to incorporate them into integrated pest management and reduce the use of agrochemicals. Plant extracts have shown satisfactory results in controlling postharvest diseases by providing fungitoxic action and inducing plant resistance. The aim of this study was to evaluate C. paradoxa severity by the application of plant extract of Mormodica charantia, determinate effect of the plant extract on post-harvest quality, and induction of enzymes linked to host resistance induction. The following treatments were studied: plant extract of bitter melon at concentrations of 10, 100, 500 and 1000 ppm, the commercial products acibenzolar-S–Methyl and fungicide (thiabendazole) in the dosages recommended by the manufacturers, and distilled water as the control, with five replications and two plants per plot. The extract of M. charantia reduced the severity of black rot caused by C. paradoxa, compared to the control treatment, showing similar efficacy to the treatments with commercial inductor Acibenzolar-S-methyl and the fungicide. The enzymatic analysis showed increase of the peroxidase by the use of the bitter melon extract along the evaluation period. The enzymes polyphenol oxidase and phenylalanine ammonia lyase showed greater activity by treatment with M. charantia extract at 500 ppm. It was observed that the plant extract did not affect most of the variables analyzed related to postharvest quality of treated fruits. The use of the extract reduced the fruit weight loss, being a positive aspect under the studied conditions.

Ethnopharmacological Significance of Eclipta alba (L.) Hassk. (Asteraceae)

International Scholarly Research Notices
Volume 2014 (2014), Article ID 385969, 22 pages
http://dx.doi.org/10.1155/2014/385969

Review Article
Ethnopharmacological Significance of Eclipta alba (L.) Hassk. (Asteraceae)
Rownak Jahan,1 Abdullah Al-Nahain,2 Snehali Majumder,3 and Mohammed Rahmatullah2,4

1Department of Biotechnology & Genetic Engineering, University of Development Alternative, Dhanmondi, Dhaka 1209, Bangladesh
2Department of Pharmacy, University of Development Alternative, Dhanmondi, Dhaka 1209, Bangladesh
3Department of Microbiology and Serology, NH Health, Bangalore 560099, India
4Faculty of Life Sciences, University of Development Alternative, House No. 78, Road No. 11A (new), Dhanmondi, Dhaka 1209, Bangladesh

Received 29 June 2014; Accepted 8 September 2014; Published 29 October 2014

Academic Editor: Jagadananda Ghosh

Copyright © 2014 Rownak Jahan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Eclipta alba can be found growing wild in fallow lands of Bangladesh where it is considered as a weed by farmers. Traditional medicinal systems of the Indian subcontinent countries as well as tribal practitioners consider the plant to have diverse medicinal values and use it commonly for treatment of gastrointestinal disorders, respiratory tract disorders (including asthma), fever, hair loss and graying of hair, liver disorders (including jaundice), skin disorders, spleen enlargement, and cuts and wounds. The plant has several phytoconstituents like wedelolactone, eclalbasaponins, ursolic acid, oleanolic acid, luteolin, and apigenin. Pharmacological activities of plant extracts and individual phytoconstituents have revealed anticancer, hepatoprotective, snake venom neutralizing, anti-inflammatory, and antimicrobial properties. Phytoconstituents like wedelolactone and ursolic and oleanolic acids as well as luteolin and apigenin can form the basis of new drugs against cancer, arthritis, gastrointestinal disorders, skin diseases, and liver disorders.

In-vivo and In-vitro Activities of Medicinal Plants on Ecto, Endo and Haemoparasitic Infections: A Review

Authors: Mbaya, Albert Wulari; Ogwiji, Mathew

Source: Current Clinical Pharmacology, Volume 9, Number 3, August 2014, pp. 271-282(12)
Publisher: Bentham Science Publishers

Abstract:

Crude methanol, pet. ether, ethyl acetate, n-hexane, dichloromethane and aqueous extracts of various species of medicinal plants have shown significant in-vivo and in-vitro pharmacological activities against ecto, endo and haemoparasites. The scientific evaluations of the use of the plants as antiparasitic agents were based on the claims of folklore drawn from traditional healers from various communities across the world. The pharmacological activities of these plants were associated with the presence of various bioactive compounds such as alkaloids, flavonoids, saponins, glycosides, allicinine, harmala, harmaline, harman, tetrahydroharman, ursolic acid, terapines, tannins, phenolic compounds, embelin and brucine. These compounds were either found in the leaves, seeds, bulbs, flowers, stem, root barks or entire plant. In the in-vivo studies, plant extracts were tested using animal models such as mice, sheep, goats, cattle and dogs. The in-vivo anthelmintic activities of the plants were assessed by faecal egg out puts and post-mortem worm counts which in most instances achieved 70-90% priori levels with some of the plants. For haemoparasitic infections, parasitaemia clearance levels were used, while larvae and adult deaths were used to measure the activity of the plants against ectoparasites. For in-vitro activities, inhibitory concentration IC50 values using the brine lethality test, micro dilution, flow cytometery and larval packet test were used to assess the efficacy of the plant extracts on the parasites in various cell cultures. Significant in-vitro activity of 99.8% at 3.1mg/ml was achieved with the ethanolic extract of Azadirachta indica. Many of the plants were found to be more potent and possessed similar mechanism of action as their novel synthetic drugs. Such breakthroughs have led to the development of less toxic, cheaper and readily available drugs. Worthy of note is the use of the fruit of the Thai plant Piper longum (PIPERACEAE) as part of a drug formulation used in India for the treatment of clinical giardiosis in human patients.

Remèdes vétérinaires traditionnels utilisés dans les élevagesdechèvres à Lubumbashi et proche périphérie, RD Congo http://link.springer.com/article/10.1007/s10298-014-0873-3 #springerlink

Remèdes vétérinaires traditionnels utilisés dans les élevagesdechèvres à Lubumbashi et proche périphérie, RD Congo http://link.springer.com/article/10.1007/s10298-014-0873-3 #springerlink

Effect of whole soy and purified daidzein on ambulatory blood pressure and endothelial function—a 6-month double-blind, randomized controlled trial among Chinese postmenopausal women with prehypertension

Effect of whole soy and purified daidzein on ambulatory blood pressure and endothelial function—a 6-month double-blind, randomized controlled trial among Chinese postmenopausal women with prehypertension

European Journal of Clinical Nutrition , (18 March 2015) | doi:10.1038/ejcn.2015.24

Z-M Liu, S C Ho, Y-M Chen, B Tomlinson, S Ho, K To and J Woo

Abstract

Background/objectives:

Although observational studies suggest that soy foods or isoflavones are cardio-protective, clinical trials on whole soy or isoflavone daidzein (one major isoflavone and the precursor of equol) on blood pressure (BP) and endothelial function (EF) are few and have not been specifically conducted among equol producers, a population most likely to benefit from soy treatment.

Subjects/Methods:

We performed a 6-month double-blind, randomized, placebo-controlled trial to examine the effect of whole soy (soy flour) or purified daidzein on BP and EF in prehypertensive or untreated hypertensive postmenopausal women verified to be equol producers. A total of 270 eligible women were recruited and randomized to either one of the three treatment groups, 40 g soy flour (whole soy group), 40 g low-fat milk powder+63 mg daidzein (daidzein group) or 40 g low-fat milk powder (active control group) daily, each given as a solid beverage powder for 6 months. The primary outcome measures were 24 h ambulatory BP (ABP) and EF assessed by flow-mediated dilation using brachial artery ultrasound.

Results:

A total of 253 subjects completed the study according to protocol. Urinary isoflavones indicated good compliance with the interventions. Intention to treat and per-protocol analysis indicated that there was no significant difference in the 6-month changes or % changes in parameters of ABP and brachial flow-mediated dilation among the three treatment groups. A further subgroup analysis among hypertensive women (n=138) did not alter the conclusions.

Conclusions:

Whole soy and purified daidzein had no significant effect on BP and vascular function among equol-producing postmenopausal women with prehypertension or untreated hypertension.

Eating green leafy vegetables keeps mental abilities sharp http://www.sciencedaily.com/releases/2015/03/150330112227.htm

Eating green leafy vegetables keeps mental abilities sharp http://www.sciencedaily.com/releases/2015/03/150330112227.htm

Elizabeth May and Bruce Hyer announce 60 amendments to Bill C-51 – elizabethmaymp.ca – March 31, 2015

Elizabeth May and Bruce Hyer announce 60 amendments to Bill C-51 – elizabethmaymp.ca – March 31, 2015

NDP Bill Would Ban Tankers off BC's North Coast (The Tyee)

NDP Bill Would Ban Tankers off BC's North Coast (The Tyee)
http://thetyee.ca/News/2014/09/24/NDP-Bill-North-Coast-Tankers/

5 things to know about today’s Northern Gateway oil pipeline decision (Toronto Star) 
http://www.thestar.com/news/canada/2014/06/17/5_things_to_know_about_todays_northern_gateway_oil_pipeline_decision.html 

Enbridge staff ignored warnings in Kalamazoo River spill (CBC) 
http://www.cbc.ca/news/canada/edmonton/enbridge-staff-ignored-warnings-in-kalamazoo-river-spill-1.1129398

ABC Registered User Advisory

For Friends of the American Botanical Council

GNC and NY AG Announce Agreement on DNA Testing of Herbal Supplements

New York Times story discusses arrangement where GNC will use DNA testing for all plant materials in dietary supplements

ABC's Blumenthal interviewed and quoted in article

(AUSTIN, Texas, March 25, 2015) The New York Times today published front-page article by reporter Anahad O’Connor that describes elements of an agreement between New York Attorney General (NY AG) Eric T. Schneiderman’s office and GNC of Pittsburgh, PA, related to the AG’s previous investigation into herbal dietary supplements sold by GNC. In the agreement GNC has agreed to employ DNA barcode testing in all of its crude botanical ingredients to be used in herbal dietary supplements within 18 months.¹

Mr. O’Connor initially broke the story with his article on February 3 describing the NY AG’s having sent cease-and-desist letters to GNC as well as Walgreens, Walmart, and Target for allegedly selling house brands of herbal dietary supplements that the NY AG had determined the listed ingredients were absent from about 80% of the products.²

This morning’s New York Times article quotes GNC CEO Michael G. Archbold as stating, “As our testing demonstrated, and this agreement affirms beyond any doubt, our products are not only safe and pure but are in full compliance with all regulatory requirements.” Mr. Archbold was referring to an announcement that GNC had made earlier this month that its independent testing had confirmed the authenticity of the ingredients in the effected herbal supplements.^3,4 This morning GNC issued a press release announcing the agreement although specific details were not given.⁵

The American Botanical Council’s Founder and Executive Director Mark Blumenthal was interviewed by Mr. O’Connor on Sunday. The one-hour interview covered many aspects of the background of this issue, including the capabilities, limitations, and problems associated with DNA barcoding in testing the authenticity of botanical extracts, the need for confirmatory testing by using other scientifically valid test methods, and much more.

Blumenthal emphasized to Mr. O’Connor that highly reputable experts in industry, academia, independent nonprofit research and standards-setting organizations, and government agencies have cast significant and serious doubt on the AG’s reliance on the DNA barcoding method used at Clarkson University as the sole test method utilized, particularly when such methods are unreliable for testing botanical extracts, of which about 76% of the tested products were labeled. Thus, said Blumenthal on several occasions in the interview, without any full disclosure and transparency on the part of the NY AG, serious scientists and others cannot rely on the unusual and non-credible test results upon which the NY AG based its legal actions.

A key point made by Blumenthal in the interview with Mr. O’Connor, but not mentioned in the article, is that there appeared to be a false presumption that GNC had agreed to what are most likely the AG’s stipulated terms because GNC had been seen as selling substandard herbal dietary supplements. In fact, in the agreement there is reportedly no admission of wrongdoing on behalf of GNC and, as Blumenthal pointed out, analytical experts and others have raised serious doubts about the conclusions that have been drawn on the faulty application of DNA barcode technology to GNC’s products, which were all botanical extracts.

Blumenthal told O’Connor that despite this growing view that the NY AG’s tests results were not valid, that independent testing of products similar to those that were tested by the AG would likely reveal problems with some of the products, as ABC is aware of adulteration problems associated with some of the herb categories, particularly ginkgo extract and St. John’s wort extract.

The article includes the following statements based on the interview:

Mark Blumenthal, the executive director of the American Botanical Council, a nonprofit organization that promotes the use of herbal products, said he believed that the supplements included in the investigation most likely suffered from some level of adulteration — an established problem in the industry — but not to the extent suggested by the attorney general. He criticized the attorney general’s testing procedures and said his study data should be made public.

Mr. Blumenthal said he applauded GNC for reaching an agreement with New York and putting new procedures into effect, but he also worried that actions at the state level could create “a patchwork quilt of different quality standards and requirements” across the country.

“I’m concerned that this can potentially lead to various standards being set up in different states and that’s a legitimate issue for the industry and consumers,” he said.

In the interview, Blumenthal also noted the following points, among others, that were not mentioned in the Times article:

• NY AG appears to be attempting to establish new testing requirements beyond what is required by the US Food and Drug Administration’s (FDA) Good Manufacturing Practices (GMP) rule. This would be the responsibility of the FDA, not an individual state agency.
• It appears that the NY AG is doubling or perhaps tripling down on its intransigence with respect to its unscientific and untenable position on relying on only one DNA barcoding test upon which it has made its entire case and publicity campaign. By stipulating that GNC adopt and adhere specifically to DNA barcoding-based testing methods, the NY AG appears to be trying to validate his conclusions based solely on inadequate DNA testing of herbal dietary supplements without further verification of the results by standard chemically-based methods, which has been criticized in several recently published white papers, and also by inference by a letter from the FDA.  
• It is not clear at this time as to which herbal dietary supplements products of GNC’s, if any, may be out of compliance with label claims regarding their identity, which would give the AG sufficient leverage to force GNC to agree to the terms of the reported agreement. Thus, it is not clear to ABC why GNC has agreed to the stated terms.
• He stressed that, of the four companies to which cease-and-desist letters were sent by the NY AG in February, GNC is the only company to ABC’s knowledge that purchases its own raw materials and extracts and processes them into dietary supplements in its own facilities. Therefore, GNC is not dependent on third-party contract manufacturers and has significantly more control over its purchasing and manufacturing processes than the other companies, allowing GNC to negotiate terms with the NY AG to which it believes it can adhere and comply.
• GNC’s agreement to utilize DNA testing on botanical raw materials to determine their identity prior to their being extracted suggests that the NY AG now appears to have realized that DNA testing is not suitable or fit for purpose for the extracts themselves, even though the NY AG’s office has not publicly acknowledged such a now well-known fact.
• GNC does not produce its own botanical extracts. The requirement that GNC stipulate that its extract suppliers utilize DNA test methods on botanical raw materials to authenticate their identity can help ensure that the extractor is using correct botanical materials for extraction. However, Blumenthal noted that it is quite possible that some high-quality extractors in the international market may already use DNA barcoding with other analytical methods. But, he emphasized, it does not mean that if this were to become a standard industry practice, as the NY AG appears to be promoting, that a potentially unscrupulous extractor could still subsequently manipulate the extract in such a way as to create a false sense of value, i.e., adulterate it with other extracts and/or additional unlabeled chemical compounds, natural or synthetic, from other sources. DNA testing will not be able to detect such changes in the chemical composition; only appropriate chemical testing methods, as is currently used in many parts of the botanical industry, can do this. In addition, DNA barcode testing can be easily fooled by adding a few sprinkles of authentic plant material to an otherwise unacceptable material and, therefore, cannot guarantee by itself the quality of an ingredient or supplement.

ABC has not yet been able to obtain a copy of the final agreement. According to material supplied to ABC by the New York Times, some of the terms of the GNC agreement with the NY AG are as follows:

• GNC agrees to routinely test for various potential contaminants (which is a normally required provision of the current GMP rule), specifically those that may cause allergic reactions;
• GNC agrees to the stipulation that “all herbal ingredients used in its products are manufactured in facilities that are certified as good manufacturing compliant by a third-party accreditation body, such as ISO, USP, or NSF”;
• “Within 18 months, GNC will begin utilizing DNA barcoding to confirm the authenticity of all plants used as sources for its herbal supplements products prior to processing. GNC will contribute any new barcodes, and the scientific methods used to identify them, to a publicly accessible database within 24 months”;
• “GNC will prominently display signs in stores across the country and on its website that explain the difference between whole herbs and extracts”;
• “GNC will provide semiannual reports to the Attorney General’s Office, detailing all plant species sourced after authentication using DNA barcoding; the name and address of all facilities in which DNA barcode authentication was performed; a list of materials rejected as a consequence of the results of the barcoding and the results of the randomized testing for common allergens.”

Other persons quoted in the article include Pieter Cohen, MD of Harvard, and David Schardt of the Center for Science in the Public Interest.
The new GNC agreement was also the subject of this morning’s “Marketplace” segment on National Public Radio’s Morning Edition, and, presumably, other media has been and will be reporting and misreporting on this development for at least the next few days, or longer.
A comprehensive article on this issue since the NY AG’s February 3 announcement was published in ABC’s monthly e-newsletter HerbalEGram in March, available here.⁶
References
1. O’Connor A. GNC to Strengthen Supplement Quality Controls. New York Times. March 30, 2015. Available at: http://well.blogs.nytimes.com/2015/03/30/gnc-to-strengthen-supplement-quality-controls/?&hp&action=click&pgtype=Homepage&module=first-column-region&region=top-news&WT.nav=top-news. Accessed March 30, 2015.
2. O’Connor A. 4 Retailers Told to Stop Selling Supplements: Tests Find Fillers, Not Medicinal Herbs [print version]; New York Attorney General Targets Supplements at Major Retailers [online version]. New York Times, Feb 3, 2015. Available at: http://well.blogs.nytimes.com/2015/02/03/new-york-attorney-general-targets-supplements-at-major-retailers/?_r=0. Accessed March 30, 2015.
3. GNC Holdings. GNC Refutes New York State Attorney General’s Claims in Full and Robust Response. Business Wire. February 9, 2015. Available at: www.businesswire.com/news/home/20150209006388/en/GNC-Refutes-York-State-Attorney-General’s-Claims#.VRk2GULQBJ1. Accessed March 30, 2015.
4. Armental M. GNC Says Outside Testing of Nutritional Supplements Found No Improper Labeling. Wall Street Journal. March 10, 2015. Available at: www.wsj.com/articles/gnc-says-outside-testing-of-nutritional-supplements-found-no-improper-labeling-1426033941. Accessed March 30, 2015.
5. GNC Holdings. GNC Reaches Agreement with New York Attorney General. Business Wire. Available at: http://www.businesswire.com/news/home/20150330005372/en/GNC-Reaches-Agreement-York-Attorney-General#.VRkzuELQBJ0. Accessed March 30, 2015.
6. Smith T. A Review of the New York Attorney General's Recent Actions against Herbal Dietary Supplements. HerbalEGram. March 2015;12:3. Available at: http://cms.herbalgram.org/heg/volume12/03March/NYAG_HerbalSupplement_InvestigationReview.html. Accessed March 30, 2015.

Antibiotic-Resistant Bacteria Are No Match For Medieval Potion | Popular Science

Antibiotic-Resistant Bacteria Are No Match For Medieval Potion | Popular Science

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Journal of Ethnopharmacology

Available online 19 January 2015

In Press, Corrected Proof — Note to users

Editorial

Food and medicine: Old traditions, novel opportunities

• Rob Verpoorte (Editor in Chief)

 Show more

  Open Access

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Since ancient times people have been observing nature carefully and were able to find plants for food, medicine, clothes, shelter and fuel in their direct environment. This process still continues and is now known as bioprospecting. Particularly in biotechnology and in the development of medicines there have been quite some activities in finding novel products from nature. Particularly in the past years interests in exploring nature has increased in an endeavor to make our presence and activities in this world more sustainable. Much of the industrial bioprospecting is based on fast screening methods for e.g. a pharmacological effect or an enzyme activity. These methods are very efficient and in some molecular based bioassays all plants of the world could probably be screened in just a few days. But at the same time it becomes clear from basic research that human health is very complex and that it is not likely that a single molecule will be able to cure diseases which often have multifactorial causes. Obviously infectious and parasitic diseases might be cured by single compounds, as it concerns exogenous organisms that invade the body. Though in that case development of drug-resistance of these organisms is becoming a major problem.

Health is a complex, but robust state of the body. It is determined by external factors (the environment), genetic factors and age. The homeostasis, typical for health, is maintained as well as affected by food and medicines. Prevention and controlling risk factors are important aspects in keeping homeostasis. With the rapid development of centralized food preparation and fast food, old traditions and common knowledge of “what to eat when” are disappearing. The fast spreading of, for example, diabetes type 2 and obesitas are signs that we should look for novel approaches to stay healthy and reach the 125 years of age that some experts predict as being feasible for humans. Besides basic research on health and diseases, we may return to the knowledge of our ancestors as a valuable resource of tens of thousands years of observations of nature and human health. The personalized medicine is, for example, an interesting approach. The remark of Allen Roses (Anonymous, 2014), vice-president of genetics at GlaxoSmithKline: “The vast majority of drugs – more than 90 per cent – only work in 30 or 50 per cent of the people” shows that a personalized treatment also in western pharmacotherapy might be of interest. Apparently the number game of screening large numbers of compounds to find novel leads for drug development results in medicines that for many users do not work.

Considering the present problems to develop methods for prevention and novel treatments of diseases I am very happy with this interesting special issue series of international experts give a historical, cultural and scientific background to food, medicinal, or ritual (usually affecting the CNS) plants. Reading the various papers in this issue will be a source of inspiration and new ideas for future research. Our ancestors found important plants like poppy and cannabis; a number of plants containing caffeine; and plants containing alkaloids with a curare effect, without the aid of any of our …scopy and …omics tools, just by common sense and observations made by their own senses. With all the scientific knowledge and tools we have now really great perspectives for discovering novel information concerning the role of food and medicinal plants for our health by starting from what our ancestors already discovered.

References

1. • Anonymous, 2014
   • Anonymous, 2014. 〈http://www.independent.co.uk/news/science/glaxo-chief-our-drugs-do-not-work-onmost-patients-575942.html〉 (accessed 01.07.14).
   • 
     

Copyright © 2015 Elsevier Ireland Ltd.

Editorial

Journal of Ethnopharmacology

Available online 27 March 2015

In Press, Uncorrected Proof — Note to users

A network pharmacology approach to determine active ingredients and rationality of herb combinations of Modified-Simiaowan for treatment of gout

• Fangli Zhao^a,
• Li Guochun^b,
• Yanhua Yang^c,
• Le Shi^a,
• Li Xu^a,
• Lian Yin^{a, ,}

Under a Creative Commons license

 

  Open Access

---

Abstract

Ethnopharmacological relevance

Modified Simiaowan (MSW) is a traditional Chinese medicine (TCM) formula and is widely used as a clinically medication formula for its efficiency in treating gouty diseases.To predict the active ingredients in MSW and uncover the rationality of herb combinations of MSW.

Materials and methods

Three drug-target networks including the “candidate ingredient-target network” (cI-cT) that links the candidate ingredients and targets, the “core ingredient-target-pathway network” connecting core potential ingredients and targets through related pathways, and the “rationality of herb combinations of MSW network”, which was derived from the cI-cT network, were developed to dissect the active ingredients in MSW and relationship between ingredients in herb combinations and their targets for gouty diseases. On the other hand, herbal ingredients comparisons were also conducted based on six physicochemical properties to investigate whether the herbs in MSW are similar in chemicals. Moreover, HUVEC viability and expression levels of ICAM-1 induced by monosodium urate (MSU) crystals were assessed to determine the activities of potential ingredients in MSW.

Results

Predicted by the core ingredient-target-pathway network, we collected 30 core ingredients in MSW and 25 inflammatory cytokines and uric acid synthetase or transporters, which are effective for gouty treatment through some related pathways. Experimental results also confirmed that those core ingredients could significantly increase HUVEC viability and attenuate the expression of ICAM-1, which supported the effectiveness of MSW in treating gouty diseases. Moreover, heat-clearing and dampness-eliminating herbs in MSW have similar physicochemical properties, which stimulate all the inflammatory and uric acid-lowing targets respectively, while the core drug and basic prescription in MSW stimulate the major and almost all the core targets, respectively.

Conclusion

Our work successfully predicts the active ingredients in MSW and explains the cooperation between these ingredients and corresponding targets through related pathways for gouty diseases, and provides basis for an alternative approach to investigate the rationality of herb combinations of MSW on the network pharmacology level, which might be beneficial to drug development and applications.

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Graphical abstract

Figure options

Abbreviations

• MSU, monosodium urate;
• NSAIDs, non-steroidal anti-inflammatory drugs;
• UA, uric acid;
• TCM, traditional Chinese medicine;
• MSW, Modified-Simiaowan;
• EM, Ermiao;
• SM, Simiao;
• AR, Atractylodes chinensis (DC.) Koidz.;
• PC, Phellodendron chinense Schneid.;
• AB, Achyranthes bidentata Bl.;
• CS, Coix lacryma-jobi L. var. mayuen (Roman.) Stapf;
• LJ, Lonicera japonica Thunb.;
• SG, Smilax glabra Roxb.;
• ICAM-1, intercellular cell adhesion molecule;
• OB, oral bioavailability;
• DL, drug-likeness;
• MW, molecular weight;
• nHDon, number of donor atoms for H-bonds;
• nHAcc, number of acceptor atoms for H-bonds;
• MLOGP, Moriguchi octanol–water partition coeff. (logP);
• DMEM, Dulbecco’s modified Eagle’s medium;
• FBS, fetal bovine serum;
• MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
• SD, standard deviation;
• IL-1β, interleukin -1 beta;
• TNF-ɑ, tumor necrosis factor-alpha;
• PTGS2, prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase);
• COX, cyclooxygenase;
• P13K, phosphotylinosita l3 kinase;
• MAPK1, mitogen-activated protein kinase-1;
• TGFB1, transforming growth factor, beta 1;
• XOD, xanthine oxidase;
• OAT1, organic anion transporter 1;
• URAT1, urate-anion transporter1;
• GLUI9, glucose transporter 9;
• LEP, leptin;
• ABCG2, ATP-binding cassette sub-family G member 2;
• NF-ƙB, nuclear factor-kappa B;
• TLR, toll-like receptor.

Keywords

• Modified Simiaowan;
• Gout;
• Network pharmacology;
• Active ingredients;
• Herb combinations

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1. Introduction

Hyperuricemia is predictive for the development of gout, renal dysfunction, hypertension and hyperlipidemia, which is one of the most common and extensive metabolic diseases in populations (Richette and Bardin, 2010). Gout is a common metabolic disorder in human that afflicts about 8.3 million people in the United States, 6.4 million in the European Union, and 2.9 million in Japan in 2008 (Miao et al., 2008, Doherty, 2009 and Zhu et al., 2011). The disease is also rapidly rising in China probably due to recent changes in dietary habits. Preventing and treating the occurrence of hyperuricemia and gout become imperative in contemporary clinical therapy, but there are lack of effective means to control acute gouty arthritis.

Gout is a type of inflammatory arthritis induced by deposition of monosodium urate (MSU) crystals in the joints and kidneys, where MSU crystals stimulate monocytesmacrophages and neutrophils to produce different pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α (Tausche et al., 2004) and monocyte chemotactic factor (Inokuchi et al., 2006). Despite advances in the application of anti-gout drugs for the treatment, non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin, as first-line agents, are commonly used for acute inflammation. However, according to epidemiological study, NSAIDs, which play an important role in terms of anti-inflammation by inhibiting the activity of cyclooxygenase (COX) (Yuan et al., 2000), show some adverse effects including renal toxicity, gastrointestinal toxicity and gastrointestinal bleeding (Sabina et al., 2011). Besides, Colchicine inhibits the propagation of inflammation by suppressing several genes encoding Caspase-1 such as phosphotylinosita l3 kinase (P13K) and mitogen-activated protein kinase-1 (MAPK1), which could reduce the release of mature IL-1 (Ben-Chetrit et al., 2005). Nevertheless, it is still limited and often poorly tolerated because of severe gastrointestinal reactions and toxicity (Borstad et al., 2004). Besides, underexcretion of urate has been implicated to produce gouty diseases (Rott and Agudelo, 2003 and Lioté, 2003). Ways to reduce uric acid (UA) levels can be classified into two types: decreasing UA synthesis with inhibitory activities of xanthine oxidase (XOD) and promoting UA excretion by stimulating some transporters such as organic anion transporter 1 (OAT1), urate-anion transporter 1 (URAT1), glucose transporter 9 (GLUI9) and leptin (LEP), which are important for drugs to treat Hyperuricemia (Sha and He, 2009 and Tong et al., 2013). Allopurinol is a frequently used XOD inhibitor in clinical use (Horiuchi et al., 2000 and Chen et al., 2005), and benzbromarone is an uricosuric agent by inhibiting the main renal UA transporter URAT1 (Kunishima et al., 2007). Nevertheless, both of them may result in allergic, sever hypersensitivity reactions, and nephropathy (Hande et al., 1984 and Kumar et al., 2005). Therefore, it underlines much impetus for urgent need of available anti-gout agents, especially herbal medicine (Ahmad et al., 2008 and An et al., 2010).

Traditional Chinese medicine (TCM) is a comprehensive medicinal system that has been used in clinical practice for thousands of years, whose applications have been raised dramatically around the world in recent decades because of their moderate treatment effects and lower side effects (Tang et al., 2009). Modified-Simiaowan (MSW) is widely used clinically as a formula for its prominent efficiency in terms of TCM for treating gouty diseases, such as gout, hyperuricemia and inflammatory arthritis (Yin and Si, 2006), which is a patented invention of the Chinese (Yin et al., 2008). It is made up of six herbs: Phellodendron chinense Schneid. (PC), Atractylodes chinensis (DC.) Koidz. (AR), Achyranthes bidentata Bl. (AB), Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (CS), Smilax glabra Roxb. (SG) and Lonicera japonica Thunb. (LJ) and has been used in treating gouty diseases for its efficiency in anti-inflammation, analgesia and the reduction of UA levels (Shi et al., 2008).

In this formula, Ermiao (EM), also called as the core drug, which is composed of PC and AR, is a classical formula and recorded in State Pharmacopoeia of People’s Republic of China at all times for successful treatment of gout and hyperuricemia. According to Danxi’s China, EM is described to treat acute gout through clearing heat and eliminating dampness in terms of TCM. Simiao (SM), a classical formula, which is derived from EM with additions AB and CS. According to Cheng-Fang-Bian-Du (a Chinese medicine book named Conventional Prescriptions for Easily Reading), SM is commonly used to treat gout through clearing heat, eliminating dampness and strengthening the liver and kidney in terms of TCM (Shi et al., 2008; Hu et al., 2010; Hua et al., 2012).

Recent researches have demonstrated that MSW could inhibit the occurrence of acute gout by suppressing the expression of intercellular cell adhesion molecule (ICAM-1), protecting HUVECs by reducing cell apoptosis, increasing cell viability in MSU-induced HUVECs, and weakening adhesion between neutrophil and endothelial cells (Shi et al., 2013). Moreover, MSW could significantly reduce UA levels in serum and urine in hyperuricemic model, together with decreasing liver XOD activity and IL-1β, IL-6 levels (Zeng et al., 2014) and has a better anti-gouty inflammation effect compared with indomethacin in the clinical study (Shi et al., 2008). All the results indicate that MSW is a comprehensive therapeutic formula with multi-approaches for gouty diseases. However, the active ingredients and the rationality of herb combinations of MSW through integrated multiple pathways for gouty treatment are still unclear.

With the rapid progress of bioinformatics, systems biology and polypharmacology, network-based drug discovery is considered a promising approach toward more cost-effective drug development (Keith et al., 2005, Schadt et al., 2009, Jia et al., 2009 and Levinson, 2010). Network pharmacology can reveal the underlying complex relationship between a herbal formula and the whole targets. Besides, this sustainable development platform coupling with rich experience of TCM is hopeful of shifting the paradigm “one target, one drug” to the “network targets, multi-components” strategy (Li and Zhang, 2013).

Our previous studies have shown that “network target” as a key concept of TCM network pharmacology can help us to decipher active ingredients in MSW and rationality of herb combinations of MSW. In present study, we first manually collected the information of targets reported for main active ingredients in MSW and targets of gouty diseases from OMIM, and selected those gouty genes that are targeted by the corresponding ingredients as the candidate targets. After oral bioavailability (OB) screening and drug-likeness (DL) evaluation, potential ingredients and targets could be obtained. In order to predict the active ingredients in MSW and uncover the rationality of herb combinations of MSW, drug-target networks have constructed to this formula, which offers an opportunity for deep understanding of the efficiency and rationality of TCM in ways for the prevention of gouty diseases.

2. Material and methods

2.1. Chemical ingredients database building

A total of 631 chemical constituents of all six individual herbs in MSW were retrieved from Traditional Chinese Medicine Systems Pharmacology Database (TcmSP™, http://tcmspnw.com) and related literatures. TcmSP™ is a unique systems pharmacology platform designed for herbal medicines, from which 49 ingredients in AR, 58 in PC, 176 in AB, 38 in CS, 74 in SG, and 236 in LJ were all collected.

2.2. The properties of ingredients in MSW

In order to investigate whether the six individual herbs in MSW are similar or different in chemicals, herbal ingredients comparisons based on chemical properties were performed. The important pharmacology-related properties including molecular weight (MW), the number of donor atoms for H-bonds (nHDon), the number of acceptor atoms for H-bonds (nHAcc) and Moriguchi octanol–water partition coeff. (logP) (MLOGP) are also collected from TcmSP™ for each ingredient. These four parameters reflect the basic characteristic of a molecule ( Lipinski et al., 1997). Next, OB and DL properties of each herb ingredient were collected from the same database, which describe the physicochemical property and pharmacological feature of every ingredient. Therefore, the histograms of the physicochemical properties of all ingredients accompanying with t-test were carried out to analyze the variables in the property spaces.

2.3. Data preparing

2.3.1. Predicting target profiles of main active ingredients in MSW

Based on our pervious study and literature, the main ingredients in MSW were all obtained, such as the volatile oil in AR, alkaloids in PC, saponins in AB and flavonoids in LJ. We collected all their targets profiles from PubMed or SciFinder, and then removed the ones with no targeting information. After that, we extracted the relevant targets profiles of important ingredients. For the reason that some contain protein and gene files from different species, the demand for standardization of the information is necessary. We limited the species as “Homo sapiens” when entering the gene or protein target with the genetic search function in the NCBI database (http://www.ncbi.nlm.nih.gov/gene), which could obtain all the targets having revised to their official names, including multiple subtypes of genetic information. After the above retrieval and transformation, we gained distinct target files associated with main active ingredients.

2.3.2. Gout-associated targets

We collected different genes associated with gout from the following resources. (1) The Online Mendelian Inheritance in Man (OMIM) database, which catalogues all known diseases with a genetic component and when possible links them to the relevant genes in the human genome and provides references for further research and tools for genomic analysis of a catalogued gene (Hamosh et al., 2005). We searched the OMIM database with a keyword “gout” and found a set of genes, such as ABCG2, GLUT9, TNF-α, IL-8. (2) Genecards (http://www.genecards.org), it is a database about genes, their products and biomedical applications, which is maintained by Israel’s Weizmann Institute of science. (3) Literatures, we could collect some significant targets from gouty related literatures.

Based on the above three methods, we collected 277 distinct targets associated with gouty diseases.

2.4. Candidate ingredients and their corresponding targets prediction

Compared the target files of ingredients in MSW collected from literature with gouty genes from databases, we selected those gouty genes that could be targeted by the corresponding ingredients as the candidate targets. The candidate targets and corresponding ingredients are useful for the following study.

2.5. Oral bioavailability screening and drug-likeness evaluation

Generally, a TCM formula covers various chemicals, bioactive ingredients can contribute to its therapeutic effects. Hence, it is essential to conduct the OB screening and DL evaluation for the goal to identify the potential active ingredients in this formula.

2.5.1. OB screening

A robust in-house system OBioavail1.1 (Xu et al., 2012), is applied to calculate the OB value, which is efficient in screening out the potential ingredients. In this work, the OB threshold was defined as 30% and those ingredients with OB≧30% were selected as the ingredients for further analysis.

2.5.2. DL prediction

In this study, DL index of a new compound is calculated by Tanimoto similarity defined as

equation(1)

$ƒ\left(A,B\right)=\frac{A.B}{|A{|}^2+|B{|}^2-A\cdot B}$

where A represents the new ingredient, and B is the average molecular properties of all ingredients in Drug-Bank database. According to this result, we removed those ingredients with DL<0.1.

Briefly speaking, ingredients that meet both of the requirements OB≧30% and DL≧0.1 were selected as the potential ingredients.

2.6. Construction of network

In our present study, the content for construction of network was performed as following, (1) the “candidate ingredient-target network” was established by connecting the candidate ingredients and all their corresponding targets, (2) the “core potential ingredient-target-pathway network” was also constructed after OB screening and DL evaluation, by linking the potential ingredients and targets with high degree through related pathways, (3) the “rationality of herb combinations of MSW network”, which describes the complex relationship between herbs combined in MSW based on herbal ingredients comparisons. All the networks were created using network visualization software Cytoscape 2.8.2 (http://www.cytoscape.org/), which is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework, in order to facilitate scientific interpretation of complex relationships between medicines involved in formula orchestration (Shannon et al., 2003).

2.7. Experimental validation

2.7.1. Drugs and preparation

Chlorogenic Acid (no. 110753200413), Berberine (no. 713 9906), Astilbin (with a purity of 98% determined by HPLC), Caffeic acid and Ferulic acid were purchased from National Institute for the Control of Biological Products. Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA), Fetal bovine serum (FBS) (Hangzhou Sijiqing Co., Ltd., China), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (AERESCO, USA), Uric acid (Sigma St. Louis, MO, USA), Indomethacin (no. 030901, Shanghai Yanan Vientiane Pharmaceutical Co., Ltd.) were used in this experiment. Other reagents were of analytical grade from commercial suppliers and manufactured in the People’s Republic of China.

Berberine (2.5 mg), Astilbin (2.5 mg), Chlorogenic Acid (2.5 mg), Ferulic acid (2.5 mg) and Caffeic acid (2.5 mg), DMSO was dissolved in every solution at concentration less than 0.02%, Serum-free DMEM culture medium was added into the five solutions at concentration of 2.5 µg/ml, 25 µg/ml and 250 µg/ml. Approximately 4 g of uric acid was dissolved and heated in 800 ml of H₂O with NaOH (9 ml/0.5 N), adjusted to pH 8.9 at 60 1 C, cooled overnight in a cold room, washed, and dried. MSU crystals, needle-like crystals were recovered and were suspended in sterile saline (20 mg/ml) (Denko and Whitehouse, 1976).

2.7.2. Cells culture and treatment

HUVECs were provided by basic medical college from Nanjing university of Chinese medicine, and were isolated as described previously (De Martin et al., 1993). The cells were grown in DMEM culture medium containing 10% FBS at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air after digested by 0.25% trypsin, neutralized with the same DMEM, then centrifuged 6 min at 1000 r/min and discarded the supernate. Cells were then serially passaged with 0.25% trypsin/1 mM.

2.7.3. Detection on HUVECs activity stimulated by MSU

HUVECs were washed three times with DMEM culture medium containing 10% FBS after digested by 0.25% trypsin, centrifuged, and modulated the cell suspension at density of 4×10⁴/ml with the same DMEM, then seeded onto a 96-well culture plate at density of 200 μl/well and incubated at 37 °C for 24 h. The medium was replaced with the following groups (8 well/group), then incubated at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air for 48 h, and collected the supernatant. The rest of endothelial cells were used to measure cell viability using MTT assay (Welde, 1992). MTT was added to cell cultures at concentration of 0.5 mg/ml. After 4 h, MTT was discarded and the formazan crystals were dissolved by adding 200 μl of dimethyl sulfoxide.

The absorbance at 490 nm was read on a microplate reader (Bio-Rad, Hercules, CA, USA). Absorbance, which was used as a measurement of cell viability, was normalized to cells incubated in the control medium. The cells were considered 100% viable. The percentage of cell viability was calculated as follows: cell viability %=b/a×100%. Here, a is the absorbance of the control, and b is the absorbance of cells with MSU crystals. Groups: blank group (DMEM culture medium 200 μl), model group (MSU 100 µg/ml), Indomethacin (MSU 100 µg/ml and Indomethacin 20 µg/ml), five low dosage groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 2.5 µg/ml); five middle groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 25 µg/ml), five large groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 250 µg/ml)

2.7.4. Detection on expression of ICAM-1

HUVECs were seeded into tissue culture flask under logarithmic growth phase after digested by 0.25% trypsin, and prepared into cell suspension at the density of 5×10⁹ l⁻¹. After about 24 h, the supernatant was discarded. Groups: control group, model group (MSU 100 µg/ml), Indomethacin (MSU 100 µg/ml and Indomethacin 2.5 µg/ml), five drug groups (MSU 100 µg/ml and Berberine, Astilbin, Chilrogenic acid, Caffeic acid, Ferulic acid 2.5 µg/ml).

HUVECs were cultured for 24 h and collected by PBS. The supernatant was removed by centrifugation and then CD54 monoclonal antibody was added in. HUVECs were washed with PBS and resuspended after 30 min, Detect the percentage of positive cells by flow cytometry (FCM) and repeated for 3 times. The percentage of the expression of ICAM-1 was calculated as follows: Inhibition rate %=b/a×100%. Here, a is the value of the control, and b is the value of cells with MSU crystals.

3. Results

3.1. Herbal ingredients comparisons in MSW

MSW consists of six herbs, including AR, PC, AB, CS, LJ and SG. In order to investigate whether the six herbs are similar or different in chemicals, herbal ingredients comparisons were conducted based on six significant properties: MW, nHDon, nHAcc, MLogP, OB and DL. (1) From the average number of MWs (Fig. 1 and Table 1), we could see that the values of MWs are similar (p=0.73) for AR (300.42) and CS (361.74), as well as the values (p=0.93) for PC (363.57) and SG (367.04), while AB (405.94) is significantly higher than that of PC (p=0.0026) and LJ (p=0.0212). (2) The ingredient in CS is most hydrophobic with the average number of MlogP of 7.16, which is higher than AR of 4.27, followed by AB, PC and SG, which share the similar values. While for LJ, its average number of MLogP exhibits the lowest degree. (3) The average number of H-bond donors of AR is similar with CS (p=0.053), while AB (3.80) is larger than that of other herbs, especially CS of 0.76 (p=0.00058). (4) The average of H-bond acceptors (HAcc) of AB (7.98) is significantly larger than AR of 2.51 (1.14E−15) and CS of 2.5 (2.12E−14). (5) The OB and DL analysis indicate much difference among these six herbs. For OB, we could realize that SG exhibits the highest value of 33.24, followed by AR and CS, which possess the similar OB values, and AB exhibits the lowest (22.28), which is significantly different from others. (6) For DL, unlike OB distribution, PC possesses the highest average value of 0.49, followed by CS of 0.32 and AB of 0.31. Whereas LJ shows the lowest average DL index (0.23). Besides, AR and SG exhibit the similar values (AR=0.29, SG=0.25), which display no significant difference (p=0.42).

Fig. 1. 

The profile distributions of six significant properties for all ingredients in six herbs of MSW including Atractylodes chinensis (DC.) Koidz. (AR), P. chinense Schneid. (PC), Achyranthes bidentata Bl. (AB), Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (CS), Lonicera japonica Thunb. (LJ) and Smilax glabra Roxb. (SG) to analyze the molecular diversity of ingredients. The significant properties consist of molecular weight (MW), Moriguchioctanol–water partition coeff. logP (MLogP), number of donor atoms for H-bonds (nHDon), number of acceptor atoms for H-bonds (nHAcc), oralbioavailability (OB) and drug-likeness (DL).

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Table 1. Comparison of ingredient properties between AR, PC, AB, CS, LJ and SG (mean±SD).

Index	AR	PC	AB	CS	LJ	SG
MW	300.42	363.57 (136.00#)	405.94 (200.10##,270.91*)	361.74 (157.94, 156.44)	307.7 (189.50, 190.22)	367.04 (129.68, 223.87)
ALogP	4.27	3.29(2.37)	3.505 (2.63,2.63)	7.16 (2.59##, 3.78**)	2.5 (3.16##,3.11)	3.25 (2.93, 2.89)
Hdon	1.31	1.62(1.85)	3.9 (3.27##, 3.27**)	0.76 (1.25, 1.70*)	2.38 (2.90#,2.92)	3.08 (2.19##, 3.20**)
Hacc	2.51	5.03(3.08##)	7.98 (5.43##, 6.11**)	2.5 (2.16, 2.82**)	4.87 (5.12##,5.03)	6.08 (3.38##, 5.07)
OB	32.90	32.3(14.51)	22.28 (17.32##, 17.71**)	32.90 (13.98, 15.43)	30.01 (20.02, 20.20)	33.24 (20.08, 20.49)
DL	0.29	0.49(0.31##)	0.31 (0.30, 0.32**)	0.32 (0.28, 0.30**)	0.23 (0.26, 0.28**)	0.25 (0.27, 0.30**)

SD, standard deviation; AR, Atractylodes chinensis (DC.) Koidz.; PC, Phellodendron chinense Schneid.; AB, Achyranthes bidentata Bl.; CS, Coix lacryma-jobi L. var. mayuen (Roman.) Stapf; LJ, Lonicera japonica Thunb.; SG, Smilax glabra Roxb

##

p<0.01.

#

p<0.05 when compared with AR.

⁎⁎

p<0.01.

⁎

p<0.01 when compared with PC.

Table options

In general, all the results show that although there are diverse ingredients in six herbs, many of them still have similar chemical properties. To some extent, as dampness-eliminating herbs, the properties of AR are similar with CS, the same as PC and LJ, which play an important role in clearing heat. What’s more, SG has somewhat similar properties with AR.

3.2. Targets prediction in the cI-cT Network

It could be meaningful to bridge ingredients in MSW and gouty diseases via their common targets. We constructed the candidate ingredient-target network (cI-cT), which contains active ingredients in this formula and their corresponding targets, and then mapped these nodes onto the network. As depicted in Fig. 2, 44 candidate ingredients in this formula yielded 71 targets with anti-inflammatory and UA-lowering therapeutic effects. Targets in the outer circle show much less interactions with the candidate ingredients than those in the inner, which also indicate that many candidate targets are hit by only one candidate ingredient, but some could be modulated by multiple rather than single one. For example, some inflammatory cytokines such as IL-6, PTGS2, MAPK1, are activated by multiple ingredients including Wogonin, Berberine, Luteoline. Besides, some UA synthetase XOD and transporters OAT1, GLUT9, could also be modulated by more than one. Thus, we could have a rough observation on the relationships between candidate ingredients and corresponding targets from the cI-cT network.

Fig. 2. 

Candidate ingredient-target network of MSW on treating gout. It was built by linking 44 candidate ingredients (M) and 71 corresponding targets (G and P) which were validated in published literatures. Diamond represents ingredients; Circle represents targets. Targets in the inner circle show much more interactions with candidate ingredients than those in the outer circles. (AR, A. chinensis (DC.) Koidz.; PC, P. chinense Schneid.; AB, A. bidentata Bl.; CS, C. lacryma-jobi L. var. mayuen (Roman.) Stapf; LJ, L. japonica Thunb.; SG, S. glabra Roxb.).

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3.3. OB prediction and DL evaluation of candidate ingredients in MSW

OB represents a subcategory of absorption and is the fraction of the orally-administered does that reaches the systemic circulation unchanged (Xu et al., 2012). It is one of the essential used pharmacokinetic parameters in drug screening cascades. While DL is a qualitative concept used in drug design for how “drug-like” a substance is with respect to factors like OB. It is estimated from the molecular structure before the substance is even synthesized and tested. In order to further screen out the potential active ingredients from the candidate ingredients in MSW, OB prediction and DL evaluation were conducted. As listed in Table 2, the OB and DL calculation show that 31 of 44 candidate ingredients in MSW possess proper values.

Table 2. Chemical information of the potential ingredients.

No.	Compound	OB	DL	Structure
ARM1	Beta eudesmol	29.97	0.1
ARM2	Palmitic acid	19.3	0.1
ARM3	AtractylenolideI	37.37	0.15
ARM4	Atractylenolide III	31.66	0.17
ARM6	Wogonin	30.68	0.23
ARM8	Vanillic acid	35.47	0.04
PCM1	Syringin	14.64	0.32
PCM2	Berberine	36.86	0.78
PCM3	Jatrorrhizine	19.65	0.59
PCM5	Obacunone	43.29	0.77
PCM6	Tetrahydroberberine	53.83	0.77
ABM2	Beta sitosterol	36.91	0.75
ABM4	Ecdysterone	6.94	0.82
ABM5	Betaine	40.92	0.01
CSM1	Coixol	63.01	0.05
CSM2	Coixenolide	32.4	0.43
CSM3	Oleic acid	33.13	0.14
CSM4	Triglyceride	33.61	0.03
LJM1	Luteoline	36.16	0.25
LJM5	Chlorogenic acid	13.61	0.31
LJM6	Caffeic acid	54.97	0.05
LJM7	Ferulic acid	40.43	0.06
LJM8	Rutin	11.7	0.683
SGM1	Quercetin	46.43	0.28
SGM2	Astilbin	36.46	0.74
SGM3	Taxifolin	60.51	0.27
SGM4	Epicatechin	48.96	0.24
SGM6	Kaempferol	41.88	0.24
SGM7	Resveratrol	19.07	0.11
SGM8	Oxyresveratrol	109.29	0.13
SGM9	Dioscin	17.75	0.06

Table options

3.3.1. AR

The rhizome of A. chinensis (DC.) Koidz. has been used widely in Chinese traditional medicine for various indications such as rheumatic diseases, digestive disorders, night blindness, and influenza. These traditional uses are explained by the compound’s ability to eliminate dampness, strengthen the spleen, expel wind-cold from the superficial parts of the body, and clear away the common cold ( Koonrungsesomboon et al., 2014). Of 11 ingredients with corresponding targets in which 70.45% have appropriate OB (≧30%) and DL values (DL≧0.1), including β-Eudesmol, Palmitic acid, Wogonin, Atractylenolide І, Atractylenolide III, Vanillic acid. As seen in Table 2, almost all the ingredients have relatively moderate bioavailability (OB≈30%). However, Vanillic acid, as an active ingredient in AR, exhibits low DL value (OB=35.47%, DL=0.04). Considering its UA-lowing effect through targeting uarte synthetase and transporters ( Oskoueian et al., 2011 and Wang and Sweet, 2012), it was also selected for the further study.

3.3.2. PC

Cortex Phellodendri, known as “Huang Bai”, is derived from the dried bark of P. chinense Schneid. or Phellodendron amurense Rupr. (Family Rutaceae) according to Chinese Pharmacopoeia Commission 2010. It is one of the most commonly used traditional Chinese medicinal plants, which is widely used to remove damp heat, quench fire, counteract toxicity, relieve consumptive fever and also effective in curing dysentery, diarrhea and other syndromes ( Yen, 1994). Among the six active ingredients in PC, five of them
have suitable OB and DL values, including Berberine, Jatrorrhizine, Obacunone, Tetrahydroberberine and Syringing. However, Jatrorrhizine and Syringing, the main active components in PC ( Zhang et al., 2012), exhibit low DL values (19.65%, 14.64%). Considering their potential pharmacological effects ( Hu et al., 2009 and Gong et al., 2014), they were still adopted for the further targeting.

3.3.3. AB

A. bidentata Blume (Amaranthaceae family), a perennial herbaceous plant, is widely distributed and grown in China. The roots of A. bidentata Blume have proven to possess many pharmaceutical properties, including immunostimulant, anti-inflammatory, antitumor, analgestic, cognition-enhancing, anti-osteoporotic, anti-bacterial, uteri-excitant and antifertility activities ( Li et al., 2007). However, only three from the candidate ingredients could be used for further targeting. Although Ecdysterone has poor OB value (OB=6.94), it is one of the representative ingredients of AB ( Shen et al., 2011). As for Betaine, which exhibits low DL value (DL=0.01), it is also one of the major ingredients for lowering UA, and could protect the occurrence of inflammation by inhibiting many inflammatory cytokines ( Fan et al., 2014). In view of considerations above, we selected the two ingredients as potential ingredients.

3.3.4. CS

CS is the dry and ripe seeds of C. lacryma-jobi L. var. mayuen (Roman.) Stapf, which first sets out in “Shen Nong’s Herb”, and has the effects of clearing damp and promoting dieresis, invigorating the spleen and stopping diarrhea, resolving toxin and dissipating binds. Modern pharmacology study shows that it is commonly used Chinese medicine in treatment of anti-cancer, boosting immunity, anti-hypertensive, anti-oxidation, anti-inflammatory ( Liu et al., 2010). The details were shown in Fig. 3, four ingredients in CS including Coixol, Coixenolide, Oleic acid and Triglyceride have suitable OB values. Although some of them exhibit low DL values, they were still included for further targeting, since they are the active ingredients in CS.

Fig. 3. 

Core ingredient-target-pathway network of MSW on treating gout. There were 30 core potential ingredients (M) and 25 corresponding targets (G and P) which were validated in published literatures. Node size of targets and pathways are proportional to the number of ingredients associated with the nodes. This network depicted a clear three-level structure of the mode of core ingredients of MSW, that is multi-ingredient targeted groups of genes through multi-pathway to show their pharmacological effects. Diamond represents ingredient; Circle represents target; Vee represents Gout related pathway. The purple circles represent inflammatory cytokines and the yellow represent uric acid synthetase and transporters. (AR, A. chinensis (DC.) Koidz.; PC, Phellodendron chinense Schneid.; AB, A. bidentata Bl.; CS, C. lacryma-jobi L. var. mayuen (Roman.) Stapf; LJ, L. japonica Thunb.; SG, S. glabra Roxb.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure options

3.3.5. LJ

L. japonica Thunb. (family Caprifoliaceae) are perennial arching shrubs or twining vines that are commonly found as dried buds or early-opened flowers. It is commonly used in traditional Chinese medicine for the treatment of various diseases, including arthritis, diabetes mellitus, fever, infections, sores, and swelling. Pharmacological studies have shown that extracts of LJ flower buds have a broad spectrum of biological activity, including antibacterial, anti-inflammatory, antioxidant, antiangiogenic, antipyretic, antiviral, and hepatoprotective effects ( Qian et al., 2007). It has nine candidate ingredients in which over half show proper OB values (>30%). Interestingly, Caffeic acid and Ferulic acid, the representative active ingredients in LJ ( She and Zhu, 2008), exhibit low DL values, although their OB values meet the standards. They show potent antisepsis and anti-inflammatory effects ( Ma et al., 2010, Zhang et al., 2014 and Tang et al., 2014) and could be selected for further evaluation. Based on all these considerations, it was reasonable to believe that five could be listed as potential active ingredients.

3.3.6. SG

SG, belonging to the Smilacaceae family, Smilax genus, is described to be effective in xeransis, detoxification, and easing joint movement in traditional Chinese medical literature, which include the Compendium of Materia Medica and the State Pharmacopoeia of the People’s Republic of China. Extract of SG has also been studied for multiple pharmacologic activities, such as immunomodulatory (Jiang and Xu, 2003), anti-inflammatory (Jiang et al., 1997). It has nine valid ingredients in which almost all of them have appropriate OB and DL values. Those ingredients that meet the selection criteria are selected for further study, including Quercetin, Astilbin, Taxifolin, Epicatechin, Oxyresveratrol, Kaempferol, Resveratrol, Dioscin. Notably, Oxyresveratrol has significantly high OB value (109.29%), which means it is more easily to be absorbed into our bodies. Another active ingredient Resveratrol, exhibits low OB value (19.07%). However, as the homolog of Oxyresveratrol, it is rather effective for anti-inflammation (Chen et al., 2011) and especially benefit for lowering UA by reducing the expression of UA synthetase XOD or transporters such as OAT1, GLUT9 (Shi et al., 2012). Therefore, it was added as potential ingredients for further analysis.

3.4. Network construction and analysis

A drug-target network is defined as a bipartite network for the drug-target associations consisting of two disjoint sets of nodes. We constructed the networks to find out the active ingredients in MSW, as well as to understand rationality of herb combinations of MSW.

3.4.1. The core potential ingredients-target-pathway network

Based on the cI-cT network which illustrates the relationships between candidate ingredients and targets, the potential ingredient-target network (pI-pT) was generated by connecting potential ingredients and corresponding targets. In order to clearly distinguish the relationship between ingredients and their targets, we constructed a core potential ingredients-target-pathway network (Fig. 3), that depicts the relationship between 30 core ingredients and 25 corresponding targets through related pathways. In this study, we selected those whose degree values are more than four as the core ingredients from the pI-pT network for further research.

Furthermore, multiple gouty related pathways were listed as follows to reveal possible mechanism involved in gouty treatment. A central feature of gout is inflammation, Table 3 includes several possible pathways related to inflammatory signaling, in which the most important one is the Toll-like receptor (TLR) signaling pathway. Some other pathways, such as NF-kappa B (NF-ƙB), TNF signaling pathways, have been known to be associated with gouty treatment. Another prominent feature of gout is enhanced uric acid, which implies that the synthesis and excretion of UA pathways are also associated with the function of lowing UA.

Table 3. The core targets and their network degrees and related pathways.

No.	Target	Degree	Description	Possible pathway
G5	PTGS2	23	Prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)	NF-kappa B, TNF signaling pathways
G56	TNF	23	Tumor necrosis factor	Toll-like receptor, NF-kappa B, mTOR, MAPK, TNF signaling pathways
G57	IL6	22	Interleukin 6	Toll-like receptor, P13K-Akt, TNF signaling pathways
G46	MAPK1	17	Mitogen-activated protein kinase 1	Toll-like receptor, MAPK, mTOR, P13K-Akt, TNF signaling pathways
G58	IL1B	16	Interleukin 1, beta	Toll-like receptor, MAPK, NF-kappa B, TNF signaling pathways
G8	BAX	11	BCL2-associated X protein	p53 Signaling pathway
G10	BCL2	11	B-cell CLL/lymphoma 2	NF-kappa B, P13K-Akt signaling pathways
G47	MAPK3	10	Mitogen-activated protein kinase 3	MAPK signaling pathway
G4	PIKC3G	9	Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit gamma	P13K-Akt signaling pathway
G31	HSP90AA1	9	Heat shock protein 90 kDa alpha (cytosolic), class A member 1	P13K-Akt signaling pathway
G50	MAPK14	9	Mitogen-activated protein kinase 14	MAPK signaling pathway
G52	IL8	9	Interleukin 8	Toll-like receptor, NF-kappa B signaling pathways Rheumatoid arthritis
G6	MTOR	8	Mechanistic target of rapamycin (serine/threonine kinase)	mTOR, P13K-Akt signaling pathways
G2	PIK3CA	6	Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha	P13K-Akt signaling pathway
G3	PIK3CB	6	Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit beta	P13K-Akt signaling pathway
G23	PPARA	5	Peroxisome proliferator-activated receptor alpha	PPAR signaling pathway
G7	RPS6KB1	4	Ribosomal protein S6 kinase, 70 kDa, polypeptide 1	MAPK, P13K-Akt, mTOR signaling pathways
G15	TGFB1	4	Transforming growth factor, beta 1	MAPK signaling pathway Rheumatoid arthritis
G37	JAK2	4	Janus kinase 2	P13K-Akt signaling pathway
G55	IL10	4	Interleukin 10	JAK-STAT signaling pathway
G59	IL4	4	Interleukin 4	P13K-Akt signaling pathway
P1	XOD	17	Xanthine oxidase	Uric acid synthesis
P3	OAT1	7	Solute carrier family 22 (organic anion transporter), member 6	Uric acid excretion
P5	GLUT9	5	Solute carrier family 2 (facilitated glucose transporter), member 9	Uric acid excretion
P2	URAT1	4	Solute carrier family 22 (organic anion/urate transporter), member 12	Uric acid excretion

Core targets are those whose degree values are more than four from the potential ingredient-target network. The possible pathways are collected from KEGG database.

Table options

As listed in Table 3, the expression of IL-6, TNF, PTGS2, one of the most core targets with highest degrees, could be inhibit by more than 20 distinct ingredients in this formula via TLR, NF-ƙB or mTOR signaling pathways. IL-1β, the major mediator that induces acute gouty arthritis (Schlesinger et al., 2012 and Terkeltaub et al., 2013), is also suppressed by 16 ingredients through TLR, NF-ƙB and MAPK signaling pathways. Moreover, XOD, a key enzyme to generate UA as well as the decisive factor for regulating generation of UA, is intimately connected with the potential ingredients. To be specific, over half potential ingredients are able to inhibit the expression of XOD. It indicates that XOD inhibitory activity might be one of anti-gout mechanisms, and ingredients inhibiting the expression of XOD are effective in reducing UA for gouty treatment. Besides, OAT1 is a potential molecular component in the first step of renal urate secretion, which can mediate the active uptake of organic anions and controls the final exit into the urine via ATP-powered transporters and bidirectional exchangers (EI-Sheikh et al., 2008). More than five potential ingredients in MSW could increase renal OAT1 expression, resulting in the increased urate secretion.

In conclusion, these anti-inflammatory and UA-lowing properties of MSW account, at least in most part, for its pharmacological efficacy against gouty diseases. However, their detailed mechanism involved and clinical significance activity require further study.

3.4.2. Explore the rationality of herb combinations of MSW

The core drug (EM) is descried to treat acute gout through clearing heat and eliminating dampness in terms of TCM (Kong et al., 2004). Basic prescription (SM), which is described in a famous TCM monograph Danxi Xinfa (comprehensive medical book), has been used for hundreds of years in Chinese medicine for treating gouty arthritis with eliminating dampness, clearing heat and promoting blood circulation (Hua et al., 2012 and Shi et al., 2013). Therefore, the network (Fig. 4) was displayed to reveal rationality of herb combinations between ingredients and corresponding targets.

Fig. 4. 

Rationality of herb combinations of MSW network. It contains 31 potential ingredients (M) and 67 corresponding targets (G and P). The diagram of the combined rationality of the core drug (EM), basic prescription (SM) and Modified-Simiaowan (MSW). The targets in the inner circle represents core targets, while those in the outer represents the minor ones. Diamonds in green series on the left represent heat-clearing herbs, while those in yellow series on the right represent dampness-eliminating herbs, and the one in red at the top represents blood-activating herb. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Figure options

EM has both anti-inflammatory and uricosuric effects through hitting most core targets including inflammatory cytokines such as IL-6, TNF-α, COX-2, IL-1β, and UA synthetase XOD or several transporters URAT1, OAT1, OAT3, ABCG2. SM is able to stimulate almost all the core targets, owing to the additional herbs AB and CS, which could hit more inflammatory cytokines and UA transporters, such as IL-10, TGFB1, IL-4, PPARA, GLUT9, hUAT and LEP. Mostly important, MSW, which is derived from SM, could activate all the inflammatory cytokines and UA-lowing targets. The results show that EM plays a key role, while SM plays an important role in MSW for the treatment of acute gout.

According to the nature of Chinese herbs, most of inflammatory cytokines such as IL-6, TNF-α, IL-1β, MAPK1, could be hit by those ingredients such as Berberine, Syringing, Luteoline, Chlorogenic acid, which are just important ingredients in PC and LJ. As antipyretics, PC and LJ play the major role of clearing heat on anti-inflammation for gouty treatment. While AR, CS and SG, used for eliminating dampness in TCM, could reduce UA levels through targeting UA synthetase and transporters. Equally important, AB, as a hemorheologic agent, promotes blood circulation and could be an adjuvant therapy for gouty diseases. These results preliminarily show that the nature of TCM is consistent with the clinical therapeutic effects.

3.5. Experimental validation for anti-gout effects of ingredients in MSW

Berberine, Astilbin, Chlorogenic Acid, Ferulic acid and Caffeic acid are the representative ingredients PC, LJ and SG, which actually play a crucial role in clearing heat on anti-inflammation. Besides, they are highly connected with the core targets. Therefore, they were selected to investigate therapeutic effects for treating acute gouty arthritis in MSU-induced HUVECs.

3.5.1. Effect on cell viability stimulated by MSU

To determine whether ingredients in MSW have protective effects on HUVECs damaged by MSU crystals, cell viability was assessed using MTT assay. Results (Fig. 5) confirmed that all ingredients exhibit significant protective effect on HUVECs compared with the model. To be specific, compared with the model, effect of Berberine has a significant difference (p<0.05) with dose-effect relationship. While effect of Astilbin exhibits an extremely significant difference (p<0.01) with does-effect relationship varying from low to middle dosage. The same as Caffeic Acid, Ferulic Acid and Chlorogenic Acid (p<0.01), and Chlorogenic Acid shows a dose-effect relationship.

Fig. 5. 

Effects of five representative ingredients in MSW on the cell viability in MSU crystal-induced HUVECs. Cells were culture for 48 h in MSU crystal (100 μg/ml), indomethacin (20 μg/ml) and different ingredients (Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid and Ferulic Acid) at various concentrations (2.5 μg/ml to 250 μg/ml). Cell viability was assessed by MTT assay. Values were expressed as mean ±S.D. (n=6). ^#p<0.01 represents a significant difference compared with the blank, ^⁎p<0.01 and ^⁎⁎p<0.05 represent a significant difference compared with the model groups. MSU: monosodium urate.

Figure options

3.5.2. Effect on expression of ICAM-1 stimulated by MSU

As depicted in Fig. 6, the expression level of ICAM-1 in HUVECs was a lot in untreated HUVECs and was augmented by stimulation with MSU crystals (100 μg/ml) for 24 h. Treatment of HUVECs with Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid, Ferulic Acid (2.5 μg/ml) and Indomethacin (2.5 μg/ml) for 24 h blocked the increase of ICAM-1 expression induced by MSU crystals to different extents compared with the model.

Fig. 6. 

Effects of five representative ingredients in MSW and Indomethacin on MSU crystal-induced ICAM-1 expressions in HUVECs. Cells were pretreated with different ingredients (Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid and Ferulic Acid) at concentrations of 2.5 μg/ml and Indomethacin (2.5 μg/ml) for 24 h. MSU: monosodium urate.

Figure options

In general, we could draw such a conclusion that Berberine, Astilbin, Chlorogenic Acid, Caffeic Acid and Ferulic Acid are the effective ingredients for acute Gout. This study has also demonstrated for the first time that ingredients in MSW exhibit significant does-dependent inhibitory effects in experimental gouty arthritis models in vivo induced by MSU crystals.

4. Discussions

Gout is a type of inflammatory arthritis induced by the deposition of MSU crystals in the tissues or a joint, and is a polygenetic disorder induced by the increase of UA synthesis or decrease of excretion through UA synthetas XOD and transporters, accompanying with inflammation caused by inflammatory cytokines (Sha and He, 2009 and Tong et al., 2013).

Given the intensity of inflammatory reactions characterizing the acute attack, NSAIDs and Colchicine, as appropriate first-line agents in clinical use (Neogi, 2010), could stimulate several targets such as COX-2, MAPK1, in a” single-target” paradigm (Yuan et al., 2000 and Ben-Chetrit et al., 2005). However, Colchicine’s toxicity was wildly reported (Finkelstein et al., 2010). NSAIDs also present some adverse effects such as gastrointestinal toxicity, renal toxicity and gastrointestinal bleeding (Sabina et al., 2011). Besides, Benzbromarone, an uricosuric agent, has been used for more than 25 years to control hyperuricemia by inhibiting the main renal UA transporter URAT1, which could suppress the reabsorption of UA (Kunishima et al., 2007). It was withdrawn from the European market due to the risk of severe hepatotoxicity (Kumar et al., 2005). Allopurinol, as a frequently used XOD inhibitor in clinical use (Horiuchi et al., 2000 and Chen et al., 2005), could cause severe hypersensitivity and was restricted in patients with renal insufficiency (Hande et al., 1984).

Predicted by network pharmacology, many ingredients in MSW are able to stimulate MAPK1, COX-2, XOD, URAT1 through inflammatory and synthesis or secretion of UA pathways, which are just the targets of classical anti-gout drugs. Besides, those genes such as IL-8, TNF-α, IL-1β, OAT1, GLUI9 (Rott and Agudelo, 2003, Lioté, 2003 and Tausche et al., 2004), that may induce the occurrence of gout, could also be targeted by various ingredients in MSW. Experimental results show that MSW could play an important role in terms of anti-inflammation by inhibiting expression of ICAM-1, preventing neutrophil infiltration and apoptosis of HUVECs (Shi et al., 2013) and significantly reducing serum UA levels, together with decreasing liver XOD activity and IL-1β, IL-6 levels (Zeng et al., 2014). Thus, we consider that MSW may be effective in treating gouty diseases by cooperation of multiple targets and multiple pathways.

The study also indicates the rationality of herb combinations of EM and SM in MSW for gouty treatment. To be specific, EM is a classical formula used for clearing heat and eliminating dampness to treat gouty diseases through stimulating most core targets such as XOD, URAT1, IL-6, TNF-α, which are consistent with those collected from published literatures (Kong et al., 2004 and Kao and Dong, 2013). As a classic formula derived from EM, SM is commonly used to treat gout and hyperuricemia in clinical (Hu et al., 2010 and Hua et al., 2012), eliminating dampness, clearing heat and promoting blood circulation in terms of TCM through targeting UA synthetase, transporters and inflammatory cytokines. Literatures also suggest that SM could suppress inflammation and inhibit the release of IL-1β, TNF-α (Xu et al., 2013 and Liu et al., 2014). Besides, it could also significantly inhibit XOD activities in serum and liver, and reverse oxonate-induced alterations in renal URAT1, GLUT9, OAT1 mRNA and protein levels, resulting in the enhancement of renal urate excretion (Hua et al., 2012), which are consistent with those predicted from the gouty network. Mostly important, MSW, which develops based on TCM theory, clinical research, and pharmacodynamic findings (Yin and Si, 2006), is prepared with two additional agents on the basis of SM and does a more comprehensive and synergetic effect than EM and SM on gouty diseases.

Acute gouty arthritis is caused by MSU crystals in and around the joints, which causes acute inflammation that manifests as intense pain, swelling, and reddening of the skin (Chia et al., 2008). TCM believes that a formula with clearing heat, eliminating dampness and promoting blood circulation is useful for treating acute gout. Chemical ingredients in six herbs (AR, PC, AB, CS, LJ and SG) of MSW were compared based on those important drug-associated descriptors including MW, ALogP, Hdon, Hacc, OB and DL. Results from herbal ingredients comparisons (Table 1) and networks (Fig. 4) suggested that AR and CS have similar chemical properties on most of the descriptors except ALogP. Both of them are used for eliminating dampness by targeting most UA synthetase and transporters through synthesis or excretion of UA. Besides, PC and LJ, which are similar with each other on those descriptors including MW, ALogP, Hdon, Hacc and OB, play the role of clearing heat on anti-inflammation by targeting many inflammatory cytokines such as TNF-α, IL-6, IL-1β via TLR, NF-ƙB and other signaling pathways. While SG, having somewhat similar properties with AR and PC, is useful in both clearing heat and eliminating dampness. AB, a hemorheologic agent, which is different from others, could promote blood circulation by activating inflammatory cytokines and UA-lowing targets. The study based on network pharmacology confirms herb combinations of MSW including heat-clearing, dampness-eliminating and blood-activating drugs is rational for gouty treatment. Experimental results first demonstrated that core ingredients in MSW could protect HUVECs by reducing cell apoptosis and inhibiting expression of ICAM-1 in MSU-induced HUVECs for anti-acute gout. Besides, these ingredients such as Quercetin, Resveratrol, Palmitic acid, Astilbin, down-regulated hepatic XOD and enhanced renal urate excretion in hyperuricemic mice by up-regulating OAT1 and down-regulating URAT1 and GLUT9 mRNA and protein levels in the kidney (Wang and Sweet, 2012, Shi et al., 2012 and Hu et al., 2012). Researches confirm that various ingredients in MSW are effective for gouty treatment by targeting inflammatory cytokines and UA synthetas or transporters through different related pathways, which indicates a whole regulation with the paradigm of “multi-ingredient, multi-target, multi-pathway”.

5. Conclusion

Combination therapy is a fundamental principle of TCM, which is developed for the purpose of maximizing the efficacy and minimizing the adverse effects or toxicity (Sucher, 2013). An herbal formula is not a simple quantitative addition of different herbs (Jia et al., 2004), instead, all the herbs of a defined prescription should correspond exactly to the related diseases, showing a significantly better effect than the constituent herb used alone (Scholey and Kennedy, 2002). We integrate our previous methods into a TCM network pharmacology platform to illustrate network connections between multiple targets of ingredients in herbal formula and multiple targets of a specific disease such as gout. Our study has successfully predicted the active ingredients in MSW and uncovered the rationality of herb combinations of MSW. Therefore, such a network pharmacology strategy and platform is expected to make the systematical study of herbal formulae achievable and make the TCM drug discovery predictable.

Conflict of interest

All authors have no financial or scientific conflict of interest with regard to the research described in this manuscript.

Acknowledgement

This study is supported by Natural Science Foundation of Jiangsu Province, China (no. BK2002033), Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China and Research Foundation of Nanjing University of Traditional Chinese Medicine, China.

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