The 1st Euro-Mediterranean Workshop:Natural Products in Health and Diseases:Cairo,Egypt, March 2,2015

Kamal D.Moudgil*,Ashraf A.Khalil**

aUniversity of Maryland School of Medicine,Baltimore,USA

bCity of Scientifc Research and Technological Applications,Borg Elarab,Alexandria,Egypt

The 1st Euro-Mediterranean Workshop:Natural Products in Health and Diseases:Cairo,Egypt, March 2,2015

Kamal D.Moudgila,*,Ashraf A.Khalilb,**

aUniversity of Maryland School of Medicine,Baltimore,USA

bCity of Scientifc Research and Technological Applications,Borg Elarab,Alexandria,Egypt

A R T I C L EI N F O

Article history:

Available online 10 February 2016

BioNat

Natural products

Workshop

Health

Disease

EMALS

Natural plant products have been used for centuries for health maintenance and treatment of a variety of diseases in various traditional systems of medicine in Egypt and other African countries,India,China,and other countries around the world.Over the past several decades,the popularity of herbal and other natural products has gradually increased in many western countries.Despite the increasing popularity of natural products,both the public and the professionals are skeptical about the use of these products.Reasons for this skepticism include,but are not limited to,the lack of proper documentation about the source and formulations used;standardization of the composition,batch-to-batch consistency,documented safety of herbal products;and information about the mechanisms of action of these products.The1st Euro-Mediterranean Workshop:Natural Products in Health and Diseaseswas organized by the Euro-Mediterranean Association of Life Sciences(EMALS)and various other local organizations to review and discuss the above-mentioned strengths and limitations of the use of natural products.The aim of this workshop was to share best practices and expertise about natural products,and to identify opportunities for collaboration among researchers,health professionals,and business personnel in a productive atmosphere.

?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical

University.This is an open access article under the CC BY-NC-ND license(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

For thousands of years,natural products have played a very important role in health care and prevention of diseases.The ancient civilizations of the Chinese,Indians and North Africans provide written evidence for the use of products from natural sources for curing various diseases[1].The earliest known written document is a 4000-year-old Sumerian clay tablet that records remedies for various illnesses[2].For example, mandrake was used for pain relief,turmeric for blood clotting properties,roots of the endive plant for gall bladder disorders,and raw garlic for circulatory disorders.These agents are still being used in several countries as alternative medicines.

According to recent studies conducted by the World Health Organization(WHO),about 80%of the world’s population relies on traditional medicine[3].About 121 drugs prescribed in the USA today are from natural sources,90 of which are derived either directly or indirectly from plant sources[4].Furthermore,forty-seven percent of the anticancer drugs in the market are from natural products or natural product mimics[4].

It is well documented that most natural products are enriched with bioactive components that have protective action against potentially disease-inducing events.There is currentlyagrowingbodyofevidencethatsupplementingthehuman dietwithnaturalproductsoffersmajorbeneftsforhumanhealth and well-being.The use of complementary/alternative medicine,functional foods,and other natural products has been increasing rapidly worldwide,mostly because of the perceivedsafetyofsuchproducts.Theissuesregardingtheeffcacy andsafetyofcurrentlyavailablemodern(allopathic)drugshave prompted the search for safer and more effective alternatives. As elaborated below,this workshop was focused on the use of natural products for health and disease prevention.

The aim of the 1st BioNat International Workshop was to share best practices and expertise,and to identify opportunities for collaboration.Well-known researchers,health professionals,and business personnel presented their expertise in a productive atmosphere.It was organized by the Euro-MediterraneanAssociation of Life Sciences(EMALS)and various other local organizations.EMALS aims to contribute effectively to the development of international and national programs of scientifc research;to provide strategic services that affect the lives of scientists,biomedical researchers and their assistants;to develop their professional and research competence;and to enlist their participation in increasing the awareness of various segments of the society about science in general and natural products in particular.

The above-mentioned BioNat workshop considered a range of issues,such as(1)classifcation and distribution of wild medicinal plants in Egypt;(2)separation and identifcation of bioactive compounds from plants;(3)natural products in the prevention and treatment of human diseases;(4)antimicrobial activity of natural products;(5)search for microbial sources of natural and unnatural products;(6)natural products chemistry,chemosystematics and quality control;and(7)patentability in pharmacognosy,plant extraction and dietary supplements.

To begin with,Dr.Ashraf A Khalil introduced the speakers,presented a brief synopsis of the importance of the workshop and discussed the day’s program in detail.During the period of this workshop,the participants met with experienced professionals from diverse disciplines,but all connected with natural products research and applications.Thus,professors,project leaders of R&D,post-doc researchers,CEOs, technical managers,heads of departments,group leaders,PhD students,and research scientists from both developed and developing countries were brought together on this occasion to share current knowledge and future strategies for the use of medicinal and herbal products for the prevention and treatment of human diseases.

The format of the workshop consisted of lectures and discussions aimed at reviewing and exchanging basic information about herbal products and their use in different diseases,and sharing information via a questionnaire completed by the participants.The titles of lectures,the names of the presenters, and the abstracts summarizing the major fndings are given below.A copy of the questionnaire is also enclosed.The responses to questions were read,analyzed,and utilized to guide the discussions within and between lectures.

2.Presentations

2.1.Nanoencapsulation of natural products for their enhanced anti-cancer activity

The introductory lecture was presented by Prof.Hasan Mukhtar (School of Medicine and Public Health,Department of Dermatology,University of Wisconsin,Madison,WI,USA).He enlightened the participants with the information that the practical applicability of the bioactive food components for the prevention and treatment of human cancer has met with limited success largely due to ineffcient systemic delivery and bioavailability of promising agents despite highly encouraging results in preclinical settings.His group was the frst to employ nanotechnology to improve the outcome of cancer chemoprevention by natural agents and to introduce the concept of,as well as the term,Nanochemoprevention[5]. To establish the proof-of-principle,they encapsulated epigallocatechin-3-gallate(EGCG)in PLA-PEG nanoparticles(NPs) and demonstrated a ten-fold dose advantage over native EGCG in human prostate cancer(PCa)cells bothin vitroandin vivo. Later,theyreportedtheeffcacyassessmentofa nanotechnology-based oral formulation of chitosan NPs encapsulating EGCG in a PCa tumor xenograft mouse model.Prof. Mukhtar’s lab demonstrated that nanoformulated EGCG signifcantly induced apoptosis and reduced the expression of markers of cell proliferation in the tumors[6].Recently,they proposed targeted NPs as novel prototypes for the delivery of EGCG,thereby exploiting small molecular entities able to bind to prostate-specifc membrane antigen(PSMA).PSMA-specifc cellular binding and uptake of the nanosystems were detected,and EGCG polymeric NPs showed an increased antiproliferative activity in both PSMA-negative and PSMA-positive PCa cells,with better effcacy in PSMA-positive cells. The study revealed a better response to targeted NPs in inhibiting the tumor growth as compared to non-targeted EGCG. In a parallel study,the team of Prof.Mukhtar tested the effcacy of EGCG encapsulated in chitosan NPs and surfacefunctionalized withA10 2′-fuoropyrimidine RNA aptamers(chit-EGCG-Apt)that recognize the extracellular domain of PSMA. They detected PSMA-specifc internalization and accumulation along with a sustained and constant release of EGCG from the nanobioconjugates.Chit-EGCG-Apt also led to a PSMA-specifc enhanced anti-proliferative activity in PCa cells with a signifcant decrease in IC50.EGCG encapsulated in chit-EGCGApt also retained its mechanistic identity for induction of apoptosis,modulation of cell cycle,and inhibition of invasion and migration.They also designed novel polymeric NPs composed of PLGA-PEG-COOH encapsulating resveratrol(nano-RSV)to enhance its bioavailability and to improve the intracellular penetration.These NPs were able to control the RSV release at both acidic and physiological conditions.Fluorescence microscopy revealed that NPs were effciently takenup by all PCa cell lines.Furthermore,nano-RSV signifcantly improved the cytotoxicity compared to that of free RSV toward PCa cells proving a consistent sensitivity toward both the androgen-independent and hormone-sensitive cells.Prof. Mukhtar ended his lecture with a note that additional work is required to make this approach useful in preventing and/ or treating cancer in the human population.

2.2.Anti-arthritic activity of traditional medicine herbal products

The second keynote lecture was presented by Prof.Kamal D. Moudgil(University of Maryland School of Medicine,Baltimore,USA).He started his talk by introducing adjuvant arthritis (AA)as an experimental model of human rheumatoid arthritis(RA)[7].This model has extensively been used by his group and many other investigators for testing potential antiarthritic agents,including herbal products.AA shares several features with RA.The disease is inducible in Lewis rats by subcutaneous injection of heat-killedMycobacterium tuberculosisH37Ra(Mtb).Arthritis appears within about 10 d of Mtb injection,peaks about 18 d,and then gradually regresses in the next 7–10 d[7].Prof.Moudgil and colleagues have tested a multiherbal traditional Chinese medicine(TCM)formula,Huo-luoxiao-ling(HLXL)dan,in the AA model to determine its arthritissuppressive effects as well as the immunological and biochemical mechanisms involved[7,8].HLXL consists of 11 defned herbs.This herbal extract was characterized by HPLCMS using defned compounds as biomarkers[7,8].The herbal mixture was also subjected to quality control and toxicity testing.The feeding of arthritic Lewis rats with HLXL(2.3 g/ rat/d)by gavage was begun at the onset of AA and then continued throughout the course of disease.Arthritis severity was graded on a scale from 0 to 4 per paw.The draining lymph node cells of rats were subjected to immunological tests to measure the production of cytokines,whereas serum was tested for levels of antigen-specifc antibodies and nitric oxide (NO).Also tested was metalloproteinase-9(MMP-9)activity.HLXL reduced the severity of AA,altered the balance between proinfammatory and anti-infammatory cytokines in favor of the latter,and reduced NO and MMP-9.Through this prototypic study,they not only highlighted the importance ofTCM for arthritis therapy,but also emphasized the signifcance of product characterization,quality control,toxicity assessment,and mechanistic studies to offer scientifc validation of the use of herbal products for the treatment of autoimmune diseases.Prof. Moudgil emphasized that using state-of-the-art scientifc methods in natural products research is essential to gain the confdence of the public as well as the professionals in eventual application of natural products as therapeutic agents for human diseases.

2.3.Classifcation and distribution of wild medicinal plants in Egypt

The third lecture was presented by Dr.Mona Mohamed Marzouk (Department of Phytochemistry and Plant Systematic,National Research Centre,Cairo,Egypt).Egypt has always been one of the most important countries in the feld of exportation of medicinal plants.More than one thousand species of medicinal and aromatic plants are growing in Egypt,which create a base of raw natural materials for the folkloric medicine,the pharmaceutical industry,and for phytochemical research.The large numbers and variability of medicinal plants and their importance in the feld of folk medicine in Egypt encourage chemists to investigate their chemical and biological constituents.Medicinal plants are classifed in many ways,for example,according to their life period(annual,biennial or perennial),their life form(tree,shrub or herb),their usage (medicinal,culinary,aromatic,or ornamental),or their active constituents(aromatic;containing volatile oils,astringents;containing tannins,bitter;containing phenol compounds,saponins, and alkaloids,mucilaginous;containing polysaccharides and nutritive).Dr.Marzouk explored the principles ofTaxonomy of Medicinal Plants,which involve three steps,namelyplant identifcation(the identifcation of an unknown plant by a professional taxonomist using identifcation reference books and by comparison with previously collected specimens),plant nomenclature(once a plant specimen has been identifed,its name and properties are registered:family,genus and species) andplant classifcation(it is the placement of known plants into groups or categories to show some relationship within the plant kingdom).Finally,Dr.Marzouk discussed the distribution of wild medicinal plants in Egypt.The Egyptian fora is composed of many common species that cover most of the area and also many rare species that have limited distribution.Over 1000 medicinal species belonging to 340 genera and 78 families are growing wild in Egypt.Most of these plants are belonging to the familiesCompositae,Labiatae,Brassicaceae,Leguminosae,Umbelliferae,Roseaceae,Rutaceae,Solanaceae,Liliaceae,Caryophyllaceae,Boraginaceae,Ranunculaceae,Papaveraceae,Malvaceae,Cucurbitaceae,Verbenaceae,ScrophulariceaeandPlantaginaceae.Mediterranean coastal district(350 species),Nile region(189 species),North Sinai(141 species)and South Sinai (132 species,including 12 endemic species)possess the highest number of species,while Eastern desert(70 species),Western desert(57 species)and Oasis(46 species)have relatively lesser number of species.

2.4.Separation and identifcation of plant phenolics

The fourth lecture was presented by Nesrine M.Hegazi(Researcher of Phytochemistry,National Research Center).Plant phenolics share a common structural feature:an aromatic ring bearing at least one hydroxyl substituent.Several classes of natural products are therefore considered as phenolics,including phenolic acids and phenyl propanoids,favonoids, tannins,coumarins,lignans and stilbenes.Phenolic metabolites are extracted from the plant material with solvent extraction,soxhlet extraction or maceration.Newly developed methodologies include ultrasound-assisted extractions, pressurized liquid extraction,supercritical fuid extraction and microwave-assisted extraction.Fractionation and purifcation of the extracts thus obtained are achieved by column chromatography and/or liquid–liquid extraction.For separation and detection of the phenolic metabolites,paper chromatography,thin layer chromatography,gas chromatography or high performance liquid chromatography is performed. Identifcation and structure elucidation involve conventional chemicalmethodsandthemoreadvancedspectraltechniques(mass spectrometry,UV spectrophotometry,and NMR spectroscopy).

2.5.Patentability in pharmacognosy,plant extraction and dietary supplements

Dr.Saly Odah(Egyptian Innovation Offce,The Egyptian Academy of Science andTechnology,Cairo,Egypt)gave a comprehensive presentation covering practical(patentability)aspects of natural products research.Dr.Odah overviewed and explained in details the following topics:(1)What is a patent? (2)Why should I apply for a patent?(3)Benefts of a patent application,(4)What happens if I do not apply?(5)What does a patent application include?(6)Types of pharmaceutical applications,(7)Discussion of each type of pharmaceutical application in detail,and(8)Case studies.

3.Questionnaire

The questionnaire(Table 1)addressed two important aspects of natural products research:(I)research aspect of natural products,and(II)practical use of natural products.In regard to research aspects,majority of participants had used one or more natural products in their research work.These products varied from crude/purifed plant extract to isolated single/multiple bioactive components of medicinal plants.To name some,green tea polyphenolic extract,EGCG,pomegranate,Huo-luo-xiaoling(HLXL)dan,Celastrus,Celastrol,Naja haje crude venom, Jojoba,Moringa,Morus alba,Berberine,triterpenoids from plants, favonoids from plants,phenolics fromL.nobilis,Punica granatum,and other plants,essential oils,and polysaccharides from plants and algae.Participants emphasized upon several diffculties that they had faced in doing research with a natural product.For question 3,the choices in descending order ranked as follows:b,a,c,and e.(Choice‘d’was confgured into other choices when analyzing results.)These corresponded to diffculties in maintaining batch-to-batch consistency(quality control),obtaining and/or characterizing it,and publishing the results using natural products,and others.The last category included two items:diffculty in getting research grants funded, in part because some reviewers do not believe in the results obtained with,or the effcacy of,natural products;and diffculty in scaling up the separation techniques economically. For question 4,participants’responses brought out some of the major limitations that are presently hindering their research work.Different participants specifcally identifed the lack of the following facilities and other resources:HPLC/MS, NMR,biological assays,cell culture for anti-cancer effects,establishing product quality and composition,obtaining a reliable product for research,characterization of the separated components to be used in assays,and limited amount of isolated compounds such that the amount is not enough for analysis and biological testing despite best effort invested in purifcation of compounds from natural sources.Accordingly, appropriate resources in the form of proper instrumentation and methodology are required to advance the feld of natural products research.

In regard to part II of the questionnaire pertaining to the practical use of natural products,over half of the participants shared that they had used a natural product for treatment of common ailments or any other disease.This included prescribed folk medicine as well as self-procured natural products. The biological activities of natural products,either extracts or purifed compounds,included anti-infammatory,anti-microbial, anti-viral,anti-cancer,and anti-hyperlipidemic effects.Some of the products named were green tea,pomegranate,Morus alba, berberine,and snake venom.Among the participants who had not used a natural product,there was no indication of a bias against natural products use.Instead,one participant positively emphasized trust in using natural products.In question 4,participants were also asked to name a few advantages/ positives and a few disadvantages/negatives of using natural products over allopathic remedies for therapy.Both advantages and disadvantages were identifed.The advantages mentioned were as follows:fewer side effects,high antioxidant contents,organic in nature,biodegradability in the body, and less expensive,which is a critical consideration in the developing countries.The disadvantages mentioned were short preservation time or shelf-life,inadequate marketing,lack of information about their mechanism of action,not clear whether the observed effect might be a placebo effect,and being less effective than synthetic compounds.

Table 1–Workshop’s surveys.

R E F E R E N C E S

[1]Phillipson JD.Phytochemistry and medicinal plants. Phytochemistry 2001;56:237–243.

[2]Kong JM,Goh NK,Chia LS,et al.Recent advances in traditional plant drugs and orchids.Acta Pharmacol Sin 2003;24:7–21.

[3]Benowitz S.As war on cancer hits 25-year mark,scientists see progress,challenges.Scientist 1996;10:1–7.

[4]Newman DJ,Cragg GM.Natural products as sources of new drugs over the last 25 years.J Nat Prod 2007;70:461–477.

[5]Siddiqui IA,Adhami VM,Bharali DJ,et al.Introducing nanochemoprevention as a novel approach for cancer control:proof of principle with green tea polyphenol epigallocatechin-3-gallate.Cancer Res 2009;69:1712–1716.

[6]Khan N,Bharali DJ,Adhami VM,et al.Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model.Carcinogenesis 2014;35:415–423.

[7]Yang YH,Rajaiah R,Lee DY,et al.Suppression of ongoing experimental arthritis by a Chinese herbal formula(huo-luoxiao-ling dan)involves changes in antigen-induced immunological and biochemical mediators of infammation. Evid Based Complement Alternat Med 2011;2011:642027.

[8]Zhang RX,Fan AY,Zhou AN,et al.Extract of the Chinese herbal formula Huo Luo Xiao Ling Dan inhibited adjuvant arthritis in rats.J Ethnopharmacol 2009;121:366–371.

*< class="emphasis_italic">Corresponding author.

.University of Maryland School of Medicine,Baltimore,USA.Tel.:+1 410 706 7804;fax:+1 410 706 2129.

E-mail address:kmoudgil@som.umaryland.edu(K.D.Moudgil).

**Corresponding author.City of Scientifc Research and Technological Applications,Borg Elarab,Alexandria,Egypt.Tel.:+203-459-9384; fax:+203-459-9384.

E-mail address:ashraf_khalil@msn.com(A.A.Khalil).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2016.02.001

1818-0876/?2016 Production and hosting by Elsevier B.V.on behalf of Shenyang Pharmaceutical University.This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).