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Susceptibility of Helicobacter pylori to the Antibacterial Activity of Honey

Submitted to Prof. Bernie Abestano Casera BS-Bio, .!., ".A.!.

#n Partial $ulfillment %f the e&uirement for the 'e(ree Bachelor of Science in !ursin(

esearchers) "endo*a, +unice C. estor, 'enn Anthony Christian A. ,ap, "ar- Allen .. /apanta, 0ri1ia .oy !.

Chapter # 0H+ P %B2+" A!' #0S SC%P+

Introduction 0here have been several reports of upper (astrointestinal dyspepsia, includin( (astritis, duodenitis and ulceration, bein( successfully treated 3ith oral dosa(e of honey. 0his treatment reported is a return to traditional therapy, honey havin( been used as a medicine since ancient times. #n the 3or- that has been carried out in #slamic countries it is also based on a fundamental belief in the 3ords re(ardin( honey in the Holy Quran 3herein is healin( for man-ind.

0hese reports are of interest because conventional treatment of (astric and duodenal ulceration is far from satisfactory, as most dru(s used suppress but do not cure the ulceration, 3ith slo3 healin( rates and a very hi(h rate of relapse 4 567 at 8 year and 8667 at 9 years. +ven 3ith maintenance therapy 3ith anitidine 8:6 m( at ni(ht, the relapse rate is ;57. "edication for (astric and duodenal ulcers is of hi(h cost) therapy 3ith H9-receptor anta(onists is usually maintained for 8 year, and some patients need permanent treatment.

0reatment 3ith honey is much less e1pensive and appears to need less time. Ho3ever, there is a tendency of some practitioners to prematurely re<ect alternative medicine if it does not have a rational basis. Ho3ever, the recent findin( that (astritis and duodenal ulceration appear to be caused by infection 3ith Helicobacter pylori

raised a testable scientific hypothesis that the healin( effect of honey on (astritis and ulcers occur throu(h its 3ell established antibacterial activity.

+radication of Helicobacter pylori (ives a lon(-term cure of duodenal ulcers, preventin( the hi(h rate of relapse seen other3ise. Currently a combination of t3o or three antimicrobials is necessary for a reasonable de(ree of eradication of Helicobacter pylori, one of these bein( a bismuth compound.

0he antibacterial properties of honey have been established by a lar(e amount of laboratory research, 3hich has recently been comprehensively revie3ed. #n many reports honey has been sho3n to be effective in inhibitin( a 3ide ran(e of bacterial species 3hen diluted to concentrations at 3hich its osmolarity 3ould not be effective. Ho3ever, there have been no reports of any species of Campylobacter or Helicobacter pylori could e1pected be to be affected by the concentrations of honey that 3ould be achieved in the stomach by the dosa(e of honey (iven. 0herefore, the present study 3as underta-en to find the susceptibility of Helicobacter pylori to the antibacterial activity of honey.

0he antibacterial activity of honey varies very mar-edly, and depends on the floral source of the honey. 0he ma<or antibacterial factor in most honeys is hydro(en pero1ide, produced in the honey by the action of (lucose o1idase 3hich is added to the honey by the bee, but some antibacterial activity is due to substances 3hich are derived from the flo3ers. #f honey heals (astritis and ulcers by affectin( Helicobacter pylori, it may be the phytochemical content if the honey that is involved rather than

the osmolarity or the hydro(en pero1ide content of the honey. 0his 3as ta-en into account in the present study.

0he occurrence of phytochemical antibacterial factors in honey has been studied in a survey of the antibacterial activity of =;: samples of commercially available !e3 /ealand honey from 9> different floral sources. 0his 3as done by testin( 3ith catalase added to distin(uish bet3een antibacterial activities due to hydro(en pero1ide (enerated in the honey, and activity due to non-pero1ide factors. An a(ar 3ell diffusion assay 3ith Staphylococcus aureus 3as used, that 3as not susceptible to the osmotic effects of the honey. Honey from only one floral source, manu-a ?Leptospermum scoparium@, sho3ed a hi(h level of this type of activity in a si(nificant proportion of the samples, the fairly hi(h antibacterial activity of manu-a honey ?up to the e&uivalent of ==7 vAv phenol@ bein( in many cases due entirely to this non-pero1ide component.

#n vie3 of these findin(, the present study on the susceptibility of Helicobacter pylori to honey 3as carried out 3ith both manu-a honey and another honey in 3hich the antibacterial activity 3as due primarily to hydro(en pero1ide. 0he antibacterial activity of both these honeys 3as compared 3ith that of an artificial honey formulated to match the content of su(ars and (luconic acid in honey, 3hich could possibly also affect the bacterium.

Statement of the Problem 0his research study is desi(ned to evaluate the efficiency of honey if used as an alternative medicine for prescribed antibiotics and H 9-receptor anta(onists in treatment and mana(ement of (astric and duodenal ulcerations, and 3hether it offers other contributory benefits to the field of medicine and to human health.

0his study sou(ht to ans3er the follo3in( &uestions) 8. Bhat are the components that are present in honey, that 3hen applied to ulcerations, it 3ould treat and accelerate the healin(C 9. Bhat are the possible side effects and adverse reactions that 3ould occur 3ith the application of this substanceC =. Bould the harm out3ei(h the benefitsC ;. Bould the substance be potent enou(h in eradicatin( the bacteria H. pyloriC

Theoretical Framework Conceptual Paradigm 'etermination of bacterial resistance to antimicrobials is an important part of the mana(ement of infections in patients. 0he dis- diffusion method of Dirby and Bauer has been standardi*ed and is a viable alternative to broth dilution methods for laboratories 3ithout the resources to utili*e the ne3er automated methods for broth microdilution testin(.

Bhen a >-mm filter paper dis- impre(nated 3ith a -no3n concentration of an antimicrobial compound is placed on a "ueller-Hinton ?"H@ a(ar plate, immediately 3ater is absorbed into the dis- from the a(ar. 0he antimicrobial be(ins to diffuse into the surroundin( a(ar. 0he rate of diffusion throu(h the a(ar is not as rapid as the rate of e1traction of the antimicrobial out of the dis-, therefore the concentration of antimicrobial is hi(hest closest to the dis- and a lo(arithmic reduction in concentration occurs as the distance from the dis- increases. 0he rate of diffusion of the antimicrobial throu(h the a(ar is dependent on the diffusion and solubility properties of the dru( in "H a(ar and the molecular 3ei(ht of the antimicrobial compound. 2ar(er molecules 3ill diffuse at a slo3er rate than lo3er molecular 3ei(ht compounds. 0hese factors, in combination, result in each antimicrobial havin( a uni&ue brea-point *one si*e indicatin( susceptibility to that antimicrobial compound.

#f the a(ar plate has been inoculated 3ith a suspension of the patho(en to be tested prior to the placin( of dis-s on the a(ar surface, simultaneous (ro3th of the bacteria and diffusion of the antimicrobial compounds occurs. Ero3th occurs in the presence of an antimicrobial compound 3hen the bacteria reach a critical mass and can overpo3er the inhibitory effects of the antimicrobial compound. 0he estimated time of a bacterial suspension to reach critical mass is ; to 86 hours for most commonly recovered patho(ens, but is characteristic of each species, and influenced by the media and incubation temperature. 0he si*e of the *one of inhibition of (ro3th is influenced by the depth of the a(ar, since the antimicrobial diffuses in three dimensions, thus a shallo3 layer of a(ar 3ill produce a lar(er *one of inhibition than a deeper layer.

0he point at 3hich critical mass is reached is demonstrated by a sharply mar(inated circle of bacterial (ro3th around the dis-. 0he concentration of

antimicrobial compound at this mar(in is called the critical concentration and is appro1imately e&ual to the minimum inhibitory concentration obtained in broth dilution susceptibility tests.

Independent Variable Natural honey (NH) Commercial honey (CH) Home-made honey (HH)

Dependent Variable Helicobacter pylori samples cultured on three (3) Mueller-Hinton Agar plates

Intervening Variable Time Temperature and humidity Sunlight exposure

Figure 1. Schematic Diagram of the Variables

Hypothe e !"ull# Ho8) Honey has no healin( effect to3ards (astritis and duodenal ulcerations. Ho9) Antibacterial activity of honey is not potent enou(h to eradicate the bacteria Helicobacter pylori.

Significance of the Study

To the Researchers: 0his study 3ill enable the researchers to apply the theories learned in nursin( research and microbiolo(y thus helpin( them hone their s-ills. 0he findin(s of the study could be a means to improve the basic -no3led(e concernin( the use of honey as an alternative treatment and thus can provide a3areness and recommendations to the health care providers especially to the people 3ho canFt afford advanced treatment.

To the

ursing Stu!ents: 0hey 3ill be (iven more -no3led(e on the proper

procedures in usin( honey as a conventional treatment of (astric and duodenal ulceration. 0hey 3ill be able to learn some alternative techni&ues or actions on ho3 to at least alleviate the ulceration usin( the honey. !ursin( students 3ill someho3 be ready in usin( this honey as a conventional treatment in treatin( several reports. !ursin( students 3ill have more -no3led(e concernin( the use of honey as an alternative treatment.

To the Clinical "nstructors: 0his study 3ill (ive them -no3led(e on the efficiency of honey to clients 3ith ulceration. 0he clinical instructors 3ill then be able to su((est honey as a conventional home-treatment especially in clients 3ho canFt afford buyin( medications in treatin( ulcerations.

To the #uture Researchers: 0his study 3ill serve as (uide and source of information to the future researchers thus ma-in( their paper more accurate and efficient.

Scope and $imitation of the Study 0he study 3ill utili*e three types of honey 3hich 3ill be used in the antibacterial activity testin(. $irst is the natural honey ?!H@, to be ac&uired from a nearby local bee farm and homo(eneity of the honey 3ill be ensured by thorou(h a(itation at the time of e1traction from the combs. Second is a commercial honey, to be bou(ht from bee farm dealers and 3ill be identified by the apiarists supplyin( them, identification bein( based on the flavor, color and aroma of the honey, also the season and location of its production. 0he identity of the honeys selected 3ill be confirmed by pollen analysis. 0he third one is the Gartificial honeyF 3hich can be made at home. 0he test sub<ect that 3ill be used is Helicobacter pylori that 3ould be isolated from biopsy samples 3hich are ta-en from a mar(in of (astric ulcers. 0he researchers see to it that the biopsy samples are indeed fresh to prevent any alterations or complications 3ith the specimen ?e.(. invitation of other microbes and bacteria@. 0he honeys 3ill be stored in the dar- at :HC in polyethylene buc-ets 3ith ti(ht-fittin( lids. Specific e1perimental tests to be done are the Dirby-Bauer 'is- 'iffusion Assay and the "ueller-Hinton ?"H@ A(ar culture. esearch instruments to be used are

stated in the Chapter = of this paper. 0he emphasis on this e1periment is to e1amine 3hether honey could be a promisin( conventional and alternative medicine for antibiotics and H9-receptor anta(onists in treatment and mana(ement of (astric and duodenal ulcerations, and 3hether it offers other contributory benefits to the field of medicine and to human health. 0he e1perimental procedures 3ill be done only either at the laboratories in the 'epartment of Biolo(y, Colle(e of Science and "athematics or at the "icropara 2ab, Colle(e of !ursin( or "indanao State Iniversity 4 #li(an #nsitute of 0echnolo(y. 0his study is limited only to the testin( of the antibacterial

capacity of honey and it does not concern the actual inta-eAapplication of the substance and also its positive and ne(ative effects on the human anatomy.

%efinition of Term 0he follo3in( are operational terms defined by the researchers to ensure clearer understandin( and appreciation of the study)

$gar a (elatinous substance obtained from certain red sea3eeds and used in biolo(ical culture media and as a thic-ener in foods.

$septic techni%ue refers to a procedure that is performed under sterile conditions. 0his includes medical and laboratory techni&ues, such as 3ith cultures. #t includes techni&ues li-e flame sterili*ation.

&iopsy an e1amination of tissue removed from a livin( body to discover the presence, cause, or e1tent of a disease.

&ismuth the chemical element of atomic number 5=, a brittle reddish-tin(ed (rey metal.

Duo!enal ulcer a peptic ulcer of the duodenum.

Duo!enum the first part of the small intestine immediately beyond the stomach, leadin( to the <e<unum.

Dyspepsia indi(estion.

#asti!ious very attentive to and concerned about accuracy and detail.

'astritis inflammation of the mucosal linin( of the stomach.

'lucose o(i!ase

is an o1ido-reductase that catalyses the o1idation of (lucose to hydro(en pero1ide and '-(lucono-J-lactone. #n cells, it aids in brea-in( the su(ar do3n into its metabolites.

'ram) egati*e &acteria (ram-ne(ative bacteria are bacteria that do not retain crystal violet dye in the Eram stainin( protocol. #n a Eram stain test, a counterstain is added after the crystal violet, colorin( all (ram-ne(ative bacteria 3ith a red or pin- color.

Helicobacter pylori a curved, Eram-ne(ative bacillusK capable of coloni*in( the stomachK a common cause of stomach and duodenal ulcers.

Homogeneity the &uality or state of bein( homo(eneous.


a s3eet, stic-y yello3ish-bro3n fluid made by bees and other insects from nectar collected from flo3ers.

Hy!rogen +ero(i!e a colorless viscous unstable li&uid 3ith stron( o1idi*in( properties, used in some disinfectants and bleaches.

H), receptor antagonist are a class of dru(s used to bloc- the action of histamine on parietal cells ?specifically the histamine H9 receptors@ in the stomach, decreasin( the production of acid by these cells.

"noculation the action of inoculatin( or of bein( inoculatedK vaccination.

-anu.a Honey is a monofloral honey produced in !e3 /ealand and Australia from the nectar of the manu-a tree. #t has demonstrated antibacterial properties in vitro, but there is no conclusive evidence of benefit in medical use.

-onofloral Honey monofloral honey is a type of honey 3hich is valued because it has a distinctive flavor or other attribute due to its bein( predominantly from the nectar of one plant species.

+eptic ulcer a lesion in the linin( ?mucosa@ of the di(estive tract, typically in the stomach or duodenum, caused by the di(estive action of pepsin and stomach acid.

+hoto!egra!ation de(radation of a photode(radable molecule caused by the absorption of photons, particularly those 3avelen(ths found in sunli(ht, such as infrared radiation, visible li(ht, and ultraviolet li(ht. Ho3ever, other forms of electroma(netic radiation can cause photode(radation.

+hytochemicals any of various biolo(ically active compounds found in plants.

+ollen $nalysis the analysis of samples of pollen to determine the species presentK palynolo(y.

/lcer an open sore on an e1ternal or internal surface of the body, caused by a breain the s-in or mucous membrane 3hich fails to heal. Ilcers ran(e from small, painful sores in the mouth to bedsores and serious lesions of the stomach or intestine.

Chapter ## +L#+B %$ +2A0+' 2#0+ A0I +

&ICF$'() 'F TH* +)ST('I"T*STI")$ T()CT 0he (astrointestinal ?E#@ tract ?or di(estive tract@ consists of a lon( tube 3ith many e1panded areas desi(ned for di(estion of food, absorption of nutrients, and elimination of undi(ested materials. +1cludin( the oral cavity and pharyn1, 3hich have already been discussed, the E# tract includes the esopha(us, stomach, small intestine, lar(e intestine ?colon@, and anus. Accessory (lands and or(ans of the Ei system include the salivary (lands, pancreas, liver, and (allbladder.

Eastric en*ymes and the e1tremely acidic pH ?appro1imately pH 8.:@ of the stomach usually prevent (ro3th of indi(enous microflora, and the most transient microbes ?i.e., microbes consumed in foods and bevera(es@ are -illed as they pass throu(h the stomach. 0here is one bacterium, a Eram-ne(ative bacillus names Helicobacter pylori, that lives in some peopleFs stomachs and is a common cause of ulcers. A fe3 microbes, enveloped by food particles, mana(e to pass throu(h the stomach durin( periods of lo3 acid concentration. Also, 3hen the amount of acid is reduced in the course of diseases such as stomach cancer, certain bacteria may be found in the stomach.

$e3 microflora usually e1ist in the upper portion of the small intestine ?the duodenum@ because bile inhibits their (ro3th, but many are found in the lo3er parts of the small intestine ?the <e<unum and ileum@.

0he colon contains the lar(est number and variety of microor(anisms of any coloni*ed area of the body. #t has been estimated that as many as :66 to >66 different species, primarily bacteria, live there. Because the colon is anaerobic, the bacteria livin( there are obli(ate, aero tolerant and facultative anaerobes. Bacteria found in the (astrointestinal tract include species of $ctinomyces, &acterio!es, Clostri!ium, 0nterobacter, 0nterococcus, 0scherichia, 1lebsiella, Lactobacillus, +roteus,

+seu!omonas, Staphylococcus, and Streptococcus.

Also, many fun(i, proto*oa, and viruses can live in the colon. "any of the microflora of the colon are opportunists, causin( disease only 3hen they (ain access to other areas of the body ?e.(., urinary bladder, bloodstream, or lesion of some type@, or 3hen the usual balance amon( the microor(anisms is upset. 0. coli is a (ood e1ample All humans have 0. coli bacteria in their colon. 0hey are opportunists, usually causin( us no problems at all, but they can cause urinary tract infections ?I0#s@ 3hen they (ain access to the urinary bladder. #n fact, 0. coli is the most common cause of I0#s.

"any microbes are removed from the E# tract as a result of defecation. #t has been estimated that about :67 if the fecal mass consists of bacteria.8

)$T*()TI'"S '" TH* +)ST('I"T*STI")$ T()CT A. Bacterial Eastritis and Eastric Ilcers #nfection 3ith Helicobacter pylori can cause chronic bacterial (astritis and duodenal ulcers. Eastritis is suspected 3hen a person has upper abdominal pain 3ith nausea or heartburn. People 3ith duodenal ulcers may e1perience (na3in(, burnin(, achin(, mild-to-moderate pain <ust belo3 the breastbone, an empty feelin(, and hun(er. 0he pain usually occurs 3hen the stomach is empty. 'rin-in( mil-, eatin(, or ta-in( antacids (enerally relieves the pain, but usually returns 9 or = hours later. Eastric ulcers and (astric adenocarcinoma are also epidemiolo(ically associated 3ith H. pylori infection. Eastric ulcers can cause s3ellin( of the tissues leadin( into the small intestine, 3hich prevents food from easily passin( out of the stomach. 0his, in turn, can cause pain, bloatin(, nausea, or vomitin( after eatin(. Eastric ulcers and duodenal ulcers are types of peptic ulcers. Complications of peptic ulcers include penetration, perforation, bleedin( and obstruction.

B. Patient Care Ise Standard Precautions for hospitali*ed patients.

!dition "#$T%N&S M'C$%"'%(%)* +%$ H!A(TH SC'!NC!S ,y -aul ). !ngel/ir/ 0 1anet 2u,en-!ngel/ir/ (Chapter 13 4Micro,ial !cology and Micro,ial "iotechnology56 -age 173)


C. Patho(en Helicobacter pylori is a curved, microaerophilic, capnophilic, Eram-ne(ative bacillus that is found on the mucus-secretin( epithelial cells of the stomach. !o other bacteria are -no3n to (ro3 in the e1tremely acidic stomach.

'. eservoirs and "ode of 0ransmission #nfected humans serve as reservoirs. 0ransmission probably occurs via in(estionK presumed to be either oral-oral or fecal-oral transmission.

+. 2aboratory 'ia(nosis 'ia(nostic techni&ues include stainin( and culturin( of (astric and duodenal biopsy specimens, the urea breath test, the !H ; e1cretion test, molecular dia(nostic procedures, and immunodia(nostic procedures. #n the urea breath test, the patient in(ests radioactively labeled urea and his or her breath is analy*ed >6 minutes later for radioactively labeled C%9. 0he en*yme urease, produced by H. pylori, splits the urea into ammonia and C%9K hence, the presence of radioactively labeled C%9 indicates the presence of H. pylori. #n the !H; e1cretion test, the patient consumes urea containin( radioactively labeled nitro(en. 0he ammonia produced in the stomach by H. pylori is absorbed into the blood, e1creted in the urine, and the amount of radioactively labeled !H; in the urine is measured.9

!dition "#$T%N&S M'C$%"'%(%)* +%$ H!A(TH SC'!NC!S ,y -aul ). !ngel/ir/ 0 1anet 2u,en-!ngel/ir/ (Chapter 1 4"acterial 'n9ections56 -age 33:6 Ta,le 1 -;)


FI,* &)-'( .*"*FITS 'F H'"*/ 8. Anti-microbial and anti-o1idant properties Honey ?in particular propolis-containin( ra3 honey@ is reno3ned for its antiseptic properties. #t is simultaneously anti-bacterial, anti-microbial and anti-fun(al, providin( all-round protection from micro-or(anisms.

Honey is also an anti-o1idant, protectin( the body from the carcino(enic effects of free radicals and boostin( the immune system.

9. Bound care Honey is often used in the first aid treatment of cuts and burns and is no3 used in modern medicine to aid 3ith the healin( of 3ounds. Applyin( honey to a 3ound stimulates the dama(ed tissues. #t also minimi*es scarrin(, reduces s3ellin( and helps in deodori*in( foul-smellin( 3ounds. Honey promotes a process -no3n as autolytic debridement, the painless self-di(estion of dead s-in.

=. Bei(ht loss and s-in care Honey is one of natureFs o3n s3eetenersK typically consistin( of over t3othirds (lucose and fructose, it can be added to anythin( you 3ould normally add su(ar to. #t may seem a contradiction, but despite containin( over four times the calories of su(ar ?over >6 calories per tablespoon@, honey can actually help 3ith 3ei(ht loss. 0his is due, in part, to its fat-di(estin( properties.

Honey is also effective in improvin( the endurance and all-round performance of athletes. !ot only is it rich in ener(y ?both &uic--release (lucose and slo3-release fructose@, it also maintains healthy blood su(ar levels. Honey efficiently restores (lyco(en levels ?the 3ay the body stores su(ar@ and aids 3ith muscle recuperation, leadin( to less muscle fati(ue. %f course, you donFt have to be an athlete to benefit from these advanta(es) anyone can ta-e honey, or (ive it to their children, to boost fla((in( ener(y levels.

Honey has also been lauded for its s-in-care properties. Ised in various health spas, honey and mil- in e&ual combination both cleanses and protects the s-in, leavin( it loo-in( radiant.

;. Eentle on the di(estive system Honey is a 3ell--no3n tonic for the di(estive system, helpful in controllin( a host of di(estive complaints, from diarrhea and indi(estion to (astroenteritis and stomach ulcers.

:. Litamins and minerals 0he actual vitamin and mineral content in honey depends on 3hich flo3ers have been visited and ho3 the honey has been processed. +1cess processin( strips nutrients, especially phytochemicals, so ra3 honey is al3ays a better choice. %r(anic

honey also claims to be free of pesticides and antibiotics, althou(h the e1tent to 3hich bees can really be protected from contaminated flo3ers is a sub<ect for debate. 0he main nutrients to be found in honey are vitamin C, Calcium and #ron.

Bith so many valuable properties, it is no 3onder that the list of ailments that honey has been used to treat is so e1tensive. AthleteFs foot, halitosis, sleep disorder, acne, con<unctivitis, alopecia, arthritis and yeast infections are <ust some of the complaints that honey is claimed to alleviate. Christie Hospital, near "anchester, ID, is even trialin( the use of honey in assistin( the recovery of cancer patients.=

"0T()C*0TIC)$ ,)$0*S 'F H'"*/ 0he use of natural honey ?!H@ as a nutraceutical a(ent is associated 3ith nutritional benefits and therapeutic promises. !H is 3idely accepted as food and medicine by all (enerations, traditions and civili*ations, both ancient and modern. 0he nutritional profiles, includin( its use in infant and children feedin( reported in different literatures as 3ell as health indices and biomar-ers observed by various researchers are illustrated in this manuscript. 0he revie3 documents fol- medicine, e1perimentation 3ith animal models, and orthodo1 medical practices sho3n by clinical trials. 0his covers virtually all human or(ans and body systems e1tensively studied by different 3or-ers. 0he sources and adverse effects of !H contamination, as 3ell as the preventive methods are identified. 0his could promote the availability of residue free honey and a 3holesome natural product for domestic consumption and international mar-et. 0his could also help to prevent health problems associated 3ith

http<==>>>.>,ene9its-o9-honey= (,y $omona @eston)

!H poisonin(. #n addition, apicultural practices and the economic importance of honey are 3ell documented. 0his report also includes information about a relatively un-no3n and uncommon South American stin(less bee species. Be concluded this revie3 by identifyin( important roles for +thno-entomolo(ists, other Scientists and Apiculturists in the development of stin(less bees to boost honey production, consumption and economic earnin(s.

H'"*/ )S F''% A. Ero3th $ood is eaten for nourishment, metabolic activities, (ro3th and healthy livin(. e(ular consumption of natural honey (ives all these benefits. #n fact, honey is a complete meal, as sho3n in 0ables8 4 =. #t contains ma<or components of a meal, and micronutrients that 3ill enhance the di(estion and absorption of these ma<or dietary components, as 3ell as those re&uired for metabolism and body functions. Be recorded enhanced body 3ei(ht (ain by our rats fed blossom honey in t3o separate studies at different laboratories in !i(eria and South Africa. #n 9665, Chepulis and Star-ey fed honeyde3 honey to 5-3ee- old rats for :9 3ee-s to assess 3ei(ht (ain. 0hese 3or-ers sho3 that the (ro3th influence of honey in rodents is partly due to increased bone (ro3th and minerali*ation, probably due to the calcium content of honey. %ur unpublished data from very recent study on !H supplemented rats confirmed this linear (ro3th influence of honey. #n his e1tensive revie3 of the literature, "olan ?9668@ confirmed the (ro3th stimulatin( property of honey. He opines from his histolo(ical studies on 3ounds that stimulation of cell (ro3th by honey also enhances !H healin( properties.

B. Source of antio1idants 0he presence of free radicals and reactive o1y(en species ? %S@ is culpable in the processes of cellular dysfunction, patho(enesis of metabolic and cardiovascular diseases ?CL's@ as 3ell as a(in(. 0he consumption of foods and substances rich in antio1idant can protect a(ainst these patholo(ical chan(es and conse&uently prevent the patho(enesis of these and other chronic ailments. esearches indicate that !H

contains several important compounds, and these include antio1idants. 0he &ualitative and &uantitative composition of honey ?includin( the antio1idants constituent and the other phytochemical substances@ is a reflection of the floral source as 3ell as the variety of the particular honey. 0he color of honey also influences its antio1idant content, as dar-er honeys are -no3n to have hi(her amount than li(hter honeys. #n their analysis of the phytochemical composition of monofloral Cuban honeys, Alvare*-Suare* and co-3or-ers, a(reed 3ith this submission and concluded that Cuban honeys contain important phenolic, flavonoid and carotenoid concentrations 3ith hi(h substantial antio1idant capacity. esearchers in California also submitted

that human bein(s can be protected from the dama(in( effects of free radicals and %S. 0he protection is throu(h the absorption of the antio1idants from foods such as honey hi(hly-rich in this important substance called antio1idant. 0he report of their study in 3hich t3o buc-3heat honey treatments 3ere administered to =M healthy human adults at the rate of 8.:(A-( body 3ei(ht, 3ith corn syrup as control, sho3 increased ?pN6.6:@ plasma total-phenolic content and plasma antio1idant. 0hus, supportin( the concept that phenolic antio1idants from processed honey are bioavailable, and they increase antio1idant activity of plasma. 0hey advocated for the

substitution of honey in some foods as traditional s3eetener for enhanced antio1idant defense system in healthy human adults.

C. +1ercise and athletic performance 0he consumption of ener(y (ivin( substances before, durin( and after any form of physical e1ercise improves the individualFs performance and increases the re<uvenation of muscles. 0his is also associated 3ith dietary supplementation 3ith !H, 3hich provides up to 8M ( of carbohydrates for every tablespoon consumed and (ives the much needed ener(y, thus servin( as an ine1pensive substitute to commercially available sportin( activities enhancers. 0he data obtained from the Sports !utrition and +1ercise 2aboratory of one Iniversity sho3 that honey can be used effectively instead of (lucose for ener(y replenishment durin( physical e1ercise. 0he physiolo(ical actions of !H observed durin( this performance 3ere a si(nificant increase in heart fre&uency and a fairly constant blood (lucose level. 0hese su((est honey as a better substitute to (lucose. +arnest and co-3or-ers improved on this preliminary investi(ation in another trial by administerin( lo3 ?honey@ or hi(h ?(lucose@ (lycemic inde1 ?E#@ carbohydrate (els on athletes, and testin( them on the performance of cyclists travellin( a distance of about >: -m. 0he results of the cyclin( event sho3 that both the lo3 ?honey@ and the hi(h ?(lucose@ E# substances caused increase in performance. Ho3ever, the effect produced by eatin( !H surpassed that observed in the athletes fed 3ith (lucose. 0his ali(ns 3ith other previous studies that !H consumption does not compromise metabolic and physical activities. !H has been sho3n to decrease blood (lucose level in hyper(lycemia such as diabetic sub<ects, plausibly due to the beneficial effects of fructose, and more

importantly honeyFs several phytochemical constituents. A very recent revie3 of the hypo(lycemic effect of honey by some 3or-ers conclude that, the syner(istic effect of fructose and (lucose constituents of honey mi(ht contribute to the lo3 (lycemic response after a honey meal. 0hese e1perimental and clinical trials sho3 that honey is a 3ell-tolerated li&uid food.

$urthermore, honey can be an effective carbohydrate source and a better substitute to (lucose for e1ercise and athletic performance, due to its constituent of various classes of su(ars. People favor slo3-burnin( su(ars for sustenance as ener(y source durin( physical e1ercise. Honey is beneficial in this re(ard as it releases fructose slo3ly into the blood stream to produce a sustained ener(y boost and maintain homeostasis. 0he other ma<or component of !H apart from fructose is (lucose. $ructose and (lucose are -etose and aldose su(ars respectively 3ith chemical structural differences, and conse&uently different patterns of metabolism, despite both bein( monosaccharides 3ith &uic- burnin( tendency. #t is important to note that (lucose is rapidly metaboli*ed for absorption into the blood system for ener(y provision. %n the other hand, fructose absorption is slo3, and 3ill continue to sustain the individual 3ith ener(y, 3hile the (lucose moiety burns out. 0he various phytochemical constituents of honey also contribute to the pro(ressive slo3 rate of fructose metabolism. #n addition, honey contains disaccharides such as sucrose and oli(osaccharides as 3ell as other trisaccharides that are slo3 burnin( su(ars. 0hese could facilitate ener(y replenishment, muscle recuperation and enhancement of performance in athletes nourished 3ith honey, 3hile those relyin( on (lucose for an ener(y boost mi(ht have been e1hausted.

'. 'i(estion and absorption !atural honey contains several en*ymes 3hich enhance the di(estion of food substances especially carbohydrates such as su(ars and starch. 0he additional benefit of eatin( honey as a source of ener(y over the commonly used artificial su(ar is that, the ma<or su(ar constituents of !H are present as monosaccharides ?simple su(ars@. Inli-e the refined su(ar ?sucrose@ 3hich normally has to under(o the processes of di(estion into simpler forms prior to their absorption, these su(ar molecules in !H are in pre-di(ested forms, and can be directly absorbed into the human system. Apart from (ivin( nutrition, the use of honey as a s3eetenin( a(ent in s3eets and desserts is also beneficial. As a s3eetener, honey has nutritional advanta(es over su(ar, providin( some amount of small nutrients, 3hich act to aid di(estive processes in the body. 0he (astrointestinal tract ?E#0@ contains lot of essential and beneficial bacteria, especially Bifidobacteria for the maintenance of life and (ood health. #t has been su((ested that one can increase the Bifidobacteria populations in the E#0 by consumin( foods 3ith rich supply of prebiotics such as natural honey. Prebiotics are substances that facilitate the enhanced (ro3th and the biolo(ical activity of these (ood and beneficial bacteria. 0he consumption of honey is important in human di(estion, and this effect is produced by the honeyFs constituents of oli(osaccharides. Several e1perimental trials involvin( both in vitro and in vivo studies have been documented on the importance of dietary supplementation 3ith natural honey on the (ro3th of the beneficial bacteria ?bifidobacteria and lactobacilli@ and their prebiotic effects in the E#0. %ne comparative study on natural ?honey@ and artificial ?sucrose@ su(ars sho3s that honey increased both in vitro the beneficial bacteria, lactobacilli, as 3ell as in vivo ?3ithin the small and the lar(e intestines of e1perimental rats@, 3hile

sucrose had no effect. #n some cases, the consumption of relatively lar(e amounts of !H ?bet3een M6 to about O: (@ can produce a mild la1ative effect in people 3ith fructose malabsorption or inade&uate absorption. Eenerally, honey has a la1ative effect on the di(estive system of individuals. Another nutraceutical function of honey is provision of calcium. Honey consumption provides calcium, 3hich is readily absorbable and stren(then bone mass development. 0his can help reduce the ris- of osteoporosis or lo3 bone mass ?causative a(ent of fractures@ in old individuals. esearch in animal models sho3 that calcium absorption increased correspondin(ly 3ith increased honey inta-e.

$. Children nutrition 0here are anecdotal evidences encoura(in( the feedin( of honey to ne3 born babies by some customs and traditions. #t is no3 an established fact that feedin( honey to infants 3ill improve memory and (ro3th, reduce an1iety and enhance the childrenFs performance in later life. #n 966O, Chepulis and co 3or-ers (ave scientific credence to this beneficial practice in their !e3 /ealand behavioral study in animals. 0hey fed 53ee-s old rats 3ith diet supplemented 3ith either honeyde3 honey or sucrose, and control (roup 3ith su(ar-free diet ?all diets patterned after typical !e3 /ealand human diet@. 0hese 3or-ers noted improved spatial memory and reduced an1iety in the honey-fed rodents better than the other (roups over the t3elve months trial period. 0he authors concluded that early introduction of honey diet is beneficial and can improve memory loss and co(nitive decline associated 3ith a(in(.

0he application of honey in human infant nutrition also revealed some interestin( and beneficial observations. 0he palatability of honey for infants 3as investi(ated by amen(hi and others in 9668, and these 3or-ers reported that honey 3as 3ell tolerated and si(nificantly reduced the cryin( phases of babies than sterile 3ater. #n a revie3 on the importance of honey relative to sucrose in childrenFs nutrition, honey fed infants 3ere found to have improved hematolo(ical profiles and calcium upta-e, no di(estion problem, li(hter and thinner faces, better s-in color, less susceptibility to diseases, and steady 3ei(ht (ain. 0hese beneficial effects produced by !H 3hen included in infant formula are attributable to its effects in enhancin( the (astrointestinal function 3hich include the di(estion process. 0he possible cause is the effect of the carbohydrate constituents, oli(osaccharides in !H on intestinal flora of these children. %ne 3ill be doin( 3ell to children by (ivin( honey to replace s3eets and other su(ary substances they are often inclined to eat.

H'"*/ )S &*%ICI"* A. Hematolo(y and immunity Honey has been found to be beneficial to people sufferin( from anemia. A<ibola et al. ?966M@ reported enhanced blood profiles in floral honey-fed adult rats. 0he study recorded improved hemo(lobin concentration ?iron constituent of !H played an important role in this@, increased erythrocyte count and elevated hematocrit in the honey eaters. #n another laboratory, Chepulis ?966M@ also documented enhanced hematolo(y and immune response in rats fed 867 honeyde3 honey supplemented diet. 0he author noted hi(her lymphocyte count and increased neutrophil pha(ocytosis in !H-fed rats than control. 0his ali(ned 3ith previous research that

prebiotics can enhance immune function and !H is -no3n to contain the prebiotics, oli(osaccharides. Human sub<ects administered 3ith t3o honey treatments in a Californian study sho3 that honey eaters have the benefit of haematoprotection in addition to blood proliferation. 0he researchers observed that the a&ueous portion of the blood ?plasma@ is protected by honey. 0his is in a(reement 3ith the fact that most of the antio1idant components in processed honey are 3ater soluble. #n summari*in( the facts that honey can be considered to be a satisfactory immuno-nutrient, some 3or-ers opine that the oral administration of natural honey can stimulate and increase the production of antibody durin( primary and secondary immune responses a(ainst the 0-cells of the thymus-dependent as 3ell as the thymus independent anti(ens.

B. %ral health 0he use of !H can promote oral health and 3ellness. "olan opines that honey 3ith hi(h level of antibacterial activity has the potential to reduce the ris- of dental caries. #n addition to the carioprotective effect of !e3 /ealand manu-a honey ?a very potent antimicrobial honey@, "olan and co-3or-ers have sho3n from his e1tensive 3or- on the influence of honey on oral health that honey prevents dental pla&ue, (in(ivitis, periodontics. %ther 3or-ers in different laboratories have also sho3n that honey is non-cario(enic or less cario(enic than sucrose. 0he carioprotective effect of honey has been adduced to its antibacterial property, 3hich prevents the (ro3th of the bacteria that can cause dental caries. #n one electron microscopy study, honey consumption 3as found to be safer and less inimical to oral health than drin-in( fruit <uice. 0here 3as a report of the tooth enamel bein( eroded <ust ten minutes after the consumption of fruit <uice, 3hile honey in(estion delayed this observation till half an

hour after the inta-e of !H, and this erosion of teeth 3as not even as prominent or visible as that observed in the fruit <uice eaters. 0he plausible e1planation for the less cario(enic effect of honey is the protective role of !H constituents 3hich include calcium, fluoride, phosphorous and other colloidal honey components. #n summary, it can be concluded that honey has constituents 3ith cario-protective effect.

C. Eastroenterolo(y Anecdotal evidences advocate the medicinal use of !H as therapeutic a(ent a(ainst the ailments of the E#0 in the past. 0hese are presently bein( supported by the (lobal medicinal use of !H for the prevention, cure and the treatments of some E#0 disorders such as ulcers, (astritis and (astroenteritis. Honey has been sho3n to be a (astroprotective a(ent. #ts potency in inhibitin( the activity of Helicobacter pylori, that causes (astritis and peptic ulcers have been 3ell documented. #n e1perimental rats, !H miti(ated (astrointestinal assaults caused by alcohol, ammonia, aspirin and indomethacin. 03o mechanisms have been proposed to be responsible for this protective action of honey. 0he first su((ests that this effect is due to the antio1idant properties of honey. !H 3as found to maintain or enhance the level of non-protein sulfhydyl substances ?such as (lutathione@ in (astric tissue sub<ected to factors inducin( ulceration. Similar observation 3as made 3hen An*er honey pre-treatment 3as used to prevent !-ethylmaleimide ?!+"@ -induced liver dama(e in rats. 0he findin(s imply that depletion of (lutathione concentration plays an aetiolo(ical role in !+"-induced liver in<ury, and that the hepatoprotective effect of An*er honey may be mediated throu(h the sulfhydryl sensitive processes. 0he authors concluded that honey possess antio1idant properties.

Accordin( to some authors, the second mechanism of action bein( proposed sho3s that honey inta-e stimulates the sensory nerves in the stomach, and this proprioceptive effect is in response to capsaicin. 0his mechanism involves the reduction of ulcer inde1, vascular permeability, and muscular activity of the stomach. %ther authors also e1plained this phenomenon by reportin( the miti(atin( effect of dandelion honey inta-e a(ainst (astric <uice acidity by more than :67. %ne study reported a slo3er passa(e rate of (astric content of saccharides after the inta-e of !H than that after in(estion of a mi1ture simulatin( honey that is (lucose and fructose mi1ture, and thus, miti(atin( diarrhea. 0he clinical uses of honey in infants and children revealed shorter duration of diarrhea caused by bacteria. #n the same vein, !H also reduced the patho(enesis and duration of viral diarrhea unli-e that associated 3ith the use of conventional antibacterial therapy. Honey 3as also found to be beneficial in maintainin( blood-su(ar levels. #n honey, there is little 3ater available to promote the (ro3th of bacteria and yeast. #n addition, honeyPs natural acidity inhibits some patho(ens, as it has an amount of hydro(en pero1ide and other substances contributin( to its antibacterial effect.

'. %phthalmolo(y 0he use of honey in the treatment of eye diseases is 3ell documented. 0he ancient people used honey from Attica, and #ndian lotus honey as curative substances for eye disorders. 0he #ndian locals still use !H as eye drops to cure eye disease till today. 0he "alian people used !H as a tradomedicinal therapeutic a(ent a(ainst measles. 0he !H 3as usually applied on the eyes to prevent the scarrin( effect of the

cornea 3hich occurs as a complication of the measles infection. 0here 3as an astoundin( success reported from !H application in clinical trials of 869 patients 3ith different ophthalmolo(ical disorders such as ?blepharitis, con<unctivitis and -eratitis@, hitherto not respondin( to conventional treatment. After the !H application under the lo3er eyelid li-e an eye ointment, improvement 3as seen in 5:7 of the cases, 3ith no deterioration seen in any of the other 8:7. Ho3ever, a transient stin(in( sensation and redness of the eye 3as observed soon after puttin( honey in the eye, but not enou(h to halt the treatment in all the 869 cases in the trial. 0he use of !H application in ophthalmolo(y in Asia and +astern +urope has also been reported by "olan in the revie3 on !H use in ophthalmolo(y. #n one #ndian "edical Colle(e, an %phthalmic Sur(eon successfully treated bacterial caused corneal ulcers 3ith the topical application of !H. 0he same paper documented "eier referrin( to !H bein( used for the treatment of eyes dischar(in( pus. "olan ?8OOO@ also cited ussian authors,

"o*heren-ov and Pro-ofPeva that document a revie3 on the use of honey in ophthalmolo(y. 0he ussian authors observed antibacterial, antifun(al and anti-

inflammatory actions 3ith the honey application to the eye under the lo3er eyelid. #t has been used for the treatment of burns to the eye caused by chemical and thermal a(ents, as 3ell as con<unctivitis, and corneal infections. 0his is usually done by the topical application of the undiluted !H on the affected eyes, or alternatively as a solution containin( about :67 3ater, 3ithout any loss of potency.

+. "etabolic and cardiovascular effects #t has been sho3n that honey inta-e ameliorates ris- factors of metabolic and cardiovascular diseases in patients and healthy individuals at ris-. Inli-e refined

su(ars, diabetic patients can safely and harmlessly eat this natural and s3eetest su(ar ?fructose@-containin( product, natural honey. Accordin( to Costa-!eto ?8OOO@ reported by Santos and Antonini, the Pan-ararQ tribe of Bra*il even recommended honeyFs use for the treatment of diabetes mellitus, bronchitis, mycosis, and throat aches amidst other ailments. ecently in our laboratory, 3e fed male and female rats 3ith !H or su(ar ?(olden syrup, ES@ supplemented diet for 89 3ee-s from Mdays of a(e to compare their metabolic response, and see if !H is protective a(ainst metabolic syndrome ?"etS@. 0his "etS is a condition characteri*ed by abdominal obesity, hyper(lycemia, hypertension and dyslipidemia, and thus increased susceptibility to diabetes, -idney and heart diseases. #n male rats, ES si(nificantly increased ?p N6.6:@ blood levels of metabolic substrates ?(lucose and tri(lycerides, 0Es@K and caused enhanced ?pN6.668@ visceral adiposity, hypercholesterolemia, hyperinsulinemia, hepatome(aly and fatty liver. 0hese CL's and metabolic diseasesF ris- factors 3ere not observed in the !H-fed rats in this trial. Be concluded that honey is cardioprotective, and its ?!H@ consumption could not induce "etS Runpublished resultsS. 0hese results confirmed the conclusion dra3n from earlier study that substitutin( honey for refined carbohydrates 3as beneficial. +arlier researches from other laboratories and clinical trials further affirmed the metabolic and cardiovascular health si(nificance of eatin( honey by recordin( some health profiles. 0hese 3ere reduction in the plasma levels of ris- factors 3hich include total cholesterolK lo3 density lipoprotein ?2'2@-cholesterolK 0EsK (lucose in normal and diabetic patientsK C-reactive proteinK 3hile the health indices elevated in the blood 3ere hi(h density lipoprotein ?H'2@ cholesterol. #n addition, other 3or-ers recorded hi(her plasma antio1idants levels in rats nurtured 3ith natural honey relative to fructose-fed rats, and conse&uently lo3 susceptibility of these sub<ects to CL's.

$. Chemotherapy and 3ounds mana(ement Honey has antiseptic properties, (ood for treatin( burns, infected sur(ical 3ounds and ulcers. Elenys ound, a Cancer Specialist and .ulie Betts in Bai-ato

Hospital, !e3 /ealand reported e1cellent results of the therapy from patients 3ith fun(atin( 3ounds, recalcitrant le( ulcers and pressure sores usin( the Ini&ue "anu-a $actor ?I"$@-containin( honey -no3n as manu-a honey. Accordin( to the Specialist, the application of honey dressin(s 3as used in these patients ?includin( those 3ith cancer bro-en throu(h the s-in@, after failure of healin( from conventional treatments 3hich include radiation therapy. Another esearcher successfully treated

e1perimental sur(ical 3ounds in !i(erian '3arf (oats 3ith blossom honey. He observed epithelialisation and si(nificantly hi(her contraction of the !H treated 3ounds relative to untreated 3ounds. %ther several 3or-ers had also used honey for the same purpose in human sur(ical 3ards and on e1perimental animals. A comprehensive revie3 of effect of honey on 3ounds is available else3here.;

Chapter ### "+0H%'%2%E,

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(e earch %e ign 0he study entitled Susceptibility of Helicobacter pylori to the Antibacterial Activity of Honey is an e1perimental research that aims to determine the capacity of antibacterial activity of honey and 3hether it can perform an important role in bacteria-(ro3th inhibition in the (astrointestinal system and in treatment of infection caused by the bacteria H. pylori. 0he researchers 3ill be usin( H. pylori as the test sub<ect, to be cultured from a (astric biopsy specimen obtained at a local dia(nostic centerAlaboratoryAclinic 3ith permission and clearance from the research advisers and colle(e dean. After satisfactory culture of the bacteria is achieved, inoculation of the three different types of honeys 3ill then be done after to test the e1tent of antibacterial activity. Specific tests to be conducted in this e1periment are the Dirby-Bauer 'is'iffusion Assay and "ueller-Hinton A(ar Platin(, as these t3o are best for determinin( the sensitivity or resistance of patho(enic aerobic and facultative anaerobic bacteria to various antimicrobial compounds used by many pharmaceuticals and laboratories in order to assist physicians in selectin( treatment options for their patients.

0his study is a true e1perimental research because the researchers consider the four fundamental properties of an e1perimental research and the study is to be conducted in a laboratory. H. pylori is decided to be the test specimen as the researchers are focused 3ith the (astrointestinal system and this bacteria is popularly -no3n for causin( most (astric problems in the human anatomy. Honey is decided as the sub<ect to be proven by the researchers as claims about this substance for treatment of different -inds of ailments has been lon( -no3n, but up until no3 there

is minimal laboratory testin( or evidences for its medicinal properties. Tuantitative methods 3ill be employed to determine specific results necessary for (eneratin( the desired research outputs.

$ocale of the Study

Figure 1. Location of -S/)""T

0he study 3ill be conducted in #li(an City, specifically at "indanao State Iniversity 4 #li(an #nstitue of 0echnolo(y. 0he a(ar culture and antibacterial activity testin( 3ill be done at the 'epartment of Biolo(y, Colle(e of Science and "athematics.

%ata +athering Procedure $. Selection of honey 0hree ?=@ types of honeys 3ill be used in the study. $irst is the natural honey ?!H@, to be ac&uired from a nearby local bee farm and homo(eneity of the honey 3ill be ensured by thorou(h a(itation at the time of e1traction from the combs. Second is a commercial honey, to be bou(ht from bee farm dealers and 3ill be identified by the apiarists supplyin( them, identification bein( based on the flavor, color and aroma of the honey, also the season and location of its production. 0he identity of the honeys selected 3ill be confirmed by pollen analysis. 0he honeys 3ill be stored in the dar- at :HC in polyethylene buc-ets 3ith ti(ht-fittin( lids. 0he third one is the Partificial honeyP ?home-made honey@ and is to be prepared by addin( (luconic acid lactone to distilled 3ater until a pH of =.O 3as achieved, after allo3in( hydrolysis of the lactone to occur. 0o 8M.: ml of this solution 3ill be added ;6.6 ( fructose, =>.9 ( (lucose and 9.5 ( sucrose. 0he solution 3ill then be heated briefly to :6HC to aid the dissolvin( of the su(ars.

&. +reparation of research instruments &.2 -aterials A(ar plates A(ar plu(s #noculatin( loop

Sterile needle 0est tubes &., 0%uiments Colorimeter Cor- borer Elass tissue (rinder &.3 Chemicals ChristensenPs urea medium Eluconic acid lactone Sterile distilled 3ater 6.: "c$arland standard 87 Acridine oran(e

C. +reparation of honey samples for testing Solutions of honey for testin( 3ill be handled aseptically, and 3ill be protected from bri(ht li(ht to prevent photode(radation of the (lucose o1idase that (ives rise to hydro(en pero1ide in honey. 0he honey solutions 3ill be prepared <ust before inoculation to ensure that there 3ill be no loss of hydro(en pero1ide in the solutions to be used to test total activity. 0en (rams of honey 3ill be 3ei(hed out and mi1ed into 86 ml of distilled 3ater to achieve a ;67 ?vAv@ solution. 0his solution is to

be sterili*ed by filtration throu(h a 6.9 <tm membrane, and 3ill be further diluted 3ith sterile distilled 3ater if re&uired.

D. Culturing of bacteria Helicobacter pylori 3ill be isolated from biopsy samples ta-en from the mar(in of (astric ulcers. 0hese samples 3ill then be processed usin( a (lass tissue (rinder, and 3ill be plated onto three ?=@ "ueller-Hinton a(ars. ?Cultures are easily contaminated o3in( to the lon( incubation in humid atmospheric conditionsK therefore "ueller-Hinton a(ars 3ere used throu(hout the subse&uent study because of its selectivity.@ Also from each processed sample a slope of Christiensen urea medium 3ill be inoculated and a slide prepared. 0he plates and urea slope is to be incubated at =MHC in a microaerophilic atmosphere for M days. 0he slide preparation 3ill then be stained usin( 87 acridine oran(e and e1amined for the presence of curved or Sshaped bacilli.

0. +reliminary screening of susceptibility of bacteria to honey A Dirby-Bauer diffusion assay 3ill be used to determine if Helicobacter pylori is susceptible to the antibacterial activity of the various types of honey. Isin( an inoculatin( loop, a colony of each isolate of Helicobacter pylori 3ill be pic-ed from a plate and a confluent la3n spread onto a fresh plate. Bells 3ill be cut in the a(ar 3ith a cooled flamed cor- borer of 5 mm diameter, and the a(ar plu(s 3ill be removed 3ith a sterile needle. 0he si1 3ells in each plate 3ill then be filled 3ith 8:6 8A of 967 ?vAv@ and ;67 ?vAv@ solutions of the Partificial honeyP ?home-

made honey@, natural honey and the commercial honey. 0he plates are to be incubated at =MHC in a microaerophilic atmosphere and observed daily.

#. Determination of -inimum "nhibitory Concentration 4-"C5 of honey Plates 3ill be prepared from "ueller-Hinton ?"H@ a(ar and mi1ed <ust before pourin( 3ith an appropriate volume of ;67 ?vAv@ solution of natural honey to achieve a ran(e of plates containin( natural honey at concentrations from 9.:-867 ?vAv@. Control plates 3ill be prepared 3ith no honey added. Isin( a colorimeter, suspensions of Helicobacter pylori isolates 3ill be made in 6.;:7 saline, e&uivalent to a 6.: "c$arland standard ?appro1imately 865C$IAml@. A confluent la3n of each isolate of Helicobacter pylori 3ill be spread onto each plate as in the preliminary screenin(. 0he plates are to be incubated at =MHC in a microaerophilic atmosphere and observed after M9 h.

*2perimental Procedure "c$arland Standard $. "n)house preparation of 6.7 -c#arlan! Stan!ar! Add a 6.:-ml ali&uot of a 6.6;5 molAliter BaCl9 ?8.8M:7 3tAvol BaCl9 U 9H96@ to OO.: ml of 6.85 molAliter H9S%; ?87 volAvol@ 3ith constant stirrin( to maintain a suspension. Lerify the correct density of the turbidity standard by measurin( absorbance usin( a spectrophotometer 3ith a 8-cm li(ht path and matched cuvette. 0he absorbance at >9: nm should be 6.65 to 6.8= for the 6.: "c$arland

standard. 0ransfer the barium sulfate suspension in ;- to >-ml ali&uots into scre3-cap tubes of the same si*e as those used in standardi*in( the bacterial inoculums. 0i(htly seal the tubes and store in the dar- at room temperature.

&. /se of the -c#arlan! stan!ar! in the 1irby)&auer proce!ure Prior to use, vi(orously a(itate the barium sulfate standard on a mechanical vorte1 mi1er and inspect for a uniformly turbid appearance. eplace the

standard if lar(e particles appear. #f usin( a standard composed of late1 particles, mi1 by invertin( (ently, not on a vorte1 mi1er. As the student adds bacterial colonies to the saline in the preparation of the inoculum step of the procedure, he or she should compare the resultin( suspension to the "c$arland standard. 0his is done by holdin( both the standard and the inoculum tube side by side and no more than 8 inch from the face of the Bic-erham card ?3ith ade&uate li(ht present@ and comparin( the appearance of the lines throu(h both suspensions. 'o not hold the tubes flush a(ainst the card. #f the bacterial suspension appears li(hter than the 6.: "c$arland standard, more or(anisms should be added to the tube from the culture plate. #f the suspension appears denser than the 6.: "c$arland standard, additional saline should be added to the inoculum tube in order to dilute the suspension to the appropriate density. #n some cases it may be easier to start over rather than to continue to dilute a bacterial suspension that is too dense for use.

Dirby-Bauer 'is- 'iffusion Assay $. +reparation of research instruments

$.2 -aterials Antibiotic dis85- to 9;-hour old pure culture of the or(anism to be tested Bic-erham card "ueller-Hinton a(ar plates, 866 mm or 8:6 mm Caliper or ruler $orceps Sterile s3abs #noculatin( loop or needle Bact-cinerator or Bunsen burner Alcohol pads or isopropyl alcohol in a tube $., 0%uipments Lorte1 mi1er =:HC to =MHC non-C%9 incubator $.3 Chemicals Barium sulfate standard Sterile saline in 9-ml tubes 6.: "c$arland standard

&. +reparation of -ueller)Hinton $gar plate Allo3 a "H a(ar plate ?one for each or(anism to be tested@ to come to room temperature. #t is preferable to allo3 the plates to remain in the plastic sleeve 3hile they 3arm to minimi*e condensation. #f the surface of the a(ar has visible li&uid present, set the plate inverted, a<ar on its lid to allo3 the e1cess li&uid to drain from the a(ar surface and evaporate. Plates may be placed in a =:HC incubator or in a laminar flo3 hood at room temperature until dry ?usually 86 to =6 minutes@. Appropriately label each "H a(ar plate for each or(anism to be tested.

C. +reparation of inoculums Ise a sterile inoculatin( loop or needle to touch four or five isolated colonies of the or(anism to be tested. Suspend the or(anism in 9 ml of sterile saline. Lorte1 the saline tube to create a smooth suspension. Ad<ust the turbidity of this suspension to a 6.: "c$arland standard by addin( more or(anism if the suspension is too li(ht or dilutin( 3ith sterile saline if the suspension is too heavy. Ise this suspension 3ithin 8: minutes of preparation.

D. "noculation of -H$ plate 'ip a sterile s3ab into the inoculum tube. otate the s3ab a(ainst the side of the tube ?above the fluid level@ usin( firm pressure, to remove e1cess fluid. 0he s3ab should not be drippin( 3et. #noculate the dried surface of a "H a(ar plate by strea-in( the s3ab three times over the entire a(ar surfaceK rotate the plate appro1imately >6 de(rees each time to ensure an even distribution of the inoculum.

im the plate 3ith the s3ab to pic- up any e1cess li&uid. 'iscard the s3ab into an appropriate container. 2eavin( the lid sli(htly a<ar, allo3 the plate to sit at room temperature at least = to : minutes, but no more than 8: minutes, for the surface of the a(ar plate to dry before proceedin( to the ne1t step.

0. +lacement of the antibiotic !is.s Place the appropriate antimicrobial-impre(nated dis-s on the surface of the a(ar, usin( either forceps to dispense each antimicrobial dis- one at a time, or a multidis- dispenser to dispense multiple dis-s at one time. ?See steps a. throu(h d. for the use of the multi-dis- dispenser or steps e. throu(h (. for individual dis- placement 3ith forceps. 0o use a multidis- dispenser, place the inoculated "H a(ar plate on a flat surface and remove the lid. Place the dispenser over the a(ar plate and firmly press the plun(er once to dispense the dis-s onto the surface of the plate. 2ift the dispenser off the plate and usin( forceps sterili*ed by either cleanin( them 3ith an alcohol pad or flamin( them 3ith isopropyl alcohol, touch each dis- on the plate to ensure complete contact 3ith the a(ar surface. 0his should be done before replacin( the Petri dish lid as static electricity may cause the dis-s to relocate themselves on the a(ar surface or adhere to the lid. 'o not move a dis- once it has contacted the a(ar surface even if the dis- is not in the proper location, because some of the dru( be(ins to diffuse immediately upon contact 3ith the a(ar. 0o add dis-s one at a time to the a(ar plate usin( forceps, place the "H plate on the template provided in this procedure. Sterili*e the forceps by cleanin( them 3ith a sterile alcohol pad and allo3in( them to air dry or immersin( the forceps in alcohol then i(nitin(. Isin( the forceps carefully remove one dis- from the cartrid(e. Partially remove the lid of the

Petri dish. Place the dis- on the plate over one of the dar- spots on the template and (ently press the dis- 3ith the forceps to ensure complete contact 3ith the a(ar surface. eplace the lid to minimi*e e1posure of the a(ar surface to room air.

Continue to place one dis- at a time onto the a(ar surface until all dis-s have been placed as directed in steps f. and (. above. %nce all dis-s are in place, replace the lid, invert the plates, and place them in a =:HC air incubator for 8> to 85 hours. Bhen testin( Staphylococcus a(ainst o1acillin or vancomycin, or +nterococcus a(ainst vancomycin, incubate for a full 9; hours before readin(.

#. "ncubation of the plates A temperature ran(e of =:HC V 9HC is re&uired. 'o not incubate plates in C%9 as this 3ill decrease the pH of the a(ar and result in errors due to incorrect pH of the media. esults can be read after 85 hours of incubation.

'. -easuring of the 8one si8es $ollo3in( incubation, measure the *one si*es to the nearest millimeter usin( a ruler or caliperK include the diameter of the dis- in the measurement. Bhen measurin( *one diameters, al3ays round up to the ne1t millimeter. All measurements are made 3ith the unaided eye 3hile vie3in( the bac- of the Petri dish. Hold the plate a fe3 inches above a blac-, nonreflectin( surface illuminated 3ith reflected li(ht. Lie3 the plate usin( a direct, vertical line of si(ht to avoid any paralla1 that may result in misreadin(. ecord the *one si*e on the recordin( sheet. #f the

placement of the dis- or the si*e of the *one does not allo3 you to read the diameter

of the *one, measure from the center of the dis- to a point on the circumference of the *one 3here a distinct ed(e is present ?the radius@ and multiply the measurement by 9 to determine the diameter. Ero3th up to the ed(e of the dis- can be reported as a *one of 6 mm. %r(anisms such as +roteus mirabilis, 3hich s3arm, must be measured differently than non-s3armin( or(anisms. #(nore the thin veil of s3armin( and measure the outer mar(in in an other3ise obvious *one of inhibition. 'istinct, discrete colonies 3ithin an obvious *one of inhibition should not be considered s3armin(. 0hese colonies are either mutant or(anisms that are more resistant to the dru( bein( tested, or the culture 3as not pure and they are a different or(anism. #f it is

determined by repeat testin( that the phenomenon repeats itself, the or(anism must be considered resistant to that dru(.