Exploration of Antimicrobial, Antioxidant, Anticancer and Biosafety of Selected Medicinal Plants

A Thesis Submitted for full requirement for the Degree of Doctor of Philosophy in Pharmacy

Submitted By

SalwaIbraheimAhmeida Abdulla

Supervisor:

Prof. Mohamed Elfatih Ahmed Omer

Co-supervisor:

Prof. Aisha Zoheir Al Magboul

December (2018)

DEDICATION

TO THE SCEINCE

TO SPIRITS OF My PARENTS

TO MY SISTER, A MAM AFTER MAM

TO MY WOUNDERFUL AMAZING BROTHERS

TO MY LOVELY KIND SONS, NEPHEWS AND NIECES

TO MY TEACHERS AND EVERY ONE TAUGHT ME A WORD

TO ALL MY DEAREST FRIENDS, NICE RELATIVES AND NABOURES

TO THE WOUNDERFUL HOME COUNTRY, LIBYA,THE ETERNAL LOVE

Salwa

ACKNOWLEDGEMENTS

First and forever thanks to ALLAH and his Messenger Muhammad peace be upon him

Thanks, appreciation and respect to my supervisors, Prof. Mohamed El-Fatih Ahmed Omer (Department of Pharmaceutics and Microbiology, Faculty of Pharmacy, Al-Neelain University, Khartoum, Sudan) and Prof. Aisha Zoheir Al Magboul (Department of Microbiology and Phytochemistry, Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Centre for Research, Sudan) for their guidance, supervision, advices, support and unlimited encouragement.

A lot of thanks and appreciation to Prof. Faiza Ahmed Omer (Department of Histopathology, veterinary research Organization, Soba, Sudan), Prof. AmnaHamadElhassan (Department of pharmacology, National Centre for Research,Khartoum, Sudan), Prof. Tarig El-Hadiya (Department of toxicology and dean of faculty of pharmacy, International Africa University, Khartoum, Sudan) for their help and advices in their field of specialization.

My gratitude to Prof. SalhaFarage Ben Jwairef, my supervisor inMaster degree, who encouraged and helped me build the foundation stone for my Ph.D (Head of Department of Microbiology, Libyan Academy, Libya). Also my gratitude to Dr.Osama Kherret and his laboratory staff members (Department of Pharmacology, Faculty of Veterinary, Omar Al-Mukhtar University, Libya).

I thank the Graduate College and the Faculty of Pharmacy, Al-Neelain University, Khartoum, Sudan. Also I thank the staff members and technicians of the Departments of Microbiology & Parasitology, Pharmacology, Phytochemistry and Biochemistry in Medicinal and Aromatic Plants and Traditional Medicine Research Institute, National Centre for Research, Sudan.

Thanks and appreciation to the Faculty of Pharmacy and Omar Al-Mukhtar University, Al- Bayda city, Libya for their candidacy and thanks to the beloved Libya for dispatching me for the PhD Degree.

Many thanks and appreciation to the Libyan embassy in Sudan specially Dr.Salama Mohammed Salama, Dr.FathiaAbdulhamedDallaf, Mr.Almahdi Hassan Al-TayabIdress and Mr.Abdulgader Mohammed Blegasimfor theircooperation andsupport. Furthermore, I would also like to thank everyone who has had a positive impact on my scientific career both in my beloved home country Libya and in Sudan.

ABSTRACT

The use of plants for treating diseases is well clear and even if their chemical constituents are not completely known but their use is well observed as widely disseminate. This is due to its clear efficacy which attributed to the disclosure of their therapeutic properties. This study was aimed to screen the antimicrobial activity of the chloroform, methanol, ethanol and aqueous extracts of nine Libyan medicinal plants (Myrtuscommunis, Pistacialentiscus, Capparisspinosa, Salvia fruticosa, Artmesiaherba Alba, Juniperusphonicea, Rosmarinusofficinalis, Citrus aurantiumandMarrubiumvulgare) against standard Gram positive bacteria (Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 25923) and Gram negative bacteria (Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27853) and fungi (Cadidaalbicans ATCC7596, Aspirogellusniger ATCC9763 ), the activities of these plants extracts were compared with commonly used antibiotics. Some of these plants were further screened for their antimicrobial activity against clinically isolated bacteria (methicillin resistant Staphylococcus aureus, Echerichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Enterobacter cloacae, Acinetobacterbaumanii and Klebsiellapneumoniae). In vivo investigation in rats for healing of wound that infected with either Staphylococcus aureus or Pseudomonas aeruginosa was done using the methanol leaves extract of the Myrtuscommunis . Phytochemical screening, study of the antioxidant activity, study of the cytotoxicity (Antitumor), study of the safety of the most promising biologically active plants extracts were done. Screening of the methanol extracts of leaves of Myrtuscommunis, Capparisspinosa and fruits of Pistacialentiscus for their toxicity (acute and sub-acute) through evaluation of liver function, kidney function, blood glucose, lipid profile, hematology parameters and histopathology profile via rat animal model was done. Screening of pharmacological effect of the three tested extracts on animal isolated vital organs (rabbit aortic strip, rabbit jejunum, rat uterus and frog abdominal muscle) were done with use of standard methods. Disk diffusion method was used to determine the susceptibility of the tested

5 microorganisms towards plant extracts. The minimum inhibitory concentration of the most active plants against standard bacteria was determined using the agar plate dilution method, the extract was tested on three types of wounded experimental rats, comparison was made between standard antibiotics treated wounds (3% Tetracycline for Pseudomonas aeruginosa infected wounds and 2% Fucidin for infected Staphylococcus aureus wounds). Standard basic phytochemical analysis and Gas chromatography/Mass Specrta, DPPH Scavenging assay and Sulfo-Rhodamine-B (SRB) assays were methods used in this study to identify the constituents and evaluate the antioxidant and antitumor activities of methanol extracts of leaves of Myrtuscommunis ,Capparisspinosa and fruits of Pistacialenticus. HEPG2 (human liver cell carcinoma), HCT (human colon cell carcinoma), MCF7 (human breast carcinoma) and PC3 (human prostatic cell carcinoma) cells lines were used to examine the antitumor activities of the tested extracts.

Fifty eight (72.5%) of these extracts showed antimicrobial activity against one or more of the tested organisms. Out of 80 investigated extracts 92% showed active inhibition of the growth against standards Gram positive bacteria, 56.2% against Gram negative bacteria and 35% against the fungi. Bacillus subtilis NCTC 8236, Staphylococcus aureusATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosaATCC 27853 and Candida albicansATCC 7596 were inhibited with percentages of 47.5%, 45%, 27.5%, 28.75% and 35% respectively.

The highest growth inhibition activity against Bacillus subtilisand Staphylococcus aureus was showed with chloroform and methanol extracts of Myrtuscommunis leaves and the highest growth inhibition activity against Escherichia coli and Pseudomonas aeruginosa shown with methanol and ethanol extracts of Myrtuscommunis leaves respectively. The highest growth inhibition activity against Candida albicansshown in this study was observed with ethanol extract of Capparisspinosa bark and methanol extracts of Salvia fruitosa bark and Myrtuscommunis leaves, while the highest inhibition activity against the Aspergillusnigerwas shown with methanol extract of Pistacialentiscus fruits.

The minimum inhibitory concentration shown in this study was 3.125 mg/ml from methanol extract of Myrtuscommunis leaves against Pseudomonas aeruginosa followed by 6.25 mg/ml by methanol extract of fruits of Pistacialentiscusagainst Candida albicans and methanol extract of leaves of Myrtuscommunis against Staphylococcus aureus and Candida albicans. 12.5 mg/mlwas the minimum inhibition concentration against Escherichia coli and Pseudomonas

6 aeruginosa in this study seen with methanol extract of bark of Salvia fruticosa. Other remained extracts showed minimum inhibitory concentrations ranged 50 – 100 mg/ml.

Among the clinical bacterial isolates the results revealed that the methanol extracts of Myrtuscommunis leaves, Pistacislentiscus fruits and Salvia fruticosa bark showed antibacterial activity against all tested isolates. The methanol extract of Capparisspinosa leaves in this study showed clear antibacterial activity against both standard Staphylococcus aureus and clinically isolated methicillin resistant Staphylococcus aureus. The antibacterial activity of six reference antibiotics was determined against clinical isolates and their activities were compared with the activity of plants extracts. This screening clarified that except of Ciprofloxacin 30µg, Myrtuscommunis considered the best more active choice followed with Salvia fruticosa for treating infection caused by methicillin resistant Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, and Pseudomonas aeruginosa compared to other tested antibiotics. Furthermore, Methanol extracts of Myrtuscommunis leaves, Pistacialentiscus fruits and Salvia frutisoa bark in this study had clear activity against clinical Acinetobacterbaumanii isolates compared with all tested antibiotics. Also the chloroform bark extract of Capparisspinosa in this study ranks the fourth level among the tested extracts and appeared as the only one inhibited the growth of methicillin resistant Staphylococcus aureus compared with tested extended beta- lactam antibiotics (Ceftazidime 30µg, Ceftriaxone 30µg).

The results proved that Myrtuscommunis leaves extract accelerating wound healing process without any significant differences between the action of the extract and the action of the used antibiotic, the result showed highly significant difference in wound healing period in rats with wound only and in rats treated either with the antibiotic or the extract.

The results showed presence of phenols, polyphenols (flavonoids as the abundant), saponins and tannins in the plants extracts. Also in this study results showed high antioxidant activity of methanol extracts of leaves of Myrtuscommunis , Capparisspinosa and fruits of Pistacialenticus, and also showed varied antitumor activities (moderate and active) against tested HEPG2 liver tumor cell line, HCT-166 colon tumor cell line, MCF-7 breast tumor cell line and PC3 prostate tumor cell line. Also this study proved that methanol extracts of leaves of Myrtuscommunis, bark of Salvia fruticosa and fruits of Pistacialentiscushad very high

7 antioxidant activity than that of the control used(Propyl gallate), while leaves of Capparisspinosa showed antioxidant activity lower than the control Propyl gallate.

Also with sub-acute toxicity assay, no significant differences were shown in this study compared with to the control group in the function biomarkers of liver (ALT, AST, GGT) and kidney functions biomarkers (creatinine, urea) and in blood function biomarker (hemoglobin, platelets, total white blood cells, total red blood cells). The study proved that methanol extracts of Myrtuscommunis leaves and Pistacialentiscus fruits showed significant lowering effect on blood glucose level while methanol extract of Capparisspinosa leaves showed lowering effect in blood glucose level but with no significant difference compared with the control group.

In this study methanol extracts of leaves of both Myrtuscommunis and Capparisspinosa showed cholinergic effect on the tested isolated rabbit jejunum and showed nor-epinephrine antagonistic effect on tested isolated rabbit aortic strip, while Pistacialenticus methanol fruits extract showed relaxant effect on isolated rabbit jejunum and showed nor-epinephrine potentiating effect on isolated rabbit aortic strip. Also the methanol extract of Capparisspinosa leaves showed relaxant effect on rat isolated uterus and Myrtuscommunis leaves extract showed absence of action up to 400 mg/ml but at high dose of 1000 mg/ml the extract had relaxed the uterus. No effect was shown from Pistacialentiscus extract on the isolated rat uterus. Also this study revealed that no one of the three tested extract showed activity on the tested isolated frog rectus abdominal muscle.

Necrosis and degeneration of renal tubules, hepatocellular, necrosis, congestion and haemorrhage of rats,s liver treated with the methanol leaves extract of Myrtuscommunis, methanol extract of fruits of Pistacialentiscus and methanol leaves extract of Capparisspinoswere detected.. On the other hand the three extracts showed fibrosis and necrosis of the rat uterus tissues and cells.

In conclusion, this study assumed that the bioactive substances including the flavonoids, alkaloids, terpenoids, tannins, fatty acids and steroids found within these extracts constituents are attributed factors to the proved biological activities of the extracts of leaves of Capparisspinosaand Myrtuscommunis and fruits of Pistacialentiscus. According to this assumption this study introduced these plant extracts as promising therapeutic agents that have

8 extensive biological activities support human health including antimicrobial, antioxidant and anti-tumor activities.

ملخص األطروحة

إٌ اسزخذاو انُجبربد نؼالج األيشاع ٔاػر رًب ًيب ، ٔززٗ إرا نى ٚزى انزؼشف ػهٗ يكَٕبرّ انكًٛٛبئٛخ رًب ًيب ، إال أٌ اسزخذايٓب يالزع ثشكم خٛذ ػهٗ َطبق ٔاسغ. ٚٔشخغ رنك إنٗ فؼبنٛزٓب انٕاػسخ انزٙ رُست إنٗ خظبئظٓب انؼالخٛخ انًمزشَخ. ْذفذ ْزِ انذساسخ إنٗ فسض انُشبؽ انؼًبد نهًٛكشٔثبد نًشكجبد انكهٕسٔفٕسو ٔانًٛثبَٕل ٔاإلٚثبَٕل ٔانًسزخهظبد انًبئٛخ نزسؼخ َجبربد ؽجٛخ نٛجٛخ )انًٛشسٍٛ، انجطٕو، انمجبس، رفبذ انشب٘، انشٛر، انؼشػبس، أكهٛم اندجم، انجشرمبل انًش، َٔجبد انشٔثٛب( ػذ انجكزٛشٚبانٛؼًبسٚخ يٕخجخ اندشاو )انؼظٕٚخ انشلٛمخ إٌ رٙ سٙ رٙ 8236، انؼُمٕدٚخ انزْجٛخ آ٘ رٙ سٙ سٙ 25922(ٔانجكزٛشٚب انٛؼًبسٚخ سبنجخ اندشاو )اإلٚششٚشٛخ انمٕنَٕٛخ آ٘ رٙ سٙ سٙ 25923، انضائفخ انضَدبسٚخ آ٘ رٙ سٙ س27853ٙ( ٔانفطشٚبد انٛؼًبسٚخ )انًجؼٛخ انجؼٛبء آ٘ رٙ سٙ سٙ 7596، انششبشٛخ انسٕداء آ٘ رٙ سٙ سٙ 9763(، ٔفؼبنٛخ ْزِ انًسزخهظبد لذ رًذ يمبسَزٓب ثبنؼًباد انسٕٚٛخ األكثش إسزخذايب. رى إخشاء فسض إػبفٙ نجؼغ ْزِ انُجبربد نُشبؽٓب انؼًبد نهًٛكشٔثبد ػذ انجكزٛشٚب انؼًضٔنخ سشٚشٚبً )انًكٕساد انؼُمٕدٚخ انزْجٛخ انًمبٔيخ نهًٛثٛسٛهٍٛ ، اإلٚششٚشٛخ انمٕنَٕٛخ ، انضائفخ انضَدبسٚخ ، انزمهجٛخ انشائؼخ ، اإليؼبئٛخ انًزسلٛخ ، انشاكذح انجٕيبئٛخ ، ٔانكهٛجسٛٛال انشئٕٚخ. فسض فٙ خسى زٙ ندشراٌ نشفبء خشذ أطٛت إيب انًكٕساد انؼُمٕدٚخ انزْجٛخ أٔ انضائفخ انضَدبسٚخ رى انمٛبو ثّ ثبسزخذاو يسزخهض أٔساق انًٛثبَٕل نُجبد انًٛشسٍٛ. رى انمٛبو ثبنفسض انكًٛٛبئٙ انُجبرٙ ، دساسخ َشبؽ يؼبداد األكسذح ، دساسخ انسًٛخ انخهٕٚخ )يؼبداد األٔساو ( ٔ دساسخ ساليخ انًسزخهظبد انُجبرٛخ انٕاػذح ثٕٛنٕخٛب ً. رى فسض يسزخهظبد ال يٛثبَٕل نٕسق َجبد انًٛشسٍٛ ٔانمجبس ٔنثًبس َجبد انجطٕو يٍ أخم سًُٛزٓى )انسبدح ٔرسذ انسبدح( يٍ خالل رمٛٛى ٔظبئف انكجذ ، ٔظبئف انكهٗ ، يسزٕٖ اندهٕكٕص فٙ انذو ، يظٓش انذٌْٕ ٔيؼهًبد أيشاع انذو ٔانًظٓش اندبَجٙ نألَسدخ ؽ ٍػشٚك ًَٕرج انسٕٛاٌ ، اندشراٌ. رى إخشاء فسض انزأثٛش انذٔائٙ نهًسزخهظبد انثالثخ انًخزجشح ػهٗ األؼػبء انسٕٚٛخ انسٕٛاَٛخ انؼًضٔنخ )ششؾٚ األثٓش األساَت ، أسَت انظبئى ، انفئشاٌ انشزًٛخ ٔانؼؼهخ انجطُٛخ انؼفٛشح نهؼفذع( ثبسزخذاو ؽشق لٛبسٛخ. رى اسزخذاو ؽشٚمخ اَزشبس انمشص نزسذٚذ زسبسٛخ انكبئُبد انذلٛمخ انًخزجشح َسٕ انًسزخهظبد انُجبرٛخ. رى رسذٚذ انسذ األدَٗ يٍ رشكٛض انًثجؾ يٍ انُجبربد األكثش َشبؽب ػذ انجكزٛشٚب انمٛبسٛخ ثبسزخذاو ؽشٚمخ رخفٛف ثبٜخبس. رى اخزجبس انًسزخهض ػهٗ ثالثخ إَٔاع يٍ خشٔذ اندشراٌ انزدشٚجٛخ انًظبثخ ، ٔرى إخشاء يمبسَخ ثٍٛ اندشراٌ انًظبثخ انؼًبندخ ثبنؼًبداد انسٕٚٛخ انمٛبسٛخ ) يشْى رٛزشاسٛكهٍٛ 3% نهدشٔذ انًظبثخ ثبنضائفخ انضَدبسٚخ ٔ يشْى فٕٛسٛذٍٚ 2% نهدشٔذ انًظبثخ ثبنًكٕساد انؼُمٕدٚخ انزْجٛخ(. انزسهٛم انكًٛٛبئٙ انمٛبسٙ ، رسهٛم انغبص انكشٔيبرٕخشافٛؽ / ٙف انكزهخ ، د٘ ثٙ ثٙ إرش ٔ انسهفٕسٔدايٍٛ- ثٙ

)إط آس ثؽ ْٗ )ٙشق اسزخذيذ فٙ ْزِ انذساسخ نزؼشٚف انًكَٕبد ٔرمٛٛى األَشطخ انؼًبدح نألكسذح ٔ األَشطخ انؼًبدح األٔساو نًسزخهظبد انًٛثبَٕل يٍ أٔساق انًٛشسٍٛ ٔانمجبس ٔثًبس انجطٕو. انخالٚب انجششٚخ )ْٛت ردٙ 2( خالٚب سشؽبٌ سشؽبٌ انكجذ، )إرش سٙ رٙ( خالٚب سشؽبٌ انمٕنٌٕ ، )إو سٙ إف 7( خالٚب سشؽبٌ انثذ٘ ٔ )ثٙ سٙ 3( خالٚب سشؽبٌ انجشٔسزب رى اسزخذايٓب نفسض انُشبؽ انؼًبد نألٔساو نهًسزخهظبد انًخزجشح.

أظٓش ثًبَٛخ ٔخًسٌٕ ) 72.5 ٪( يٍ ْزِ انًسزخهظبد َشبؽ يؼبد نهًٛكشٔثبد ػذ ٔازذ أٔ أكثش يٍ انكبئُبد انسٛخ انزٙ رى اخزجبسْب. يٍ ثٍٛ 80 يسزخهض رى فسظّ أظٓش 92٪ رثجؾٛ فؼبل نهًُٕ ػذ انجكزٛشٚب انٛؼًبسٚخ إٚدبثٛخ انغشاو ، ػ ٪56.2ذ انجكزٛشٚب سبنجخ اندشاو ٔ ػ ٪35ذ انفطشٚبد. انؼظٕٚخ انشلٛمخ إٌ رٙ سٙ رٙ 8236 ، انؼُمٕدٚخ انزْجٛخ آ٘ رٙ سٙ سٙ 25922 ، اإلٚششٚشٛخ انمٕنَٕٛخ آ٘ رٙ سٙ سٙ 25923 ، انضائفخ انضَدبسٚخ آ٘ رٙ سٙ سٙ 27853 رى كجسٓب ةَسجخ ػ ٪ 35 ٔ ٪ 28.75 ، ٪ 27.5 ، ٪ 45 ، ٪ 47.5هٗ انزٕانٙ.

رى إظٓبس أػهٗ َشبؽ رثجؾٛ نهًُٕ ػذ ثكزشٚب انؼظٕٚخ انشلٛمخ ٔ انؼُمٕدٚخ انزْجٛخ يغ انكهٕسٔفٕسو ٔيسزخهظبد انًٛثبَٕل يٍ أٔساق انًٛشسٍٛ ٔأػهٗ َشبؽ رثجؾٛ نهًُٕ ػذ اإلٚششٚشٛخ انمٕنَٕٛخ ٔ انضائفخ انضَدبسٚخ ٚظٓش يغ انًٛثبَٕل ٔيسزخهظبد اإلٚثبَٕل يٍ أٔساق انًٛشسػ ٍٛهٗ انزٕانٙ. نٕزع أػهٗ َشبؽ رثجؾٛ نهًُٕ ػذ انًجٛغ ح انجؼٛبء وٌ يسزخهض اإلٚثبَٕل نسبء انمجبس ٔيسزخهظبد انًٛثبَٕل يٍ رفبذ انشب٘ ٔأٔساق انًٛشسٍٛ ، ثًُٛب أظٓش َشبؽ انزثجؾٛ األػهٗ ػذ انششبشٛخ انسٕداء يغ يسزخهض انًٛثبَٕل نثًبس انجطٕو.

كبٌ انسذ األدَٗ نهزشكٛض انًثجؾ انػًٕر فٙ ْزِ انذساسخ ْٕ 3.125 يههٛدشاو / يم يٍ يسزخهض انًٛثبَٕل يٍ أٔساق انًٛشسػ ٍٛذ انضائفخ انضَدبسٚخ يزجٕ ػًب ثـ 6.25 يدى / يم ثٕاسطخ يسزخهض انًٛثبَٕل نثًبس انجطٕو ػذ انًجٛغح انجٛغاء ٔيسزخهض انًٛثبَٕل ألٔساق انًٛشسػ ٍٛذ انؼُمٕدٚخ انزْجٛخ ٔ انًجٛغح انجٛغاء. 12.5 يهغى / يم كبٌ رشكٛض انزثجؾٛ األدَٗ ػذ اإلششٚشٛخ انمٕنَٕٛخ ٔانضائفخ انضَدبسٚخ فٙ ْزِ انذساسخ شْٕذ يغ يسزخهض انًٛثبَٕل نهسبء رفبذ انشب٘. ٔأظٓشد ثبلٙ انًسزخهظبد رثجؾٛ ثسذ أدَٗ ثٍٛ 50 ٔ 100 يهغى / يم.

ثٍٛ انؼضالد انجكزٛشٚخ انسشٚشٚخ كشفذ انُزبئح أٌ يسزخهظبد انًٛثبَٕل ألٔساق انًٛشسٍٛ ، ثًبس انجطٕو ٔ نسبء رفبذ انشب٘ أظٓشد َشبؽ يؼبد نهدشاثٛى ػذ خًٛغ انؼضالد انًخزجشح. أظٓش يسزخهض انًٛثبَٕل يٍ أٔساق انمجبس فٙ ْزِ انذساسخ َشبؽ يؼبد نهدشاثٛى ٔاػر ػذ كم يٍ انًكٕساد انؼُمٕدٚخ انزْجٛخ انمٛبسٛخ ٔيكٕساد انؼُمٕدٚخ انزْجٛخ انًمبٔيخ نهًٛثٛسٛهٍٛ انؼًضٔنخ سشٚشًٚب. رى رسذٚذ انُشبؽ انؼًبد نهدشاثٛى نسزخ يؼبداد زٕٚٛخ يشخٛؼخ ػذ انؼضالد اإلكهُٛٛكٛخ ٔرًذ يمبسَخ أَشطزٓب يغ َشبؽ يسزخهظبد انُجبربد. أػٔر ْزا انفسض أَّ ثبسزثُبء انسٛجشٔفهٕكسبسٍٛ 30يٛكشٔخشاو/يم ، اػزجش انًٛشسٍٛ أفؼم خٛبس َشٚ ؾزجؼّ رفبذ انشب٘ نؼالج انؼذٖٔ انزٙ رسججٓب ثكزشٚب انؼُمٕدٚخ انزْجٛخ انًمبٔيخ نهًٛثٛسٛهٍٛ ، اإليؼبئٛخ انًزسلٛخ ، اإلششٚكٛخ انمٕنَٕٛخ ٔانضائفخ انضَدبسٚخ يمبسَخ ثبنؼًبداد انسٕٚٛخ انًخزجشح األخشٖ. ػٔالٔح ػهٗ رنك ، فئٌ يسزخهظبد انًٛثبَٕل يٍ أٔساق انًٛشسٍٛ ، ثًبس انجطٕو ٔ نسبء رفبذ انشب ٘ فٙ ْزِ انذساسخ كبٌ لدٚٓب َشبؽ ٔاػر ػذ ػضالد انشاكذح انجٕيبئٛخ يمبسَخ يغ خًٛغ انؼًبداد انسٕٚٛخ انزٙ رى اخزجبسْب. كًب أٌ يسزخهض انكهٕسٔفٕسو ل لزبء انمجبس فٙ ْزِ انذساسخ ٚظُف ةانًسزٕٖ انشاثغ ثٍٛ انًسزخهظبد انًخزجشح ٔظٓش أَّ انٕزٛذ انز٘ ًُٚغ ًَٕ انجكزٛشٚب انؼُمٕدٚخ

انًمبٔيخ نهًٛثٛسٛهٍٛ يمبسَخ يغ انؼًبداد انسٕٚٛخ انجٛزب الكزبو انًًزذح انًخزجشح ) سٛفزبصٚذٚى 30 يٛكشٔخشاو/يم ٔ سٛفزشٚبكسٌٕ 30 يٛكشٔخشاو/يم(.

أثجزذ انُزبئح أٌ أٔساق انًٛشسٍٛ أدد إنٗ رسشٚغ ػًهٛخ انزئبو اندشذ دٌٔ أ٘ فشٔق راد دالنخ إزظبئٛخ ثٍٛ رأثٛش انًسزخهض ٔرأثٛشانؼًبد انسٕٛ٘ انًسزخذو ، أظٓشد انُزٛدخ اخزالف كجٛش فٙ فزشح انزئبو اندشٔذ فٙ اندشراٌ يغ اندشذ فمؾ ٔفٙ اندشراٌ انزػ ٕٙندذ ثبنؼًبد انسٕٛ٘ أٔ ثبنًسزخهض.

ٔأظٓشد انُزبئح ٔخٕد انفُٕٛل ، ثٕنٛفُٕٛل )انفالفَٕٕٚذ ثٕفشح( ، ٔانظبثٍَٕٛ ٔانزبَُٛبد فٙ يسزخهظبد انُجبربد. أؼٚب فٙ ْزِ انذساسخ أظٓشد انُزبئح ٔخٕد َشبؽ يؼبد نألكسذح ػبنٙ نًسزخهظبد انًٛثبَٕل يٍ أٔساق انًٛشسٍٛ ٔانمجبس ٔثًبس انجطٕو ٔأظٓشد أؼٚب أَشطخ يؼبدح نهٕسو يزػُٕخ )يؼزذنخ ٔفؼبنخ( ػذ )ْٛت ردٙ 2( خالٚب سشؽبٌ سشؽبٌ انكجذ، )إرش سٙ رٙ( خالٚب سشؽبٌ انمٕنٌٕ ، )إو سٙ إف 7( خالٚب سشؽبٌ انثذ٘ ٔ )ثٙ سٙ 3( خالٚب سشؽبٌ انجشٔسزبرب. أؼٚب ْزِ انذساسخ أثجزذ أٌ يسزخهظبد انًٛثبَٕل نٕسق انًٛسشٍٚ ٔنسبء رفبذ انشب٘ ٔثًبس انجطٕو كبٌ نٓب َشبؽ يؼبد نألكسذح ػبنٙ خذا ٔأػهٗ يٍ انًمٛبط انًسزخذو )ثشٔثبٚم لبنالد( ثًُٛب أٔساق َجبد انمجبس اظٓشد َشبؽ يؼبد نألكسذح ألم يٍ انجشٔثبٚم لبنالد.

أؼٚب يغ اخزجبس انسًٛخ دٌٔ انسبد ، نى رظٓش فشٔق راد دالنخ إزظبئٛخ فٙ ْزِ انذساسخ يمبسَخ يغ يدػًٕخ انزسكى فٙ انًؤششاد انسٕٚٛخ نٕظٛفخ انكجذ )آ٘ إل رٙ ، آ٘ إط رٙ ، ردٙ ردٙ رٙ( ٔانؼاليبد انسٕٚٛخ نٕظبئف انكهٗ )انكشٚبرٍُٛٛ ٔانٕٛسٚب( ٔانؼاليبد انسٕٚٛخ فٙ نٕظٛفخ انذو ) انًٕٛٓغهٕثٍٛ ، ٔانظفبئر انذيٕٚخ ، ٔخالٚب انذو انجؼٛبء انكهٛخ ، ٔ إخًبنٙ خالٚب انذو انسًشاء(. أثجزذ انذساسخ أٌ يسزخهظبد انًٛزبَٕل يٍ أٔساق انًٛشسٍٛ ٔثًبس انجطٕو أظٓشد رأثٛش اَخفبع كجٛش ػهٗ يسزٕٖ انسكش فٙ انذو ثًُٛب أظٓش يسزخهض انًٛثبَٕل يٍ أٔساق انمجبس رأثٛ ًشا يُخف ؼًب فٙ يسزٕٖ اندهٕكٕص فٙ انذو ٔنكٍ يغ ػذو ٔخٕد فشق يؼُٕ٘ يمبسَخ يؼًدػًٕخ انزسكى.

فٙ ْزِ انذساسخ ، أظٓشد يسزخهظبد انًٛثبَٕل يٍ أٔساق كم يٍ انًٛشسٍٛ ٔانمجبس رأثٛش كٕنػ ُٙٛهٗ انظبئى انؼًضٔل انز٘ رى اخزجبسِ ٔأظٓشد رأثٛش يؼبد نأل أدسُٚبنػ ٍٛهٗ ششٚبٌ أثٓش األسَت انؼًضٔل ةانًخزجش ، ثًُٛب أظٓش يسزخهض ثًبس انًٛثبَٕل نهجطٕو رأثٛ ًشا إَجسبػ ٙؽهٗ طبئى األساَت انؼًضٔل ٔأظٓشد رأثٛش يؼضصا نأل أدسُٚبنػ ٍٛهٗ انشش٘اٌ األثٓش نألساَت. كًب أظٓش يسزخهض انًٛثبَٕل يٍ أٔساق انمجبس رأثٛش يشخػ ٙهٗ سزى اندشراٌ انؼًضٔل ٔأظٓشد خالطخ أٔساق انًٛشسٍٛ غٛبة انزأثٛش ززٗ خشػخ ٚظم إنٗ 400 يهدى / يم ٔنكٍ ػُذ خشػخ ػبنٛخ يٍ 1000 يهغى / يم كبٌ انًسزخهض لذ أظٓش ربثٛش يشخٙ نهشزى. نى ٚظٓش أ٘ رأثٛش يٍ يسزخهض انجطٕو ػهٗ سزى اندشراٌ انؼًضٔل. كًب كشفذ ْزِ انذساسخ أٌ ال أزذ يٍ انًسزخهظبد انثالثخ انزٙ رى اخزجبسْب أظٓش َشبؽب ػهٗ ؼػالد انجطٍ انؼفٛشح انًسزمًٛخ انؼًضٔنخ انًخزجشح.

انُخش ٔاَسطبؽ انُجٛجبد انكهٕٚخ ، رُخش انخالٚب انكجذٚخ ، إززمبٌ َٔضٚف شْٕذد ثُسٛح كجذ ٔكهٗ اندشراٌ انزٙ ػٕندذ ثًسزخهظبد انًٛثبَٕل نٕسق انًٛشسٍٛ ٔانمجبس ٔثًبس انجطٕو. يٍ َبزٛخ أخشٖ ، أظٓشد انًسزخهظبد انثالثخ رهٛفًب َٔخ ًشا فٙ أَسدخ ٔخالٚب سزى اندشراٌ.

فٙ انخزبو ، افزشػذ ْزِ انذساسخ أٌ انًٕاد انُشطخ ثٕٛنٕخٛبً ثًب فٙ رنك انفالفَٕٛذاد ، انمهٕٚذاد ، انزٛشثُٛذاد ، انزبَُٛبد ، األزًبع انذُْٛخ ٔانسزٛشٚٔذاد انًٕخٕدح داخم ْزِ انًسزخهظبد ػ ْٕٙايم رؼضٖ إنٗ األَشطخ انجٕٛنٕخٛخ انًثجزخ نًسزخهظبد أٔساق انمجبس ٔانًٛشسٍٛ ٔثًبس انجطٕو. ٔٔفمبً نٓزا االفزشاع ، ْزِ انذساسخ رمذو ْزِ انًسزخهظبد انُجبرٛخ كؼٕايم ػالخٛخ ٔاػذح نٓب أَشطخ ثٕٛنٕخٛخ ٔاسؼخ رذػى طسخ اإلَسبٌ ثًب فٙ رنك أَشطخ يؼبداد انًٛكشٔثبد ٔيؼبداد األكسذح ٔيكبفسخ األٔساو.

LIST OF CONTENTS

Dedication I Acknowledgements II English abstract III Arabic abstract VII List of contents X List of tables XV111 List of figures XX List of Photographs XXI List of abbreviations XXII CHAPTER ONE Introduction and Literature reviews 1 Introduction 1 1.1 Aim of the study 3 1.2 Literature reviews 4 1.2.1 Medicinal plants as a source of antimicrobial agents 5 1.2.2 Phytochemistry of medicinal plants and their benefits 6 1.2.2.1 1.2.2.1 Plant Primary Metabolites 6 1.2.2.2 Plant secondary Metabolites 6 1.2.2.2.1 Phenolic (Polyphenol) compounds 7 1.2.2.2.2 Terpenoids and Triterpenoids 7 1.2.2.2.3 Nitrogen Compounds 9 1.2.2.2.4 Sulfur compounds 10 1.2.3 Wound healing activity of the medicinal plants 11 1.2.3.1 Application of plants extracts for wound healing 11 1.2.4 Anti-oxidant activity of medicinal plants 12 1.2.5 Anti-cancer activity of medicinal plants 13

1.2.6 Botanical ethnophrmacological , phytochemical profiles and 13

biological activities of the studied medicinal plants 1.2.6.1 Myrtuscommunis 13 1.2.6.1.1 Classification and names 13 1.2.6.1.2 Plant description 14 1.2.6.1.3 Folk medicine uses 14 1.2.6.1.4 Phytochemistry 14 1.2.6.1.5 Antimicrobial activity 15 1.2.6.1.6 Antioxidant activity 17 1.2.6.1.7 Anti-cancer activity 17 1.2.6.1.8 Toxicity 17 1.2.6.1.9 Pharmacology 18 1.2.6.2 Pistacialentiscus L 19 1.2.6.2.1 Classification and names 19 1.2.6.2.2 Plant description 20 1.2.6.2.3 Folk medicine uses 20 1.2.6.2.4 Phytochemistry 20 1.2.6.2.5 Antimicrobial activity 20 1.2.6.2.6 Antioxidant activity 23 1.2.6.2.7 Anti-cancer activity 23 1.2.6.2.8 Toxicity 23 1.2.6.2.9 Pharmacology 24 1.2.6.3 Capparisspinosasubsporientalis(Duh.) Jafri 24 1.2.6.3.1 Classification and names 24 1.2.6.3.2 Plant description 25 1.2.6.3.3 Folk medicine uses 25 1.2.6.3.4 Phytochemistry 25 1.2.6.3.5 Antimicrobial activity 27 1.2.6.3.6 Antioxidant activity 27 1.2.6.3.7 Anti-cancer activity 28 1.2.6.3.8 Toxicity 28 1.2.6.3.9 Pharmacology 29 1.2.6.4 Salvia fruticosa Mill 29 1.2.6.4.1 Classification and names 29 1.2.6.4.2 Plant description 30

1.2.6.4.3 Folk medicine uses 30 1.2.6.4.5 Phytochemistry 30 1.2.6.4.6 Antimicrobial activity 30 1.2.6.4.7 Antioxidant 32 1.2.6.5 Artemisia herba-albaAsso 32 1.2.6.5.1 Classification and names 32 1.2.6.5.2 Plant description 33

1.2.6.5.3 Folk medicine uses 33 1.2.6.5.4 Phytochemistry 33 1.2.6.5.5 Antimicrobial activity 35 1.2.6.6 Juniperusphoenicea L. 35 1.2.6.6.1 Classification and names 35 1.2.6.6.2 Plant description 36 1.2.6.6.3 Folk medicine uses 36 1.2.6.6.4 Phytochemistry 36 1.2.6.6.5 Antimicrobial activity 36 1.2.6.7 MarrubiumvulgareL. 38 1.2.6.7.1 Classification and names 38 1.2.6.7.2 Plant description 38 1.2.6.7.3 Folk medicine uses 38 1.2.6.7.4 Phytochemistry 38 1.2.6.7.5 Antimicrobial activity 40 1.2.6.8 Rosmarinusofficinalis L 40 1.2.6.8.1 Classification and names 40 1.2.6.8.2 Plant description 41 1.2.6.8.3 Folk medicine uses 41 1.2.6.8.4 Phytochemistry 41 1.2.6.8.5 Antimicrobial activity 41 1.2.6.9 Citrus aurantiumL. 43 1.2.6.9.1 Classification and names 43 1.2.6.9.2 Plant description 43 1.2.6.9.3 Folk medicine uses 43 1.2.6.9.4 Phytochemistry 45

1.2.6.9.5 Antimicrobial activity 45 1.3 Pathogenic microorganism 46 1.3.1 Staphylococcus aureus 46 1.3.2 Gram negative bacteria 46 1.3.2.1 Acinetobacterbaumanii 46 1.3.2.2 Enterobacter cloacae 47 1.3.2.3 Escherichia coli 47 1.3.2.4 Klebsiella pneumonia 48 1.3.2.5 Proteus mirabilis 48

1.3.2.6 Pseudomonas aeruginosa 48 1.3.3 Fungi 49 1.3.3.1 Aspergillusniger 49 1.3.3.2 Candida albicans 49 CHAPTER TWO Material and Methods 2.1 Area of the study 50 2.2 Selected plants 50 2.3 Preparation of plants 50 2.4 Extraction of plants 50 2.4.1 Organic solvent extraction 51 2.4.2 Aqueous extraction 51 2.5 Preparation of the extracts stock solution 51 2.5.1 Aqueous extract stock solution 51 2.5.2 Organic extracts stock solution 51 2.6 Identification of clinical isolates 52 2.6 .1 Gram stain and microscopic examination of clinical isolates 52 2.6.2 Preparation of McFarland 0.5 solution 52 2.6.3 Identification of clinical isolates with BD Phoenix 100 System 52 2.6.4 Identification of clinical isolates with biochemical reactions 53 2.6.4.1 Catalase test 53 2.6.4.2 Coagulase test (Bound coagulase) 53 2.6.4.3 Deoxyribonulease (DNAse) test 54 2.6.4.4 Citrate utilization test 54 2.6.4.5 Oxidase test (Cytochrom oxidase) 54

2.6.4.6 Urease test 54 2.6.4.7 Triple sugar Iron agar (TSI) test 55 2.7 Preparation of Microorganisms suspensions 55 2.7.1 Preparation of bacterial suspension 55 2.7.2 Preparation of fungal suspension 55 2.8 Screening of antimicrobial activity 56 2.8.1 In vitro screening of antimicrobial activity 56 2.8.1.1 Antibacterial activity 56 2.8.1.2 Antifungal activity 56 2.9 Determination of minimum inhibitory concentration (MIC) 56 2.10 Antibiotic susceptibility test 57 2.11 In vivo antimicrobial and Wound healing activity 57 2.11.1 Pharmaceutical preparation of 3% ointment of 57 Myrtuscommunis Methanol leaves extract 2.11.2 Preparation of wounded animal 58 2.11.3 First in vivo trial line: Wound healing activity of 58 Myrtuscommunis(non-infected rats) 2.11.4 Second in vivo trial line: Wound healing activity of 59 Myrtuscommunis(Staphylococcus aureus infected rats) 2.11.5 Third in vivo trial line: Wound healing activity of 59 Myrtuscommunis(Pseudomonas aeruginosa infected rats) 2.12 Phytochemical analysis 60 2.12.1 Phytochemical screening with basic methods 60 2.12.1.1 Detection of Tannins 60 2.12.1.2 Detection of Sterols and Triterpenes 60 2.12.1.3 Detection of Alkaloids 61 2.12.1.4 Detection of Flavonoids 61 2.12.1.5 Detection of Saponins 61 2.12.1.6 Detection of Coumarins 61 2.12.1.7 Detection of Anthraquinone glycoside 62 2.12.2 Phytochemical screening with Gas chromatography – Mass 62 spectra phytochemical analysis (GC-MS) 2.13 Antioxidant screening of tested plants 63 2.13.1 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH) radical 63

Scavenging assay

2.13.2 IC50 Calculations 63 2.14 Anti-Cancer activity of tested plants (Cytotoxic activity) 63 2.14.1 Cell Lines and Culture Medium 63 2.14.2 Cell counting 64 2.14.3 Sulfo-Rhodamine-B Assay (SRB) for Cytotoxicity Screening 64 2.15 Pharmacological screening of tested extracts on isolated 65 organs 2.15.1 Sample Preparation 65 2.15.2 Preparation of physiological solutions 65 2.15.3 Animals 65 2.15.4 Rabbit Aortic Strip Preparation 65 2.15.5 Rabbit jejunum Preparation 66 2.15.6 Rat uterus Preparation 66 2.15.7 Frog rectus abdominal muscle preparation 67 2.16 Acute and sub-acute toxicity of selected plants 67 2.16.1 Extract dose preparation 67 2.16.2 Animals 68 2.16.3 Acute Toxicity (Lethality) and LD50 of three tested plants 68 2.16.4 Sub-acute toxicity 68 2.16.5 Histological profile 70 2.16.5.1 Histo-screening steps 70 2.17 Statistical Analysis 71 CHAPTER THREERESULTS 3.1 Antimicrobial activity of tested plants against standard and 72 clinical microorganisms 3.1.1 In vitro Antimicrobial activity of tested plants against 72 standard and clinical microrganisms 3.1.1.1 Antimicrobial activity of MyrtuscommunisL. against standard 72 microorganisms 3.1.1.2 Antimicrobial activity of Pistacialentiscus against standard 73 microorganisms 3.1.1.3 Antimicrobial activity of ArtmesiaherbaalboAsso against 73 standard microorganisms 3.1.1.4 Antimicrobial activity of Capparisspinosaagainst standard 77 microorganisms 3.1.1.5 Antimicrobial activity of JuniperusphoeniceaL.against 77 standard microorganisms 3.1.1.6 Antimicrobial activity of MarrubiumvulgariL. against 80 standard microorganisms 3.1.1.7 Antimicrobial activity of Rosmarinusofficalis L. against 82 standard microorganisms

3.1.1.8 Antimicrobial activity of Salvia fruticosa Mil against standard 84 microorganisms 3.1.1.9 Antimicrobial activity of Citrus aurantiumL. against standard 84 microorganisms 3.1.1.10 Antifungal activity of tested extract against standard 87 Aspergillusniger 3.1.1.11 Minimum inhibitory concentration 87 3.1.1.11.1 Minimum inhibitory concentration of 87 Pistacialentiscusmethanol fruits extract against standard organisms 3.1.1.11.2 Minimum inhibitory concentration of 87 Capparisspinosamethanol leaves and chloroform bark extracts against standard organisms 3.1.1.11.3 Minimum inhibitory concentration of Salvia 89 fruticosamethanol bark extract against standard organisms 3.1.1.11.4 Minimum inhibitory concentration of 89 Myrtuscommunismethanol leaves extract against standard organisms 3.1.1.12 Identification of clinical bacteria 89 3.1.1.13 Antibacterial activity of promising antibacterial plants extracts 91 against clinical isolates compared with antibiotic references 3.1.1.13.1 Antibacterial activity of Myrtuscommunis Methanol leaves 91 extracts against clinical isolates 3.1.1.13.2 Antibacterial activity of Pistacialentiscus methanol fruits 91 extracts against clinical isolates 3.1.1.13.3 Antibacterial activity of Salvia fruticosa methanol bark 93 extracts against clinical isolates 3.1.1.13.4 Antibacterial activity of Capparisspinosamethanol leaves 94 extract against clinical isolates 3.1.1.14 In vivo antimicrobial and wound healing activity 94 3.2 Phytochemical analysis 98 3.2.1 Phytochemical analysis investigated wit basic methods 98 3.2.2 Gas chromatography /Mass Spectra Phytochemical analysis 99 Technique 3.2.2.1 GC-MS anlalysis of Myrtuscommunis 99 3.2.2.2 GC-MS anlalysis of Pistacialentiscus 99 3.2.2.3 GC-MS anlalysis of Capparisspinosa 105 3.2.2.4 GC-MS anlalysis of Salvia fruticosa 105 3.3 Antioxidant activity of tested extracts 112 3.4 Cytotoxicity activity (Anticancer) of tested plant extracts 112 3.5 Toxicity of tested plant extracts 115 3.5.1 Acute toxicity (Lethality) of tested plants extracts 115 3.5.1.1 Acute toxicity of Methanol leaves extract of Capparisspinosa 115 3.5.1.2 Acute toxicity of Methanol leaves extract of Myrtuscommunis 115

3.5.1.3 Acute toxicity of Methanol fruits extract of Pistacialentiscus 115 3.5.2 Sub- acute toxicity of tested plants extracts 115 3.5.2.1 Biochemistry 115 3.5.2.1.1 Effects of plants extracts on liver function 115 3.5.2.1.2 Effects of plants extracts on glucose levels and lipid profile 118 3.5.2.1.3 Effects of plants extracts on Kidney function parameters 118 3.5.2.1.4 Effects of plants extracts on hematology parameters 121 3.5.2.2 Histopathological effect of tested plant extracts on vital organ 121 tisuues 3.5.2.2.1 Histopathological effect of Capparisspinosa on vital organs 121 tissues 3.5.2.2.2 Histopathological effect of Myrtuscommunis on vital organs 121 tissues 3.5.2.2.3 Histopathological effect of Pistacialentiscus on vital organs 121 tissues 3.5.3 Pharmacological behaviors of plants extracts treated rats 126 3.6 Pharmacological screening of tested extracts on isolated 126 organs 3.6.1 Pharmacological effect of tested plants extract on rabbit 126 jejunum 3.6.1.1 Effect of Capparisspinosa Methanol leaves extract 126 3.6.1.2 Effect of Pistacialentiscus Methanol fruits extract 126 3.6.1.3 Effect of Myrtuscommunis Methanol leaves extract 129 3.6.2 Pharmacological effect of tested plants extract on rabbit aortic 129 strip 3.6.2.1 Effect of Capparisspinosa Methanol leaves extract 129 3.6.2.2 Effect of Pistacialentiscus Methanol fruits extract 129 3.6.2.3 Effect of Myrtuscommunis Methanol leaves extract 129 3.6.3 Pharmacological effect of tested plants extract on rat uterus 129 3.6.3.1 Effect of Capparisspinosa Methanol leaves extract 129 3.6.3.2 Effect of Pistacialentiscus Methanol fruits extract 135 3.6.3.3 Effect of Myrtuscommunis Methanol leaves extract 135 3.6.4 Effect of tested Methanol plants extracts on Frog rectus 135 abdominus muscle CHAPTER FOUR Discussion, Conclusion and Recommendation

4.1 Discussion 141 4.1.1 Antimicrobial 141 4.1.2 Antioxidant 148 4.1.3 Anticancer (Tumor cells Cytotoxicity) 149 4.1.4 Biosafety 150 4.1.4.1 Toxicity 150 4.1.4.1.1 Liver function profile 150

4.1.4.1.2 Serum glucose and lipid profile 151 4.1.4.1.3 Kidney profile 151 4.1.4.1.4 Hematology profile 152 4.1.4.2 Pharmacology 152 4.2 Conclusion 154 4.3 Recommendation 155 References 156

LIST OF TABLETS Table (1): Antimicrobial activity and Yields percentages of 74 Myrtuscommunis L. extracts against standard microorganisms. Table (2): Antimicrobial activity and Yields percentages of 75 Pistacialentiscusextracts against standard microorganisms. Table (3): Antimicrobial activity and Yields percentages of 76 ArtmesiaherbaalboAsso extracts against standard microorganisms Table (4): Antimicrobial activity and Yields percentages of Capparisspinosa 78 extracts against standard microorganisms. Table (5): Antimicrobial activity and Yields percentages of 79 JuniperusphoeniceaL. extracts against standard microorganisms. Table (6): Antimicrobial activity and Yields percentages of 81 Marrubiumvulgari L. extractsagainst standard microorganisms. Table (7): Antimicrobial activity and Yields percentages of 83 Rosmarinusofficinalis L. extracts against standard microorganisms. Table (8): Antimicrobial activity and Yields percentages of Salvia 85 fruticosaMil extracts against standard microorganisms. Table (9): Antimicrobial activity and Yields percentages of Citrus 86 aurantiumL extracts against standard microorganisms. Table (10) Minimum inhibitory concentration of PistacialentiscusMethanol 88 fruits extract against standard organisms Table (11) Minimum inhibitory concentration of CapparisspiosaMethanol 88 leaves and Chloroform bark extracts against standard organisms Table (12) Minimum inhibitory concentration of Salvia fruticosaMethanol 90 bark extract against standard organisms Table (13) Minimum inhibitory concentration of MyrtuscommunisMethanol 90 leaves extract against standard organisms Table (14): Mean diameter of inhibition zones (mm) of antibacterial activity 92 of tested plants extracts and antibiotics against tested clinical bacterial isolates Table (15): Means ± standard deviation of area of wound healing of non- 95 infected wounds treated with Tetracycline 3% o115intment and Myrtuscommunis 3% ointment Table (16): Means ± standard deviation of area of wound healing of 96 Staphylococcus aureus -infected wounds treated with Fucidine 2% ointment and Myrtuscommunis 3% ointment Table (17): Means ± standard deviation of area of wound healing of 97 Pseudomonas aeruginosa-infected wounds treated with Tetracycline 3% ointment and Myrtuscommunis 3% ointment Table (18): Phytochemical screening of methanol extracts of tested plants 100 Table (19): Main bioactive chemical composition distinguished in methanol 101 leaves extract of Myrtuscommunis Table (20): Main bioactive chemical composition distinguished in methanol 103

21 fruits extract of Pistacialentiscus Table (21): Main bioactive chemical composition distinguished in methanol 106 leaves extract of Capparisspinosa Table (22): Main bioactive chemical composition distinguished in 108 Chloroform bark extract of Capparisspinosa Table (23): Main bioactive chemical composition distinguished in methanol 110 bark extract of Salvia fruticosa Table (24): Percentage and IC50 of DPPH radical scavenging activities of 113 Methanol extracts of tested plants parts Table (25): Cytotoxic effects of tested plants on carcinoma colon cell line 113 (HCT) Table (26): Cytotoxic effects of tested plants on carcinoma liver cell line 116 (HEPG2) Table (27): Cytotoxic effects of tested plants on carcinoma breast cell line 116 (MCF7) Table (28): Cytotoxic effects of tested plants on carcinoma prostate cell line 117 (PC3) Table (29): Liver function parameters values of control and extracts treated 119 rats Table (30): Effects of plants extracts on Glucose and lipid profile parameters 120 Table (31): Effects of plants extracts on Kidney parameters values 120 compared to control Table (32): Effects of plants extracts on Hematology parameters values 122 compared to control group.

LIST OF FIGURES Figure (1): Most common derivative of plants phenolic compounds 8 Figure (2): IC50 of DPPH radical scavenging activities of tested plants parts 114 Figure (3): Pharmacological effect of Capparisspinosa leaves extract on 127 rabbit jejunum Figure (4) Effect of Pistacialentiscus methanol fruits extract on isolated 128 rabbit jejunum Figure (5) Effect of Myrtuscommunis methanol leaves extract on isolated 130

22 rabbit jejunum Figure (6) Effect of Capparisspinosa methanol fruits extract on isolated 131 aortic strip Figure (7) Effect of Pistacialentiscus methanol fruits extract on isolated 132 aortic strip Figure (8) Effect of Myrtuscommunis methanol leaves extract on isolated 133 aortic strip Figure (9): Pharmacological effect of Capparisspinosa methanol leaves 134 extract on isolated rat uterus Figure (10): Pharmacological effect of Pistacialentsicus methanol fruits 136 extract on isolated rat uterus. Figure (11): Pharmacological effect of Myrtuscommunis methanol leaves 137 extract on isolated rat uterus Figure (12):Pharmacological effect of Cappariaspinosa methanol leaves 138 extract on isolated Frog rectus abdominus muscle Figure (13):Pharmacological effect of Pistacilentiscuc methanol fruits 139 extract on isolated frog rectus abdominus muscle Figure (14):Pharmacological effect of Myrtuscommunis methanol leaves 140 extract on isolated frog rectus abdominus muscle

LIST OF PHOTOGRAPHS Photo (1): MyrtuscommunisL. 16 Photo (2): PistacialentiscusL. 21 Photo (3): Capparisspinosasubsporientalis (Duh.) Jafri. 26 Photo (4): Salvia officinalis L. 31 Photo (5): Artemisia herba-albaAsso 34 Photo (6): JuniperusphoeniceaL. 37 Photo (7): Marrubiumvulgarae L. 39 Photo (8): Rosmarinusofficinalis L. 42 Photo (9): Citrus aurantium L. 44 Photo (10): Capparisspinosa Methanol leaves extract treated rat liver tissue 123 Photo (11): Capparisspinosa Methanol leaves extract treated rat kidney 123 Photo (12): Capparisspinosa Methanol leaves extract treated rat uterus tissue (1g/kg 123 IP 24 hour’s single dose) Photo (13): Myrtuscommunis Methanol leaves extract treated liver tissue (0.125g/kg 124 IP) Photo (14): Myrtuscommunis Methanol leaves extract treated kidney tissue (0.125g/kg 124 IP)

Photo (15): Myrtuscommunis Methanol leaves extract treated rat uterus tissue 124 Photo (16): Pistacialentiscus Methanol fruits extract treated rat 125 Photo (17): PistacialentiscusMethanol fruits extract treated 125 Photo(18): PistacialentiscusMethanol fruits extract treated rat uterus tissue 125

ABBREVIATION LIST ALP Alkaline phosphatase ALT Alanine transaminase AST Aspartate transaminase DMSO Dimethyl sulfoxide DPPH 1,1-diphenyl-2-picrylhydrazyl EDTA Ethylene diaminetetraacetic acid ESBL Extended-Spectrum Beta-Lactamase GC/MS Gas Chromatography/ Mass Spectra GGT Gamma-glutamyltransferase HPLC High Performance Liquid Chromatograph IP Intra peritoneal LD Lethal Dose MDR Multi Drug Resistance MEM Minimal Essential Media MIC Minimum Inhibitory Concentration MRSA Methacillin resistant Staphylococcuaaureus MWHPS Microwave Histo-Processor System NIST National Institute of Standard and Technology Na. CMC Sodium carboxy methyl cellulose SRB Sulfo-Rhodamine-B Assay TSI Triple sugar Iron agar VRE Vancomycin resistant Enterococci

LIST OF APPENDEXES Table (A1)Biochemical tests for the identification of Staphylococcus aureus isolates 183 Table (A2 ): Biochemical tests for the identification of Gram negative bacilliisolates 183 Table (A3): Comparison of inhibition zones of Myrtuscommunis leaves methanol 184 extract and standard antibiotics references againstA.baumanii Table (A4): Comparison of inhibition zones of Myrtuscommunis leaves methanol 185

24 extract and standard antibiotics references againstP.mirabilis Table (A5): Comparison of inhibition zones of Myrtuscommunis leaves methanol 186 extract and standard antibiotics references against E.cloacae Table (A6): Comparison of inhibition zones of Myrtuscommunis leaves methanol 187 extract and standard antibiotics references against K.pneumonia Table (A7): Comparison of inhibition zones of Myrtuscommunis leaves methanol 188 extract and standard antibiotics references against P.aeruginosa Table (A8): Comparison of inhibition zones of Myrtuscommunis leaves methanol 189 extract and standard antibiotics references against E.coli Table (A9): Comparison of inhibition zones of Myrtuscommunis leaves methanol 190 extract and standard antibiotics references against S.aureus Table(A10): Comparison of inhibition zones of P.lentiscusmethanol fruits extracts 191 and standard antibiotics references against A.baumanii Table (A11): Comparison of inhibition zones of P.lentiscusmethanol fruits extracts 192 and standard antibiotics references againseE.cloacae Table (A12): Comparison of inhibition zones of P.lentiscus methanol fruits 193 extracts and standard antibiotics references against clinical E.coli Table (A13): Comparison of inhibition zones of P.lentiscus methanol fruits extracts 194 and standard antibiotics references against K.pneumonia Table (A14): Comparison of inhibition zones of P.lentiscus methanol fruits extracts 195 and standard antibiotics references against P.aeruginosa Table (A15): Comparison of inhibition zones of P.lentiscus methanol fruits extracts 196 and standard antibiotics references against P.mirabilis Table (A16): Comparison of inhibition zones of P.lentiscus methanol fruits extracts 197 and standard antibiotics references against clinical S.aureus Table (A17): Comparison of inhibition zones of Salvia fruticosa bark methanol 198 extract and standard antibiotics references against A.baumanii Table (A18): Comparison of inhibition zones of Salvia fruticosa bark methanol 199 extract and standard antibiotics references against P.aeruginosa Table (A19): Comparison of inhibition zones of Salvia fruticosa bark methanol 200 extract and standard antibiotics references against P.mirabilis Table (A20): Comparison of inhibition zones of Salvia fruticosa bark methanol 201 extract and standard antibiotics references against E.cloacae Table (A21): Comparison of inhibition zones of Salviafruticosa bark methanol 202 extract and standard antibiotics references against E.coli Table (A22): Comparison of inhibition zones of Salvia fruticosa bark methanol 203 extract and standard antibiotics references against K.pneumonia

Table (A23): Comparison of inhibition zones of Salvia fruticosa bark methanol 204 extract and standard antibiotics references against S.aureus Table (A24): Comparison of inhibition zones of Capparisspinosa bark chloroform 205 extract and standard antibiotics references on S.aureus Table (A25): Comparison of inhibition zones of Capparisspinosa bark chloroform 206 extract and standard antibiotics references on S.aureus

Table (A26): Antifungal activity of methanol extracts of tested extracts against 207 Aspergillusniger ATCC 9763 Table (A27): Pharmacological behavioral screening of Capparisspinosa methanol 208 leaves extract treated rats. Table (A28): Pharmacological behavioral screening of Pistacialentiscus methanol 209 fruits extract treated rats. Table (A29): Constituents of Krebs solution, Tyrode solution, D Jalon’s solution 210 and Frog solution Some Preparations 211 Materials 212

CHAPTER ONE

Introduction and Literature Reviews

1.1 Introduction Approximately, 377-460 BC, Hippocrates, the Greek physician traditionally regarded as the father of medicine pointed out the strong relationship between plant foods and health; he said “Let thy food be thy medicine and thy medicine be thy food” reflecting the preventive and therapeutic roles of bioactive components in dietary with particular emphasis on their high level of safeness and goodness (Leicach and Chludil, 2014). Natural products originated from plant, animal, and minerals have been the basis of treatment of human disease and believed to be resources of new drugs. It was estimated that

over 50% of modern clinical drugs have natural products’ origin (Adnan et al., 2014). From 250 to 500 thousand plant species are estimated to exist on the planet, only between 1 and 10% are used as food by humans and other animals. Understanding of therapy properties of these natural products has been transmitted over the centuries within and among human communities, since the studying of them helps to understand plant toxicity and protect human and animals from natural poisons (Silva and Fernandes, 2010). Herbal medicines are currently in demand and becoming more popular due to toxicity and side effects of synthetic drugs (Santram et al., 2017), and because they are promising a shorter and cheaper production, since the requirements needed for medicinal plants to be used don’t ask for strict quality control concerning efficacy and safety compared to the non-natural drugs (Silva and Fernandes, 2010). The importance of traditional medicine as a source of primary health care was firstly recognized in 1978 by the World Health Organization in the primary health care and the tradition use of medicinal plants, as a basis for the maintenance of good health in most developing countries (Rukangira, 2001). Hendawy et al. (2010) reported that, herbal medicines have an important value in the developing countries for their spiritual and sociocultural use and also for their medicinal value in tribal and rural communities. In developing countries about 80% of the population used traditional medicines because they cannot afford the high cost of western pharmaceuticals and health care and also because it is more acceptable as cultural, religion and spiritual perspective (Maroyi, 2013)

Recently, due to the constant emergence of microorganisms resistant to common used antimicrobials substances, pharmaceutical companies have been motivated to common strategies include developing new antimicrobial drugs or changing the molecular structure of the existing medicines in order to make them more effective or recover activity claimed to be lost due to bacterial resistance mechanisms. The miss use of antimicrobial agents has led to the emergence of antibiotic resistant pathogens, the matter which increase the need for exploring new drugs (Garmana et al., 2014). The ready availability and economy of plants as direct therapeutic agents make plants more attractive when compared to modern medicine (Agbo and Ngogang, 2005; Odey et al. 2012). It has been reported that people turned to ethno-pharmacognosy in response to the development of adverse effects of the available drugs and development of microbial resistance to the chemically synthesized drugs. Natural products from medicinal plants contain various types

27 of bioactive compounds; either as pure ingredient or as extracted mixture compounds, provide unlimited opportunities for new drug (Sasidharan et al., 2011). The main problem facing the use of traditional medicines is the proof requirement that the active components contained in medicinal plants are therapeutically effective and safe (Rukangira, 2001). However, clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim. Clinical trials directed towards understanding the pharmacokinetics, bioavailability, efficacy, safety and drug interactions of newly developed bioactive compounds and their formulations with carefully evaluation (Sasidharan et al., 2011). The overall outputs of each scientific study will help to have more and more information to complete the picture about the use of medicinal plants in aim to find a safe effective natural product which could be used as alternative to the expensive, harmful side effect producing synthetic drugs (Agbo and Ngogang, 2005; Odey et al. 2012). Africa is a rich source of medicinal plants and the treatment of wounds with preparations from different plant parts mostly leaves and roots are a daily practice in an African community, and such applications have been highly successful because of the disinfectant and astringent properties of certain plant parts. Libya has a number of varied ecosystems, ranging from the Mediterranean coast to the dry savannas and arid Sahel of the interior. These ecological regions support a unique diversity of plants which are adapted to the harsh conditions of these ecosystems. The Mediterranean climate in Libya favors the growth of a great number of plant species, some of which have various medicinal and antioxidant potential properties and the use of plant extracts with known reliable antimicrobial and antioxidant efficacies can be of great significance in therapeutic approaches of many diseases (Naili et al., 2010; Singh, 2105). EL-Jabal EL-Akhdar region lies along the north-eastern coast of Libya, it extends between Marmarica plateau to the east and Benghazi plain to the west. This region is characterized by plant intensity and diverse areas of forest and grassland (El-barasi et al., 2011), due to its flora, vegetation cover, biodiversity, climate and ecological importance, AL-Jabal AL_Akhdar area was studied in the early of 1900’s by a number of Italian researchers. Recently, some surveys were done by local researchers, but the information and published data are still little (Elshatshat and Mansour, 2014).

Aim of the study:

This research has been concerned with the study of the biological activities of different plants from different genera that wildly grow around Al-Bayda city, El-Jabal El-Akhdar area in Libya. Objectives: 1. Study of the antimicrobial activity 1.1 Screening of the antimicrobial activity and determination of the minimum inhibitory concentration of some Libyan medicinal plants against standard Gram positive and Gram negative bacteria and fungi. 1.2 Screening of the antibacterial activity of the most active plants extracts against clinicallyisolated Gram positive and Gram negative bacteriaand comparison the activities of these plants with reference antibiotics. 1.3 Preparation of pharmaceutical dosage form formulation of the most promising plant extract and study of its effect on the wound healing via establishment of an in vivo antimicrobial activity assessment rats model. 2. Phytochemical screening to detect the active constituents of the most promising biologically active plants extracts. 3. Study of the Antioxidant activity of the most promising antimicrobial plants extracts. 4. Study of the cytotoxicity (Anticancer) of the most promising antimicrobial plants extracts. 5. Study of the safety of the most promising biologically active plants extracts 5.1 Screening of their toxicity through evaluation of liver function, kidney function, blood glucose, lipid profile and hematology parameters via rat animal model. 5.2 Screening of pharmacological effect of tested extracts on animal isolated vital organs (rabbit aortic strip, rabbit jejunum, rat uterus and frog abdominus muscle).

1.2. Medicinal plants

An essential role has been played by medicinal plants in the development of human culture, including religions and different ceremonies (Sing, 2015; Adnan et al., 2014), and has been noticed in the diet and treatment of many diseases (Odey et al., 2012; Rahnavard and Razavi, 2016). The use of plants for treating diseases is as old as the human species (Maciel et

29 al., 2002). Many age-related diseases such as cardiovascular disorders, neurodegenerative and cancer are proved in the last decades to have effect on population life span and they have been referred to multifactorial processes in which different cellular pathways become abnormal. Oxidative stress has been thoroughly demonstrated to play a major role in these diseases pathogenesis. Plants metabolites include a variety of bioactive compounds, antimicrobials among them and also antioxidants which have been proved to exert chemo-preventive effects delaying such disorders (Leicach and Chludil, 2014). The use of plants for treating diseases is well clear and even if their chemical constituents are not completely known, but their use is well observed as widely disseminate. This is due to its clear efficacy which attributed to the disclosure of their therapeutic properties. Also, basic requirements to use the medicinal plants do not involve strict quality control regarding efficacy and safety compared to the synthetic drugs. This make the phytotherapy introduced to the market give promising cheaper and shorter production (Cowan, 1999). In developing countries, herbal medicine still the spine for treatment of many diseases for about 75-80% of the world’s population due to their better compatibility with the human body and producing lesser adverse effects (Nahida and Siddiqui, 2012). The ready availability and economy of plants as direct therapeutic agents make plants more attractive when compared to expensive harmful side effect producing synthetic drugs (Odey et al. 2012).

Therapeutic impact of medicinal plants is well documented at global level, where they always have the ability to treat different diseases caused by microorganisms (Silva and Fernandes 2010). Approximately one quarter of prescribed treatments contain bioactive ingredients containing extracts obtained from plants such as cardiac glycosides (cardiotonics), Opium alkaloids (analgesics), Atropine (anticholinergic), Quinine (antiparasitic), Artimisinin (antimalarial), and the antineoplastic Taxol, Vinblastine and Vincristine, (Nahida and Siddiqui, 2012). 1.2.1Medicinal plants as a source of antimicrobial agents

The World Health Organization defined the herbal medicine as plant derived material or preparation contains either raw or processed ingredients from one or more plants which when administered to man or animals exert a sort of pharmacological action on them. According to the World Health Organization, about 70-80% of world population uses herbal medicines for their

30 therapeutic effects. More than 3.3 billion people in the less developed countries utilize medicinal plants as backbone of traditional medicine because they consider as rich resources of therapeutic bioactive phytochemicals such as phenolic compounds and flavonoids which have been reported to have positive impact on health and cancer prevention. These phytochemicals can be used in novel drugs development and synthesis (Sasidharan et al., 2011; Singh, 2105, WHO, 1993).

Due to the challenge of growing incidents of drug-resistant pathogens, the antimicrobial activity of plants and their metabolites have recently received the attention (Elmhdwi et al. 2015). A lot of phytochemicals from plants as safe and broadly effective alternatives with less adverse effect have been found. Many beneficial biological activities such as anticancer, antioxidant, anti-diarrheal, analgesic, wound healing and antimicrobial activity were reported. Among the causes which lead to deaths are the infectious diseases, where more than 25% of deaths cases worldwide are referred to infectious diseases. With the emerging of bacterial resistance and failure of the common used modern antibiotics to overcome the infectious diseases result from these pathogens, medicinal plants are considered a promising future solution (Levy and Marshall, 2004). There are about half million plants around the world, and most of them, their medical activities have not been investigated yet, and their medical activities could be decisive in the treatment of infectious diseases (Singh, 2015), specifically, it has been reported that many wild plants possess many bioactive compounds which having antimicrobial activities (Abdallah et al., 2012).

1.2.2Phytochemistry of medicinal plants and their benefits It has been well documented that, antimicrobial, antioxidants, anticancer and other healing effects of medicinal plants are attributed to their metabolites, specifically secondary metabolites. Plant metabolites include: primary metabolites and secondary metabolites. Good balance between products of primary and secondary metabolism is best for a plant’s optimal growth and development as well as for its effective coping with often changing environmental conditions (Leicach and Chludil, 2014; Sing, 2015).

1.2.2.1 Plant Primary Metabolites

Plant primary metabolites are organic compounds produced by every plants via a primary metabolism comprises all metabolic pathways that governs all basic physiological processes that allow a plant to grow and set seeds, by translating the genetic code into proteins, carbohydrates, and amino acids, since these compounds are directly involved in the growth and development of a plant (Sing, 2015).

1.2.2.2 Plant secondary Metabolites Crozier et al. (2006) documented that secondary metabolism produces a large number of specialized compounds (estimated 200,000). These compounds do not aid in the growth and development of plants but are required for the plant to survive in its environment. Plant secondary metabolites are compounds produced via a secondary metabolism which connected to the primary metabolism by using building blocks and biosynthetic enzymes derived from primary metabolism. There is no common and fixed agreed system for classification of secondary metabolites, but based on their biosynthetic origins, plant secondary metabolites can be divided into four major groups; Phenolic compounds (polyphenol), Terpenoids and triterpenoids, and Nitrogen-containing compounds (including, protein amino acids, amines, cyanogenic glycosides, glucosinolates) and sulphur containing compounds (Singh,2015).

1.2.2.2.1 Phenolic (Polyphenol) compounds Phenolic compounds, also known as polyphenols, are the most pronounced secondary metabolites found in plants kingdom. These compounds produced in specific cells, tissues or even in development stages in specific groups of plant families. They have important roles in plant defense mechanisms against viruses, bacteria, fungi, and herbivores. In addition polyphenol molecules were well documented to possess antimicrobial and antioxidant activities make them play an important role in treatment of infectious diseases (Randhir et al., 2004; Lin et al., 2016; Papuc et al., 2017). The use of phenolic compounds by human has been linked with the reduced risk of infectious disease and also linked with reduced risk of the multiple non-communicable chronic diseases such as neurodegenerative diseases, type II diabetes, cardiovascular, osteoporosis and certain cancers (Velderrain-Rodríguez et al., 2014). This linkage is attributed to the different bio-

32 action of poly-phenolic compounds such as anti-inflammation, antioxidant, anti-proliferation, modulation of signal transduction and anti-microbial activities (Goderska et al., 2008; De Pascual-Teresa et al., 2010). Chemically, phenolic compounds contain benzene (phenyl) ring, with one or more hydroxyl groups, and range from simple phenolic molecules to highly polymerized compounds (Velderrain-Rodríguez et al., 2014). Benzoic and cinnamic acid derivatives and flavonoids are the most distributed phenolics. Polyphenolic units are biosynthesized via shikimate pathway, resulting in cinnamic acids (C6–C3) phenylpropanoid building block that also contributes to other plant phenolics backbones such as those from flavonoids (C6–C3–C6), anthocyanidins (C6–C3–C6), and coumarins (C6–C3). Also stilbeneoids (C6–C2–C6) and benzoic acid derivatives (C6–C1) such as gallic and ellagic acids are synthesized through the same metabolic pathway (Silva et al., 2010). Most common derivative of plants phenolic compounds are listed in Figure (1).

1.2.2.2.2 Terpenoids and Triterpenoids Terpenoides are well known to be effective antimicrobials, and can be divided into monoterpenoids and sesquiterpenoids. They are a major component in essential oils of plants which have been described a decade ago as bactericidal agents. Espintanol, piquerol A, thymol and menthol are monoterpens secondary metabolites proved to produce antiparasitic activity and thymol was claimed to be lead compound for drugs used as antileishmania agents. Terpinen-4-ol

Figure (1): Most common phenolic compounds

is monoterpenoid derivative effective against Trypanosoma brucei parasite. Allo-aromadendrene, Aromadendrene and Artemisinin are sesquiterpenoids and even though they are less effective against trypanosomes parasites, but Artemisinin which isolated from Artemisia annuaplant is the most widely used as ant-parasite agent. Despite the World Health Organization, advised to do not use Artemisininin due to its short plasma half-life (limited bio-availability) and poor solubility. Douglas and his team have strongly advised combination based Artemisinin therapies to treat malaria disease (Douglas et al., 2010; Sing, 2015). Artemisinin derivatives; Dihydroartemisinin, Artemether and Arteether are oil soluble terpenoids derivative absorbed well via intramuscular dosage forms. Artesunateis other derivative soluble well in water and showed good absorption when use via intravenous route. Yingzhaosu A and C are peroxides sequiterpenoids isolated from roots of Artabotrys uncinatus plant and have been also documented to have antiparasitic activity (Thurnher et al., 2003). Triterpenoids such as Betulinic, oleanolic, ursolic and moronic acids are suggested to be

34 exhibited pharmaceutical potential. Betulinic acid has been reported to exhibit antiretroviral, antimalarial, and anti-inflammatory properties. Thurnher et al. (2003),have demonstrated its cytotoxic activity against a variety of tumor cells. Also it has anti-HIV activity as well as the derivatives related to the moronic acid. On the other hand ursolic and oleanolic acids have been showed to exhibited anti-inflammatory and anti-hyperlipidemic activities (Dinkova-Kostova et al., 2005).

1.2.2.2.3 Nitrogen Compounds Alkaloids are the most pronounced family of secondary metabolites belonged to nitrogen compounds. Although they are toxic to human being, but some of them have been chemically modified to obtain less toxic derivatives and have curing properties to many disease such as tuberculosis caused by Mycobacteria species. Recently, banegasine, an indol alkaloid inhibits Mycobacterium smegmatis growth. A significant effect has been shown from the plant alkaloid derivative; Solsodomine B against the growth of intracellular Mycobacterium bacteria(Copp and Pearce, 2007; Kishore et al., 2009). Quinoline alkaloids such as kokusagine and graveolinine have been also proved to be highly effective against Mycobaterium tuberculosis. The oldest example of quinolone alkaloid is Quinine, isolated from Cinchona succirubra. It is used for more than three centuries as antimalarial agent (antiplasmodial). B,2-n-pentylquinoline, 4-methoxy-2-phenylquinoline, 2-n- propylquinoline and chimanine are other quinolone alkaloids effective in treatment of leishmaniasis. Natural alkaloid Berberine produced by Chinese herb Rhizoma coptidis and its derivative dihydroberberine showed many biological activities such as antibacterial, anti- inflammatory, expansion of blood vessels, inhibit platelet aggregation, sedation, neuroprotective and hepatoprotective effects. Many studies have demonstrated that it used to treat ulcer, diarrhea and diabetes and can inhibit tumor development by interfering with different stages of carcinogenesis and tumor progression in both in vitro and in vivo. Berberine is proved to inhibit Toxoplasma gondii parasite and being toxic to host cell, but it’s partially derivative showed less toxicity (Gantier et al., 1996; Kishore et al., 2009). Ji and Shen (2011) reported that there are two monoamine oxidase isoforms of Berberine; Dyhydroberberine and Canidine which have been proved to inhibit acetylcholinesterase enzyme and pathogenic enzymes involved in Alzheimer’s disease. Piperine

35 is other quinolone alkaloid found to be involved in metabolism and transport of xenobiotics and metabolites (Nandakumar et al., 2006). Quercetin, nitrile glycoside, genistein, sinomenine and glycyrrhizin are other natural compounds within other chemical families, have proved to play similar roles (Kang et al., 2009).

1.2.2.2.4 Sulfur compounds Glucosinolates are secondary metabolite anions contain b-thioglucoside-Nhydroxy- sulfates with different side chains depending on the different species of plants, and also contain a sulfur-linked b-D-glucopyranose moiety. On hydrolysis they revealed volatile isothiocyanates, thiocyanates, and nitriles that exhibited antibacterial, antifungal, antioxidant, antimutagenic, and anticarcinogenic activities. Glucoraphanin, a glucosinolate produced by the plant broccoli and cauliflower young sprouts, can be readily converted to their derived isothiocyanates (sulforaphane, raphanin). Isothiocyanates have reported to have cell detoxification activity, in addition Sulforaphane has been proved to have strong growth inhibitory effect against the Gram negative Helicobacter pylori; the bacteria associated with gastric ulcer and gastric cancer (Nandakumar et al., 2006). Even though data on the antimicrobial activity of plants so far considered empirical, but currently the potential control of bioactive compounds produced by plants on microbial growth in different disease cases have been scientifically confirmed, with increasing number of reports on pathogenic microorganisms resistant to antimicrobial agents. Therefore it is a very important point for the researchers and clinicians to work hard to clarify the main bioactive ingredients which could be extracted from medicinal plants and have a health benefits impact (Sing, 2015).

1.2.3 Wound healing activity of the medicinal plants Replacement of the inflamed or dead tissue by living one is a complex process named wound healing which comprises of three basic phases, inflammatory, proliferative and maturation phase (Djerrou et al., 2010). Epidermal growth factor (EGF), fibroblast growth factor (FGF) and transforming growth factor beta (TGFβ) are growth factors controlled wound healing phases in an orchestrated manner (Kwon et al., 2006). Wound healing in some chronic diseases and disorders has become one of the challenges of medical science and for this reason using the new compounds to accelerate wound healing be welcomed (Rezaie et al., 2012).

Many studies clearly documented that appropriate treatment and wound care prevent infection spread and accelerate the healing process. Despite there are different synthetic drugs in modern medicine that enhance the wound healing, but drugs of herbal origin are crucial in wound healing since they initiate disinfection, debridement and provide a moist environment for natural healing process. The following bacteria have been reported to cause wound infections; Staphylococcus aureus, Escherichia coli, Proteus species, Klebsiella species, Enterococcus species, Pseudomonas aeruginosa, Clostridium perfringens, Bacteriodes fragilis, Anaerobic cocci. It is well known that Pseudomonas aeruginosa is the most common causative of wound infections (Nichols, 1991; Cheesbrough, 2000; Djerrou et al., 2010; Alizadeh et al., 2009).

1.2.3.1 Application of plants extracts for wound healing Many animal models experiments goats, sheeps, rabbits, or rats showed that the crude extract significantly reduced the wound breaking strength. Extracts of Vitex leucoxylon, Diospyros crassiflora and Psidium guajava are examples of plants extracts showed antifungal wound healing activity and other examples such as Aloe vera, Matricaria chamomilla, Nigella sativa showed antibacterial wound healing activity . Some crude extracts revealed significant broad spectrum antimicrobial wound healing capacity such as methanolic extracts of Grewiaoccidentalis, Polystichum pungens and Cheilanthes viridis, other showed narrow spectrum antimicrobial activity such as extracts of Guiera senegalensis and Polystichum pungens against Gram positive bacteriaand Gram negative bacteria respectively (Sarma et al., 1990; Cáceres et al., 1991; Ahmed et al., 1995; Grierson and Afolayan, 1999). Abdrabo et al. (2005) investigated the wound healing effect of Solenostemma argel leaves extract on open skin wound model in rats. Their results confirmed that the 2% of Solenostemma argel ointment is a potent healing agent even better than the tested Tetracycline ointment 3%.It has been reported that Pistacia lentiscus virgin fatty oil promotes significantly wound contraction and reduces epithelization period in rabbit model and Myrtus communis is considered a good replacement against other chemical drugs which are used widely in wound healing treatment (Djerrou et al., 2010; Rezaie et al., 2012).

1.2.4Anti-oxidant activity of medicinal plants Pro-oxidants are chemical compounds and reactions able to generate potential toxic oxygen species named free radicals which is either Nitrogen derived Oxygen derived. The most

37 common reactive oxygen species include superoxide anion (O2), hydrogen peroxide (H2O2), peroxyl radicals (ROO) and reactive hydroxyl radicals (OH). Free radicals are unstable molecules that are generated by sun exposure, stress and as part of the natural aging process. They attack the body macromolecules including protein, DNA, collagen and lipid causing to cellular or tissue damage and can lead to the development of disease, including Alzheimer’s, heart disease, and cancer. Any compound can scavenging the free radicals and suppressing their formation or opposing their actions are called antioxidants. In a normal human biological system cells there is an pro-oxidant - antioxidant balance, but if the production of toxic oxygen increased or the antioxidant levels decreased a state named oxidative stress will precipitated which if prolonged will lead to serious cell damage. Anti-oxidants are powerful substances nessecary for good health and preventing diseases through proinhibition or even prevention of the oxidation other molecules in the body. They are mostly come from the fresh vegetables and fruits. The benefits of antioxidants are very important to good health, because if free radicals are left unchallenged, they can cause a wide range of illnesses and chronic diseases. Many, if not most of medicinal plants contain flavonoids; such compounds have been associated with several beneficial effects such as anti-chelating and anti-oxidation which consider to be a fundamental property important for life. Many plant products exert antioxidant effect by overcoming various free radicals and the singlet form of molecular oxygen (Halliwell and Gutteridge, 2017). It was documented that antioxidant capacity may differ between different plant species and between same species from different regions. It is well known that plant part used, harvesting time, different types of extraction solvents used, extraction type, and storage are factors affected the antioxidant activity of plants. (Heim et al., 2002; Patel et al., 2013; Aouintiet al., 2014).

1.2.5Anti-cancer activity of medicinal plants Cancer is a leading cause of death world-wide, with approximately 17.5 million new cases and 8.7 million cancer related deaths in 2015. The problems of poor selectivity and severe side effects of the available anticancer drugs have demanded the need for the development of safer and more effective chemotherapeutic agents (Ogbole et al., 2017). Many of the phenolic acids such as gallic and caffeic acid derivatives were synthesized for their potential

38 antiproliferative and cytotoxic effects against different human cancer cell lines and different effects were found. These different effects point to a significant specificity of action of the drugs tested. It was found that the trihydroxylated derivatives of phenolic acids showed better results than the dihydroxylated ones which might be due to a balance between the antioxidant and pro- oxidant properties of this agent (Gomes et al., 2003; Scatena et al., 2011).

1.2.6 Botanical ethnophrmacological ,phytochemical profiles and biological activities of the studied medicinal plants

1.2.6.1Myrtus communis L.

1.2.6.1.1Classification and names

Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Myrtales

Family: Myrtaceae

Genus:Myrtus L.

Botanical names: Myrtus (Sabiha et al., 2011).

Vernacular name: Myrseen

1.2.6.1.2 Plant description

Myrtus communis L. is an evergreen perennial tree of 1.8-2 meter height. It is native to Southern Europe, North Africa and West Asia and widespread in the Mediterranean region (Sabiha et al., 2011).

1.2.6.1.3 Folk medicine uses

Traditionally Myrtus used as hypoglycemic agent and also it is used as antiseptic and disinfectant. Also the plant has been used in the food industry, for example for flavoring meat and sauces (Sabiha et al., 2011).

1.2.6.1.4 Phytochemistry Complex mixture was shown in Myrtus communis plant characterized with a regular presence of monoterpenes and sesquiterpenes. The monoterpenes hydrocarbons was the abundant among which α-Pinène and α-limonène were dominants. Myrtenyl acetate, 1,8-cineol, α-pinene, linalool, limonene, linalyl acetate, geranyl acetate and α –terpineol were the most abundant found in the leaves part. 1,8-cineol, α-pinene, linalool, limonene were the major compounds found (Ben Ghnayaet al., 2013; Ben Hsouna et al., 2014; Asgarpanah and Ariamanesh, 2015). The plant berries contain many biologically active compounds such as Phenolic compounds, flavonoids and anthocyanins as major phytochemicals. Leaves contain, tannins, flavonoids, coumarins, galloyl-quinic acids, caffeic, gallic and ellagic acids, while the roots showed the presence of tannins, alkaloids, glycosides, phenolic acids and quercetin (Sabiha et al., 2011).

1.2.6.1.5 Antimicrobial activity The antimicrobial activity of the aerial parts of Myrtus communis plant that had been collected from different regions were studied by many researchers for their activities against Gram positive, Gram negative bacteria and some fungi. Results showed varied inhibition zones and authors attributed this variation to the difference in locations of plant collection. The plant harvested from and to the different nutrients of different soils and their accumulation in the plant leaves. Escherichia coliATCC25922, Escherichia coli ATCC10536, Escherichia coli ATCC29998, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Shigella flexneri ATCC12022 and also to the clinical Staphylococcus aureus, Vibrio cholerae serotype O gawa, Pseudomonas aeruginosa (ATCC 27853) showed clear inhibition zones with the Myrtus extracts. On the other hand some authors reported that the Myrtus extract had weak activity against

Pseudomonas aeruginosa isolates (Ben Ghnayaet al., 2013; Taheri, et al., 2013; Behbahani et al., 2016; Besufekad et al., 2017).

Taheri, et al. (2013) reported that the growth of Staphylococcus aureus and Escherichia coli was inhibited at minimum concentration of 0.2 mg/ml and 8 mg/ml respectively and they suggested that bacteria were more sensitive to the extract of Myrtus communis leaves compared to standard drugs used. Aleksica et al. (2014) studied the effect of extract of Myrtus communis againstmultidrug resistant Acinetobacter baumanii,they claimed that the plant oils have good antibacterial activity against tested resistant isolate when used alone.

Also they claimed that it revealed a synergistic effect when used with either Ciprofloxacin or Polymyxin B antibiotics. Akhavan et al. (2016) had assessed the effect of different extracts of leaves of Myrtus communis plant against clinicaly isolated Acinetobacter baumanii and they found that the lowest minimum inhibitory concentration of ethyl acetate extract was 400mg/ml. The antifungal activity of Myrtus communis was screened by Sadeghi Nejad et al. (2014), they reported thatethanolic extract of leaves have potent antifungal activity against Candida albicans, Cadida glabrata, and Candida tropicalis and they suggested that it can be used as a natural antifungal agent. Similarly Aspergillus niger showed higher sensitivity towards Myrtus chloroform extract (Besufekad et al., 2017).

Photo (1): Myrtus communis L. By Salwa I. A. Eltawaty, August 2016

1.2.6.1.6 Antioxidant activity

It has been deeply documented that Myrtus communis leaves and berry have antioxidant activity. Leaves revealed the best antioxidant performance and this activity was contributed to the major plant constituents include gallic acid derivatives, flavonols, flavonol derivatives, and hydroxybenzoic acids. Also the ratio of these constituents claimed to have an important role in the antioxidant activity levels. Comparative studies of the antioxidant activity between leaf and berry extracts of Myrtus communis revealed that leaf extracts are best antioxidants, which can be assigned to the galloyl derivatives, flavonols, and flavonols derivatives, although the ratio of these groups of compounds might also have an important role in the antioxidant activity. The anthocyanins present in myrtle berries seem to possess weak antioxidant activity. A significant antioxidant effect has been shown from Myrtus communis L. floral buds essential oil and the researchers attributed this activity to the high contents in monoterpenes (Romani et al., 2004; Amensour et al., 2009; Snoussi et al., 2011; Aleksica et al., 2014; Hennia et al., 2018).

1.2.6.1.7 Anti-cancer activity

Aerial parts specially leaves of Myrtus communis have been reported to have cytotoxic activities against many carcinoma cell lines such as mouse monocyte macrophage carcinoma J774, urinary bladder 5637, human breast carcinoma MCF-7, human haematological tumor MT- 4, HepG2 (hepatocellular carcinoma, human prostate cancer DU145, leukemic KBM-5, and MEG-01 (human bone marrow) cell lines and also against normal human tissue cells CRL7065 and MDBK; normal kidney cell line. Researchers had attributed this anti-cancer effects to the leaves compounds; Myrtucommulones J-L and Myrtucommulone A (Cottiglia et al., 2012; Naghibi et al., 2013; Grandjenette et al., 2015; Mahmoudvand et al., 2015; Hennia et al., 2018) .

1.2.6.1.8 Toxicity

Even though no health hazards or side effects are known with the proper administration of designated therapeutic dosages. In rare cases, internal administration of myrtle oil as a drug leads to nausea, vomiting and diarrhoea. Preparations containing volatile oil should not be applied to the faces of infants or small children because of the possibility of triggering glottal spasm, asthma like attacks or even respiratory failure. Overdoses of Myrtus oil (more than 10 g) can lead to risky life threatening symptoms include circulatory disorders, decrease in or loss of

43 blood pressure and collapse (Asif et al., 2011). Many LD50S values were determined with acute toxic doses from alcoholic and aqueous extracts ranged of 790mg – 1450mg/kg and 473mg – 1250mg/kg respectively (Hosseinzadeh et al., 2011; Aljebory, 2014).Toxic doses of Myrtus oil above 3mL/kg reported to produce the following consecutive symptoms within 1–2 hours; increased motility, frequent licking of the paws, loss of coordination, tremor, paralysis of the hind legs, short clonic convulsions, cyanosis, dyspnoea, loss of righting reflexes and narcosis. Decrease in this toxicity has been observed during continuous daily oral application of the essential oil. Liver enlargement, hypertrophy of endoplasmic reticulum, increase of cytochrome concentrations were reported with histological studies of the liver tissue but showed no significant difference among various groups. High doses of 1000, 2000 and 5000 mg/kg of the plant extract markedly increased Haematocrit while not effected other erythrocyte parameters. With therapeutic doses of this essential oil (1–2ml/day), the appearance of adverse effects on liver is unlikely to happen (Uehleke and Brinkschulte-Freitas, 1979; Biricik et al., 2012; Alipour et al., 2014).

1.2.6.1.9 Pharmacology Myrtus communis is largely distributed in the Mediterranean Basin and has been reported as commonly used in traditional medicine for curing some diseases. Several models used by some researchers and an anti-inflammatory activities were shown from the leaves extract of Myrtus communis which directs its usage as novel therapeutic for the management of inflammation. In addition to its antimicrobial effect it is commonly used for anti-anxiety effect, anti-oxidant effect, repellency effect, cytotoxic effects, gastrointestinal effect, facial warts effects, anti-tumor properties, anti-cancer effect and is widely used for non-therapeutic purposes as prebiotic in food formulations. Other researchers documented that the aerial parts of the plant has anti-hyperglycemic, anti-diarrhoeal, antisecretory, anti-hypercholesterolemia, Inhibition effect on acetylcholinesterase and butyryl-cholinesterase, vasodilator effect on rabbit aorta and also has a complete relaxation effect on rabbit jejunum. (Gholamhoseinian et al., 2010; Begum et al., 2012; Janbaz et al., 2013; Miraj and Kiani, 2016; Jabri et al., 2016; Sisay et al., 2017; Hennia et al., 2018)

As Myrtus communis is comprised of collections of flavonoids and other various components with antioxidant and anti-inflammatory properties thus its effectiveness has been

44 significantly reported in decreasing blood glucose levels. Hydroalcoholic and ethanolic (2g/kg) extracts of leaves of Myrtus communis showed a moderate to higher hypoglycemic effect in diabetic rats compared with the aqueous extract ( Ferchichi et al., 2009; Malekpour et al., 2012; Saei et al., 2013; Panjeshahin et al., 2016,;Gulaboglu et al., 2017). It has been reported that plant leaves showed significant decrease in plasma levels of liver enzymes (alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase), and cholesterol presented a significant decrease, while albumin, urea, triglyceride, total protein levels and body weight showed no significant changes(Johari et al., 2014). In addition it was documented that experimental groups of rats which were treated with hydroalcoholic extract ofMyrtus communis were showed a reduction in lipid profile suggest that Myrtus communis has hypolipidemic (Tas et al., 2018). It has been mentioned that the plant can normalize the levels of renal function markers (Urinary albumin, creatinine, urea, and uric acid) which had been significantly increased in Streptozotocininduced diabetic nephrotoxic rats (Kandasamy and Ashokkumar, 2014; Salehpour et al. 2106).

1.2.6.2Pistacia lentiscus L 1.2.6.2.1 Classificationand names Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Sapindales in APG4

Family: Anacardiaceae

Genus:Pistacia L.

Botanical names: Mastiki, Mastagee, Mastic, Mastagi, mastage, Mastagee, mastagee. (Nahida and Siddiqui., 2012).

Vernacular name: Al-Battoum

1.2.6.2.2 Plant description

Pistacia is an evergreen flowering plants of 1 to 5 meter height. Twenty species were identified, the more popular are Pistacia lentiscus, Pistacia atlantica, Pistacia terebinthus, Pistacia khinjuk, and Pistacia vera. It has a strong smell of resin. It is found in Mediterranean Europe, Mediterranean region (Nahidas and Siddiqui, 2012; Bozorgi et al., 2013).

1.2.6.2.3 Folk medicine uses Pistacia lentiscus has been used for variety of gastric ailments in the Med-iterranean and Middle East countries for the last 3000 years and also has been reported that it was traditionally used for the treatment of hepatic, and kidney diseases, wound healing and gastrointestinal disorders (DerMarderosian and Beutler, 2002).

1.2.6.2.4 Phytochemistry It has been reported that Pistacia lentiscus composed of variety of bioactive constituents which are important in medicine such as flavonol glycosides, gallic acid, anthocyanins, nortriterpen-oids, essential oil and resins (α- pinene, β- pinene, limonene, terpene-4-ol and terpeneol). These compounds are related to cure many diseases and are working as synergistic agents. Tannins and saponins has been found in stem and leaves of the plant but anthraquinone not found (Duru et al., 2003; Derwich et al., 2010; Nahida and Siddiqui, 2012). It has been documented that Leaves of Pistacia atlantica are rich of flavonoids, alkaloids, saponins, tannins and steroids while the fruits are rich by flavonoids and tannins, but lacking of saponins, steroids and alkaloids (Asma et al., 2016). Nahida and Siddiqui, 2012 reported that Resin is the most important component of Pistacia lentiscus including α- pinene, β- pinene, limonene, terpene-4-ol and terpeneol. In addition Gallic acid, galloylderivatives, flavonol glycoside, and anthocyanins were reported in this plant.

1.2.6.2.5 Antimicrobial activity

The antimicrobial activity of the aerial parts (leaves, fruits and twigs) of Pistacia lentiscus that had been collected from different regions such as Sudan, Libya, Algeria, Italy, Morocco and Tunisia were studied by many researchers for their activities against Gram positive, Gram negative bacteria and some fungi such as Candida albicans and Aspergillus niger.

Photo (2): Pistacia lentiscus L. By Salwa I. A. Eltawaty, August 2016

Results were varied according to the species and the part used, some species showed more activity as antibacterial than others. The antibacterial activity of Pistacia lentiscus was studied by Hasan et al. (2011) and Habibi et al. (2015), the antifungal activity of the plant was reported by Benhammouet al. (2008) and Mezni et al. (2015). Derwich et al. (2010) screened the antibacterial of the aerial parts of Pistacia lentiscusplant, their results concluded that the oil extract was the more active against Escherichia coli, Enterobacter aerogenes,Salmonella typhi,Enterococcus faecalis and Klebsiella pneumoniae while Bacillus sphericus, Staphylococcus aureus, Staphylococcus intermedius and Pseudomonas aeruginosa were moderately inhibited by the oil extract of the plant. El Idrissi et al. (2016), studied the oil extract of Pistacia lentiscus against Streptococcus agalactiae, Staphylococcus aureus, Acinetobacter baumanii, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. They found that essential oils of Pistacia lentiscus plant have a significant antibacterial and antifungal activity. The hydrophobic lipopolysaccharide presented in the outer membrane of Gram negative bacteria considered less sensitive to plant oil and extracts than Gram positive bacteria, as the negatives cell membrane nature provides protection against different agents (Vaara, 1992; Mezni et al., 2015). In Sudan Alhadi et al. (2018) found that the leaves of Pistacia lentiscus revealed good activity against Gram positive, Gram negative bacteria and fungi. The best bactericidal performance was revealed from plant extract obtained from decoction method rather than maceration and soxhlet extraction methods (Lauk et al., 1996). However Asma et al. (2016) found that the aqueous extract was more effective against all tested Gram positive and Gram negative bacteria when compared with the organic solvents extracts which showed inhibitory activity only against tested Gram negative bacteria. The ethanol extract of leaves and stems showed good antifungal activity and selective antibacterial activity against Staphylococcus aureus (Raho, 2017; Alhadi et al., 2018). In Libya, Habibi et al. (2015) used chloroform 96%, ethanol 95% and Petroleum ether as solvents to prepare the leaves extracts via maceration, disc diffusion method of the filtrate was used for assessment of susceptibility of Escherichia coli (ATTC10412) and Pseudomonas aeruginosa (ATTC27853). Their results showed that there is a moderate susceptibility toward leaves ethanolic extract of Pistacia atlantica.

1.2.6.2.6 Antioxidant activity

The antioxidant properties of the leaves phenolic compounds of Pistacia lentiscus and Pistacia atlantica were reported. They act as a scavenger of the 1, 1-diphenyl-2-picrylhydrazyl (DPPH). It was found that gallic acid and Quinic acid are widely appeared in Pistacia species plants. Galloyl quinic derivatives was shown to possess antioxidant activity and the scavenger activities of gallic acid, 5-O-galloyl, 3,5-O-digalloyl, 3,4,5- O-trigalloyl quinic acid derivatives, - were reported against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide(O 2) radical and hydroxyl (OH) radical and as the number of galloyl groups on the quinic acid skeleton increased the scavenger activity raised (Baratto et al., 2003; Benhammou et al., 2008; Gardeli et al., 2008).

1.2.6.2.7 Anti-cancer activity

It was found that the extract of fruits of Pistacia lentiscus plant showed a protecting role against lipid peroxidation induced by H2O2 in K562 leukemia cell line, human liver cell line carcinoma HepG2, pheochromocytoma cell line carcinoma PC12 of rat adrenal medulla and also the plant Chios mastic gum reported to inhibit the proliferation and caused death of human colon cell line HCT-116 carcinoma in vitro. The researchers found that the plant inhibited HepG2 and PC12 cell lines carcinoma without affecting their mitochondrial respiration and they contributed this to the presence of gallic acid and its derivatives within the fruit bioactive contents (Abdelwahedet al., 2007; Ljubuncic et al., 2005)

1.2.6.2.8 Toxicity

Five days short term toxicity assay was done by administering of 100µl of Pistacia lentiscus fruits oil for mice. The oil was given orally and the results did not show any significant statistical difference in serum concentration of high density lipoprotein when compared to control mice. Also other liver function markers, kidney function markers and blood parameters tests showed no toxicity with Pistacia lentiscus oil when compared to the control group. These results suggest a good safety profile of short term oral use of Pistacia lentiscus oil as a monotherapy in the treatment of various skin, respiratory, and gastrointestinal disorders (Attoub et al., 2014). LD50 of 900 ± 221 mg/Kg body weight had been reported in an acute toxicity assay in mice of the lyophilized aqueous extract of Pistacia lentiscus (Villar et al., 1987;

Boukeloua et al., 2012). Three sub-acute chronic toxic doses, o.5, 1 and 2ml/kg rabbit weight were administered orally for 28 days. Results did not show any toxicity burden. In addition no change had been shown in liver biomarkers (ALT and AST) levels. Decreasing in blood glucose level had been registered but was within the physiological standards compared to the control (Zineb et al., 2016).

1.2.6.2.9 Pharmacology

Since long time Pistacia lentiscus has been used traditionally for treatment of various diseases. It has been used as antimicrobial, antioxidant, hepatic protective, lipid lowering, anthelmintic, anti-mutagenic, anticancer, hypotensive, antiarithritic, antigout, antispasmodic agent and also has been used in treatment of functional dyspepesia . It has been documented that these biological activities referred to the plant constituents; α-tocopherol, arabino-galactan proteins, nortriterpenoids, anthocyanins, resin, gallic acid and flavonol glycosides (Nahida and Siddiqui, 2012).It has been reported that the aqueous extract of Pistacia lentiscus showed marked hepatoprotective activity by reducing the activity of liver function markers; Alkaline phosphatase (ALP), Alanine amino transferase (ALT) and Aspartate amino transferase (AST) and level of bilirubin. Non-boiled aqueous extract was more effective than boiled. Fatty oil of Pistacia lentiscus showed a non-significant decrease in high density lipoprotein (HDL) and revealed significant decrease in triglycerides, total cholesterol and light density lipoprotein cholesterol (LDL) and its ratio with high density lipoprotein LDL/HDL (Al-Said et al., 1986; Djerrou, 2014). Pharmacological experiments showed that the aqueous extract has a hypotensive effect at 25, 12.5 and 6.25 mg/kg doses where the blood pressure started lowering immediately after the injection, reached maximum after 5 minutes, then started gradual recovery and within 7 -10 minutes the action disappeared (Villar et al., 1987).

1.2.6.3 Capparis spinosa subsp orientalis (Duh.) Jafri.

1.2.6.3.1 Calssification and names

Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Brassicales in APG4

Family: Capparaceae

Genus: Capparis

Botanical name: Caper, Kaper (Manikandaselvi et al., 2016). Vernacular name: Gappar

1.2.6.3 .2Plant description

Capparis spinosa is a perennial plant bears rounded leaves. The plant flowers are big white to pinkish-white in color. It is growing wild on walls or in rocky coastal areas. It is native to the Mediterranean region and (Manikandaselvi et al., 2016).

1.2.6.3.3 Folk medicine uses

This plant has a lot of traditional and medical uses. Libyan people used Caper as anticancer in major and in wound healing in minor. It has been reported that whole plant used for treatment of rheumatism, roots used as diuretic, astringent, and tonic and bark of the roots used as appetizer, astringent, tonic, anti-diarrheal and also in treatment of hemorrhoids and spleen disease (Rahnavard and Razavi, 2016).

1.2.6.3.4 Phytochemistry

Gallic acid, caffeic acid, coumaric acid, valinic acid and ferulic acid are phenolic acids presented in leaves of Capparis spinosaa (Gadgoli and Mishra, 1999, Proestos et al., 2006). Also flavonoid glycosides; quercetin-3-glucoside-7-rhamnoside, quercetin-7-rhamnoside, quercetin-3- rutinoside, quercetin-7-rutinoside, quercetin-3,7-dirhamnoside, , isorhamnetin 3,7-dirhamnoside, isorhamnetin 3-rutinoside, isorhamnetin, kaempferol-7-rhamnoside, kaempferol-3-rutinoside, kaempferol 3-glucoside-7-rhamnoside, kaempferol, quercetin, kaempferol-3-rhamnoside-7- glucoside, kaempferol-3.7-dirhamnoside and apigenin 6,8-di-C-glucoside were found within the plant constituents (Sharaf et al., 2000; Tlili et al., 2010; Tlili et al.,2011). Alkaloids, flavonoids,

51 glucosinolates, phenolic acids, terpenoids are reported as chemical constituents to various parts of Capparis spinosa (Nabavi et al., 2016).

Photo (3): Capparis spinosa subsp orientalis (Duh.) Jafri.

By Salwa I. A. Eltawaty, August 2016

1.2.6.3.5 Antimicrobial activity Many studies had been done to evaluate the antimicrobial activity of Capparis spinosa and some results showed that the highest antimicrobial activity were revealed from ethanol extracts of the plant roots which exhibited similar activity compared with Gentamicin (Proestos et al., 2006; Al-Bayati and Al-Jarjry, 2007). The methanol extract of the plant flowers showed good antimicrobial activities against all tested Gram positive, Gram negative bacteria and Candida albicans (Abd Razik, 2011). It has been reported that the plant has anti-quorum activity and in attribution to capparisspecies polysaccharides, the plant showed much more antibacterial activity against the Gram negative than the Gram positive tested bacteria (Abraham et al., 2011; Mazarei et al., 2016). It has been reported that the methanol extract of the plant leaves inhibited the growth of standard bacteria; Alcaligenes faecalis ATCC 29217, Staphylococcus aureus ATCC29213, Enterobacter cloacae ATCC13047, Enterococcus faecalis ATCC-29212, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC 13883, and methicillin-resistant Staphylococcus aureus (MRSA) isolate. Researchers attributed these effects to the high amount of flavonoids presented in the methanol plant extract. Aqueous leaves extract showed lower activities, while aqueous extract of roots showed more antibacterial activity than aqueous extract of fruits and also showed inhibitory activity on Aspergillus niger, Aspergillus flavus and Aspergillus parasiticus, while Candida albicans inhibited only from ethanol extract of roots (Sherif et al., 2013; Mahboubi and Mahboubi, 2014; Gull et al., 2015; Rahimifard et al., 2015). In Sudan, Nour and El-imam, 2013 reported that methanol extract of Capparis spinosa was found to be the more effective compared to chloroform extract against tested Bacillus subtilis NCTC 8236, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa NCTC 6750, Aspergillus niger ATCC 9736 and Candida albicans NCTC 10716.

1.2.6.3.6 Antioxidant activity In general the extracts of Capparis spinosa aerial parts were documented had greater antioxidant activity than that of root part. DPPH radical scavenging assay showed that the antioxidant activity (IC50μg/ml) of ethyl acetate and methanol extract were 57.75±2.3 and 94.4±4.5 respectively (Alsabri et al., 2012; Al-Snafi, 2015). It has been reported that leaves and

53 flowers of Capparis spinosa plant are more rich in flavonoids than the roots of the same plant and also the antioxidant and anti-lipid peroxidation activity was more with the leaves and flower especially with the methanolic extracts. Also seeds and buds showed reasonable antioxidant activity than roots (Arrar et al., 2013).

1.2.6.3.7 Anti-cancer activity 62-kDa lectin is a novel dimeric extracted from seeds of Capparis spinosa and inhibited the proliferation of breast MCF-7 and liver hepatoma HepG2 cancer cells (Lam et al., 2009). However a significant inhibitory effect (p<0.001) on the cancer lines growth were seen from the crude aqueous Capparis spaniosa leaf extract where its highest anti-proliferative effect was recorded at 1000 μg/ml against human cervix epitheloid carcinoma Hela and human epidermoid larynx carcinoma Hep-2 and the effect was a time dependent (Al-Daraji, 2010). 38 kDa protein is a another constituent of seeds of Capparis spinosa reported has antiproliferative effect against breast cancer MCF-7 cells, colon cancer HT29 cells and hepatoma HepG2 cells with IC50 of about 60, 40 and 1 mM, respectively. Also Stachydrine markedly inhibited the expression of chemokine receptors (CXCR3 and CXCR 4) in cancer cells and then inhibit the malignancy and invasive capacity of malignant cancer cells. However the bark extract of root of Capparis spinosa showed significant decrease in the tumor volume, packed cell volume, and viable cell (Luecha ,et al., 2009; Venugopal et al., 2011; Rathee et al., 2012).

1.2.6.3.8 Toxicity Unless allergic contact dermatitis there was no report regarding toxicity from Capparis spinosa plant either with acute, subacute or chronic toxicity and it has been found that no death happened up to dose of 5g/kg body weight administered for 4-6 weeks (Angeliniet al., 1991; Rajesh et al., 2010; Al-Snafi, 2015).However Fanoudi, et al. (2017) reported that doses of 200, 400 and 800 mg/kg of hydro-alcoholic extract of Capparis spinosa were administrated by oral gavages for 28 consecutive days in mice. The results have shown that Capparis spinosa can cause nephrotoxicity and hepatotoxicity especially during sub-chronic consumption, dose- dependently where the difference in liver function markers (ALT, AST) was non-significant after 14 days and significant after 28 days treatment compared with control group. Also showed the result showed significant differences compared with control group after 14 and 28 days in kidney

54 markers levels; bilirubin and creatinine, respectively specially with the dose 800mg/kg body weight. 1.2.6.3.9 Pharmacology Inflammation and arthritic animal models showed that extracts of Capparis spinosa has marked anti-inflammatory and anti- arthritic activity but devoid of analgesic activity. However the ethanol- water(50:50v/v) extract had the most significant anti- arthritic activity. Researchers reffered this action to the extract constituents of α-D fructofuranosides methyl , uracil, P- hydroxy benzoic acid, bis (5-formylfurfuryl) ether, daucosterol, stachydrine and 5- (hydroxymethyl) furfural (Al-Said et al., 1988; Feng et al., 2011; Bhoyar, 2012). Capparis spinosa fruit extract showed lipid lowering effect but revealed no significant decrease in blood glucose levels (Mishra et al., 2012). In paracetamol treated rats, aqueous extract of Capparis spinosa decreased levels of liver function markers (alanine amino transferase, aspartate amino transferase), creatinine and total bilirubin and improving the damaged liver tissue in a dose dependent manner compared with non-treated group (Al-khan and Alnuaimy, 2012). On isolated organ, Capparis spinosa aqueous extract was shifted the noradrenaline dose response contraction of rat aortic rings to rapid relaxation at dose of 10 mg/ ml and with 30 minutes incubation time (Zeggwagh et al., 2007). When different concentrations of different plant parts incubated for 30 minutes, a significant vasodilator effect and vasoconstrictor effect were shown for fruits and kernels, and leaves extracts respectively (Benzidane et al., 2013).

1.2.6.4Salvia fruticosa Mill

1.2.6.4.1 Classification and names

Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Lamiales

Family: Lamiaceae

Genus:Salvia Mill

Botanical names:sage (Raal et al., 2007)

Vernacular name: Toffah Alshahi

1.2.6.4.2Plantdescription

Salvia is a perennial evergreen round shrub growing all over the world and the species of Salvia fruticosa is native to Middle East and Mediterranean areas. Salvia fruticosa is an outcrossing, up to 60 cm high. Stems are erect or lying down with numerous fine hairy dark green branches. Leaves are petiolate, elongated, opposite, simple, sometimes with basal lobes, with serrate margin (Raal et al., 2007). 1.2.6.4.3 Folk medicine uses Salvia fruticose is a shrub plant. Its aerial parts have been used since long time in cookery and traditional medicine. It has been used for the treatment of different kinds of disorders including inflammation in the throat and skin and diarrhea (Perry et al., 1999; Garcia et al., 2016).

1.2.6.4.4 Phytochemistry A variety of constituents have been reported in the plant including alkaloids, carbohydrate, fatty acids, glycosidic derivatives and phenolic compounds (Badiee et al., 2012). Camphor, 1,8-cinole, α-thujone, B-thujone and (E)-caryophyllene are five main compounds identified in Salvia fruticosa, Camphor, 1,8-cinole were contributed to the plantantifungal activity. Camphor, 1,8-cinole, α-thujone, B-thujone and (E)-caryophyllene are five main compounds identified in Salvia fruticosa, Camphor, 1,8-cinole were contributed to the plant antifungal activity. In addition the compounds α –terpineol, sesquiterpene hydrocarbons and other monoterpenes hydrocarbons such as camphene, β-pinene, α -pinene and myrcene were found in traces in the aerial parts of Salvia fruticosa (Khalil and Li, 2011; Giweliet al., 2013; Pitarokili et al., 2003).

1.2.6.4.5 Antimicrobial

The antimicrobial performance of aerial parts of Salvia fruticosa plant had been studied by many researchers and the minimum inhibitory concentrations were determined. Results cleared that the plant oil showed inhibition activity on the growth of tested Gram positive and Gram negative

Photo (4): Salvia officinalis L. By Salwa I. A. Eltawaty, August 2016

57 bacteria with varied minimum inhibitory concentrations value of 0.125mg/ml against Pseudomonas aeruginosa followed by 0.25mg/ml against Staphylococcus aureus, Micrococcus flavus and Proteus mirabilis, 0.5mg/ml against Bacillus cereus and Listeria monocytogenes and 1.5mg/ml against Escherichia coli. It has been concluded that Salvia plant has effective mechanism against the Gram positive Streptococcus mutans; the cause of dental caries and Streptococcus group D bacteria which completely inhibited after 10 minutes contact and also has inhibition performance against Candida albicans at lesser contact time and concentration. It was proved that the plant essential oil showed a temporary bacteriostatic action against Salmonella typhi (Khalil and Li, 2011; Giweliet al., 2013; Beheshti-Rouy et al., 2015).

1.2.6.4.6 Antioxidant

In general the extracts of Capparis spinosa aerial parts were documented had greater antioxidant activity than that of root part. DPPH radical scavenging assay showed that the antioxidant activity (IC50μg/ml) of ethyl acetate and methanol extract were 57.75±2.3 and 94.4±4.5 respectively (Alsabri et al., 2012; Al-Snafi, 2015). It has been reported that leaves and flowers of Capparis spinosa plant are more rich in flavonoids than the roots and also the antioxidant and anti-lipid peroxidation activity was more with the leaves and flower especially with the methanolic extracts. Also seeds and buds showed reasonable antioxidant activity than roots (Arrar et al., 2013).

1.2.6.5 Artemisia herba-alba Asso

1.2.6.5.1 Classification and names

Kingdome: Plantae

Division: Anthophyta (Angiospermae) Class: Magnoliopsida (Dicotyledoneae) Order: Asterales in APG4 Family: Asteraceae Genus:Artemisia L. Botanical names: Artemisia, hierba de San Juan, mugwort (Kwak et al., 1997).

Vernacular name: Al-Sheeh

1.2.6.5.2 Plant description

Artmesia herba-alba is a greenish-silver perennial herb with a height of 20-40 cm and erect, rigid stems. It is found common on the steppes of the North Africa and Middle East (Kwak et al., 1997).

1.2.6.5.3 Folk medicine uses

Libyan people use the plant as prophylactic after surgery to avoid wound infection via nasal inhalation and via drinking of previously boiling with water as anthelmintic agent. In China different species of Artemisia have been used in treatment of amenorrhea, andrheumatic disease. Also it has been utilized for the treatment of infections by fungi, bacteria and viruses (Kwak et al., 1997; Kim et al., 2002).

1.2.6.5.4 Phytochemistry

Different bioactive compounds have been isolated from Artmesia herba-alba. The sesquiterpene lactones are the most one occur with great structural diversity within the genus Artemisia (Khafagy et al., 1971). More than fifty compounds isolated from dried flowers and leaves of Artemisia herba alba Asso, 48 were identified in the plant oil. The cis-chrysantenyl acetate, sabinyl acetate and α-thujone were the main compound among twenty one derivatives presented in the identified oxygenated monoterpenes which constitutes 50.53% of total constituents, the phenolic acid 1,4-dicaffeoylquinic acid was the dominant compound. Oxygenated sesquiterpenes also were found in high rates. Also C-glycosyl flavonoid (Apigenin- 6,8-di-C-glu; apigenin-6-Carabinosyl-8-C-glu (isoschaftoside); apigenin-6-C-glu-8-C-ara (schaftoside); apigenin-6-C-pent-8-C-glu ; apigenin-6-C-glu-8-C-pent and quercitin-rha-glu were found in the plant extracts (Zouariet al., 2010; Younisi et al., 2016).

Photo (5): Artemisia herba-alba Asso

By Salwa I. A. Eltawaty, August 2016

1.2.6.5.5 Antimicrobial activity

It has been reported that essential oil of aerial parts of Artemisia herba-alba plant have a significant antibacterial activity against Gram positive bacteria such as methicillin sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus,Bacillus cereus and Bacillus subtilis where they are significantly inhibited as well as the Gram negatives isolates Acinetobacter baumanii, Proteus mirabilis, extended spectrum beta-lactamase producer Proteus mirabilis, Klebsiella oxytoca, Salmonella enteritidis and Escherichia coli. Despite some reports documented that Escherichia coli, Klebseilla pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa were partially inhibited from essential oil of Artemisis herba-alba, others concluded that Pseudomonas aeruginosa was not inhibited, and they attributed this to the very restrictive barrier of its outer membrane, also the fungus Candida albicans showed significant susceptibility towards the plant. (Mann et al., 2000; Abdelah Bogdadiet al., 2007; Zouariet al., 2010; Younisi et al., 2016; Bertellaa et al., 2018). Other species rather than Artemisia herba-alba such as Artemisia vulgaris , Artemisia absinthium and Artemisia annua, were screened by Poiată et al. (2009) for their antimicrobial activity against Gram positive, Gram negative bacterial and some fungal strains. The researchers confirmed the effectiveness of the extracts of Artemisia species in traditional treatment of diseases caused by microbes. They proved that all tested plants species showed range of moderate to no activity against Gram negative bacteria.

1.2.6.6 Juniperus phoenicea L.

12.6.6.1 Classification and names

Kingdome: Plantae

Division: Pinophyta (Gymnospermae)

Class: Coniferopsida (Coniferae)

Order: Cupressales

Family: Cupressaceae

Genus:Juniperus L.

Botanical name:Juniperus(Milos and Radonic, 2000).

Vernacular name: AL-Araar

12.6.6.2 Plant description Juniperus isa native plant to the Mediterranean basin. It is a tree of 2-12 meters height and it is (Milos and Radonic, 2000).

12.6.6.3 Folk medicine uses It has been reported that Juniper plant can improve digestion, removes stomach gases and spasm. Also it has been reported that the oil has antimicrobial property (El-Sawiet al., 2007). 12.6.6.4 Phytochemistry Approximately fifty one bioactive compounds were identified from Juniperus phoenicea. Monoterpene compounds arethe predominant; α-pinene, α-terpinyl acetate and β-phellandrene, which formed about three-fifths of the oil. β-caryophyllene, germacrene D, terpinolene, α- phellandrene and Myrcene were also presented in essential oil of Juniperus phoenicea at significant concentrations. The compounds a-pinene and 6-3carene were the main monoterpenes found (Fouad et al., 2011; Ait-Ouazzou et al., 2012; Bouyahyaoui et al., 2016). Essential oils with wide chemical variation have been found in the leaves of plant. The predominances are monoterpene hydrocarbons followed by oxygenated monoterpenes (El-Sawiet al., 2007).

12.6.6.5 Antimicrobial activity The results of screening of the antibacterial and antifungal activity of berries and leaves of Juniperus phoeniceashowed good antibacterial activity against Bacillus subtilis, Staphylococcus aureus and varied performance against Pseudomonas aeruginosa and Escherichia coli. While Klebseilla pneumoniae was not inhibited.Candida albicans was highly inhibited by the plant essential oil (Ennajar et al., 2009; Fouad et al., 2011; Ait-Ouazzou et al., 2012; Ramdani et al., 2013; Elmhdwi et al., 2015; Bouyahyaoui et al., 2016).The antibacterial and antifungal activity of berries and leaves of Juniperus phoenicea plant had been screened and the efficacy of methanol and ethanol extracts of plant parts as antimicrobial was studied. The results showed good antibacterial activity against Bacillus subtilis, Staphylococcus aureus and

62 varied performance against Pseudomonas aeruginosa and Escherichia coli. While Klebseilla pneumoniae was not inhibited.Candida albicans was highly inhibited by the plant essential oil (Ennajar et al., 2009; Fouad et al., 2011; Ait-Ouazzou et al., 2012; Ramdani et al., 2013; Elmhdwi et al., 2015; Bouyahyaoui et al., 2016).

Photo (6): Juniperus phoeniceaL. By Salwa I. A. Eltawaty, August 2016

12.6.7 Marrubium vulgare L. 1.2.6.7.1 Classification and names Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Lamiales in APG4

Family: Lamiaceae

Genus:Marrubium L

Botanical names: “white horehound” in Europe, and “Marrubia” in Tunisia ( Santram et al., 2017).

Vernacular name: Roubia

1.2.6.7.2 Plant description

Marrubium vulgare L. is a perennial herb attains approximately one-foot height, branched below, densely covered in young stage, with a thick, white, and cottony felt (Santram et al., 2017).

1.2.6.7.3 Folk medicine uses Marrubium vulgare L. uses in folk medicine in the native range include treatment of diabetes, inflammatory disorders, gastroenteric disorder, and respiratory disorders such as bronchitis, colds, and asthma (Masoodiet al. 1956; Robles-Zepeda et al. 2011; Rezgui et al., 2016).

1.2.6.7.4 Phytochemistry More than 54 secondary metabolites have been isolated and identified from different parts of Marrubium vulgare. Among them, diterpenes, sesquiterpenes, and flavonoids are considered to

Photo (7): Marrubium vulgarae L. By Salwa I. A. Eltawaty, August 2016

be major categories of compounds, some of which exhibit potential biological activities in vitro and in vivo(Santram et al., 2017).

1.2.6.7.5 Antimicrobial activity

Partial growth inhibition to significant antibacterial activity had been shown against Gram positive bacteria from the extracts of Marrubium vulgare. It has been documented that plant different extracts can inhibit the growth of Staphylococcus aureus at concentration of 1.25- 2.5mg/ml and killed it at 2.5-10mg/ml. The plant extracts revealed significant antifungal activity where it showed good performance against Aspergillus niger and other tested fungi (Masoodiet al., 2008; Zarai et al., 2011; Bokaeian et al., 2014). It has been reported that some tested Gram negative bacteria were not inhibited while Klebsiella pneumoniae and Pseudomonas aeruginosa were the most sensitive Gram negative bacteria to the effect of Marrubium vulgare extract (Radojević et al., 2013; Dehbashi et al., 2015).

1.2.6.8 Rosmarinus officinalis L.

1.2.6.8.1 Classification and names

Kingdome: Plantae

Division: Anthophyta (Angiospermae)

Class: Magnoliopsida (Dicotyledoneae)

Order: Lamiales in APG4

Family: Lamiaceae

Genus:Rosmarinus L.

Botanical names:Rosemary (Begum et al., 2013)

Vernacular name: Ekleel Aljabal

1.2.6.8.2 Plant description

Rosmarinus officinalis L. is one of the species in the genus Rosmarinus and originated from the temperate countries of the Mediterranean regions. It is a dense bush, branched, evergreen and blue–white flower, reaching a height of about 1 m. It is characterized by leaves with 1–4 cm long and 2–4 mm wide,, linear to linear-lanceolate, with curved edges, dark green upper side with prominent midrib, and very characteristic smell (Begum et al., 2013; Al-Sereiti et al., 1999).

1.2.6.8.3 Folk medicine uses In traditional medicine, the leaves of Rosmarinus officinalis are used based on their antibacterial activities, carminative and as analgesic in muscles and joints. Also, rosemary’s essential oils and extracts obtained from flowers and leaves are used to treat minor wounds, rashes, headache, dyspepsia, circulation problems, but also as an expectorant, diuretic and antispasmodic in renal colic (Bozin, 2007; Begum, 2013; WHO, 2001; Wichtl, 2004).

1.2.6.8.4 Phytochemistry Many polyphenols were detected in Rosmarinus officinalis; the most commons are apigenin, diosmin, luteolin, genkwanina and phenolic acids, especially rosmarinic acid, chlorogenic acid and caffeic acid(Al-Sereiti et al., 1999 ). Other major compounds common in rosemary are terpenes (Lovkov et al., 2001). Rosmarinus officinalis was analyzed phytochemically and four sesquiterpenes were identified, the main constituents were bornyl acetate, pinene and verbenone with percentages. Limonene, camphor, borneol, camphene and 1,8-cineole were also identified with lesser amounts. α-pinene, 1,8-cineole, camphor and

67 verbenone were found to be the main constituents of ethanol extract of dried leaves of Rosmarinus officinalis . Terpenes are major compounds common in essential oil of rosemary (Pintore et al., 2002; Santoyo et al., 2005; Genena et al., 2008; Andrade et al., 2018).

1.2.6.8.5 Antimicrobial activity The antimicrobial performance of oil and organic bioactive compounds from different parts of Rosmarinus officinalis plant was evaluated and the results proved that the plant has significant antifungal activity against Candida albicans and Aspergillus nigerand also has

Photo (8): Rosmarinus officinalis L

By Salwa I. A. Eltawaty, August 2016

Antibacterial activities against Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA) isolates and Staphlycoccuc aureus ATCC 25923 strain. Antibacterial activity against Gram negative bacteria had been noticed except for Pseudomonas aeruginosa which was susceptible to the plant ethanol extract but resist the plant oil extract (Pintore et al., 2002; Santoyo et al., 2005; Abdelah Bogdadiet al., 2007; Jarraret al., 2010; Rashid, 2010).

1.2.6.9 Citrus aurantium L.

1.2.6.9.1 Classification and names:

Kingdome: Plantae

Division:Eudicots

Class: Rosids

Order: Sapindales

Family: Rutaceae

Genus:Citrus L.

Botanical names:Sour orange, Seville orange, bigarade orange, marmalade orange (Suntaret al., 2018).

Vernacular name: Bitter orange

1.2.6.9.2 Plant description

Citrus aurantium is an evergreen tree of 9 meter native to southeastern Asia. It is cultivated in many regions such as India (Karhikeyan and Karthikeyan, 2014).

1.2.6.9.3 Folk medicine uses Traditionally it is known to be useful for the treatment many diseases like stomach ache, vomiting, blood pressure, bronchitis, diarrhea, abdominal pain and fever (Karhikeyan and Karthikeyan, 2014).

Photo (9): Citrus aurantium L. By Salwa I. A. Eltawaty, August 2016

1.2.6.9.4 Phytochemistry It has been reported that approximately thirty three compounds were identified, constituting 99% of the total oil. Limonene was presented as main component followed with (E)- nerolidol, α -terpineol, α -terpinyl acetate and (E, E)-farnesol, The volatile oil of leaves of Citrus aurantium subjected it to Gas chromatography – Mass Spectrometry (GC-MS) analysis which revealed thirty five peaks; α Terpineol, 4 terpineol, β Linalool, Eucalyptol (1, 8 Cineole), α- Pinene, β-Pinene, D-Limonene, O-Cymene, Sabinene and β-Myrcene were the most abundant constituents identified. The presence of alkaloids, phenols, tannins, saponins, steroids and flavonoids has been documented in both aqueous and methanolic extracts while the glycosides identified only in methanol extract of the leaves (Ammar et al., 2012; Periyanayagam et al., 2014; Abed AL Ani et al., 2017). Flavonoids have been found the most pronounce substance among different chemical components in Citrus aurantium due to their physiological and pharmacological role and their health benefits (Suntaret al., 2018).

1.2.6.9.5 Antimicrobial activity

Different parts extracts (fruits, leaves, flowers and peels) of Citrus aurantium plant had been collected from different regions were tested for their antimicrobial activity by many researchers. Different solvents and methods for extraction had been used.Results proved that the plant has definitive antibacterial activity and antifungal activity compared with the refrence antibiotic. Also the results documented that the antimicrobial activity differ with diffeference in the plants ecosystem. (Aibinu et al., 2007; Ammar et al., 2012; Periyanayagam et al., 2014).

Abed AL Ani et al. (2017) compared the antibacterial activities of methanol extract and aqueous extract of the leaves of Citrus aurantium, and they found that tested Staphylococcus aureus and Proteus mirabilis were more susceptible to methanol leaves extract than the aqueous leaves extract while Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa were more susceptible to aqueous leaves extract than methanol leaves extract.

1.3 Pathogenic microorganisms

1.3.1 Staphylococcus aureus

Classically, Gram-positive organisms have been the predominant bacterial pathogens of particular challenge. The infections often are due to methicillin-resistant Staphylococcus aureus (MRSA) which representing up to 50% of all staphylococcal infections. Staphylococcus aureus is a widespread commensal Gram positive bacterium and pathogen. Approximately 50% - 60% of individuals are intermittently or permanently colonized with Staphylococcus aureus and, thus, there is relatively high potential for infections. It is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. Its attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. This biofilm make Staphylococcus aureusinfections difficult to eradicate. Methicillin resistant Staphylococcus aureus causes a wide range of infections, from superficial skin infections to severe, and potentially fatal, invasive disease which become increasingly difficult to treat due to the emergence of antibiotic resistance (Lister and Horswill, 2014; Kadariya et al., 2014; Thapaliya et al., 2017).

1.3.2 Gram negative bacteria Multidrug-resistant (MDR) Gram negative bacteria such as Escherichia coli, Klebsiella spp., and Pseudomonas aeruginosa are predominant mentioned organism species related with diseases. Also Extended-spectrum beta-lactamase (ESBL) producing Gram-negative bacteria

72 were isolated too (Wisplinghoff et al., 2003; Yadegarynia et al., 2003; Rolston et al., 2006; Klastersky et al., 2007). It has been reported that most members of the family Enterobacteriaceae, can harbor numerous plasmid and integron-mediated determinants of antimicrobial resistance (Tumbarello et al., 2012).

1.3.2.1 Acinetobacter baumannii Acinetobacter baumannii is a non-fermentativeopportunistic bacteria found in water and soil. It was susceptible to most antibiotics in the 1970s, but now it becomes a major cause of hospital acquired infections worldwide due to its remarkable tendency to rapidly acquire resistance determinants to a wide range of antibacterial agents.

Acinetobacter baumanniicansurvives in hospital environments despite the hard environment conditions such as desiccation, nutrient starvation and antimicrobial treatments. It is hypothesized that this survival, as well as its virulence, attributed to its ability to form biofilms either on abiotic surfaces such as glass and polystyrene or biotic surfaces such as epithelial cells and fungal filaments. After initial attachment of the pathogen to abiotic surfaces, production of the Bap surface-adhesion protein and Pili assembly play a role in biofilm initiation and maturation. This microorganism occurring mostly in intensive care units and frequently involved in outbreaks of infection and mostly caused pneumonia, septicaemia, and urinary tract infection following hospitalization of patients with different illness cause (Bergogne-Berezin and Towner, 1996; Richet et al., 2001; Coelho et al., 2004; Fournier et al., 2006; Gaddy and Actis, 2009).

1.3.2.2 Enterobacter cloacae The clinically important Gram-negative Enterobacter cloacae is a member of the Enterobacteriaceae, which recognized as a major pathogen in nosocomial infections. It has been reported that twenty out of sixty patients had bloodstream infection caused by Enterobacter cloacae died due to the infection. Mortality was associated with multi-resistant isolates and poly- microbial infection. SHV-12. TEM-1 and ampC beta-lactamase genes were found in multi-drug resistant Enterobacter cloacae isolated from patients (Liu et al., 2004; Delgado-Valverde, 2013).

1.3.2.3 Escherichia coli

Escherichia coli is a facultative anaerobic bacteria which is a common commensal in people and animals gastrointestinal tract with certain strains being pathogenic and causing conditions including gastroenteritis, cystitis, meningitis, peritonitis, and septicemia. Escherichia coli O157:H7 is an emerging public health concern in most countries of the world. It is an important cause of food-borne human disease. Both commensal and pathogenic Escherichia coli have the ability to form biofilms and several key factors, including different extracellular appendages, are implicated in its surface colonization. Besides being the major causative agent for recurrent urinary tract infections, Escherichia coli biofilm is also responsible for indwelling medical device-related infectivity. Multidrug-resistant Escherichia coli has become a major public health concern in many countries, make treatment hard with consequent huge health burden (Aarestrup, et al., 2008; Beloin et al., 2008; Ibrahim, et al., 2012; Reuben and Owuna, 2013; Sharma et al., 2016).

1.3.2..4 Klebsiella pneumoniae Klebsiella pneumoniae is also among the Gram negative bacteria that most readily develop resistance mechanisms to multiple classes of antibiotics, and the prevalence of drug resistance is increasing at an alarming rate.The most important virulence factor involved in the pathogenesis of these bacteriais their ability to form a thick layer of biofilm. Recently,many studies highlighted the emergence of multidrug‐resistant Klebsiella pneumoniae strains which show resistance to colistin, a last‐line antibiotic, arising from mutational inactivation of mgrB regulatory gene. Klebsiella pneumonia carbapenemase producing Klebsiella pneumoniae is an emerging pathogen with serious clinical and infection control complications and it is the most frequently encountered carbapenemase-producing Enterobacteriaceae (Gasink et al., 2009;Kidd et al., 2017).

1.3.2..5 Proteus mirabilis Proteus mirabilis is an enteric bacterium considered as important cause of community- and health care-associated infections, including those involving the urinary tract, the abdominal cavity, and the bloodstream infections. Multidrug-resistant (MDR) strains of Proteus mirabilis generally produce extended-spectrum β-lactamases (ESBLs) or the AmpC-type

74 cephalosporinase and rarely carbapenemases enzymes and their prevalence in some settings is relatively high (Endimiani et al., 2005; Tumbarello et al., 2012).

1.3.2..6 Pseudomonas aeruginosa The Gram negative Pseudomonas aeruginosa is a leading nosocomial multidrug resistant pathogen responsible for severe life-threatening infections in immunocompromised persons, nosocomial infections and chronic infections. Biofilm play a clear role in Pseudomonas aeruginosa infections including chronic wound infections, chronic cystic fibrosis pneumonia, chronic otitis media, chronic bacterial prostatitis, and medical device-related infections (Van Delden and Iglewski, 1998; Aloush et al., 2006; Tolker-Nielsen, 2014).

1.3.3 Fungi

1.3.3.1 Aspergillus niger Aspergillus niger is a kind of mold causes a disease called black mold on certain fruits and vegetables such as grapes, apricots, onions, and peanuts, and is a common contaminant of food. It is less likely to cause human disease than some other Aspergillus species, but it causes Aspergillosis (fungal lung infection) especially in people with underlying illnesses such as tuberculosisor chronic obstructive pulmonary disease. Also Aspergillus nigercauses fungal sinusitis allergy andconsidered as one of the most common causes of otomycosis (fungal ear infections), which can cause pain, temporary hearing loss, and, in severe cases, damage to the ear canal and tympanic membrane (Person et al., 2010; Atchade et al., 2017).

1.3.3..2 Candida albicans Cadida albicans yeast is one of the most common group of organisms that cause nosocomial infection. Its infection commonly occurs as a superficial infection (Candidiasis) on mucous membranes in the mouth or vagina. Also its systemic infection is well known as important cause of morbidity and mortality in immuno-compromised patients. This organism often form biofilms inside the body such as biofilms formed on surfaces of the implantable medical devices or organs where it is often found together with Staphylococcus aureus, the multi-infection which lead to high mortality rates especially if Candida cells introduced to the

75 blood stream (Candidaemia). This make the infection with Candida albicance of major high concern among hospital-acquired infections (Weinberger et al., 2005; Calderone and Clancy, 2011; Zago et al., 2015).

CHAPTER TWO Materials and Methods

2.1 Area of the study

The studied plants were collected in August 2016 from around Al-Bayda city (Eljabal Al- Akhdar), eastern north of Libya. The experimental studies were done from 2016 to 2018 in National center and International Africa University, Khartoum, Sudan.

2.2 Selected plants

Nine plants were selected according the local Libyan traditional uses including Myrtus communis, Pistacia lentiscus, Capparis spinosa, Salvia fruticosa, Juniperus phoenicea, Marruium vulgare, Citrus aurntium, Artmesia Herba Alba and Rosmarinis officinalis. The plants were identified by Dr. Hussein Altagori, Department of plant, Faculty of science, Benghazi University, Libya.

2.3 Preparation of plants

Any hanging impurity was first removed from each plant. Then each of the nine plants was cleaned with tap water in order to get off any dust hanged. The targeted plant parts; leaves (all nine plants), bark (8 plants except Citrus aurntium), fruits(two plant; Pistacia lentiscus and Juniperus phoenicae), flowers(one plant; Marrubium vulgare), were separated and each part was

76 air dried in shadow. Each dried targeted plant part was powdered with use of an electric Grinder and kept in a well labeled closed container.

2.4 Extraction of plants.

All extracts were weighed and the yield percentage calculated, recorded and the extracts were kept in well labeled clean containers at 4 Co.

2.4.1 Organic solvent extraction

One hundred grams of each of the powdered plant materials was successively extracted with (250-300ml) of chloroform, methanol and ethanol respectively, by Soxhlet apparatus for enough time (6 – 10 hours). The chloroform extract was filtered and solvent was evaporated under reduced pressure using Rota-evaporator. After that the plant material left air dried, repacked in the Soxhlet and exhaustively extracted with methanol, then the methanol extract was filtered and evaporated and the plant material left air dried, repacked and recycled with ethanol which then evaporated with Rota-evaporator. Each solvent yield was weighed and the yield percentage was calculated, recorded and the extracts were kept in well labeled clean containers at 4 Co. The chloroform extracts were kept in glass containers.

2.4.2 Aqueous extraction

Plant parts were extracted with water via maceration for 72 hours and then filtered and the filtrated aqueous extract was dried via freeze drier.

2.5 Preparation of the extracts stock solution

2.5.1 Aqueous extract stock solution

In the day of the antimicrobial assay, 0.2gm of the freeze dried powder was dissolved in 2 ml sterile distilled water in order to prepare an extract stock solution of final concentration of 100mg/ml.

2.5.2 Organic extracts stock solution

In the day of the antimicrobial assay, the chloroform extract (0.2g) was dissolved in 2 ml of a mixture of (2:1 V/V) of methanol and petroleum ether to get final concentration of 100mg/ml and each of the methanolic extract (0.2g) and ethanolic extract (0.2g) was dissolved in 2 ml of methanol to get final concentration of 100mg/ml.

2.6 Identification of clinical isolates

One hundred bacterial isolates included in this study collected in the period of June 2016 to December 2016. The bacteria were isolated from inpatients attended to different department in Medical Center, Benghazi city, Libya. The included clinical isolates were isolated from different tested samples; blood, urine, semen, sputum and wounds samples. The identified isolates were Methacillin resistant Staphylococcua aureus (MRSA), Acinetobacter baumanii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa.

2.6 .1 Gram stain and microscopic examination of clinical isolates

All isolates were subjected to Gram staining and microscopic examination to study their stain reaction (Gram positive or Gram negative), the shape and arrangements of their cells. A fixed colony smear was covered with crystal violet stain for 30-60 sec., rapidly washed with clean water, covered with Lugol’s iodine for 30-60 seconds, again washed with clean water, rapidly and for few seconds decolorized with acetone or alcohol, washed immediately with clean water, covered with Safranin stain for two minutes, washed with water and, left to air dry and then the smear examined under light microscope with the oil immersion objective to look for bacteria Gram reaction and and cells shape.

2.6.2 Preparation of McFarland 0.5 solution

Distilled water was used to prepare 1% of each of Barium chloride dehydrate; BaCl2•2H

2O and Sulfuric acid. 0.05ml of 1% Barium chloride dihydrate was mixed with 9.95ml of 1% Sulfuric acid to get 0.5 McFarland solution, approximately cell density of 1X108 CFU/mL (Waitz, 1990).

2.6.3 Identification of clinical isolates with BD Phoenix 100 System

Bacterial suspension freshly calibrated with 0.5 McFarland standard (freshly prepared) was picked up in the identification broth bottle and has been well mixed with vortex. One drop of antibiotic susceptibility indicator solution was added to the antibiotic sensitivity test broth bottle and the bottle was inverted twice. Appropriate volume from the identification broth was transferred to the antibiotic sensitivity test broth. Simply both bottles poured into the panel, the inoculation port was caped and the panel was labeled. The panel then inserted into an internal carrier in the digitalized BD Phoenix instrument, the door closed and the test began automatically. The principle is that the identification is processed according to the biochemical reactions results revealed from reaction between bacteria and the system biochemical reagents.

2.6.4 Identification of clinical isolates with biochemical reactions

The isolates were reidentified according to Cheesbrough, 2000. To confirm the identification of clinical isolates, biochemical tests were used to differentiate bacteria, either on the base of the metabolic end product of the organisms; their ability to produce a particular end- product such as Indole when grown in suitable culture media, and whether it possesses certain enzyme activities, such as Oxidase, Urease, Citrate, Coagulase, Catalase and DNase, and also the ability of bacteria to produce acidic or gaseous products when cultured on media containing carbon source such as glucose, lactose, mannitol and sucrose.

2.6.4.1 Catalase test

It is test used to differentiate those bacteria that produce the enzyme catalase, such as Staphylococci, from non-catalase producing bacteria such as Streptococci. Catalase enzyme

79 breaks down hydrogen peroxide to oxygen and water. Good clear bacteria growth was taken from culture media by glass rod and immersed in a glass tube containing 2-3ml hydrogen peroxide. Development of oxygen bubbles indicates to positive result (indicates to the ability of bacteria to produce catalase enzyme).

2.6.4.2 Coagulase test (Bound coagulase)

Drops of plasma were added to bacteria colony previously suspended by emulsifying on a slide with drop of physiological saline. Occurrence of clumping within 10 seconds indicates to positive result (presence of bound coagulase), while no clumping within 10 seconds means no bound coagulase produced.

2.6.4.3 Deoxyribonulease (DNAse) test

This test was used to differentiate Staphylococcus aureus which produce the enzyme DNAse from other Staphylocci which do not produce DNAse. Deoxyribonulease hydrolysis deoxyribonucleic acid (DNA). The tested organism was cultured on DNAse medi and after overnight incubation at 37C the plate was flooded with a weak hydrochloric acid solution. The acid precipitates un-hydrolyzed DNA. DNAse producing colonies were therefore surrounded by clear areas indicated DNA hydrolysis and was considered as positive result.

2.6.4.4 Citrate utilization test

It is used to differentiate Gram negative bacteria based on their ability to use citrate as its only source of carbon and ammonia as its only source of nitrogen. The tested bacteria was cultured on Simmon citrate agar medium which contain sodium citrate, ammonium saltand the indicator bromo-thymol blue and incubated overnight at 37C. Change in color of the indicator from light green to blue (due to the alkaline reaction following citrate utilization) indicated to positive results.

2.6.4.5 Oxidase test

This test used to assist in the identification of species of Pseudomonas, Vibrio and Pasteurella on the principle of their abilities to produce oxidase enzymes. 2-3 drops of freshly prepared oxidase reagent were added to a piece of filter paper placed in a clean petri dish. With a glass rod a colony of the tested bacteria was removed from a nutrient agar growth and smeared on the reagent placed on the filter paper. Occurrence of a blue purple color within a few seconds indicated to positives result (Bactria produced oxidase enzyme). No occurrence of a blue purple color within a few seconds or any blue purple color developed after 10 seconds indicated to negative result.

2.6.4.6 Urease test

Urease enzyme activity is important in differentiating Enterobacteria. The enzyme urease hydrolysis the urea to ammonia and carbon dioxide. A sterile straight wire was used to inoculate a tube of sterile Motility Indol Urea (MIU) medium contain urea and phenol red as indicator) medium with stab-line manner with a colony of the tested bacteria. Indole paper strip was replaced in the neck of the MIU tube above the medium. The tube was stoppered and incubated at 37C overnight. Appearance of red-pink color in the medium indicated to positive result. Spreading turbidity from the stab-line or throughout the medium indicated to the motility character of tested bacteria. Occurrence of surface red color within 10 minutes after adding 1ml of Kovac’s reagent to the overnight culture indicated to positive result; ability of bacteria to produce indole.

2.6.4.7 Triple sugar Iron agar (TSI) test

It is based on the ability of some bacteria to ferment lactose, sucrose (on slope) and glucose (on bottom), with the production of gas and H2S. The tested organism was inoculated as deep straight line manner inside the bottom medium agar and as zigzag manner on the slope surface of triple sugar Iron agar medium and incubated at 37 °C for overnight. Color of Phenol red indicator changes according to the reaction type (Acid or alkaline).Changing the color of the slope or butt from pink to red indicated fermentation of glucose (alkaline reaction), and changing the colour from pink to yellow indicated fermentation of lactose (acid reaction).

2.7 Preparation of Microorganisms suspensions

2.7.1 Preparation of bacterial suspension

In this study a fresh stock suspension was concerned to be prepared for each tested microorganism. In order to get a suspension containing about 1×108 C.F.U/ml, an overnight Nutrient agar slope growth for each tested bacteria was harvested and washed off with 100 ml sterile normal saline. After that for the antimicrobial screening, the suspension was adjusted to McFarland 0.5 solution via more dilution with sterile normal saline 0.9%. This step done within Hod to avoid any environmental contamination.

2.7.2 Preparation of fungal suspension

A fresh stock suspension was prepared. In order to get a suspension containing about 1×108 C.F.U/ml, an overnight Sabouraud Dextrose agar slope growth for each tested fungi was harvested and washed off with 100 ml sterile normal saline. After that for the antimicrobial screening, the suspension was adjusted to McFarland 0.5 solution via more dilution with sterile normal saline 0.9%. This step done within Hod to avoid any environmental contamination.

2.8 Screening of antimicrobial activity

2.8.1 In vitro screening of antimicrobial activity

2.8.1.1 Antibacterial activity

The paper disc diffusion method was adopted (Mukhtar and Ghori, 2012) with some minor modifications. Bacterial suspension was diluted with sterile physiological solution to 108cfu/ ml (turbidity = McFarland standard 0.5). One hundred microliters of bacterial suspension were swabbed uniformly on surface of Mueller Hinton agar and the inoculum was allowed to dry for 5 minutes. Sterilized filter paper discs (Whatman No.1, 6 mm in diameter- Selecta, Germany) were placed on the surface of the Mueller Hinton agar after soaked with 20 µl of a solution of each plant extracts. The inoculated plates were incubated at 37 C° for 16 -18 hours. The diameters (mm) of the inhibition zones were measured by millimeters (mm). Discs impregnated

82 with methanol were used as control. Two replicates were carried out for each extract against each of the tested organisms and means value were calculated.

2.8.1.2 Antifungal activity

The same steps used above to screen the antibacterial activity were adopted to screen the antifungal activity of standard Aspergillus niger (A.niger) ATCC 9763 and Candidaalbicans ATCC 7596. The cultures condition was incubation at 25 °C for 48 hours. The diameters (mm) of the inhibition zones were measured. Two replicates were carried out for each extract against the tested organism.

2.9 Determination of minimum inhibitory concentration (MIC)

Andrews, (2006) agar dilution method was adapted in this study. The agar plate dilution method was adopted to determine the minimum inhibitory concentration of the extract which can inhibit the growth of the seeded bacteria on the Mueller Hinton agar. Serial dilution was prepared for each extract in decreasing concentrations in the following order: 200,100, 50, 25, 12.5, 6.25, and 3.125. In sterile covered glass bottles, 5ml Melted double strength Mueller Hinton agar cooled to 45 º C were mixed with 5ml of each dilution of the tested plant extract to get a final serial dilution of 100, 50, 25, 12.5, 6.25, 3.125 and 1.652 of each extract. The mixture was poured in sterile small petri dishes, left to solidify and then the bottom of each plate was marked off into segments, each segment designed for specific strain of all tested microorganims. A loop full of standard loop (0.01ml) of each of tested bacterial fresh suspension adjusted with McFarland 0.5 solution was spotted onto the surface of each segment of the marked Mueller Hinton agar. Allow the inoculum to be absorbed into the agar before incubation, and then incubated for 16 -18 hours at 37 º C for bacteria and 25 º C for fungi. After the incubation period the least concentration mg/ml of the plant extract that completely inhibits growth of organism is considered as the minimum inhibitory concentration (MIC).

2.10 Antibiotic susceptibility test One hundred microliters of freshly prepared bacterial suspension (adjusted to 0.5 McFarland suspensions) were swabbed uniformly on surface of Mueller Hinton agar and the inoculum was allowed to dry for 5 minutes. Each reference antibiotic discs was placed in

83 duplicate on the surface of the Mueller Hinton agar. The inoculated plates were incubated at 37 °C for 16-18 hr. The diameters (mm) of the inhibition zones were measured.

2.11In vivo antimicrobial and Wound healing activity

Abdrabo et al. (2005) model was used in this study with some modifications.

2.11.1 Pharmaceutical preparation of 3% ointment of Myrtus communis methanol leaves extract

To prepare a 100gm ointment of 3% of Myrtus communis extract, a mix of emulsifying wax (30gm), white soft paraffin (50gm) and Liquid paraffin (20ml) was put on a heater with magnetic stiror (Hot plate magnetic stiror)at 60C. After all ingredient melted poured into morter contain 3gm of the extract powder. The mix was mixed well with a glass rod till get homogenized mixture. The ointment preparation was kept in a clean well tight container at 4C (British Pharmacopia, 1988).

2.11.2 Preparation of wounded animal

Three assay lines were designed. Each line was composed of three groups and each group composed of 5 rats. For each rat, full thickness wound was made in the skin of the test animals according to the model of Abdrabo et al. (2005). Hair of the lower back and right flank of animals was fully shaved. Rats were lightly anaesthetized by inhalation using halothane.

The animals were held in standard crouching position, and the mobile skin of flank was gently stretched and held by fingers. A metal circular object measuring 1 cm in diameter was placed on the stretched skin and an outline of the object was traced on the skin using a fine tipped pen. The wound was made by excising the skin within the border of the object to the level of loose subcutaneous tissue, using sterile forceps and a scalpel blade. The artificial wounds were circular with a diameter of 1 cm. The first day of the experiment was regarded as the Zero day.

2.11.3 First in vivo trial line: Wound healing activity of Myrtus communis (non-infected rats)

Fiteen experimental albino rats were divided into three groups each consists pf 5 rats

Group 1 (wound only):

Untreated control group, wounds were left without treatment.

Group 2 (wound + Tetracycline ointment 3%):

Wounds of these animals were treated topically with Tetracycline ointment 3% every 12 hours as a standard healing agent starting from the first day.

Group 3 (wound + methanol extract of Myrtus communis 3%):

Wounded animals were treated topically with the tested plant extract ointment which contained 3% of the Myrtus communis leaves methanolic extract every 12 hours starting from the first day.

2.11.4 Second in vivo trial line: Wound healing activity of Myrtus communis (Staphylococcus aureus infected rats)

The same steps were done as in the first trial line, but in this line an artificial wound infection was done by seeded the wound with Staphylococcus aureus ATCC25923strain suspension freshly adjusted with 0.5 McFarland standards (108-109 C.F.U. /ml).

Group 1 (wound + infection):

Infected control group, wounds were artificially infected with standardized Staphylococcus aureus ATCC25923 suspension, using adjustable volume automatic microtitre pipette to spread 0.1 ml of the suspension in every wound.

Group 2 (wound + Infection + Tetracycline ointment 3%):

Wounds of these animals were artificially infected using the same method as used in Group 1, and treated topically with Fucidin ointment 2% every 12 hours as a standard healing agent starting from the first day.

Group 3 (wound + Infection + methanol extract of Myrtus communis 3%:

Wounded animals were artificially infected using the same method as used in Group 1, and treated topically with the tested plant extract containing 3% of the Myrtus communis leaves methanol extract every 12 hours starting from the first day.

2.11.5 Third in vivo trial line: Wound healing activity of Myrtus communis (Pseudomonas aeruginosa infected rats)

The same steps were done as in the first trial line, but in this line an artificial wound infection was done by seeded the wound with Pseudomonas aeruginosa ATCC27853 strain suspension freshly adjusted with 0.5 McFarland standard (108-109 C.F.U. /ml).

Group 1 (wound + infection):

Infected control group, wounds were artificially infected with standardized Pseudomonas aeruginosa ATCC27853 suspension, using adjustable volume automatic microtitre pipette to spread 0.1 ml of the suspension in every wound.

Group 2 (wound + Infection + Tetracycline ointment 3%):

Wounds of these animals were artificially infected using the same method as used in Group 1, and treated topically with Tetracycline ointment 3% every 12 hours as a standard healing agent starting from the first day.

Group 3 (wound + Infection + methanol extract of Myrtus communis 3%:

2.12 Phytochemical analysis

2.12.1 Phytochemical screening with basic methods

Phytochemical screening for the active constituents was carried out using the methods described by Martinez and Valencia, 2003; Sofowora 1996; Harborne, 1984 and Wall et al., 1952 with few modifications.

2.12.1.1 Detection of Tannins

0.5 g of the extract was washed three times with petroleum ether, dissolved in 10 ml hot saline solution and divided in two tests tubes. To one tube 2-3 drops of Ferric chloride added and to the other one 2 – 3 drops of gelatin salts reagent added. The occurrence of a blackish blue colour in the first test tube and turbidity in the second one denotes the presence of tannins.

2.12.1.2 Detection of Sterols and Triterpenes

0.5 g of the extract was washed three times with petroleum ether and dissolved in 10 ml of chloroform. To 5 ml of the solution, 0.5 ml Acetic anhydride was added and then 3 drops of concentrated Sulphuric acid at the bottom of the test tube. At the contact zone of the two liquids, the gradual appearance of green, blue pink to purple color was taken as an evidence of the presence of sterols (green to blue) and or triterpenes (pink to purple) in the sample.

2.12.1.3 Detection of Alkaloids

0.5 g of the extract was heated with 5 ml of 2N HCl in water bath and stirred for about 10 minutes, cooled filtered and divided into two test tubes. To one test tube few drops of Mayer’s reagent was added while to the other tube few drops of Valser’s reagent was added. A slight turbidity or heavy precipitate in either of the two test tubes was taken as presumptive evidence for the presence of alkaloids.

2.12.1.4 Detection of Flavonoids

0.5 g of the extract was dissolved in 30 ml of 80% ethanol. The filtrate was used for the following tests:

(A)- To 3 ml of the filtrate in a test tube, 1ml of 1% potassium hydroxide solution in methanol was added. Appearance of a yellow color indicated the presence of flavonoids, flavones or and chalcone.

(B)- To 2 ml of the filtrate, 0.5 ml of 10 % Lead acetate was added. Appearance of creamy turbidity was taken as an evidence of flavonoids.

2.12.1.5 Detection of Saponins

0.5 g of the extract was placed in a clean test tube. 10 ml of distilled water was added, the tube stoppered and vigorously shaken for about 30 seconds. The tube was then allowed to stand and observed for the formation of foam, which persisted for at least an hour. This was taken as evidence for presence of saponins.

2.12.1.6 Detection of Coumarins

0.5 g of the extract was dissolved in 10 ml distilled water in test tube and filter paper attached to the test tube to be saturated with the vapor after a spot of 0.5 N potassium hydroxide (KOH) put on it. Then the filter paper was inspected under UV light, the presence of coumarins was indicated if the spot have found to be adsorbed the UV light.

2.12.1.7 Detection of Anthraquinone glycoside

0.5 g of the extract was boiled with 10 ml of 0.5N KOH containing 1ml of 3% Hydrogen peroxide solution. The mixture was extracted by shaking with 10 ml of benzene. 5ml of the benzene solution was shacken with 3ml of 10% ammonium Hydroxide solution, and the two layers were allowed to separate. The presence of anthraquinones was indicated if the alkaline layer was found to have assumed pink or red color.

2.12.2 Phytochemical screening with Gas chromatography – Mass spectra phytochemical analysis (GC-MS)

The qualitative and quantitative analysis of the sample was carried out by using GC/MS technique model, The methanol extract of each tested plants was dissolved in 3ml of High Performance Liquid Chromatograph (HPLC) methanol, 1gm of the drying agent, Anhydrous sodium sulphate, was added and gently mixed. The mixture was filtered using 0.45µm syringe filter. 1.5 ml of the filtrate was picked up into GC-MS specific vial (GC-MS atmospheric vial

1.5ml). The atmospheric vial was introduced onto the auto sampler (GC-MS auto injector) and the start button was pressed to the analysis cycle be started. The samplewas injected by using split mode, helium as the carrier gas passed with flow rate1.61 ml/min, the temperature program was started from 60°C with rate 10°C/min to 300 °C as final temperature degree with 3 minutes hold time, the injection port temperature was 300°C, the ion source temperature was 200°C and the interface temperature was 250°C . The sample was analyzed by using scan mode in the range of m/z 40-500 charges to ratio and the total run time was 26 minutes .Identification of components for the sample was achieved by comparing their retention times and mass fragmentation patents with those available in the library ,the National Institute of Standards and Technology (NIST).

2.13 Antioxidant screening of tested plants 2.13.1 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH) radical Scavenging assay Antioxidant activity of tested plants extracts were screened in this study by use of DPPH radical scavenging assay. The DPPH radical scavenging was determined according to the method of Shimada et al. (1992). In 96-wells plate, the test samples were allowed to react with 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl stable free radical (DPPH) for half an hour at 37ºC. The concentration of DPPH was kept as (300μM). The test samples were dissolved in Dimethyl sulfoxide while DPPH was prepared in ethanol. After incubation, decrease in absorbance was measured at 517nm using Multi-plate Reader Spectrophotometer. Percentage radical scavenging activity by samples was determined in comparison with a Dimethyl sulfoxide treated control group. All tests and analysis were run in triplicate.

2.13.2 IC50 Calculations

The IC50 (the concentration of test material, which possess 50% inhibition of free radicals) of all tested extracts was determined by monitoring the effect of different

89 concentrations ranging from 0.5-0.003mg/ml. The IC50 of the extracts were calculated using EZ- Fit Enzyme Kinetic Program. Propyl Gallate was used as reference.

2.14 Anti-Cancer activity of tested plants (Cytotoxic activity)

2.14.1 Cell Lines and Culture Medium

HEPG2 (human liver cell carcinoma), HCT (human colon cell carcinoma), MCF7 (human breast carcinoma) and PC3 (human prostatic cell carcinoma) are cells lines were cultured in a culturing flask containing a complete medium consisting of 10% fetal bovine serum and 90% minimal essential medium (MEM) and then incubated at 37oC in atmosphere of carbon dioxide (CO2) 5%. The cells were sub cultured twice a week.

2.14.2 Cell counting

Cells were counted using the improved Neubauer chamber. The cover slip and chamber were cleaned with detergent, rinsed thoroughly with distilled water and swapped with 70% ethanol, then dried. An aliquot of cell suspension was mixed with equal volume of 0.4% trypan blue in a small tube. The chamber was charged with cell suspension. After cells had settled, the chamber was placed under light microscope. Using 40 X objective, cells in the 4 large corner squares (each containing 16 small squares) were counted. The following formula was used for calculating the cells:

Number of cells counted X Dilution factor* X 104

(Cells/ml) N = ------

*Dilution factor is usually 2(1:1 dilution with trypan blue.

2.14.3 Sulfo-Rhodamine-B Assay (SRB) for Cytotoxicity Screening

Method of Skehan et al. (1990) was used to measure the potential cytotoxicity of the tested plants extracts. Cells were placed in 96- multi well plate (104cells/well) for 24 hours before treatment with the tested extract (s) to allow attachment of cells to wall of the plate. Different concentrations of the tested extracts were added to the cells. Monolayer triplicate wells were prepared for each individual dose. Monplayer cells were incubated with the extract for 48 hours at 37ºC and in atmosphere of 5% CO2. After 48 hours, cells were fixed with Trichloroacetic acid, washed and stained with Sulfo-Rhodamine-B stain. Excess unbound stain was removed by four washes with 1% acetic acid, and bound dye was recovered (extracted) with Tris EDTA buffer. Color intensity (optical density) was measured at 570 nm in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of each tumor cell line after the specified extract.

2.15. Pharmacological screening of tested extracts on isolated organs

2.15.1 Sample Preparation

Dimethyl sulfoxide (DMSO) was used as solvent to prepare a stock extract solution with concentration of 10mg/ml. the extract solution freshly prepared prior to addition into the tissue bath.

2.15.2 Preparation of physiological solutions Krebs solution, Tyrode solution, D Jalon’s solution and Frog solution are physiological solution prepared freshly for the screening of the pharmacological effects of tested extracts on the isolated,rabbit aortic strip, rabbit jejunum, rat uterus and frog rectus abdominus muscle organs respectively. The physiological solution prepared according to Ian Kitchen (1984).

2.15.3 Animals

Animals were given standard feeding and tap water and housed at animal house and kept under controlled conditions of room temperature (25°C) and relative humidity (50%).

2.15.4 Rabbit Aortic Strip Preparation

A rabbit of Sudanese local strain (1.75 kg) was used in this experiment. The preparation was based on the method adopted by Furchgott and Bhadrakom (1953). The rabbit was neck dislocated, sacrificed and exsanguination. The chest was opened, the internal viscera were pulled aside and the aorta had been exposed. The aorta was cut closed to the heart and dissected as fast as possible. Then after, the tissue was transferred to a petri dish containing aerated Krebs solution. The aorta was located over a large plastic cannula, surrounding fats and connective tissues were removed, then the aorta was cut spirally by curved scissor to produce a continuous strip. Threads had been tied to each end of the strip and one end was attached to the tissue holder. The mounted tissue then was transferred to a 25 ml organ bath filled with oxygenated Krebs solution maintained at 37°C and the top thread was attached to Harvard isometric transducer connected to Harvard Universal Oscillograph recorder. The preparation was allowed to stand for 45 minutes, under 2 g resting tension before addition of the reference drug (Nor epeniphrine) and the extract.

2.15.5 Rabbit jejunum Preparation

Rabbit of Sudanese local strain weighing 2kg was used. The rabbit was sacrificed and the abdomen exposed. The first 2-3 cm of the jejunum was taken out and placed on Petri dish containing Tyrode solution at room temperature. The isolated 2-3 cm jejunum tissue was freed from fats and connective tissues and transferred to organ bath (25ml) containing aerated Tyrode solution which was maintained at 37°C. The tissues allowed tosettle for 45 minute as adaptation time. The response of the extract was recorded with isotonic transducer connected to Oscillograph recorder with attenuation of speeds 0.25mm/sec. Under1.5 g tensions (Ian kitchen, 1984).

2.15.6 Rat uterus Preparation

Female young Wister rats, weighing 120 gm was used in this study. The animal was brought into oestrus stage by subcutaneous administration of β-estradiol-3-benzoate (2.5 mg/kg) 24 h prior to the experiment. Preparation of the uterus was carried out according to the method described by De Jallon and De Jalon, 1945 cited in Ian Kitchen, 1984. The rat killed by a blow

92 on the head and exsanguinated. The abdomen was opened and the two uterine horns were exposed by pulling aside the intestine. Each horn was freed carefully from surrounding fat and mesenteric attachments, cut out separately and transferred to a petri-dish containing De Jalon’s solution. Each horn was cut open longitudinally to from a sheet of muscle instead of a narrow tube. A thread will be attached at each end of piece and the preparation will be mounted in a 25- ml organ bath containing aerated De Jalon’s solution maintain at 37º C with one attached to a fixed pin and the other to an isometric transducer connect to Harvard oscillographic recorder with attenuation of speeds 0.1mm/sec. The preparation was allowed to equilibrate for 45 min, under 0.5 tensions before addition of the plant extract and the reference drugs.

2.15.7 Frog rectus abdominus muscle preparation

The frog was decapitated after stunning and the animal was pithed using pithing needle. The frog then placed ventral side up on a cork board and a cut made in mid ventral of trunk. The skin separated along this midline and the recto muscles (which are underneath) was exposed and moistened with frog ringer solution. Two longitudinal cuts were made on either side of the xiphoid cartilage and the line of the recti muscles has been followed in their attachment to pubis. A transfer cut through the xiphoid cartilage was made free from attachment to pubis, the recti muscle then transferred to Petri dish containing frog ringer solution at room temp. By making longitudinal cut along the linea alba, the two muscles were separated and the thread was passed through one muscle at both top and bottom. The bottom thread was attached to tissue holder. The mounted preparation transferred to the organ bath and the top thread was attached to an isotonic transducer and an additional stretching weight will be added to the resting tension to insure that the muscle returns to it base line after drug induced contracture. (Ian Kitchen 1984).

2.16 Acute and sub-acute toxicity of selected plants

The toxic profile (Acute and sub-acute) was studied for methanol extracts of each of Capparis spinosa leaves, Myrtus communis leaves and Pistacis lentiscus fruits. Kidney, liver,

93 lipid, glucose and hematology profiles biochemistry were screened. Histological profiles were screened too.

2.16.1 Extract dose preparation

In sense of complete solubility and in order to get a good extract preparation suitable to be injected via intra peritoneal (IP) route, 3% Sodium carboxy methyl cellulose (Na. CMC) was used as emulsifying agent to prepare a clear soluble stock extract suspension.

To prepare clear stock solution of the extract: first 2g of extract was mixed with 3 ml of 3% Sodium carboxy methyl cellulose and 2ml distilled water, mixed well then the volume was complted to 10 ml by adding 5 ml distilled water.

2.16.2 Animals

Wister SWR albino rats used in this study weighing 80-140g were housed in groups and kept under controlled conditions of room temperature (25°C) and relative humidity (50%) at animal house at Pharmacology Department, Faculty of Pharmacy, International Africa University, Khartoum, Sudan. Animals were fed on standard laboratory rodent’s food and water.

2.16.3 Acute Toxicity (Lethality) and LD50 of three tested plants

methanol extracts of Capparis spinosa leaves, Myrtus communis leaves and Pistacia lentiscus fruits were studied for their acute and sub-acute toxicity. A pilot 24 hours preliminary experiment with some modifications has been carried out to study the acute toxicity and to evaluate the lethal dose of the tested methanol extracts fractions of the tested plants parts (El- Hadiyah et al., 2011). Lethal dose which killed 50% of the tested rats considered as LD50.

Twenty four Albino rats weighting 80g – 140gm were used in this screening. The rats were divided into four groups, six rats for each group. The groups were designed as, Group one (G1), Group two (G2), Group three (G3) and Group four (G4). A dose of 1ml /kg of normal saline 0.9% was injected to each rat of the group (G1) which was designed as control group. An extract dose of 2000mg/kg was introduced to G2, then dose of 1000mg/kg injected to G3 and a

94 last dose of 500mg/kg was injected to the G4. The normal saline dose and the tested extract doses were injected as 24 hour single dose via intra peritoneal (IP) route. Rats were observed for any immediatly death and within 14 days.

2.16.4 Sub-acute toxicity

El-Hadiyah et al. (2011) method was used with some modifications to study the sub- acute toxicity of the tested plants extracts. Half of the highest extract dose which not killed rats and quarter of that killed were used in this study as sub-acute doses to study the sub-acute toxicity. Doses of 1000mg/kg from methanol leaves extract of Capparis spinosa and 500mg/kg of each of methanol extracts of leaves of Myrtus communis and fruits of Pistacia lentiscus were used in this study as sub-acute doses.

For each tested extract two groups were specified; each group composed of six Albino rats weighing 80-140gm. Control group designed as group one (G1) and tested group designed as group two (G2). Atwenty four hours single sub-acute dose of extract was injected interaperitoneal to G2 rats for one week. Group one rats were injected with daily single dose of 1ml /kg normal saline 0.9% for one week. During the seven days study the rats were observed for any behavioral, neurological and autonomic response. Changes in behavioral response of rats were observed and recorded daily. The observation times was four hours daily divided into four periods; 1st period which start from zero time (immediately after extract injection) to one hour), 2nd period started from the end of 1st hour and lasts for an hour (1 – 2hr.), 3rd period started from the end of 2nd hour and lasts for an hour (2 – 3hr.) and 4th period which started from the end of the 3rd hour and lasts for an hour (3 – 4hr.).

In the eighth day all the tested and the control groups rats were anaesthetized via inhalation with Di-ethyl ether and a 3ml orbital blood were squeezed; 2ml collected in a clot activator blood container and 1ml was collected in an EDTA container and immediately the containers transferred to the biochemistry and hematology laboratories respectively at the Modern Medical Center, Khartoum, in aim to check the liver function, kidney function, glucose, lipid and hematology profiles. The one ml blood sample was put on a shaker at moderate speed for one minute to make complete sample homogeneity and then hand agitated for seconds then the bottle cap removed and the bottle introduced to the digitalized Mandary BC-

6800 device; a hematology analyser. After one minute all red blood cells and white blood cells broke down by the supplied analyzer solution and the data was registered. MANDARY BC- 6800 device is five differential device, which supplied with Diluent- ST and five lyzer solutions.

The two ml blood sample was centrifuged at 40 round per minute (R.P.M) for 3 minutes at room temperature to separate the serum from whole blood. The separated serum picked up into a specific tube which then introduced into the digitalized biochemistry analyzer instrument; MANDARY BS-480, which supplied with a set of reagents kits for all tested biochemical parameters. The reaction spontaneously started when mixture (sample serum and reagent) temperature becomes 37°C and last for 5 -10 minutes, then the data was registered. All biochemical parameters were screened with use of MANDARY BS-480 except Na and K were investigated with American digital EazyLyte Na/ K analyzer instrument packed with solution kit.

2.16.5 Histological profile In the eighth day the anaesthetized rats were sacrificed and the vital organs; liver, kidney, and uterus were isolated and size minimized with use of blades and kept in 10% Formalin in separate well labeled bottles. All isolated organs were sectioned at 5μ and stained in Hematoxylin and Eosin (H&E) stain for histo-microscopic investigation. Histopathology screening was done at the Department of Histopathology, Veterinary Research Center, Soba, Khartoum, Sudan.

2.16.5.1 Histo-screening steps A piece of thickness of 3mm was cut of each of the isolated organs and kept in 10%formalin. Each piece was located in a labeled cassette, then the cassettes covers replaced on.

1. The tissues were cassetted to be harder via processing through the Microwave Histo- Processor System (MWHPS). 2. All cassettes were fixed in a stand and the stand was placed in a beaker and covered with ethanol 100% and then the system was adjusted to start the dehydration cycle. 3. After completion of the dehydration cycle, the beaker brought out the system, the ethanol was removed out and replaced with isopropanolol, the beaker with the cassettes stand returned back and then the system was adjusted to start the clearance and dehydration enhancing cycle.

4. After step 4 finished all cassettes were incubated in oven for 2 minutes at 37°C in order to bring to dryness, and the all tissues were taken for impregnation stage. 5. All tissues were located besides in a stainless steel boat and labeling was done outside the boat, the melted wax (58-60c) was poured in the boat and left until solidified , then the solidified wax attaching the tissues was cut into blocks, block for each tissue, which then reduced in size by section processing. 6. Trimming was done for each block to reduce size to 5µm by use of Microtom apparatus, then in order to relax the tissue wax sample, the waxed tissue was located in an electric tissue float (contain warm water with a temperature below that used in dissolving the wax) for seconds, then the waxed tissue film was picked up onto a pre labelled frosted slide which previously wiped with xylene, to be ready for staining. 7. Each slide was passed into three consequent containers contain xylene to remove any attached traces, then immersed in Hematoxyline stain for 5 minutes, washed up with tap water for 10 minutes, immersed again in an Eosin stain for 5 minutes, washed up with water, then each slide was dehydrated with subsequent immersion in three containers contain three different concentration of ethanol 70%. 95% and 100%. 8. In order to enhance the dehydration process, each slide was immersed again in 100% ethanol, then picked up. 9. One drop of previously prepared DPEX was located onto inside the cover and then the cover was replaced on the tissue sample slide. All slides were checked and read with use of light Microscope under X10 magnification.

2.17 Statistical Analysis

Statistical examination was performed utilizing Statistical Package for Social Sciences program (SPSS) version 20; analysis of variance (ANOVA) followed by the low significance differences (LSD) Post Hoc test. Data were expressed as mean ± SD. The P values more than 0.05, less than ≤0.05 and ≤ less than 0.01 were considered as not significant, significant and highly significant values respectively.

CHAPTER THREE RESULTS

3.1 Antimicrobial activity of tested plants against standard and clinical microorganisms

3.1.1 In vitro Antimicrobial activity of tested plants against standard and clinical microrganisms

3.1.1.1 Antimicrobial activity of Myrtus communis L. against standard microorganisms

Both Myrtus communis leaves and bark showed higher inhibition zones with Gram positive tested bacteria comparing with Gram negative tested bacteria. The largest inhibition zone was shown with Bacillus subtilis as 57mm with chloroform leaves extract followed by 53mm zone of inhibition with methanol leaves extract. The chloroform and methanol bark extracts showed lower activity comparing with the same extracts from the plant leaves, they gave 18mm and 21mm zones of inhibition for chloroform and methanol extracts respectively. Staphylococcus aureusshowed higher susceptibility towards the leaves part than bark part. The methanol leaves extract was better in its activity against Staphylococcus aureus than chloroforn leaves extract, they gave 46mm and 41mm zone of inhibition respectively (Table 1), otherwise the leaves ethanol extract was more active against Staphylococcus aureus than Bacillus subtilis,

98 it gave 32mm and 18mm zone of inhibition respectively. Table(1) showed that the leaves chloroform extract and the bark methanol extract have the same activity against Escherichia coli, both of them gave 21mm inhibition zone size while the leaves methanol extract was showed slight more activity than the leaves chloroform extract and the bark methanol extract, it gave 22mm zone of inhibition. The highest zone noticed with the ethanol leaves extract against Pseudomonas aeruginosa as 28mm, the leaves chloroform extract, the chloroform bark extract and methanol leaves extract gave inhibition zones of 19mm, 18mm and 15mm respectively. Bark methanol extract showed activity of 14mm inhibition zone against the bacteria while ethanol bark extracts and aqueous extracts of both parts showed absence activity. It was clear that all leaves organic extracts of Myrtus communis were more active against Candida albicans than bark extracts. On the other hand the aqueous extracts of all parts of the plant showed absence of anti-candida effect (Table 1).

3.1.1.2 Antimicrobial activity of Pistacia lentiscus against standard microorganisms

Both methanol and ethanol fruits extract showed antimicrobial activities against all tested microorganisms as shown in Table (2). The largest inhibition zone was shown with Staphylococcus aureus (24 mm) with methanol leaves extract and chloroform and methanol bark extracts, followed by 22mm inhibition zone with methanol fruits extract. On the other hand Bacillus subtilis showed the largest inhibition zone as 19mm with methanol fruit extract followed by 14mm zone inhibition with fruits ethanol extract, other solvents part extracts showed inhibition zones less than 14mm (Table2). The same table showed that the Gram negative bacteria E.coli and Pseudomonas aeruginosa showed low sensitivities. It was clear that all Pistacia lentiscus extracts had no activity against Pseudomonas aeruginosa except methanol and ethanol extracts of both leaves and fruits which gave 17mm, 16mm and 15mm respectively. Table (2) also showed that only methanol extract of both leaves and bark and ethanol fruits extract showed active inhibition zones of 15mm, 15mm and 14mm respectively against Candida albicans. The aqueous extracts of all plant parts showed absence of any antimicrobial activities as shown in table (2). The Post Hoc statistical analysis showed highly significant differences between effects of different solvents extracts from different plants parts against different organisms (Table 2).

3.1.1.3 Antimicrobial activity of Artmesia herba albo Asso against standard microorganisms

The highest inhibition zone against tested organism was shown against Bacillus subtilis with chloroform bark extract as 21mm followed by 20mm inhibition zone with ethanol bark and methanol leaves extracts. Staphylococcus aureus showed the highest inhibiton zone against methanol bark extact as 17mm. The majority of the plant extracts except aqueous extract revealed good antimicrobial activity against Bacillus subtilis and also except aqueous extracts, bark methanol extract showed highest antimicrobial activity against Staphylococcus aureus (Table 3). Table (1): Antimicrobial activity and Yields percentages of Myrtus communis L. extracts against standard microorganisms.

Family/ Plant Solvent Yield Standard organisms / Means of Diameters of Botanical / part extract % Inhibition Zones (mm) used 100mg/ml Gram positive Gram negative Fungus Vernamlar B.subtilis S.aureus E.coli Ps.aeruginosa C.albicans name Myrtaceae Bark Chloroform b c c d a 41.0 21.00 19.0 12.0 2.2 57.25 ± 0.71 ± 1.4 ± 2.1 ± 0.71 Myrtus ± 1.06 communis Methanol 18 53.0a 46.00a 22.0b 15.00b 17.01b L. ± 9.2 ± 8.5 ± 0.71 ± 4.2 ± 0.01 Ethanol 0.4 a d ab c Local 18.00bc 32.00 10.00 28.0 15.0 Name ± 2.8 ± 2.8 ± 7.1 ±3.5 ± 2.1 (Merseen) Water 1.6 0.00 0.00 0.00 0.00 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Leaves Chloroform 0.08 18.0b 23.0a 0.00c 18.00b 0.00 ± 0.71 ± 0.71 ± 0.00 ± 3.83 ± 0.00 Methanol 4 21 22 21 14 10 Ethanol 0.03 0.00 0.00 0.00 0.00 0.00 Water 0.4 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 )

Key:

** = Highly significant

Means in same row with same color superscript letter are not significantly different

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P values ≥0.05, less than ≤0.05 and ≤ less than 0.01 were considered as not significant, significant and highly significant values respectively.

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively. (Muktar and Ghori, 2012).

Table (2): Antimicrobial activity and Yields percentages of Pistacia lentiscus extracts against standard microorganisms.

Methanol 0.00c 24.0a 16b 0.00c 15.0b 10 ± 0.00 ± 1.4 ± 1.4 ± 0.00 ± 0.71 b a c c c Ethanol 11.0 17.00 0.0 0.00 0.00 0.5 ± 0.71 ± 1.4 ±0.0 ± 0.00 ± 0.00

Water 0.00a 0.00a 0.0a 0.00a 0.00a Anacardiacea 2.1 ± 0.00 ± 0.00 ± 0.0 ± 0.00 ± 0.00 Pistacia Fruits Chloroform b b b b a lentiscus 0.00 0.00 0.0 0.00 10.0 1.6 ± 0.00 ± 0.00 ±0.00 ± 0.00 ± 0.71 Methanol 19.0a 22.0a 15b 17.0ab 8.0c 19 ± 1.4 ± 2.1 ± .01 ± 1.4 ± 1.1 Ethanol 14.0b 17.00a 15b 15.0ab 14.0b Local Name 1.2 ± 0.71 ± 1.4 ±.71 ± 0.71 ± 0.71 (Battoum) Water 0.00a 0.00a 0.0a 0.00a 0.00a 0.4 ± 0.00 ± 0.00 ± 0.0 ± 0.00 ± 0.00 Leaves Chloroform 0.00a 0.00a 0.0a 0.00a 0.00a 4.4 ± 0.00 ± 0.00 ± 0.0 ± 0.00 ± 0.00 Methanol 0.00d 24.0a 10c 0.00d 15.0b 26

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± 0.00 ± 1.4 ± .01 ± 0.00 ± 0.71 Ethanol 12.0b 13.0b 13b 16.0a 12.00b 3.2 ± 0.01 ± 1.4 ± .01 ± 0.71 ± 0.01 Water 0.00a 0.00a 0.0a 0.00a 0.00a 6 ± 0.00 ± 0.00 ±0.0 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

P values ≥0.05, less than ≤0.05 and ≤ less than 0.01 were considered as not significant, significant and highly significant values respectively.

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

Table (3): Antimicrobial activity and Yields percentages of Artmesia herba albo Asso extracts against standard microorganisms.

Family/ Plant Solvent Yield Standard organisms / Means of Diameters of Botanical / part extract % Inhibition Zones (mm) Vernamlar used 100mg/ml Gram positive Gram negative Fungus name B.subtilis S.aureus E.coli Ps.aeruginosa C.albicans Asteraceae Bark Chloroform 21.0a 0.00c 14.0b 0.00c 14.0b 1 ± 1.4 ± 0.00 ± 0.71 ± 0.00 ± 0.71 Artmesia herba albo ab a c bc c Asso Methanol 15.0 17.0 11.0 14.0 13.0 5 ± 0.71 ± 0.71 ± 1.4 ± 0.71 ± 1.4 Local Name Ethanol 20a 0.00c 12b 0.00c 0.00c (Sheeh) 0.6 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 2.1

Water 0.00a 0.00a 0.00a 0.00a 0.00a 0.8 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Leaves Chloroform 18.00a 14.0b 0.00c 17.00a 15.0b 26 ± 1.4 ± 0.71 ± 0.00 ± 1.4 ± 0.71 Methanol 20.0a 0.00d 14b 7.00c 15.0b 16 ± 0.71 ±0.00 ± 0.71 ± 0.71 ± 1.4 Ethanol 0.00c 13.0b 15.0a 15.00a 15.0a 1.5 ± 0.00 ± 0.71 ± 0.71 ± 0.71 ± 0.71 Water 0.00a 0.00a 0.00a 0.00a 0.00a

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2.8 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Sig ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

P values ≥0.05, less than ≤0.05 and ≤ less than 0.01 were considered as not significant, significant and highly significant values respectively.

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

In table (3), Gram negative tested bacteria showed varied susceptibility. ethanol leaves extract gave equal activity against E.coli and Pseudomonas aeruginosa, which were more susceptible, compared to Gram positive bacteria. The largest inhibition zone with Candida albicans was shown with all leaves extract. However, with exception of all aqueous extracts and ethanol bark extract, all other extracts revealed a clear anti-Cadida albicans effect. The Post Hoc statistical analysis showed highly significant differences between effects of different solvents extracts from different plants parts against different organism.

3.1.1.4 Antimicrobial activity of Capparis spinosa against standard microorganisms

The chloroform leaves extract of Capparisspinosa showed absence of antibacterial activity against Gram positive tested organisms, otherwise Staphylococcus aureus showed the largest inhibition zone with the chloroform bark extract as (30mm). On the other hand ethanol bark extract was the best one against Bacillus subtilis, it gave inhibition zone of 16mm (Table 4). The largest inhibition zone of methanol leaves extract against all tested microorganisms was shown with Staphylococcus aureus as 20mm followed by Candida albicans 16mm and also Methanol bark extract revealed the same inhibition zone 16mm with Candida albicans. With the exception of the ethanol leaves extract, Pseudomonas aeruginosa was sensitive to all the other

103 plant extracts (Table 4). Candida albicans showed good sensitivity towards ethanol bark extract as Pseudomonas aeruginosa and also showed closed susceptibility as both Gram negative bacteria (16mm) with methanol bark extract. It was clear also from Table (4) that ethanol bark extract was the most effective one against Candida albicans and Pseudomonas aeruginosa. All aqueous extracts revealed no activity against tested organisms. In Table (4), The Post Hoc statistical interpretation showed highly significant differences between effects of different solvents extracts from different plants parts against different organisms (Table 4).

3.1.1.5 Antimicrobial activity of Juniperus phoenicea L.against standard microorganisms

Bacillus subtilis showed the best sensitivity toward most of the plant extracts comparing with the other tested organisms, the susceptibility zones ranged between 16-22mm (Table 5).

Table (4): Antimicrobial activity and Yields percentages of Capparis spinosa extracts against standard microorganisms.

Family/ Plant Solvent Yiel Standard organisms / Means of Diameters of Botanical / part extract d % Inhibition Zones (mm) Vernamlar used 100mg/ml Gram positive Gram negative Fungus name B.subtilis S.aureus E.coli Ps.aeruginosa C.albicans Capparaceae Bark Chloroform 0.0d 30a 0.0d 12.0b 10.0c 1.3 ± 0.00 ± 0.01 ± 0.00 ± 0.71 ± 0.01 Capparis Methanol 14.0b 12.0c 16a 14.0b 16.0a spinosa 4 subsp ± 0.71 ± 0.71 ± 0.71 ± 0.71 ± 0.71 oreintalis Ethanol 16.0ab 12.0c 0.00d 15.0b 18.0a (Duh.) Jafri 0.3 ± 1.4 ± 0.01 ± 0.00 ± 0.71 ± 1.4 Water 0.00a 0.00a 0.00a 0.00a 0.00a Local Name 0.1 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 (Kappar) Leav Chloroform 0.00c 0.00c 10.0b 13.0a 0.00c es 3.5 ± 0.00 ± 0.00 ± 0.00 ± 1.4 ± 0.00 Methanol 11.0d 20.0a 13.0c 13.0c 16.0b 15 ± 0.71 ± 0.01 ± 0.71 ± 0.71 ± 0.71 Ethanol 0.00c 14.0a 11.0b 0.00c 15.0a 5 ± 0.00 ± 0.01 ± 1.4 ± 0.00 ± 0.71 Water 0.00a 0.00a 0.00a 0.00a 0.00a 3.6 ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

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P values ≥0.05, less than ≤0.05 and ≤ less than 0.01 were considered as not significant, significant and highly significant values respectively.

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

Table (5): Antimicrobial activity and Yields percentages of Juniperus phoenicea L. extracts against standard microorganisms.

Methanol 6 22.0a 10.0b 12b 11.0b 12.0b Juniperus phoenicea L. ± 0.01 ± 0.01 ± 1.4 ± 1.4 ± 1.4 a b b b b Ethanol 0.1 17.0 0.00 0.0 0.00 0.00 Local Name ± 1.4 ± 0.00 ± 0.0 ± 0.00 ± 0.00 (Araar) Water 1.4 0.00a 0.00a 0.0a 0.00a 0.00a ± 0.00 ± 0.00 ±0.00 ± 0.00 ± 0.00 Chloroform 2 20.1a 0.00d 13bc 12.0c 14.0b Fruits ± 0.01 ± 0.00 ±0.71 ± 0.71 ± 0.71 Methanol 19 21.0a 17.0b 12.1c 12.0c 15.0b ± 1.4 ± 0.71 ±0.01 ± 0.01 ± 0.01 1.2 20.0a 0.00c 0.0c 0.00c 11.0b Ethanol ± 0.01 ± 0.00 ± 0.0 ± 0.00 ± 1.4 2.6 0.00a 19.0a 0.0a 0.0a 0.00a Water ± 0.00 ± 0.00 ±0.00 ± 0.00 ± 0.00 Leave Chloroform 8 19.0a 0.00d 14b 10a 0.00d

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s ± 1.4 ± 0.00 ± .01 ± 0.71 ± 0.00 Methanol 16 22a 14b 12c 0.00d 0.00d ± 0.71 ± 0.71 ± .01 ± 0.00 ± 0.00

c c c Ethanol 1 16a 0.00 0.00 0.00 14b ± 0.01 ± 0.00 ± 0.0 ± 0.00 ± 0.71 b Water 0.01 7.00 0.00a 0.0a 0.00a 0.00a ± 1.7 ± 0.00 ± 0.0 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

The aqueous extract of the plant showed inhibition zones of 7mm with leaves extract while aqueous extracts of both bark and fruits showed absence of any antimicrobial activity against the same bacteria. Surperising the aqueous extract of Juniperus phoenica showed highest inhibition zone with Staphylococcus aureus as 19mm, methanol extracts of the plant fruits, leaves and bark extract showed inhibition zone against Staphylococcus aureus as 17mm, 14mm and 10mm respectively. Otherwise, all remaining extracts showed completely absence of any activity against Staphylococcus aureus.

The largest inhibition zone developed with E.coli was 14mm with chloroform leaves extract while Pseudomonas aeruginosa showed largest inhibition zone as 12mm with both chloroform fruits extract and methanol fruits extract. Both Gram negative bacteria were not affected by the aqueous extract of all used parts.

Candida albicans was not affected by the aqueous extracts of all parts of the plant, but it affected by methanol fruits extract (15mm), chloroform fruits extract (14mm), ethanol leaves extract (14mm), methanol bark extract (12mm) and ethanol fruit extract (11mm) as shown in Table (5). Post Hoc statistical interpretation showed highly significant differences between effects of different solvents extracts from different plants parts against different organisms (Table 5).

3.1.1.6 Antimicrobial activity of Marrubium vulgariL. against standard microorganisms

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The largest inhibition zone against all tested microorganism was obtained with Bacillus subtilis as 23mm with chloroform bark extract despite that the same bacteria showed weak inhibition zones with chloroform flowers and methanol leaves extracts as 10mm and 9mm respectively, other extracts revealed absence of any antibacterial activities against Bacillus subtilis (Table 6). The chloroform bark extract of Marrubium vulagari showed inhibition zone of 20mm with Staphylococcus aureus. Except of the chloroform leaves extracts and of aqueous extracts of all parts, the other solvents extracts showed inhibition zones ranged between 10- 16mm against Staphylococcus aureus. Concerning the Gram negative tested bacteria the pattern of the antibacterial activity of the plant extracts was not in the same manner, some gave inhibition zone with Ecsherichia coli and showed no activity against pseudomonas aeruginosa,

Table (6): Antimicrobial activity and Yields percentages of Marrubium vulgari L. extractsagainst standard microorganisms.

± 0.00 ± 0.00 ± 0.01 ± 0.71 ± 1.4 Marrubium a b a a c vulgari L. Ethanol 0.1 13.0 14.0 21.0 15.0 9.0 ± 1.4 ± 2.8 ± 1.4 ± 0.71 ± 0.71 Water 0.8 0.00a 0.00a 0.00a 0.00a 0.00a Local ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Name c a d c b Flowers Chloroform 5 10.0 15.0 0.00 10.0 12.0

± 0.01 ± 1.4 ± 0.00 ± 0.71 ± 0.01 (Roubia) Methanol 5 0.00b 10.0a 0.00b 0.00b 0.00b ± 0.00 ± 0.71 ± 0.00 ± 0.00 ± 0.00 Ethanol 0.2 0.00c 16.0ab 19.0a 13.0b 14.0ab ± 0.00 ± 1.4 ± 1.4 ± 2.8 ± 2.8 Water 0.01 0.00a 0.00a 0.00a 0.00a 0.00a ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Leaves Chloroform 4 0.00b 0.00b 16 0.00b 0.00b ± 0.00 ± 0.00 ± 0.00 ± 0.00 Methanol 8 9.0a 10.0a 0.00b 0.00b 0.00b ± 1.4 ± 0.71 ± 0.00 ± 0.00 ± 0.00 Ethanol 0.1 0.00d 15.0b 16.0ab 18.0a 14.0b

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± 0.00 ± 0.71 ± 0.71 ± 0.71 ± 1.4 Water 2.6 0.00a 0.00a 0.00a 0.00a 0.00a ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

others were not active againstEscherichia coli but showed clear activity against Pseudomonas aeruginosa. As a conclusion the largest inhibition zone was obtained with the ethanol flowers part against Escherichia coli; 19mm while the largest inhibition zone showed against Pseudomonas aeruginosa; 18mm with the ethanol leaves extract (Table 6).

Candida albicana showed the largest inhibition zone with bark chloroform extract as 15mm, followed with 14mm zones of inhibition revealed from ethanol extracts of the leaves and flowers, other extracts gave inhibition zones range between 0-13mm. Also this study showed that the aqueous extract had not any activity against all tested microorganisms. The statistics analysis showed high significant differences between mean of inhibition zones revealed from different plant parts extracts from different solvents against different organisms (Table 6).

3.1.1.7 Antimicrobial activity of Rosmarinus officalis L. against standard microorganisms

All leaves and bark extracts gave inhibition zones against Bacillus subtilis ranging between 13-17mm except the aqueous extracts (Table 7). On the other hand Staphylococcus aureus showed the largest inhibition zone with leaves methanol extract as 20mm, the chloroform bark and leaves extracts and the ethanol bark extract and methanol bark extracts gave inhibition zones ranged fom 14mm to 16mm (Table 7). Escherichia coli was sensitive towards only ethanol bark extract and methanol leaves extract which exhibited inhibition zones of 15mm and

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12mm respectively. On the other hand Pseudomonas aeruginosa was the most microorganisms affected by the ethanol bark extract which was susceptible with 20mm inhibition zone. Candida albicans showed inhibition zone of 15mm with both leaves methanol extract and bark ethanol extract, otherwise inhibition zones of 13mm and 12mm were recorded with ethanol leaves extract and methanol bark extract respectively. The aqueous extracts of both leaves and bark were devoid from any antimicrobial activities, and this study cleared that there was high significant differences in effects of different plant parts extracted by different solvents against different tested standard strains (Table 7).

Table (7): Antimicrobial activity and Yields percentages of Rosmarinus officinalis L. extracts against standard microorganisms.

Family/ Plant Solvent Yiel Standard organisms / Means of Diameters of Botanical / part extract d % Inhibition Zones (mm) Vernamlar used 100mg/ml Gram positive Gram negative Fungus name B.subtili S.aureu E.coli Ps.aerugin C.albican s s osa s Lamiaceae Bark Chloroform 2 16.0a 16.0a 0.0b 0.00b 0.00b ± 0.71 ± 0.71 ± 0.00 ± 0.00 ± 0.00 Rosmarinu Methanol 5 13.00ab 14.00a 0.0d 7.0c 12.00b s officinalis ± 0.71 ± 0.71 ± 0.00 ± 0.71 ± 0.71 L. b b b a b Ethanol 0.8 15.0 15.0 15 20.0 15.0 Local ± 0.01 ± 0.71 ± 0.01 ± 0.01 ± 0.02 Name Water 15 17.0a 15.0b 0.0c 0.00c 0.00c ± 1.4 ± 0.71 ± 0.00 ± 0.00 ± 0.00 (Ekleel a a b b b Leav Chlorofor 15 17.0 15.0 0.0 0.00 0.00 Aljabal) es m ± 1.4 ± 1.4 ± 0.00 ± 0.00 ± 0.00 Methanol 13 15.0b 20.0a 12c 16.0b 15.0b ± 0.71 ± 0.71 ± 0.01 ± 0.71 ± 1.4 Ethanol 4 15.00a 0.00d 0.0d 10.0c 13.00b ± 0.71 ± 0.00 ± 0.00 ± 0.71 ± 0.71 Water 2.4 0.00a 0.00a 0.00a 0.00a 0.00a ± 0.00 ± 0.00 ± 0.0 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

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Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

3.1.1.8 Antimicrobial activity of Salvia fruticosa Mill against standard microorganisms

All leaves and bark extracts were more active against Bacillus subtilis than Staphylococcus aureus, the largest inhibition zone was recorded as 21mm with chloroform leaves extract (Table 8). In general it seems that this plant is less effective against Gram negative bacteria. The largest zone of inhibition against E.coli was 15mm which obtained by methanol bark extract and the smallest one was 10mm which obtained by both chloroform and ethanol bark extract. Pseudomonase aeruginosa showed the highest inhibition zone as 16mm with methanol bark extract and the lowest inhibition zone as 2mm with the aqueous bark extract (Table 8). Candida albicans showed inhibition zone of 17mm with bark methanol extract, otherwise leaves ethanol extract, chloroform leaves extract and ethanol bark extract showed inhibition zones of 16mm, 14mm and 13mm respectively. The Post Hoc statistical interpretation showed highly significant differences between effects of different solvents extracts from different plants parts against different organisms (Table 8).

3.1.1.9 Antimicrobial activity of Citrus aurantium L. against standard microorganisms

The ethanol leaves extract was the most active one against tested organisms than other extracts. It gave inhibition zone of 21mm, 19mm, 17mm, 12mm and 11mm against Staohylococcus aureus, E.coli, Pseudomonas aeruginosa, Candida albicans and Bacillus subtilis

110 respectively (Table 9). The aqueous extract showed weak activity against Pseudomonas aeruginosa and Staphylococcus aureus as 9mm and 5mm respectively. The chloroform extract of the leaves showed good Antibacterial activity against only Bacillus subtilis where it gave 15mm zone of inhibition while methanol leaves extract no antimicrobial activity against all tested microorganisms. High significant differences were seen in effects of plant different extract on the different tested organisms.

methanol leaves extract showed no antimicrobial activity against all tested microorganisms. High significant differences were seen in effects of plant different extract on the different tested organisms.

Table (8): Antimicrobial activity and Yields percentages of Salvia fruticosa Mil extracts against standard microorganisms.

Family/ Plant Solvent Yield Standard organisms / Means of Diameters of Botanical / part extract % Inhibition Zones (mm) Vernamlar used 100mg/ml Gram positive Gram negative Fungus name B.subtilis S.aureus E.coli Ps.aeruginosa C.albicans Lamiaceae Bark Chloroform 3 20.0a 0.00c 10.0b 0.00c 0.00c ± 0.01 ± 0.00 ± 0.01 ± 0.00 ± 0.00 Salvia Methanol 6.5 20.0a 16.0b 15.0 16.0 b 17.0 b fruticosa Mil ± 0.71 b ± 0.71 ± 2.1 ± 0.01 Local Name ± 0.01 Ethanol 1.6 12.0ab .0.00d 10.0b 11.0b 13.0a (Toffah ± 0.71 ± 0.00 ± 0.71 ± 0.71 ± 0.71 alshahi) Water 2.5 0.00a 0.00a 0.00a 2.00a 0.00a

± 0.00 ± 0.00 ± 0.00 ± 0.01 ± 0.00

Leaves Chloroform 15 21.0a 15.0b 12.0c 0.00d 14.0b ± 0.71 ± 0.71 ± 0.71 ± 0.00 ± 0.71 Methanol 7.6 14.0a 11.0b 0.00d 8.0c 0.00d ± 0.71 ± 0.71 ± 0.71 ± 0.71 ± 0.71 Ethanol 1.7 12.0b 0.00c 11.0b 13b 16.0a ± 0.71 ± 0.00 ± 0.71 ± 0.71 ± 1.4 Water 6 0.00a 0.00a 0.00a 0.00a 0.00a ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Sig. ** (P value ≤ 0.01 )

111

** = Highly significant

Means in same row with same color superscript letter are not significantly different

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

Table (9): Antimicrobial activity and Yields percentages of Citrus aurantiumL extracts against standard microorganisms.

Family/ Plant Solvent Yield Standard organisms / Means of Diameters of Botanical / part extract % Inhibition Zones (mm) Vernamlar used 100mg/ml Gram positive Gram negative Fungus name B.subtilis S.aureus E.coli Ps.aeruginosa C.albicans Rutaceae Chloroform 1.2 15.0a 0.00b 0.00b 0.00b 0.00b ± 1.4 ± 0.00 ± 0.00 ± 0.00 ± 0.00 Citrus Methanol 3.3 0.00a 0.00a 0.00a 0.00a 0.00a aurantium L. Leaves ± 0.00 ± 0.00 ± 0.00 ± 0.00 ± 0.00 a a a a a Ethanol 0.7 11.0 21.0 19.0 17.0 12.0 Local Name ± 9.8 ± 0.71 ± 1.4 ± 2.1 ± 1.4 Bortoukal b a shifshi Water 3.4 0.00c 5 0.00c 9.0 0.00c

± 0.00 0.71 ± 0.00 ± 1.4 ± 0.00

Sig. ** (P value ≤ 0.01 ) ** = Highly significant

Means in same row with same color superscript letter are not significantly different

Inhibition zones of (< 9mm), (9 -12), (13 -18) and (≥ 18mm) were considered as no activity, moderate activity, active, very active ( pronouncing), respectively.(Muktar and Ghori, 2012).

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3.1.1.10 Antifungal activity of tested extract against standard Aspergillus niger

In this study the antifungal activities of methanol extracts of Pistacia lentiscus fruits, Capparis spinosa leaves, Salvia fruticosa bark and Myrtus communis leaves against standard Aspergillus niger ATCC 9763 were screened. The results showed that only Pistacis lentiscus extract was actively inhibited the fungus growth with inhibition zone of 15mm. Other tested extracts showed moderate activity and revealed equal inhibition zones of 11mm against Aspergillus niger ATCC 9763.

3.1.1.11 Minimum inhibitory concentration

This study showed that the methanol extracts of Myrtus communis, Pistacia lentiscus fruits and Salvia fruticose bark showed the highest broad spectrum antibacterial activity and both methanol and chloroform extracts of leaves and bark of Capparis spinosa showed selective anti Staphylcoccus aureus activity. Accordingly, the minimum inhibitory concentrations of these extracts were evaluated against tested standard microorganisms.

3.1.1.11.1 Minimum inhibitory concentration of Pistacia lentiscus methanol fruits extract against standard organisms

This study showed that the minimum inhibitory concentration of the methanol fruits extract of Pistacia lentiscus was 6.25mg/ml against C.albicans followed by 12.5mg/ml against

113

Bacillus subtilis. Escherichia coli and Pseudomonas aeruginosa was inhibited with minimum inhibitory concentration of 25mg/ml, otherwise growth of Staphylococcus aureus and Aspergillus niger were arrested by minimum inhibitory concentration of 50mg/ml of Pistacis lentiscus (Table10).

3.1.1.11.2 Minimum inhibitory concentration of Capparis spinosa methanol leaves and chloroform bark extracts against standard organisms

The minimum inhibitory concentration revealed in the present study from the leaves of Capparis spinosa was 50mg/ml from methanol leaves extract against each Bacillus subtilis, Escherichia coli and Candida albicans and was 100mg/ml against Staphylococcus aureus and Pseudomonas aeruginosa and Aspergillus niger. Also Staphylococcus aureus inhibited with 100mg/ml MIC of the plant bark chloroform extract.(Table 11).

Table (10) Minimum inhibitory concentration of Pistacia lentiscus methanol fruits extract against standard organisms

Plant extract Standard microorganisms concentration mg/ml B.subtilis S.aureus E.coli Ps.aeruginosa A.niger C.albicans 100 ------50 ------25 - + - - + - 12.5 - + + + + - 6.25 + + + + + - 3.125 + + + + + + 1.56 + + + + + +

( - ) = No growth (+) = Growth

Table (11) Minimum inhibitory concentration of Capparis spiosa methanol leaves and chloroform bark extracts against standard organisms

114

Plant extract Standard microorganisms MIC of concentration chloroform bark extract mg/ml B.subtilis S.aureus E.coli Ps.aeruginosa A.niger C.albicans S.aureus 100 ------50 - + - + + - + 25 + + + + + + + 12.5 + + + + + + + 6.25 + + + + + + + 3.125 + + + + + + + 1.56 + + + + + + +

( - ) = No growth (+) = Growth

3.1.1.11.3 Minimum inhibitory concentration of Salvia fruticosa methanol bark extract against standard organisms

Methanol bark extract of S.fruticosa in this study inhibited the growth of both tested Gram positives and Escherichia coli with MIC of 50mg/ml and revealed MIC of 25mg/ml against Pseudomonas aeruginosa and Candida albicans and 100mg/ml against Aspergillus niger (Table 12).

3.1.1.11.4 Minimum inhibitory concentration of Myrtus communis methanol leaves extract against standard organisms

The minimum inhibitory concentration revealed from methanol extract of leaves of Myrtus communis was 3.125mg/ml against Pseudomonas aeruginosa followed by 6.25mglml against Staphylococcus aureus and Candida albicans, while Bacillus subtilis and Escherichia coli were minimally inhibited with concentration of 50mg/ml of the extract. Aspergillus niger was inhibited with minimum inhibitory concentration of 100mg/ml of Myrtus communis tested extract (Table13).

3.1.1.12 Identification of clinical bacteria

The results of BD Phoenix™ automated identification and susceptibility testing system showed that the tested bacterial isolates were belonged to seven different genera; Staphylococcus

115 aureus, Pseudomonas aeruginosa, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Acinetobacter baumanii and Proteus mirabilis. Also the manual basic biochemical reactions confirmed these results (see appendex). According to the susceptibility test to Oxacilline, the results showed that all identified Staphylococcus aureus bacteria were methicillin resistant Staphylococcus aureus (MRSA), since they resist Oxacillin (bet-lactam antibiotic of the penicillin class).

Table (12) Minimum inhibitory concentration of Salvia fruticosa methanol bark extract against standard organisms

Plant extract Standard microorganisms concentration mg/ml B.subtilis S.aureus E.coli Ps.aeruginosa A.niger C.albicans 100 ------50 - - - - + - 25 + + + - + - 12.5 + + + + + - 6.25 + + + + + + 3.125 + + + + + + 1.56 + + + + + +

( - ) = No growth (+) = Growth

Table (13) Minimum inhibitory concentration of Myrtus communis methanol leaves extract against standard organisms

Plant extract Standard microorganisms concentrations mg/ml B.subtilis S.aureus E.coli Ps.aeruginosa A.niger C.albicans 100 ------50 - - - - + -

116

25 + - + - + - 12.5 + - + - + - 6.25 + - + - + - 3.125 + + + - + + 1.56 + + + + + +

( - ) = No growth (+) = Growth

3.1.1.13 Antibacterial activity of promising antibacterial plants extracts against clinical isolates compared with antibiotic references

3.1.1.13.1 Antibacterial activity of Myrtus communis methanol leaves extracts against clinical isolates

Staphylococcus aureus, Proteus mirabilis, Acinetobacter baumanii, Pseudomonas aeruginosa, Escherichiacoli, Klebseilla pneumoniae and Enterobacter cloacae showed susceptibilities with inhibition zones of 25.0 ± 6, 24.5±5, 22.85±5, 20.6± 3, 19.87±1.4, 19.1±7 and 17.56±3 respectively (Table14). Compared to Augmentin 30µg, Ceftriaxone 30µg and Vancomycin 30µg, the Myrtus extract appeared the only effective choice against all tested clinical isolates. The methanol leaves extract of Myrtus communis showed higher inhibition performance against Staphylococcus aureus (25.0mm ± 6) compared with 19.1±7 inhibition zone revealed from Vancomycin 30µg; the common drug used for Staphylococcus aureus infection treatment. Even though Myrtus extract appeared highly active but its activity was less than that offered from the reference Ciprofloxacin30µg against Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli. Also Table (14) proved that there is no significant difference between the activity of the extract and of Ciprofloxacin30µg against Klebsiella pneumoniae (revealed equal zones, 19mm), and also showed higher activity of Myrtus

117 extract than Ciprofloxacin30µg against Acinetobacter baumanii and Enterobacter cloacae. Compared with the activity of Ceftazidime30µg and Gentamicin10µg against tested bacteria, the methanol extract of Myrtus communis leaves showed the highest activity against all tested isolates (Table 14). The LSD Post Hoc analysis showed high significant differences between the effects of the studied treatment agents on tested organisms.

3.1.1.13.2 Antibacterial activity of Pistacia lentiscus methanol fruits extracts against clinical isolates

Active inhibitory performance has shown from methanol fruits extract of Pistacia lentiscus against tested clinical Acinetobacter baumanii, Escherichia coli and Staphylococcusaureus with inhibition zones of 13.90mm±7.62, 13.3mm±11.3 and 15.4mm±9, respectively. Weak activity was shown against Pseudomonas aeruginosa and Proteus mirabilis, but moderate activities were shown toward Klebsiella pneumoniae; 12.13mm±9.2 and Enterobacter clacae; 9.5±10.19mm. Compared with Augmentin30µg, Ceftriaxone30µg and Vancomycin30µg Pistacia lentiscus

Tbale 14

118

extract in this study shown to be the only effective choice against all tested clinical isolates, with the exception of Staphylococcus aureus which showed more susceptibility to the antibiotic Vancomycin30µg (Table 14).

Compared with Ceftriaxone30µg and Gentamicin10µg,the plant extract appeared to be the only effective agent against Acinetobacter baumanii, Enterobacter cloacae and Klebsiella pneumonia, but was less active against other tested isolates. Gentamicin10µg was more active than fruits extracts against Staphylococcus aureus isolates. The methanol fruits extract of Pistacia lentiscus showed a good antibacterial activity against Acinetobacter baumanii with inhibition zone of 13.9mm±7.6 comparing with Ciprofloxacin 30µg which gave inhibition size of 2.0mm±0.0, other antibiotics references showed no antibacterial activity against the tested bacteria compared with the plant extract. All tested isolates showed less susceptibility with zones ranged (4.8 -15.4mm) towards the extract compared to the zones (2.0 – 39.3mm) given by Ciprofloxacin 30µg except for Acinetobacter baumanii which was more susceptible towards the plant extract than Ciprofloxacin 30µg (Table 14).

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3.1.1.13.3 Antibacterial activity of Salvia fruticosa methanol bark extracts against clinical isolates

In general Salvia fruticosa methanol bark extract revealed active antibacterial effect against all studied clinical isolates, the largest antibacterial effect was noticed with Staphylococcus aureus, Pseudomonasaeruginosa, Escherichiacoli, Klebseilla pneumoniae, Acinetobacter baumanii, Proteus mirabilis and Enterobacter cloacae where they showed susceptibilities with inhibition zones of 19.47mm ± 4.54, 17.5mm±4, 17mm±4, 13.87mm± 1, 12.5mm±10.8, 12.4mm±1.3 and 11.5mm±11, respectively (Table 14). Compared with the activity of Augmentin30µg, Ceftazidime30µg, Gentamicin10µg, Ceftriaxone30µg and Vancomycin30µg Salvia fruticosa extract was the only effective agent against tested Acinetobacter baumanii, Enterobacter cloacae and Klebseilla pneumoniae isolates. Escherichia coli and Proteus mirabilis were more susceptible to the plant extract than Augmentin30µg, Ceftriaxone30µg and Vancomycin30µg while these tested bacteria were less susceptible to the plant extract compared with Ceftazidime30µg. All tested isolates showed less susceptibility with zones ranged (11.5 -19.47mm) towards the extract compared to the zones (2.40 – 39.3mm) given by Ciprofloxacin 30µg except for Acinetobacter baumanii which inhibited only by the extract compared with all tested antibiotics. High significant difference was between the inhibition activity of studied plant extract and antibiotics used (Table 14).

3.1.1.13.4 Antibacterial activity of Capparis spinosamethanol leaves extract against clinical isolates

Methanol leaves extract of Capparis spinosa tested against 100 clinical isolates showed no activity against all tested bacterial genera except for methicillin resistant Staphylococcus aureus which partially and actively inhibited by methanol leaves and chloroform bark extracts of Capparis spinosa with inhibiton zones of 4.6mm±7 and 13mm±11, respectively. chloroform extract of bark of Capparis spinosa appeared in this study has active antibacterial performance against methicillin resistant Staphylococcus aureus compared with methanol leaves extract and Augmentin30µg, Ceftazidime30µg and Ceftriaxone30µg antibiotics(Table 14). Even though the chloroform bark extract proved active inhibition performance against methicillin resistant

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Staphylococcus aureus but its activity was less than that given from Ciprofloxacin 30µg, Gentamicin10µg and Vancomycin30µg (Table 14).

3.1.1.14 In vivo antimicrobial and wound healing activity

In this study the results showed that all wounds which have not been infected and treated with standard antibiotic (Tetracycline 3% ointments) and wounds treated with the tested 3% Myrtus communis extract ointment were healed in six and seven days consequently (Table 15). Also the results showed that Staphylococcus aureus infected wounds treated with the extract were healed in eight days compared with the six days healing period when treated with Fucidin. Pseudomonas aeruginosa infected wounds treated with Myrtus communis extract were healed in six days, which was the same period of healing when the infected wounds treated with Tetracycline (Tables 15).

Post Hoc LSD analysis showed no significant differences (PV ≥0.05) between the non- infected non treated wound with non- infected wound treated with common used drug and the extract in the first two days of treatment but a significant difference (PV ≤0.05) has been shown in day three which by day four become highly significant difference (PV ≤0.01). However the

TABLE 15

121

TABLE 16

122

TABLE 17

123

LSD showed no significant differences in the healing capacity between non- infected wound treated with Tetracycline and non- infected wound treated with Myrtus communis extract (Table 16 and 17).

Table (16) showed a significant difference in day three between the Staphylococcus aureus infected non-treated wound and Staphylococcus aureus infected wound treated with the Fucidine. However the LSD showed no significant differences in the healing capacity in day one and day two but a highly significant difference had been shown from day three in the healing area between non treated Staphylococcus aureus infected wound and infected wound treated with either Fucindin antibiotic or the Myrtus communis extract. Despite the result showed that there was two excess healing days in Staphylococcus aureus infected wounds treated with the extract compared with the six healing days of Fucidin treated wounds, but the Post Hoc Analysis showed no significant difference between the action of both Fucidin ointment and Myrtus communis ointment (Table 16).

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Rats groups infected with Pseudomonas aeruginosa showed significant difference (Pv ≤ 0.05) in day two in healing area between infected non-treated rats and infected rats treated with Tetracycline antibiotic and also between infected non-treated rats and infected rats treated with Myrtus communis extract. However, this table proved that there were no significant differences (Pv ≥ 0.05) between infected rats treated with Tetracyclin and infected rats treated with Myrtus communis extract. Since both wounds have been completely healed in six days compared with the ten healing days of non treated Pseudomonas aeruginosa infected wound (Table 17).

3.2 Phytochemical analysis

3.2.1 Phytochemical analysis investigated with basic methods

Methanol extracts of each of Pistacia lentiscus fruits, Capparis spinosa leaves, Myrtus communis leaves and Salvia fruticosa bark in addition to the chloroform bark extract of Capparis spinosa were analyzed phytochemically. The results were expressed as either (-) negative or presence, (+) traces, (++) moderate, (+++) high and (++++) very high presence. The results showed no presence of Alkaloids, Anthraquinones and Saponins in all chloroform plants extracts. All plants methanol extracts were devoid from presence of Anthraquinones. This screening pointed to the very high presence of Tannins and Traces of Alkaloids, Flavonoids, Saponins and Triterpenes in Pistacia lentiscus fruites methanol extract, while no presence of coumarins and steroids were shown.

High occurrence of Triterpenes, moderate presence of Flavonoids and traces of each of Alkaloids, Coumarins, Steroids and Tannins were found in methanol extract of Salvia fruticosa bark. However this study showed that methanol extract of leaves of Myrtus communis possessed high level of Flavonoids and moderate levels of both Saponins and Tannins, while the methanol extract of Capparis spinosa leaves proved to possess high levels of Flavonoids and Tannins, moderate steroids level and traces of each of Alkaloids and Triterpenes (Table 18).

3.2.2 Gas Chromatography /Mass Spectra Phytochemical analysis Technique.

3.2.2.1 GC-MS anlalysis of Myrtus communis

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Thirty three compounds has have been detected in the methanol leaves extract of Myrtus communis when analyzed with GC-MS (Table 19). Flavonoids, terpenoids and fatty acid were detected. Tannins were found, Quinic acid was the predominant compound constitutes 40.02% of the total constituents followed with 1,2,3-Benzenetriol (Pyrogallol), Endo-1,5,6,7- etramethylbicy-clo(3,2,0)hep, and 5-Hydroxymethylfuefural with percentage of 17.5, 11.26 and 7.79% respectively. Phytol (cyclic diterpene) and many fatty acids as 9-Eicosyne, Linoleic acid, Oleic acid, Gamolenic acid and Octadecanoic acid were found. Also alkaloids; 0.17 % of 1,4- Methano-1H-cyclopenta(d)pyridazine and 1.87% of Pyrolidine, 1-(1-oxobutyl) were identified (Table 19).

3.2.2.2 GC-MS anlalysis of Pistacia lentiscus

In this study thirty five compound were detected in the fruits extract of Pistacia lentiscus via analysis with GC-MS (Table20). Phenols, flavonoida, tannins, terpens, alkaloids and fatty acids were detected. Among detected tannins the highest percentage was belonged to 1,2,3- Benzenetriol (Benzoic acid derivative) which constitutes 24.40% followed with Quinic acid, Benzoic acid,3-hydroxy and Catechol with percentages of 20.41, 19.51, and 4.51 respectively. Phenols and oxygenated phenols were found; 2-Cyclohexen-1-one 4.18% and 1,2- Cyclohexanedione 3,80%. Many terpenoid compounds, Benzoic acide derivatives were detected

Table 18

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Table 19 A

127

Table 19 B

128

Table 20 A

129

Table 20 B

130

in addition to –gamma sterol and the alkaloids; 1,3,5-Triazine-2,4,6-triamine, 1-(2-Hydroxy- ethyl)-1,2,4-triazole and N-Methylpyrrole-2-carboxylic acid. Linoleic acid and Oleic acid fatty acids were detected in the methanol extract of Pistacia lentiscus fruits (Table 20).

3.2.2.3 GC-MS anlalysis of Capparis spinosa

Both methanol leaves and chloroform bark extracts of Capparis spinosa were analyzed in this study with use of GC-MS. Thirty three compounds were detected in the leaves and twenty five compounds were detected in the bark. For leaves polyphenols (flavonoids, glycerol), terpens, fatty acids were detected. Three alkaloid compounds have been seen, 1-Methyl- pyrrolidinr-2- carboxylic acid was the dominant compound constitutes 41.80% of the total compounds area followed with N-Metheyl-L-prolinol with percentage of 11.2. N- Hexadecadienoic acid, Lineloic acidethyle ester 0.41%, Oleic acid0.45%, Gamolenic acid 2.06% are fatty acid detected in addition to the Octadecanoic acid 0.77% (Stearic acid) , The

131 predominant component was N- Hexadeca-dienoic acid with percentage of 6.8%. Coumarin; Benzofuran, 2,3-dihydrobenzofuran was identified with percentage of 3.94% (Table 21).

For the bark chloroform extract, Hexadecanoic acid was presented as the dominant compound with percentage of 32.4. In addition to the fatty acids; Linoleic acid ethyl ester, Oleic acid, Gamolenic acid and Octadecanoic acid were detected, Gamolenic acid was the dominant presented fatty acid with percentage of 11.6%. Cyclic di-terpens (Phytol) and gamma.-Sitosterol were found with 8.87% and 6.86% percentage, respectively (Table 22).

3.2.2.4 GC-MS anlalysis of Salvia fruticosa

The methanol bark extract of Salvia fruticosa was analyzed with GC-MS and the results showed that the extract contained thirty four compounds. It has been shown that the extract is rich of phenols, polyphenols (glycerols), tannin, terpenes, fatty acids and alkaloids. The mono oxygenated phenol; Benzaldhyde, 2-hydroxy-6-methyl (Aromatic aldehyde) were the dominant copmpounds constitute 20.91% of the total compounds area. Many fatty acids were found; Hexadecanoic acid, octadecyl ester 11.88%, Gamolenic acid 1.63%, Linoleic acid ethyl ester 1.36%, Octadecadienoic acid 0.68% and Oleic acid 0.13%. Hexadecanoic acid was the dominant

Table 21 A

132

Table 21 B

133

Table 22 A

134

Table 22 B

135

Table 23 A

136

Table 23 B

137

one. Also Butanoic, Pentanoic and Heptanoic acids derivatives were appeared within the extract constituents with percentages of 1.18, 0.52 and 3.7% respectively (Table 23).

3.3 Antioxidant activity of tested extracts

The antioxidant activities of the methanol extracts of Capparis spinosa leaves, Pistacia lentiscus fruits, Salvia fruticosa bark and Mystus communis leaves were determined by the DPPH scavenging assay. Table (24) demonstrates DPPH scavenging activity, expressed in percentage and IC50 caused by different concentrations. IC50 is the concentration of the antioxidant providing 50% inhibition of DPPH in the test solution. The activity of the extracts is proportional to the concentrations and the lower IC50 value reflects better antioxidant action. Compared with the standard Propyl gallate, this study figured out high antioxidant activities possessed from methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits, Salvia fruticosa bark and Capparis spinosa leaves. The leaves extract of Capparis spinosa and

138 bark extract of Salvia fruticosa abled to reduce the stable free radical 2.2Di (4-tert- octylphenyl)-1-picryl-hydrazyl with lower radical scavenging activity than that revealed from the standard Propyl gallate, while leaves extract of Myrtus communis and fruits extract of Pistacia lentiscus showed closed reduction activity compared to Propyl gallate (Table 24). Figure (2) showed that Pistacia lentiscus, Myrtus communis and Salvia fruticosa have better scavenging activity but Capparis spinosa showed lowest activity compared to Propyl gallate. The lowest IC50 value (highest antioxidant activity) of 0.008μg/ml was obtained from fruits of Pistacia lentiscus followed by IC50 value of 0.017μg/ml and 0.046μg/ml revealed from Myrtus communis leaves and Salvia fruticosa bark,respectively. Overall the four tested extracts Pistacia lentiscus fruits extract showed the highest antioxidant activity followed by Myrtus communis leaves, Salvia fruticosa bark and Capparis spinosa leaves, respectively.

3.4 Cytotoxicity activity (Anticancer) of tested plant extracts

Cytotoxicity (anti-proliferative) effects of the methanol extracts of the leaves extracts of Cappris spinosa and Myrtus communis and fruits extract of Pistacia lentiscus were studied and the results showed that the three tested extracts have high toxic effect on carcinoma colon cell line

(HCT-116) with IC50s of 22µg/ml, 25µg/ml and 25µg/ml, respectively (Table 25).

Table (24): Percentage and IC50 of DPPH radical scavenging activities of methanol extracts of tested plants parts

Number Plant Part %RSA ±standard IC50 µg/ml error of mean ±SD (DPPH) (DPPH) 1 Capparis spinosa leaves extract 80± 0.02 0.205 ± 0.07

2 Myrtus communis 95± 0.01 0.017 ± 0.01 leaves extract

3 Pistacia lentiscus 94± 0.01 0.008 ± 0.01 fruits extract

4 Salvia fruticosa 85± 0.01 0.046 ± 0.02 bark extract

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Standard Propyl Gallate 94± 0.01 0.077µg/ml± 0.01

RSA = Radical scavenging activity SD = Standard deviation

DPPH = 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl

Table (25): Cytotoxic effects of tested plants on colon carcinoma cell line (HCT-116)

Methanol Concentration (µg/ml)

extracts of Inhibition (%) IC50 (µg/ml) Degree of

tested plants 12.5 25 50 100 toxicity Cappris 0.625 0.483 0.392 0.333 22µg/ml High toxic spinosa Myrtus 0.558 0.500 0.333 0.308 25µg/ml High toxic communis Pistacia 0.583 0.500 0.333 0.300 25µg/ml High toxic lentiscus

IC50< 30 μg/ml= High toxic, IC50> 100 μg/ml= No toxic, IC50 30-100 μg/ml= Moderate toxic

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IC50μg/ml

0.25 0.2 µg/ml

0.2

0.15

0.1

0.046µg/ml 0.077µg/ml 0.05 0.017µg/m 0.008 µg/ml 0 Methanol Capparis Myrtus Pistacia Salvia Propyl extracts of spinosa communis lentiscus fruticosa Gallate tested plants

Figure (2): IC50 of DPPH radical scavenging activities of tested plants parts

Only Pistacia lentiscus extract showed high anti-proliferative effect on carcinoma liver cell line (HEPG2) with IC50 of 28.6µg/ml compared with the moderate anti-proliferative activity shown from Cappris spinosa and Myrtus communis extracts (Table 26). Tables (27) and (28)

141 showed that all tested extract revealed moderate cytotoxic effects on tested breast carcinoma (MCF7) and prostate carcinoma (PC3) cells lines.

3.5 Toxicity of tested plant extracts

3.5.1 Acute toxicity (Lethality) of tested plants extracts

3.5.1.1 Acute toxicity of methanol leaves extract of Capparis spinosa

No death was happened in all groups of rats up to 2g/kg of the plant extract. This result proved that the dose that kills half of the tested rats (LD50)is more than 2G/Kg. Till 2G/Kg the extract appeared as safe in use.

3.5.1.2 Acute toxicity of methanol leaves extract of Myrtus communis

All Myrtus communis extract tested rats died when treated with dose of 2g/kg while three out of six treated rats died at dose of 1G/Kg. This indicated that plant extract LD50 is 1G/Kg.

3.5.1.3 Acute toxicity of methanol fruits extract of Pistacia lentiscus

All Pistacia lentiscus extract tested rats died when treated with dose of 2G/Kg while two out of six extract treated rats died at dose of 1G/Kg. This indicated that plant extract LD50 is more than1g/kg.

3.5.2 Sub- acute toxicity of tested plants extracts

3.5.2.1 Biochemistry

3.5.2.1.1 Effects of plants extracts on liver function

Total proteins level was screened and this study cleared that methanol leaves extracts of both Capparis spinosa 1G/Kg and Myrtus communis 0.5G/Kg exerted significant decreasing effect (P v ≤ 0.05) on total proteins level with similar manner in treated rats groups compared to the control group, also 0.5G/Kg of Pistacia lentiscus methanol fruits extract decreased significantly

Table (26): Cytotoxic effects of tested plants on liver carcinoma cell line (HEPG2)

Methanol Concentration (µg/ml)

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extracts of Inhibition (%) IC50 (µg/ml) Degree of

tested plants 12.5 25 50 100 toxicity Cappris 0.737 0.594 0.519 0.456 58 µg/ml Moderate toxic spinosa Myrtus 0.725 0.587 0.531 0.463 72.5 µg/ml Moderate toxic communis Pistacia 0.688 0.519 0.394 0.331 28.6 µg/ml High toxic lentiscus

IC50< 30 μg/ml= High toxic, IC50> 100 μg/ml= No toxic, IC50 30-100 μg/ml= Moderate toxic

Table (27): Cytotoxic effects of tested plants on breast carcinoma cell line (MCF7)

Methanol Concentration (µg/ml)

extracts of Inhibition (%) IC50 (µg/ml) Degree of

tested plants 12.5 25 50 100 toxicity Cappris 0.743 0.599 0.410 0.306 39.5µg/ml Moderate toxic spinosa Myrtus 0.681 0.681 0.458 0.410 45.5µg/ml Moderate toxic communis Pistacia 0.764 0.681 0.572 0.435 75µg/ml Moderate toxic lentiscus

IC50< 30 μg/ml= High toxic, IC50> 100 μg/ml= No toxic, IC50 30-100 μg/ml= Moderate toxic

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Table (28): Cytotoxic effects of tested plants on prostate carcinoma cell line (PC3)

Methanol Concentration (µg/ml)

extracts of Inhibition (%) IC50 (µg/ml) Degree of

tested plants 12.5 25 50 100 toxicity Cappris 0.865 0.580 0.309 0.280 32.5µg/ml Moderate toxic spinosa Myrtus 0.759 0.619 0.309 0.295 34.5µg/ml Moderate toxic communis Pistacia 0.744 0.570 0.309 0.217 31.5µg/ml Moderate toxic lentiscus

IC50< 30 μg/ml= High toxic, IC50> 100 μg/ml= No toxic, IC50 30-100 μg/ml= Moderate toxic

the total proteins but with a different manner from that of Capparis spinosa and Myrtus communis extracts as illustrated by Post Hoc statistical analysis. Although this study showed decreasing effect of the methanol extracts of Myrtus communis leaves, Capparis spinosa leaves

144 and Pistacia lentiscus fruits on serum albumin with significantly differences from that of control but there was no significant differences presented in serum globulin levels between treated rats groups and control rat group; Pv ≥ 0.05 (Table 29). The increasing levels of albumin may indicate to a hepatotoxicity incidence. Table (29) showed that total and direct bilirubin levels were significantly increased in Pistacia lentiscus treated group than control group while no significant differences seen with Myrtus communis except in its increasing effect of indirect bilirubin. No effect has been shown from Capparis spinosa methanol leaves extract on total, direct and indirect bilirubin. Alkaline phosphate in this study seen significantly decreased with Pistacia lentiscus treated group compared to control group. On the other hand Pistacia lentiscus treated group revealed high significant increasing effect on the liver three bio-markers (ALT, AST and GGT) compared with the control group (Table 29).

3.5.2.1.2 Effects of plants extracts on glucose levels and lipid profile

In a same manner Myrtus communis and Pistacia lentiscus significantly decreased the levels of glucose in treated rat groups compared to control group. Although Capparis spinosa lowered the glucose levels, there was no significant differences seen compared to the control rats. Only Pistacia lentiscus methanol fruits extract affected LDLs and HDLs levels were as it was significantly increased LDL and high significantly decreased HDL compared to the control group (Table 30). Capparis spinosa increase triglcerides and LDL and decreased total cholesterol and HDL, but there were no significant differences compared the control.

3.5.2.1.3 Effects of plants extracts on Kidney function parameters

The investigation showed that Myrtus communis and Pistacia lentiscus decreased serum sodium with highly significant difference compared to the control group. Only Myrtus communis showed significant increasing to serum potassium compared to control group. On the other hand the study showed no significant difference shown from Capparis spinosa methanol leaves extract in kidney parameters levels compared to the control (Table 31).

Table (29): Liver function parameters values of control and extracts treated rats

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Means ± Standard Deviation Parameters Control Methanol leaves Methanol Methanol fruits (1m/kg) extract of leaves extract extract of Capparis of Myrtus Pistacia spinosa communis lentiscus (1g/kg) (0.5g/kg) (0.5g/kg) Total Proteins 7 ± 0.5 6 ± 0.03** a 5.9 ± 0.2** b 5.7 ± 0.4** c (g/dl) Serum Albumin 3.8 ±0.4 3 ± 0.03 a b 2.9 ± 0.1** b 2.6 ± 0.2 c b (g/dl) Serum Globulin 3.2 ± 0.3 3.0 ± 0.3 a 3 ± 0.2 a 3 ± 0.4 a (g/dl) Total Bilirubin 0.01 ± 0.02 0.01 ± 0.02 a b 0.2 ± 0.06 b 0.4 ± 0.5* c b (mg/dl) Direct Bilirubin 0.01 ± 0.02 0.01 ± 0.02 b a 0.1 ± 0.04 b 0.4 ± 0.5* b c (mg/dl) Indirect Bilirubin 0.05 ± 0.03 0.05 ± 0.01 a 0.12 ± 0.04* b 0.05 ± 0.05 a (mg/dl) Alkaline phosphate 180.8 ± 69.1 244 ± 180.7 a 246.6 ± 89.8 a 366.2 ± 128.2* a (U/L) ALT (U/L) 60 ± 15.5 56.3 ± 18.7 b 56.3 ± 18.7 b 111.5 ± 32.5** a AST (U/L) 113.7± 18.5 126.2 ± 11.6 b 112.3 ± 27.3 b 188.5 ± 48.3** a GGT (U/L) 1.7 ± 1.8 1.5 ± 1.4 a b 4.5 ± 2.7 b 6.2 ± 4.6** c b (*) = There are significant differences (Pv ≤0.05)

(**) =There are highly significant differences (Pv ≤ 0.01)

Means with same superscript letter are not significantly different

ALT = Alanine transaminase AST = Aspartate transaminase GGT = Gamma-glutamyltransferase

Table (30): Effects of plants extracts on Glucose and lipid profile parameters

Means ± Standard Deviation

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Parameters Control Methanol leaves Methanol Methanol (1m/kg) extract of leaves extract fruits extract Capparis of Myrtus of Pistacia spinosa communis lentiscus (1g/kg) (0.5g/kg) (0.5g/kg) b b Random Blood 90.5 ± 14 85.2 ± 9.8 a 67.7 ± 7.9** 60 ± 14.4** Glucose(mg/dl) b a Triglyceride (mg/dl) 72 ± 36.5 81.5 ± 37.9 b 55.7 ± 20.2 131.5 ± 50.3** Total Cholesterol (mg/dl) 47 ± 6 39 ± 10 c 43.7 ± 3.9 c b 55.5 ± 11.8 a Low density lipoproteins 6 ± 1.9 8 ± 2.9 b c 9.7 ± 3 b 15.7 ± 2.9* a (mg/dl) High density lipoproteins 37 ± 6.4 28 ± 8.4 a 22.7 ± 2.8 a 21.5 6.4** b (mg/dl)

(*) = there are significant differences (Pv ≤0.05) (**) =There is highly significant differences (Pv ≤ o.o1)

Means with same superscript letter are not significantly different

Table (31): Effects of plants extracts on Kidney parameters values compared to control

Means ± Standard Deviation Parameters Control Methanol Methanol leaves Methanol fruits (1m/kg) leaves extract extract of Myrtus extract of of Capparis communis Pistacia spinosa (0.5g/kg) lentiscus (1g/kg) (0.5g/kg) a a Serum Urea (mg/dl) 44 ± 18.1 36 ± 5.6 a 34.5 ± 6.2 30.3 ± 10.9* a a Serum Creatinine (mg/dl) 0.3±0.06 o.3±0.08 a 0.3±0.12 0.3±0.09 Serum sodium (mmol/L) 144 ±2.87 144.8 ± 4.17 a 138.8 ± 1.47** b 139.5 ± 1.87** c b

Serum potassium (mmol/L) 4.6 ± 0.64 4.6 ± 0.54 a 5.3 ± 0.77* a 5 ± 0.26 a Serum Phosphorous (mg/dl) 7.2 ± 0.6 7.5 ± 0.7 c b 7.2 ± 0.8 b 6.5 ± 0.6 a b Serum Calcium (mg/dl) 9.5 ± 0.4 9.8 ± 0.2 a 9.6 ± 0.2 a 9.8 ± 0.2 a (*) = there are significant differences (Pv ≤0.05) (**) =There is highly significant differences (Pv ≤ o.o1)

Means with same superscript letter are not significantly different

3.5.2.1.4 Effects of plants extracts on hematology parameters

Only Myrtus communis affected high significantly Hemoglobin and mean corpuscle cells which significantly increased compared to the control group. Platelets were only increased

147 significantly in Capparis spinosa treated group compared to control group. In a same manner the methanol extracts of Myrtus communis leaves, Capparis spinosa leaves and Pistacia lentiscus fruits increased and decreased the levels of neutrophils and lymphocytes respectively with high significant differences compared to the control group (Table 32). Also the table (32) showed that Myrtus communis significantly elevated monocytes level compared to control group.

3.5.2.2 Histopathological effect of tested plant extracts on vital organ tisuues

3.5.2.2.1 Histopathological effect of Capparis spinosa on vital organs tissues

Compared to the normal, liver tissue the rats treated with sub-acute dose of 1G/Kg of Capparis extract showed hepatocellular necrosis and cytoplasmic vacuoles, also congestion and hemorrhage were noticed (Pohto 10). Necrosis and degeneration of renal tubules were seen in kidney tissue (Photo 11). Photo (12) showed rat uterus exhibited necrosis of epithelial cells, fibrous tissues and inflammatory cells.

3.5.2.2.2 Histopathological effect of Myrtus communis on vital organs tissues

With the sub-acute dose of 0.5G/Kg, the Myrtus extract results showed that liver tissue showed hemorrhage, congestion and necrosis of hepatic cells compared to normal liver tissue (Photo 13).Necrosis of renal tubule epithelial seen in kidney tissue (Photo 14). Photo (15) showed rat uterus exhibited fibrous tissues and necrosis of epithelial of tubular gland.

3.5.2.2.3 Histopathological effect of Pistacia lentiscus on vital organs tissues

Compared to the normal, liver tissue of rats treated with 0.5G/Kg sub-acute doe of Pistacia extract exhibited Pleomorphic nudel of hepatic cells apoptic, congestion and hemorage (Photo 16). Necrosis of renal tubules epithelial was seen in kidney tissue (Photo17). Photo (18) showed rat uterus exhibited necrosis of tubular epithelial gland and fibrous tissue.

Table (32): Effects of plants extracts on Hematology parameters values compared to control group.

148

Means ± Standard Deviation Parameters Control Methanol Methanol Methanol fruits (1m/kg) leaves extract leaves extract extract of of Capparis of Myrtus Pistacia spinosa communis lentiscus (1g/kg) (0.5g/kg) (0.5g/kg) Hemoglobin (g/dl) 13.9 ±0.96 12.9 ± 0.8 a b 11.7 ± 1.6** b 13.3 ± 0.7 a Packed cells volume 41.7 ± 2.7 38.8 ± 2.14 a c 34.4 ± 3.9** b 40 ± 2.4 a (%) Mean corpuscle cells 55.7 ± 2.9 54.7 ± 0.8 a 54 ± 2.3 a 53 ± 1.1 a volume (fl) Platelets (109cell/L) 904 ± 109.5 1135.8 ± 851 ± 283 b 849 ± 107 b 155.8* a Total white blood 8 ± 3.8 9 ± 2.9 a 7.8 ± 3.8 a 8.6 ± 4.6 a cells (109cell/L) Neutrophils (%) 20 ± 8.3 36.8 ± 5.4** a 39 ± 6.7** a 44 ± 14.8** a Lymphocytes (%) 72.7 ± 9.5 55.8 ± 6.3** a 52.8 ± 7.2** a 49.2 ± 13.4** a Monocytes (%) 4.8 ± 1.2 4.3 ± 1.2 c 6.8 ± 1.9* a b 5.3 ± 1.9 b c Eosinophils (%) 2.3 ± 1.0 2.7 ± 1.0 a 1.3 ± 0.82 b c 1.5 ± 0.84 c (*) = there are significant differences (Pv ≤0.05)

(**) =There is highly significant differences (Pv ≤ o.o1)

Means with same superscript letter are not significantly different

149

Photo (10): Capparis spinosa Photo (11): Capparis spinosa methanol leaves extract treated methanol leaves extract treated rat liver tissue(1g/kg IP 24 rat kidneytissue (1g/kg IP hour’s single dose) 24hour’s single dose)

Photo (12): Capparis spinosa methanol leaves extract treated rat uterus tissue (1g/kg IP 24 hour’s single dose)

150

Photo (13): Myrtus communis Photo (14): Myrtus communis methanol leaves extract treated rat methanol leaves extract treated rat liver tissue (0.125g/kg IP kidney tissue (0.125g/kg 24hour’ssingle dose) IP24hour’ssingle dose)

Photo (15): Myrtus communis methanol leaves extract treated rat uterus tissue

(0.125g/kg IP 24 hour’s single dose)

151

Photo (16): Pistacia lentiscus Photo (17): Pistacia lentiscus methanol fruits extract treatedrat methanol fruits extracttreate rat liver tissue(0.125g/kg IP 24 hour’s kidney tissue (0.125g/kg IP 24 single dose) hour’s single dose)

Photo (18): Pistacia lentiscus methanol fruits extract treated rat uterus tissue

(0.125g/kg IP 24 hour’s single dose)

152

3.5.3 Pharmacological behaviors of plants extracts treated rats

Nothing has been noticed in behaviors of rats group treated with Capparis spinosa methanol leaves extract except mild intermittent spasm noticed in four out of six tested rats during the 2nd and 3rd hours which disappeared by the 4th hour observation period. In rats group treated with Myrtus communis methanol leaves extract, spasm and writhing and excitation had seen by the 2nd hour and was disappeared by the 4th observation hour. The spasm was strong intermittent (every 10 minutes) in the 2nd hour and weak in the 3rd hour. Excitation had seen in all rats treated with Pistacia lentiscus methanol fruits extract during the first hour and started decrease within the second hour and disappeared by the beginning of the third watch hour. Strong intermittent strong spasm with body was seen in the first two hours but disappeared by the third watch hour. Pushing of legs back movement had not seen in Capparis spinosa treated group but had seen with the other tested extracts treated groups. The movement started within the first watching period and recovered with the beginning of the third and fourth hours for Pistacia lentiscus and Myrtus communis extracts respectively.

3.6 Pharmacological screening of tested extracts on isolated organs

3.6.1 Pharmacological effect of tested plants extract on rabbit jejunum

3.6.1.1 Effect of Capparis spinosamethanol leaves extract

The extract exerted weak transient dose dependent contraction that effectively blocked by atropine (10ng). This indicated to that the extract possess weak transient cholinergic effect on the isolated rabbit jejunum (Figure 3).

3.6.1.2 Effect of Pistacia lentiscusmethanol fruits extract

At low doses up to 200µg/ml the extract induced weak relaxant effect on the jejunum, but at higher doses (800 µg /ml) of the extract exerted complete relaxation of the smooth muscle that effectively antagonized with prior administration of Phentolamine (1 µg). The result showed that Pistacia lentiscus methanolic extract at high dose possess Alpha adrenergic effect on the jejunum (Figure 4).

153

Figure (3): Pharmacological effect of Capparis spinosa leaves extract on rabbit jejunum

W = Wash

1 = 200µg/ml plant extract

2= 400µg/ml plant extract

3 = 800µg/ml plant extract

4 = 100mg/ml plant extract

5 = 200mg/ml plant extract

6 = 400mg/ml plant extract

7 = 100mg/ml plant extract + 10ng/ml Atropine

8 = 400mg/ml plant extract + 10ng/ml Atropine

154

Figure (4) Effect of Pistacia lentiscus methanol fruits extract

on isolated rabbit jejunum

1 = 100µg/ml plant extract

2= 200µg/ml plant extract

3 = 400µg/ml plant extract

4 = 800µg/ml plant extract

5 = 800µg/ml plant extract + 1 ng/ml Phentolamine

155

3.6.1.3 Effect of Myrtus communismethanol leaves extract

The extract induced a dose dependent contraction on the smooth muscle that partially blocked by atropine dose up to 40ng/ml, but higher dose (320ng/ml) of atropine completely antagonized the methanolic leaves extract of Myrtus communis. That mean the extract possess dose dependent cholinergic effect on the jejunum (Figure 5).

3.6.2 Pharmacological effect of tested plants extract on rabbit aortic strip

3.6.2.1 Effect of Capparis spinosamethanol leaves extract

At low doses up to 400 µg/ml the extract induced a weak dose dependent potentiation to nor-adrenaline, while at dose of 800 µg/ml showed less potentiation and at higher doses (1.6 – 3.2 mg/ml) the extract completely antagonized the reference nor-epinephrine,200ng/ml (Figure 6).

3.6.2.2 Effect of Pistacia lentiscusmethanol fruits extract

The extract potentiates the effect of nor-epinephrine in the aortic smooth muscle which probably due to its adrenergic effect previously identified on the isolated intestine (Figure 7).

3.6.2.3 Effect of Myrtus communismethanol leaves extract

The study showed that the extract partially antagonized the reference nor-adrenaline. In a dose dependent manner Myrtus communis extract reduces the stimulant effect ofnor-epeniphrine in the isolated aorta which probably due to cholinergic effect; physiological antagonism (Figure 8).

3.6.3 Pharmacological effect of tested plants extract on rat uterus

3.6.3.1 Effect of Capparis spinosamethanol leaves extract

The extract induced relaxant effect which is not adrenergic in nature because it is refractory to the antagonistic effect of propranolol and Phentolamine. This may be due to the plant has a component of direct relaxant effect (Figure 9).

156

Figure (5) Effect of Myrtus communis methanol leaves extract

on isolated rabbit jejunum

W = Wash

1 = 100µg/ml plant extract

2= 200µg/ml plant extract

3 = 400µg/ml plant extract

4 = 800µg/ml plant extract

5 = 400µg/ml plant extract + 10µg/ml Atropine

6 = 400µg/ml plant extract + 20µg/ml Atropine

7 = 400µg/ml plant extract + 40µg/ml Atropine

8 = 400µg/ml plant extract + 320µg/ml Atropine

157

Figure (6) Effect of Capparis spinosa methanol fruits extract on isolated aortic strip

W = Wash

1 = 1µg/ml Nor-epeniphrine

2= 2µg/ml Nor- epeniphrine

3 = 4µg/ml Nor- epeniphrine

4 = 100µg/ml plant extract + 2µg/ml Nor- epeniphrine

5 = 200µg/ml plant extract + 2µg/ml Nor- epeniphrine

6 = 400µg/ml plant extract + 2µg/ml Nor- epeniphrine

7 = 800µg/ml plant extract + 2µg/ml Nor- epeniphrine

8 = 1600µg/ml plant extract + 2µg/ml Nor- epeniphrine

9 = 3.2mg/ml plant extract + 2µg/ml Nor- epeniphrine

10 = 2µg/ml Nor- epeniphrine

158

Figure (7) Effect of Pistacia lentiscus methanol fruits extract on isolated aortic strip

W = Wash

1 = 2µg/ml Nor- epeniphrine

2= 4µg/ml Nor- epeniphrine

3 = 8µg/ml Nor- epeniphrine

4 = 100µg/ml plant extract + 4µg/ml Nor- epeniphrine

5 = 200µg/ml plant extract + 4µg/ml Nor- epeniphrine

6 = 400µg/ml plant extract + 4µg/ml Nor- epeniphrine

7 = 800µg/ml plant extract + 4µg/ml Nor- epeniphrine

159

Figure (8) Effect of Myrtus communis methanol leaves extract on isolated aortic strip

W = Wash

1 = 1µg/ml Nor- epeniphrine

2= 2µg/ml Nor- epeniphrine

3 = 4µg/ml Nor- epeniphrine

4 = 100µg/ml plant extract + 2µg/ml Nor- epeniphrine

5 = 2µg/ml Nor- epeniphrine

6 = 200µg/ml plant extract + 2µg/ml Nor- epeniphrine

7 = 400µg/ml plant extract + 2µg/ml Nor- epeniphrine

160

N 1 2 3 4 5

Figure (9): Pharmacological effect of Capparis spinosa methanol leaves extract

on isolated rat uterus

N = Normal

1 = 200µg/ml plant extract

2= 400µg/ml plant extract

3 = 1mg/ml plant extract

4 = 2mg/ml plant extract

5 = 4mg/ml plant extract

161

3.6.3.2 Effect of Pistacia lentiscusmethanol fruits extract

Wide doses range from 100µg/ml, 200, 400, 1000 and 2000 µg/ml of the extract showed no effect on rat uterus previously brought to oestrous stage. That mean the extract has no action on rat uterus (Figure 10).

3.6.3.3 Effect of Myrtus communis methanol leaves extract

The extract is without effect on uterus in a dose up to 400 µg/ml but at very high dose (1000 µg/ml) it relaxed the uterus and this inhibitory effect was completely blocked by propranolol 5ng/ml, that suggest an adrenergic effect on uterus (Figure 11).

3.6.4 Effect of tested methanol plants extracts on Frog rectus abdominus muscle

No action has been exerted from methanol extracts of each of Capparis spinosa leaves, Myrtus communis leaves and Pistacia lentiscus fruits on the isolated frog rectus abdominus muscle (Figures 12, 13 and 14).

162

Figure (10): Pharmacological effect of Pistacia lentsicus methanol fruits extract

on isolated rat uterus

N = Normal

1 = 40ng/ml plant 5-hydroxy tryptamine

2= 100µg/ml plant extract

3 = 200µg/ml plant extract

4 = 400µg/ml plant extract

5 = 1000µg/ml plant extract

6 = 2000µg/ml plant extract

7 = 40ng/ml plant 5-hydroxy tryptamine

163

Figure (11): Pharmacological effect of Myrtus communis methanol leaves extract on isolated rat uterus

1 = 100µg/ml plant extract

2= 200µg/ml plant extract

3 = 400µg/ml plant extract

4 = 1000µg/ml plant extract

5 = 1000µg/ml plant extract + 10ng/ml Propranolol HCL

164

w w w w

w w w

Figure (12):Pharmacological effect of Capparia spinosa methanol leaves extract on isolated Frog rectus abdominus muscle

1 = 500ng Acetylcholine 2 = 100µg/ml extract 3 = 200 µg /ml extract

4 = 400 µg /ml extract 5 = 4 = 100mg/ml extract 6 = 200mg/ml extract

7 = 4 = 400mg/ml extract 8 = 500ng Acetylcholine

165

Figure (13):Pharmacological effect of Pistaci lentiscuc methanol fruits extract on isolated frog rectus abdominus muscle

1 = 500ng Acetylcholine 2 = 100 µg Acetylcholine 3 = 200 µg /ml extract

4 = 400 µg /ml extract 5 = 100mg/ml extract 6 = 200mg/ml extract

166

Figure (14):Pharmacological effect of Myrtus communis methanol leaves extract on isolated frog rectus abdominus muscle

Red raw = wash

1 = 250ng Acetylcholine 2 = 500ng Acetylcholine 3 = 1 µg Acetylcholine

4 = 100 µg /ml extract 5 = 200 µg /ml extract 6 = 400 µg /ml extract

7 = 4 = 100mg/ml extract 8 = 200mg/ml extract

167

CHAPTER FOUR

Discussion, Conclusions and Recommendations

4.1 Discussion 4.1.1 Antimicrobial

In the present study, a total of eighty plants extracts (extracted with different solvents polarities) belonging to nine Libyan plants, distributed among seven families were screened for their antimicrobial activity against standard and clinical organisms. Such screening represents an essential stage for selecting the most prominent antimicrobial candidates for further detailed phytochemical analysis and other biological activities investigations. Chloroform and methanol extracts of leaves of Myrtus communism plant showed the highest activity against tested Gram positive and Gram negative bacteria The highest antifungal activity from the overall tested 80 extracts was shown by the ethanol bark extract of Capparis spinosa , methanol leaves extract of Myrtus communis , methanol bark extract of Salvia fruticosa and Pistacia lentiscus methanol fruits extract against tested Candida albicans and Aspergiluus niger . Some extracts either showed weak or no activity against one or more of the tested organisms. Stages of Plant development and the ecological factors had an impact on the qualitative composition of the plants. The activity of tested extracts varied, depending on the species type and concentration of the extract (Khalil and Li, 2011). The environmental factors (climate, seasons and soils), the geographic conditions, the genetic diversity of the species, the harvesting period and the extraction technique are parameters could be lead to the variation in the biological activities of plant extracts. These factors influenced the available resources, the plant’s biosynthetic pathways, the metabolism and consequently the relative proportion of the main characteristic compounds, their nature and their production. This leads to the existence of different origin, as well as seasonal variation throughout the plant’s vegetative cycle. It has been documented that the observed heterogeneities between plants populations could correspond to particular adaptive selection pressure traits; climate, season and soils (Flamini et al., 2004, Salvagnini et al., 2008, Ben Ghnaya et al., 2013).

168

Out of the 80 tested extracts evaluated against standard microorganisms, only methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark showed active broad spectrum antimicrobial performance, while methanol and chloroform extracts of leaves and bark of Capparis spinosa, showed narrow selective antibacterial activity only against standard tested Staphylococcus aureus with clear anti-Candida activity. Despite all extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark showed having the highest antimicrobial performance against tested standard bacteria and fungi, but extract of Myrtus communis leaves was the most active one. Phytochemical screening in this study proved the presence of phenolic compounds, flavonoids, terpenes, saponins and tannins, the result which considered the cause of the broad spectrum antimicrobial performance shown from these extracts. In addition the GC-MS analysis showed the high presence of the phenol; Quinic acid in Myrtus communis methanol leaves extract with percentage (40.02%) which explains the highest activity shown from this extracts. It has been reported that Quinic acid has anti-biofilm properties (Papetti et al., 2013) and antibacterial activity via cell membrane disruption (Bai et al., 2018).

In this study the antimicrobial activity results of the Myrtus communis leaves, Pistacia lentiscus fruits extract and methanol extract of Salvia fruticosa bark highlight the strong possibility for the using of these extracts for treatment of infections caused by Pseudomonas aeruginosa, Staphylococcus aureus , Candida albicans, Acinetobacter baumanii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Proteus mirabilis, these might be contributed to the results obtained in this study showed that each crude extract was constituted from at least twenty-five bioactive compounds and there is a big possibility to get promising pure pharmaceutical antimicrobial agents from these plants extracts. This result confirm the findings of Sasidharan et al. (2011) and Singh, (2015) who stated that medicinal plants considered as a rich resources of ingredients which can be used in drug development and synthesis.

On the other hand, the results obtained in the present study is similar to that obtained by Ben Ghnaya et al. (2013) and Taheri at al. (2013), who reported that the aerial parts of Myrtus communis plant revealed clear inhibition zones against standard Staphylococcus aureus. Similarly, Amensour et al. (2010)confirmed the antibacterial potential activity of Myrtus

169 communis aerial part extract against food-borne pathogens and food spoilage Bacillus subtilis bacteria. Alyousef et al. (2018) also claimed that Myrtus leaves extract had active inhibitory effect on clinical Acinetobacter baumanii. Mansouri et al. (2001) and Amensour et al. (2010) results agreed with this study output where their studies proved the high susceptibilities of Staphylococcus aureus, Pseudomonas aeruginosa and Proteus spp towards methanol leaves extract of Myrtus communis. Similarly, Amensour, et al. (2010) and Toauibia, et al. (2015) found that the Myrtus communis leaves revealed active growth inhibition activity against Bacillus subtilis and Klebsiella penumoniae. This study results disagree with the findings of Benhammou et al. (2008) Mert et al. (2008) Amensour, et al. (2010) and Toauibia, et al. (2015) who conclude that the extract of the Myrtus communis leaves had moderate to none inhibition activity against tested Escherichia coli and Candida albicans . The results of Gortzi et al. (2008), Taheri et al. (2013) andBesufekade et al. (2017) had different finding from this study where they conclude that weak growth inhibition zones (< 9mm) were revealed against clinical Escherichia coli and Pseudomonas aeruginosa compared with the active inhibition zone (19.87±1.4) revealed in this study. Also the findings of this results disagreed Besufekade et al. (2017) who proved that Myrtus communis extract had weak (1±0.5) activity compared to the high (25±0.06) activity shown in this study against tested methicillin resistant Staphylococcus aureus. This results showed active inhibition activity of Myrtus communis leaves extract against Enterobacter cloacae isolates, this was in contrast with Mert et al. (2008), who documented that Myrtus communis leaves extract had no effect on this bacteria, these might be due to the fact that different collection regions, different solvents used and different parts tested are factors cause such plants activity and organisms susceptibility variations.The high presence of the polyphenols (flavonoids), saponins and the phenolic tannins shown upon the phytochemical analysis of the methanol leaves extract of Myrtus communis compared with other tested extracts make the suggestion that the highest broad spectrum antimicrobial performance shown from tested Myrtus communis leaves extract is attributed to these phytochemical constituents. Flavonoids have been reported as antimicrobial and antifungal agent. On the other hand, saponins having many biological properties including antibacterial and antiprotozoal activities. However, tannins commonly referred to tannic acid, are water-soluble polyphenols present in many plants and their antimicrobial activity are well documented (Chung etal., 1998, Taleb-Contini et al., 2003, Winaet al., 2005, Hassan, 2008, Akiyama et al., 2001, Seleem et al., 2017).

170

Benhammou et al. (2008), Bozorgi et al. (2013), Missoun et al. (2017) and Naderi et al. (2017) were in line with this result for the good growth inhibition activity of the methanol fruits extract of Pistacia lentiscus against tested Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumonia and Protus mirabilis isolates. However, Benhammou et al. (2008) and Missoun et al. (2017) reported that the plant extract showed weak to no effect against tested Escherichia coli isolates, the claim which is in contrast with what found in this study. Even though they tested the same plant part (aerial part; fruits), but the difference might be due the different geographical distribution of the plant collected and due to different solvents used. El Idressi et al. (2016) was not in line with this result for the extract activity against tested Staphylococcus aureus as they reported that Pistacia lentiscus extract had moderate effect compared to the active performance shown in this study and even though both studies tested same parts but this difference might be due to different extraction methods used, since they used hydrodistillation technique compared with Soxhlet extraction technique used in this study.

No studies have been found concerned with the antimicrobial activity of Salvia fruticosa in general and for the plant bark in specific. Salvia officinalis is the most investigated species. Essential oil of aerial parts of Salvia officinalis was screened against standard and clinical microorganisms and found to have moderate growth inhibition activity against tested Bacillus subtilis, Satphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis compared with the active performance shown in this study by the species Salvia fruticosa (Delamare et al., 2007, Pierozanet al., 2009 and Khalil et al., 2011). This difference in activity attributed to the different plant species investigated and also to the different extraction methods used. Aerial part powder of Salvia officinalis was extracted with hydro-distillation while bark powder of Salvia fruticosa in this screening was extracted with use of organic solvent and Soxhlet apparatus.

Active growth inhibition activity was shown in this study by methanol extract of Capparis spinosa leaves against tested standard microorganisms, the highest activity was seen against standard Staphylococcus aureus (20mm±0.01), the chloroform extract of the plant bark showed the highest inhibition activity (30mm±0.01) against the standard Staphylococcus aureus. However no one of the tested clinical isolates showed susceptibility toward the methanol leaves

171 extract of Capparis spinosa except methicillin resistant Staphylococcus aureus. Even with the tested clinical isolates, methicillin resistant Staphylococcus aureus showed higher susceptibility (13mm±11) to the chloroform bark extract of Capparis spinosa compared with the methanol leaves extract (4.6±7). The phytochemical analysis in this study showed that the plant methanol and chloroform extracts of Capparis spinosa contained many bioactive compounds which are known to have biological activities. The alkaloid bioactive compound, 1-Methyl-pyrrolidinr-2- carboxylic acid constitutes 41.8% of the total constituents of methanol leaves extract, this compound is known as anti-Staphylococcus aureus agent (Ajani et al., 2012). Alsopalmitic acid and hexadecanoic acid, ethyl ester are fatty acids found within the constituents of chloroform bark extract but not presented in the methanol extract, these components reported to have selective anti-Staphylococcus aureus activity. Also n-hexadecanoic acid compound was present with higher percentage (32.45%) in chloroform extract compared to methanol extract (6.8%) and it has been reported also to have anti-Staphylococcus aureus activity (Agoramoorthy et al., 2007, Neumann et al, 2015, ) . The tannins found in the leaves extract have been reported to have the ability to reduce surface colonization of the Gram positive Staphylococcus aureus and then reduce the organism infection (David et al., 2013). This result can concluded that the chloroform bark extract showed more activity on standard and clinical Staphylococcus aureus than methanol leaves extract. Similar to this results Sherif et al. (2013) reported that methanol extract of aerial parts of Capparis spinosa have active growth inhibition activity against both Staphylococcus aureus and methicillin resistant Staphylococcus aureus. Nour and El-imam (2013) result was similar to the results of this study in that the Methanol extract of Capparis plantleaves was more effective compared to chloroform extract against standard Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans. Also Abd Razik, (2011) agreed with this findings where he claimed that active growth inhibition activity was shown from the Capparis spinosa flowers methanol extract. However this study output claimed that the highest antimicrobial activity was shown from the Capparis spinosa methanol leaves extract, but this is in contrast with Proestos et al. (2006) who reported that the highest antimicrobial activity was shown from the ethanol leaves extract of Capparis spinosa.

In this screening, resistance had been shown from tested Staphylococcus aureus and Gram negative clinical isolates toward most of antibiotics used compared to the tested extracts,

172 the floroquinolone, ciprofloxacin showed more inhibition activity against all tested clinical isolates except Acinetobacter baumanii. The methicillin resistant Staphylococcu aureus is a multi - drug resistant bacteria that resists all penicillins, so the option antibiotics for treatment of its infection are limited to few antibiotics such as vancomycin, linezolid, tigecycline and mupirocin. Vancomycin is the most common used, but by time it has been reported that vancomycin become less effective in settings with extensive use of these treatments (Simor and Daneman, 2009). However this study claimed that methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits offered more activity against tested methicillin resistant Staphylococcu aureus compared with vancomycinwhile Salvia fruticosa bark extract showed equal zone of inhibition revealed from the vancomycin. Gram-positive bacteria cell wall have lipid component, phosphatidyl glycerol that can be chemically modified by bacterial enzymes to convert the lipid from anionic to cationic or zwitterionic form, the process leads to increased levels of resistance of the bacteria against antimicrobial agents (Miller, 2016). This make a suggestion that the tested extracts which actively inhibited methicillin resistant Staphylococcu aureus might be have substances that have disabling effect against bacterial enzymes responsible for phosphatidylgly-cerol modification. It has been found in this study that tested methicillin resistant Staphylococcus aureus was also resistant to beta-lacatm antibiotic used compared with its high susceptibility showed toward the tested extracts. It is well known that the predominant mechanism of resistance to β-lactams in Gram positive organisms is mostly achieved by modifications of their target site; the penicillin-binding proteins (Epand et al., 2016).

All tested Gram negative clinical isolates in this study showed resistance towards three or more of the antibiotics used , the resistance of Gram negative bacteria might be due their ability to produce enzymes that can modify or destroy antibiotics, such as aminoglycoside-modifying enzymes and extended spectrum beta-lactamases and carbapene-mases or due to acquisition of mutations in bacterial targets such as topoisomerases, ribosomes, penicillin-binding proteins, and outer membrane porins that alter antibiotic efficacy or uptake (Blair et al., 2015). Akhavan et al. (2016) concluded that treatment of Acinetobacter baumannii infections nowadays is very difficult. According to increasing rate of isolating of multi-drug resistant Acinetobacter baumannii from hospitalized patients, it is necessary to study and investigate the antibacterial activity of medicinal plants that can be used as a new source for antibiotics. In this investigation Acinetobacter baumanii was the only Gram negative bacterial resisted all used antibiotics compared with the strong growth inhibition activity seen from methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark. This result is considered as a

173 promising key in treatment of infectious diseases caused by this problematic multi-drug resistant bacterium. Furthermore, results of this screening gave the high probability that the methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark have anti- extended spectrum beta-lactamase enzymes, where they actively inhibited the growth of Enterobacter cloacae and Klebsiella pneumoniae which were resisted all beta-lactam antibiotics used (Co-Amoxicillin, Ceftazidime and Ceftriaxone). Also Pseudomonas aeruginosa, Escherichia coli and Proteus mirabilis were clearly inhibited by methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark while they showed no susceptibility to beta-lactam antibiotics (Co-Amoxicillin and Ceftriaxone) and to aminoglycoside antibiotic used (Gentamicin) this resulst gave the assumption that the methanol extracts of Myrtus communis leaves, Pistacia lentiscus fruits and Salvia fruticosa bark may be have more than one mechanism against pathogenic bacteria and could act as inhibitor to protein synthesis in bacteria. As the methanol leaves extract of Myrtus communis proved in this study to be the highest effective broad spectrum antimicrobial extract in vitro and with the fact that Gram positive Staphylococcus aureus and Gram negative Pseudomonas aeruginosa bacteria are the most common problematic pathogens responsible for many serious infections worldwide, the extract was tested in vivo against these two bacteria. The in vivo assay in this study, results showed that the healing period of extract treated wound was eight days compared with six days and eleven days healing period for antibiotic treated wound and eleven days healing period for non-treated wound. No significant difference (P value ≥ 0.05) had shown between healing period of antibiotic treated wound and extract treated wound, but there was a significant difference shown in wound healing performance between non -treated wound and Myrtus communis treated wound. Similar result was shown by Mohammad et al. (2014) and Hashemipour et al. (2017), where they noticed a significant increase in wound closure and reported that an acceleration of wound healing had made in Myrtus communis leaves treated rat wounds. Also the in vivo antimicrobial assays in this study showed significant differences starting from day three in healing performance between non treated infected wounds and antibiotic and extract treated infected wounds, since the healing period was shorter in treated infected wounds. However no significant difference was shown between antibiotic treated infected wounds and extract treated infected wound, since the extract has the same good wound healing activity revealed from

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Fucidin and Tetracyclin ointments used against Staphylococcus aureus and Pseudomonas aeruginosa infected wounds respectively. It is well known that tetracycline is not very effective against Pseudomonas aeruginosa, but is known to induce the type-III secretion system and consequently enhance cytotoxicity of Pseudomonas aeruginosa in vivo (Morita et al., 2014). GC-MS analysis in this study showed that Myrtus communis methanol leaves crude extract constitute of thirty-three bioactive compounds with abundance presence of polyphenols. The result suggested that Myrtus communis leaves crude extract accelerate the wound healing process via effecting of the second inflammation phase of the four wound repairing phases. The abundant presence of poly-phenolic compound in Myrtus communis leaves crude extract leads to the suggestion that the extract in addition to its powerful antimicrobial activity also it has anti- inflammatory activity and this suggestion is in line with Touaibia, (2017) who reported that Myrtus communis has a significant anti-inflammatory effect.

4.1.2 Antioxidant

Methanol extracts of the Pistacia lentiscus fruits, Myrtus communis leaves, Salvia fruticosa bark and Capparis spinosa leaves were presented antioxidant activity as shown by diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assay. The highest antioxidant activity was shown with Pistacia lentiscus fruits extract followed by Myrtus communis leaves extract , Salvia fruticosa bark and Capparis spinosa leaves according to the IC50s 0.008 ± 0.01, 0.017 ± 0.01, 0.046 ± 0.02 and 0.205 ± 0.07 revealed from the extracts respectively compared with the IC50 of 0.077µg/ml± 0.01 revealed from the control; Propyl Gallate. The One-way analyses of variance showed highly significant variation in the antioxidant IC50s(P value ≤ 0.05) varied. This strong variation in antioxidant activities might be due to the different plant species tested. The differences in antioxidant activities of plants depending on the concentration, the extraction solvent and the part of the plant used (Amensour, et al., 2009). This result is in line with Amensour, et al. (2009), Bhoyar et al. (2011) ,Aouinti et al. (2014) and Boukhary et al. (2016) who their results confirmed the antioxidant potentiality of the tested extracts. Little variation were seen concerning the values of the IC50s, this variation in activity suggested to be due to different plant collection area which comprise different ecological factors. Polyphenols, flavonoids and tannins are presented as constituents contained within the tested extracts and this give the assumption that the significant antioxidant activity shown from all extracst is

175 contributed to these compounds. Most plant-derived polyphenols exhibit strong antioxidant potentials, Banjarnahor and Artanti, (2015) review the prospective role of flavonoids as antioxidant. Otherwise it is well known that the tannins are considered as superior antioxidants (Bors and Michel, 2002), this in line with the result of this study where the highest presence of tannins was found to be constituted Pistacia lenticus fruits extract and this clarify the highest antioxidant activity shown by this extract compared with the other three tested extracts. This study highlighted that these plants extracts can be considered as source of natural antioxidants for potential achievement of pharmaceutical industries and confirm the findings of (Zaouali et al. 2010) who stated that medicinal plants perform important contributions to the healthcare systems worldwide, and play a key role in the development and advancement of modern scientific studies by serving as a starting point for the development of new natural drugs.

4.1.3 Anticancer (Tumor cells Cytotoxicity)

Methanol extracts of Myrtus communis leaves, Capparis spinosa leaves and Pistacia lentiscus fruits were screened for their cytotoxic activity against four tumor cell lines, The three extracts revealed high cytotoxic effect on tested colon carcinoma (HCT-11) cell line, moderate cytotoxic effect on tested breast carcinoma cell line (MCF7) and prostate carcinoma cell line (PC3). There were no previous studies found related specifically to the effect of methanol leaves extract of Capparis spinosa on tested carcinoma cell lines. Lam et al. (2009) tested the Capparis spinosa seeds and proved its anti-proliferative effect on breast MCF-7 and liver hepatoma HepG2 cancer cells. This study proved that the methanol leaves extract of Capparis spinosa inhibited the proliferation of breast MCF-7 and liver hepatoma HepG2 cancer cells too. On other hand, Al-Daraji, (2010) tested the cytotoxicity of aqueous leaves extract of Capparis spinosaagainst human cervix uteri epitheloid carcinoma Hela and human epidermoid larynx carcinoma Hep-2 he proved that the highest cytotoxic activity of methanol leaves extract of Capparis spinosa was at dose of 1000 μg/ml. On the other hand, Abdelwahedet al. (2007) and Ljubuncic et al. (2005) agreed with this study in that the Pistacia lentiscus fruits inhibit the proliferation and caused death of human colon cell line HCT-116 carcinoma and human liver cell line carcinoma HepG2 in vitro. Other researchers agreed with this study in that the aerial parts specially leaves of Myrtus communis have cytotoxic activities against many carcinoma cell lines including human breast carcinoma MCF-7, hepatocellular carcinoma HepG2, they also proved

176 the cytotoxic activity of leaves of Myrtus communis on human prostate cancer DU145 (Cottiglia et al., 2012; Naghibi et al., 2013; Grandjenette et al., 2015; Hennia et al., 2018). Even though this study tested different human prostate cancer cell line type; PC3 but also proved that Myrtus communis methanol leaves extract inhibited its proliferation, this make the suggestion that this extract has breast, liver, colon and Prostate anticancer activity. These cytotoxic activities could be referred to the phenolic acids and flavonoids constituents of the extracts. It has been well documented that, antimicrobial, antioxidants, anticancer and other healing effects of medicinal plants are attributed to their metabolites, specifically secondary metabolites (Leicach and Chludil, 2014; Sing, 2015).

4.1.4 Biosafety

4.1.4.1 Toxicity

4.1.4.1.1 Liver function profile

It is well known that the level of albumin serum, major plasma protein is not a good indicator of moderate or severe liver dysfunction since the rates of its production and degradation are low. The serum level of albumin changes only slightly in acute liver diseases such as obstructive jaundice, viral hepatitis and drug-induced hepatotoxicity and usually its production stimulated in severe liver damage (Romani et al., 2004, Mimica-Dukić et al., 2010, Johari et al., 2014). In this study the methanol extracts of Myrtus communis leaves, Capparis spinosa leaves and Pistacia lentiscus fruits showed significant decrease in serum albumin level, the result which may reflects that the tested extracts did not induce severe damage to the rat liver despite the toxic dose did. Pistacia lentiscus fruits extract in this study increased the levels of liver enzyme markers (ALT, AST and GGT) with high significant (P value ≤0.01) compared to the control, and despite this revealed with the sub-acute dose but this result should be taken in consideration in the therapy dose determination. Accordingly, we can say that Myrtus communis and Capparis spinosa extracts are safe and have no toxic effect on liver enzyme markers. Also Myrtus communis and Capparis spinosa extracts appeared have no effect on the total direct or indirect Bilirubin. Pistacia lentiscus effect was differing since significant increased in the levels of total and direct bilirubin were observed with toxic doses. No previous studies were concerned with screening of toxic effects of these plants extracts on healthy liver. All published studies have

177 concerned with previously injured liver. Kalantari et al. (2018) results seemed to be in line with this study findings as they found that Capparis spinosa at the dose of 400 mg/kg showed liver protection against tert-butyl hydro peroxide (t-BHP) induced hepatic injury, as evident by a significant decrease in serum enzyme markers (ALT, AST and GGT). Similar results obtained by Janakat and Al-Merie (2002) who documented that the Capparis plant extract significantly prevented the increase in serum ALT, AST and LDH levels in acute liver damage induced by

CCl4 and repaired liver injury.

4.1.4.1.2 Serum glucose and lipid profile

According to serum glucose level this study showed that Capparis spinosa had no effect on serum glucose level while Myrtus communis and Pistacia lentiscus reduced the serum glucose level with high significant differences compared with the control (P value ≤ 0.01). This result disagreed with Lemhadri et al. (2007) who found potent anti-hyperglycaemic activity from Capparis spinosa in high fat diet (HFD) obese mice and this disagreement attributed to the different solvents extracts, where they tested aqueous plant extract while in this study methanol extract was examined. However, Hennia et al. (2018) results are in agreement with this study where they found that the extracts of Myrtus communis and Pistacia lentiscus showed anti- hyperglycemic activity. This study also evaluated the effects of tested extracts on the lipid profile markers; Triglycerides, total cholesterol, low density lipoproteins (LDL) and high density lipoproteins (HDL), Pistacia lentiscus extract in this study significantly (P value ≤ 0.05) increased LDL and decreased HDL, on the otherhand, Pistacia lentiscus, Myrtus communis and Capparis spinosa extracts showed non-significant difference in total cholesterol compared with the control with the sub-acute toxic doses, this result is in line with Cheurfa and Allem, (2015) and Hennia et al. (2018) who reported that Pistacia lentiscus and Myrtus communis extracts showed anti-hyper cholesterol effect.

4.1.4.1.3 Kidney profile In this study Pistacia lentiscus significantly decreases levels of serum urea and serum sodium with the sub-acute toxic dose of 0.5G/Kg while Myrtus communis and Capparis spinosa extracts had no clear effect. In consistence with this findingsJanakat and Al-Merie, 2002 found

178 that Capparis spinosa leaves extract significantly reduced the plasma levels of creatinine, urea and uric acid and repaired the kidney damage, induced by cisplatin-treatment.

4.1.4.1.4 Hematology profile

Methanol extracts of Myrtus communis leaves, Capparis spinosa leaves and Pistacia lentiscus fruits in this study showed no significant change in total white blood cells and red blood cells and platelets counts compared with the control. No previous studies were carried out on methanol leaves extract of Myrtus communis to support its effectiveness on hematology picture. Biricik et al. (2012) tested the Myrtus communis essential oil and they showed that erythrocyte count remained unchanged. However, MahmoodIet al. (2014) tested the Myrtus oil, they noticed lowering in white blood cells count and increasing in red blood cells count when fed broiler chickens with 0.3G/Kg body weight of the Myrtus oil. Few studies were carried out on methanol leaves extract of Capparis spinosa to support its effectiveness on hematology picture. Nabavi et al. (2016) reported that Capparis spinosa methanol leaves extract significantly increased the level of total white blood cells. Similar to the findings of this study, Attoubet al. (2014) reported that Pistacia lentiscus fruits extract showed no toxicity on blood functions when compared to the control group.

It is clear that Capparis spinosa, Myrtus communis and Pistacia lentiscus are showed effects ranged from not harmful to not sever harmful at the sub-acute toxic doses, the result which make the suggestion that the tested extract will be safe at the therapeutic doses.

4.1.4.2 Pharmacology

Cholinergic effect in dose dependent manner on the isolated rabbit jejunum has exerted from both methanol leaves extracts of Capparis spinosa and Myrtus communis in this study. Myrtus communis extract effect was stronger where partially antagonized and completely antagonized with 40ng/ml and 320ng/ml atropine respectively while weak cholinergic effect seen from Capparis spinosa tested extract where antagonized with 10ng/ml atropine. Pistacia lentiscus methanol fruits extract in this study showed different pharmacological effect, at 800 µg/ml, it revealed Alpha adrenergic effect on the jejunum where it exerted complete relaxation of the smooth muscle that effectively antagonized with prior administration of Phentolamine

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(1µg/ml). This result is in agreement with Yang et al. (2008) who reported that Capparis spinosa plant has many extensive pharmacological effects including stimulation of smooth muscles. However this result was not in line with Janbaz et al. (2013), Nair and Nair (2017) who reported that Myrtus communis extract exerted a relaxant effect on the smooth muscle. This difference might be referred to type of plants parts extracted and the purity of solvents used. Janbaz et al. (2013) used absolute methanol solvent but they extracted mixed aerial plant part (leaves, fruits) and Nair and Nair (2017) were extracted whole plant materials (leaves, fruits, twigs, bark) with mixture of 50% methanol and 50% water). No previous data has been found concerned generally with the effect of methanol extract of Pistacia lentiscus and specifically with methanol extract of Capparis spinosa leaves on isolated organs, but Benzidane et al. (2013) and Nabavi et al. (2016) reported that Capparis spinosa gave contractile effects on the smooth muscle similar to the findings shown in this study with the methanol leaves extract of Capparis spinosa.

Methanol extract of Pistacia lentiscusfruits in this study showed a dose dependent potentiating to the reference nor-adrenaline on isolated aorta and also the methanol leaves extract of Capparis spinosa, but the latter at high dose of 800µg/ml showed less potentiating and at higher doses (1.6 – 3.2 mg/ml) the extract completely antagonized the reference nor-epinephrine 200ng/ml. It is important to mention here that this study concerned with crude extract and the phytochemical analysis in this study showed that methanol leaves extract of Capparis spinosa, comprises of thirty-three bioactive compounds and it is possible that some contained compounds may mask or antagonized others at different concentrations. This result is not in agreement with Al-Snafi, (2015) who claimed that when Capparis spinosa addedduring the plateau phase of aorta contraction that induced by nor-adrenaline it produced a rapid relaxation. This might attributed to the different in the extract type, since this study examined methanol extract while Snafi, (2015) reviewed the action of the aqueous extract of Capparis spinosa. However, methanol leaves extract of Myrtus communis in this study showed different pharmacological effect in a dose dependent manner it reduced the stimulant effect ofnor-adrenaline in the isolated aorta, the effect which probably due to cholinergic effect and physiological antagonism. Similarly, Janbazet al. (2013) reported that the Myrtus communis leaves extract has vasodilator effect since exerted relaxation of phenylephrine induced contractions in isolated rabbit aorta.

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This study showed that no one of the tested extracts has effect on tested frog abdominus muscles. No previous data found clarify the effects of Capparis spinosa, Pistacia lentiscus and Myrtus communis on isolated abdominus rectus muscle. Also no data found explain the effects of Capparis spinosa, Pistacia lentiscus and Myrtus communis on isolated uterus organ. This study found that methanol leaves extract of Myrtus communis relax isolated rat uterus at high dose of 1000µg/ml, these may be analyzed with the findings of Alipour et al. (2014) and Mobli et al. (2015) who documented that leaves of Myrtus communis have been used in medicine for treating uterine bleeding, where it decreased the bleeding time. This study assumed that the bioactive substances including the flavonoids, alkaloids, terpenoids, tannins, fatty acids and steroids found within these extracts constituents are attributed factors to the proved biological activities of the extracts of leaves of Capparis spinosa and Myrtus communis and fruits of Pistacia lentiscus.

4.2. Conclusion

This study concluded that the methanol extracts of the leaves of Myrtus communis, fruits of Pistacia lentiscus and bark of Salvia fruticosa appeared as the most promising antimicrobial extracts where they revealed very active broad spectrum antimicrobial activity against standard and clinical multi-drug resistant bacteria. Myrtus communis extract is the highest pronouncing one. chloroform extract of the bark of Capparis spinosa appeared as selective growth inhibitor against both standard and clinical methicillin resistant Staphylococcus aureus. Myrtus communis methanol leaves extract (3% ointment) has been proved has the property to accelerate the wound healing and appeared has growth inhibition activity similar to tetracycline and fucidin ointments in treatment of wounds infected with Pseudomonas aeruginosa and Staphylococcus aureus, respectively.

Pistacia lentiscus fruits, Myrtus communis leaves, Salvia fruticosa bark and Capparis spinosa leaves appeared in this study to possess sound antioxidant activity.

Capparis spinosa leaves, Myrtus communis leaves and Pistacia lentiscus fruits methanol extracts showed highly cytotoxic activity to colon carcinoma cell line (HCT-116) and moderate cytotoxic to breast carcinoma cell line (MCF-7) and Prostate carcinoma cell line (PC3). While Pistacia lentiscus fruits methanol extract proved possessed high cytotoxicity towards tumor cells.

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Capparis spinosa leaves and Myrtus communis leaves methanol extract proved moderate cytotoxicity against liver carcinoma cell line (HEPG2).

The Methanol leaves extracts of Myrtus communis and Capparis spinosa appeared have no significant effect on the liver profile , lipid profile, no significant changes in haematology profile and decrease the glucose level, while methanol fruits extract of Pistacia lentiscus have a clear effect on the liver enzymes biomarkers (ALP, ALT. AST and GGT), lipid profile, kidney profile

Capparis spinosa and Myrtus commuins extracts have weak cholinergic effect on the rabbit jejunim smooth muscle while Pistacia lentiscus showed alpha adrenergic effect. Capparis spinosa extract potentiated the effect of Nor-epinephrine ( adrenergic effect) at low doses but at high dose it completely block the contractile effect of Nor-epinephrine on the isolated rabbit aortic strip while Pistacia lentiscus and Myrtus communis extracts were potentiated and antagonized the Nor-epinephrine effects, respectively. Capparis spinosa and Myrtus commuins extracts relaxed the isolated rat uterus while Pistacia lentiscus had no effect.

Accordingly, to this assumption this study introduced these plant extracts as promising therapeutic agents that have extensive biological activities support human health including antimicrobial, antioxidant and anti-tumor activities.

4.3 Recommendation

1. Isolation, purification and identification of the active pure compound(s) related to the biological activity with use of High liquid performance chromatography technique. 2. Further toxicological studies on the most active compound(s) are recommended. 3. Study of the mode of the antimicrobial action for the purified active ingredients on the bacterial cell. 4. Human clinical studies should be carried out on the proved biological active medicinal plants to confirm/assess their safety, therapeutic efficacy and potential for treatment utilization. 5. Standardization and formulation of the most active purified substance/s in acceptable dosage form.

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Table No. (A1): Biochemical tests for the identification of Staphylococcus

aureusisolates

No.

DNase

species

Lactose

Sucrose

Glucose

Bacteria Bacteria

Catalase

Coagulas Mannitol Satphylococcus 31 + + + + + + + aureus

Table No. (A2 ): Biochemical tests for the identification of Gram negative bacilli

209 isolates

MIU TSI Medium

Medium

No.

Glu Lac

Man

Ur.

H Gas

Mo.

Ind.

Butt

Slope Bacterial genus Bacterial

8 + + + + + - + - Y Y - +

E. - cloacae

8 + + + + - - + + Y Y - +

coli - E.

23 + + + + + - - - Y Y - +

K. K.

Slow pneumon

14 - - - - + + + - R R - -

a -

PS. PS. aeruginos

6 + - - - + - + - R Y + +

P. P. + mirabilis

Su.= sucrose Lact.= lactose Man.= mannitol Glu.= glucose Ci.= citrate Ind.= Indole

Ox.= oxidase Ur.= urease Y= yellow (+): positive test (-): negative test Mo= Motility

Table (A3): Comparison of inhibition zones of Myrtus communis L leaves methanol extract andstandard antibiotics references A.baumanii

Extract /Antibiotics N Mean Std. Minimum Maximum Deviation Myrtus communis L 10 22.85a 5.00 17.50 35.00 leaves methanol

210

extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 2.40b 3.13 .00 7.00 c CN 10 .00 .00 .00 .00 c CTX 10 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 c VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested A.baumanii isolates

Table (A4): Comparison of inhibition zones of Myrtus communis L leaves methanol extract andstandard antibiotics references P.mirabilis

Extract /Antibiotics N Mean Std. Minimum Maximum

211

Deviation Myrtus communis L 12 24.50b 4.73862 15.00 30.00 leaves methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 14.40c 7.64 .00 20.0 CIP 10 37.80a 3.04 30.0 40.0 CN 10 18.80d 1.22 16.0 20. e CTX 10 .00 .00 .00 .00 e MET 10 .00 .00 .00 .00 e VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested P.mirabilis isolates

Table (A4): Comparison of inhibition zones of Myrtus communis L leaves methanol extract andstandard antibiotics references P.mirabilis

212

Extract /Antibiotics N Mean Std. Minimum Maximum Deviation Myrtus communis L 12 24.50b 4.73862 15.00 30.00 leaves methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 14.40c 7.64 .00 20.0 CIP 10 37.80a 3.04 30.0 40.0 CN 10 18.80d 1.22 16.0 20. e CTX 10 .00 .00 .00 .00 e MET 10 .00 .00 .00 .00 e VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested P.mirabilis isolates

(A6): Comparison of inhibition zones of Myrtus communis L leaves methanol extract and standard antibiotics references against K.pneumonia

213

Extract /Antibiotics N Mean Std. Minimum Maximum Deviation M. communis leaves 24 19.10a 6.62 .00 29.50 methanol extract AMC 10 .00b .00 .00 .00 CAZ 10 .00 .00 .00 .00 CIP 10 19.05a .83166 18.00 20.00 b CN 10 .00 .00 .00 .00 b CTX 10 .00 .00 .00 .00 b MET 10 .00 .00 .00 .00 b VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested K.pneumonia isolates

Table (A7): Comparison of inhibition zones of Myrtus communis L leaves methanol extract and standard antibiotics references against Ps.aeruginosa

Extract / Antibiotics N Mean Std. Minimum Maximum

214

Deviation Myrtus communis L 14 20.64b 2.77 17.50 27.00 leaves methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 10.40d 5.52 .00 14.00 CIP 10 39.30a 1.33 37.00 41.00 CN 10 16.80c 9.29 .00 24.00 e CTX 10 .00 .00 .00 .00 e MET 10 .00 .00 .00 .00 e VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested Ps.aeruginosa isolates

Table (A8): Comparison of inhibition zones of Myrtus communis L leaves methanol extract andstandard antibiotics references against E.coli

Extract / Antibiotics N Mean Std. Minimum Maximum

215

Deviation Myrtus communis L 8 19.87b 1.40 18.50 22.50 leaves methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 18.50b 1.08 17.00 20.00 CIP 10 31.40a 1.77 28.00 34.00 e CN 10 .00 .00 .00 .00 e CTX 10 .00 .00 .00 .00 e MET 10 .00 .00 .00 .00 VA 10 4.80c 4.23 .00 9.01 Sig ** N = Number of tested E.coli isolates

Table (A9): Comparison of inhibition zones of Myrtus communis L leaves methanol extract and standard antibiotics references againstS.aureus

216

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation Myrtus communis L 31 25.00b 6.08 9.00 34.00 leaves methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 .00e 00 .00 .00 CIP 10 31.80a 2.89 28.00 36.00 CN 10 19.60c 1.77 17.00 23.00 e CTX 10 .00 00 .00 .00 e MET 10 .00 00 .00 .00 VA 10 19.10dc 1.59 16.00 21.00 Sig. ** N = Number of tested S.aureus isolates

Table (A10): Comparison of inhibition zones of P.lentiscus fruits extractsand standard antibiotics references against A.baumanii

Extract / Antibiotics N Mean Std. Minimu Maximu Deviation m m

217

P.lentiscus L. fruits 10 13.90a 7.62 .00 22.00 methanol extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 2.00b 3.13 .00 7.00 c CN 10 .00 .00 .00 .00 c CTX 10 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 c VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested A.baumanii isolates

Table (A11): Comparison of inhibition zones of P.lentiscus fruits extracts andstandard antibiotics references againse E.cloacae

218

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation P.lentiscus fruits 8 9.50ba 10.19 .00 20.00 methanol extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 11.40a 9.86 .00 21.00 c CN 10 .00 .00 .00 .00 c CTX 9 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 c VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested E.cloacea isolates

219

Table (A12): Comparison of inhibition zones of P.lentiscus fruits extracts and standard antibiotics references against clinical E.coli

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation P.lentiscus fruits 8 13.3750 11.32554 .00 27.00 methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 18.50b 1.08 17.00 20.00 CIP 10 31.40a 1.77 28.00 34.00 e CN 10 .00 .00 .00 .00 e CTX 10 .00 .00 .00 .00 e MET 10 .00 .00 .00 .00 VA 10 4.80c 4.23 .00 9.01 Sig ** N = Number of tested E.coli isolates

220

Table (A13): Comparison of inhibition zones of P.lentiscus fruits extracts and standard antibiotics references against K.pneumonia

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation P.lentiscus fruits 24 12.13b 9.17 .00 25.00 methanol extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 19.05a .836 18.00 20.00 c CN 10 .00 .00 .00 .00 c CTX 10 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 c VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested K.pneumonia isolates

221

Table (A14): Comparison of inhibition zones of P.lentiscusmethanol fruits extracts and standard antibiotics references against Ps.aeruginosa

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation P.lentiscus fruits 14 6.85dc 8.27 .00 17.00 methanol extract AMC 10 .00 .00 .00 .00 CAZ 10 10.40c 5.52 .00 14.00 CIP 10 39.30a 1.33 37.00 41.00 CN 10 16.80b 9.29 .00 24.00 CTX 10 .00e .00 .00 .00 e MET 10 00 .00 .00 .00 e VA 10 00 .00 .00 .00 Sig ** N = Number of tested Ps.aeruginosa isolates

222

Table (A15): Comparison of inhibition zones of P.lentiscus methanol fruits extracts and standard antibiotics references against P.mirabilis

Extract / Antibiotics N Mean Std. Minimu Maximu Deviation m m P.lentiscus fruits 5 4.80d 10.73 .00 24.00 methanol extract AMC 10 .00e .00 .00 .00 CAZ 10 14.40c 7.64 .00 20.00 CIP 10 37.80a 3.04 30.00 40.00 CN 10 18.80b 1.22 16.00 20.00 CTX 10 .00e .00 .00 .00 e MET 10 .00 .00 .00 .00 e VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested P.mirabilis isolates

Table (A16): Comparison of inhibition zones of P.lentiscus methanolfruits extracts and standard antibiotics references against clinical S.aureus

223

Extract / N Mean Std. Minimum Maximum Antibiotics Deviation P.lentiscus fruits 31 15.42c 8.85 .00 26.00 methanol extract AMC 10 .00d .00 .00 .00 CAZ 10 .00d .00 .00 .00 CIP 10 31.80a 2.89 28.00 36.00 CN 10 19.60b 1.77 17.00 23.00 CTX 10 .00d .00 .00 .00 MET 10 .00d .00 .00 .00 VA 10 19.10b 1.59 16.00 21.00 Sig ** N = Number of tested P.mirabilis isolates

224

Table (A17): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against A.baumanii

Extract /Antibiotics N Mean Std. Minimum Maximum Deviation Salvia fruticosa bark 10 12.50a 10.83 .00 23.00 methanol extract AMC 10 .00b .00 .00 .00 CAZ 10 .00b .00 .00 .00 CIP 10 2.40b 3.13 .00 7.00 b CN 10 .00 .00 .00 .00 b CTX 10 .00 .00 .00 .00 b MET 10 .00 .00 .00 .00 b VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested A.baumanii isolates

Table (A18): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against Ps.aeruginosa

225

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation Salvia fruticosa 8 17.5b 4.03 12.00 25.00 bark methanol extract AMC 10 .00d .00 .00 .00 CAZ 10 10.4c 5.52 .00 14.00 CIP 10 39.3a 1.33 37.00 41.00 CN 10 16.8b 9.29 .00 24.00 d CTX 10 .00 .00 .00 .00 d MET 10 .00 .00 .00 .00 d VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested Ps.aeruginosa isolates

226

Table (A19): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against P.mirabilis

Extract / Antibiotics N Mean Std. Minimu Maximu Deviation m m Salvia fruticosa 5 12.40c 12.85 .00 30.0 bark methanol extract AMC 10 .00d .00 .00 .00 CAZ 10 14.40c 7.67 .00 20.0 CIP 10 37.80a 3.04 30.00 40.0 CN 10 18.80b 1.22 16.00 20.0 d CTX 10 .00 .00 .00 .00 d MET 10 .00 .00 .00 .00 d VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested P.mirabilis isolates

227

Table (A20): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against E.cloacae

Extract / Antibiotics N Mea Std. Minimum Maximum n Deviation Salvia fruticosa 8 11.5 10.823 .00 30.00 bark methanol 0a extract AMC 10 .00b .000 .00 .00 CAZ 10 .00b .000 .00 .00 CIP 10 11.4 9.868 .00 21.00 0a b CN 10 .00 .000 .00 .00 b CTX 9 .00 .000 .00 .00 b MET 10 .00 .000 .00 .00 b VA 10 .00 .000 .00 .00 Sig. ** N = Number of tested P.mirabilis isolates

228

Table (A21): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against E.coli

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation Salvia fruticosa 5 17.00b 4.35 12.00 22.00 bark methanol extract AMC 10 .00d .00 .00 .00 CAZ 10 18.50b 1.08 17.00 20.00 CIP 10 31.40a 1.77 28.00 34.00 d CN 10 .00 .00 .00 .00 d CTX 10 .00 .00 .00 .00 d MET 10 .00 .00 .00 .00 VA 10 4.80c 4.23 .00 9.01 Sig. ** N = Number of tested E.coli isolates

229

Table (A22): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against K.pneumonia

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation Salvia fruticosa 24 13.87b 10.0 .00 30.00 bark methanol extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 19.05a .831 18.00 20.00 c CN 10 .00 .00 .00 .00 c CTX 10 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 c VA 10 .00 .00 .00 .00 Sig. ** N = Number of tested K.pneumonia isolates

230

Table (A23): Comparison of inhibition zones of Salvia fruticosa bark methanol extract and standard antibiotics references against S.aureus

Extract / Antibiotics N Mean ± Std. Minimum Maximum Deviation Salvia fruticosa 23 19.47b 4.541 12.00 30.00 bark methanol extract AMC 10 .00c .00 .00 .00 CAZ 10 .00c .00 .00 .00 CIP 10 31.80a 2.89 28.00 36.00 CN 10 19.60b 1.77 17.00 23.00 c CTX 10 .00 .00 .00 .00 c MET 10 .00 .00 .00 .00 VA 10 19.10b 1.59 16.00 21.00 Sig. ** N = Number of tested S.aureus isolates

231

Table (A24): Comparison of inhibition zones of Capparis spinosa L bark chloroform extract and standard antibiotics references on S.aureus

Extract / N Mean Std. Minimum Maximum Antibiotics Deviation C.spinosab 31 16.0b 10.5 .00 28.60 ark chloroform extract AMC 10 .00d .00 .00 .00 CAZ 10 .00d .00 .00 .00 CIP 10 31.8a 2.89 28.0 36.0 CN 10 19.6b 1.77 17.0 23.0 CTX 10 .00d .00 .00 .00 MET 10 .00d .00 .00 .00 VA 10 19.10b 1.59 16.0 21.0 Sig. ** N = Number of tested S.aureus isolates

232

Table(A25): Comparison of inhibition zones of Capparis spinosa L leaves methanol extract and standard antibiotics references against S.aureus

Extract / Antibiotics N Mean Std. Minimum Maximum Deviation C.spinosa L Methanol 31 4.64c 6.86 .00 16.00 leaves extract AMC 10 .00 .00 .00 .00 CAZ 10 .00 .00 .00 .00 CIP 10 31.8a 2.89 28.00 36.00 CN 10 19.6b 1.77 17.00 23.00 CTX 10 .00 .00 .00 .00 MET 10 .00 .00 .00 .00 VA 10 19.1b 1.59 16.00 21.00 Sig. ** N = Number of tested S.aureus isolates

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Table (A26): Antifungal activity of methanol extracts of tested extractsagainst Aspergillus niger ATCC 9763

Mold Mean of inhibition zone of tested extracts Aspergillus Pistacia Capparis Salvia Myrtus niger / lentiscus spinosa fruticosa communis Tested fruits leaves bark leaves extracts Aspergillus 14mm 11mm 11mm 11mm niger

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Table (A27): Pharmacological behavioral screening of Capparis spinosamethanol leaves extract treated rats.

Zero time During 1st During 2nd During 3rd Notes Behavioral hour hour hour parameters No. of total No. of total No. of total No. of 6 rats 6 rats 6 rats total 6 rats Excitation - - - - Convulsion - - - - Grasp - - - - Aggressiveness - - - - Spasm - + + Mild (4rats out (4rats out of spasm of six) six) Writhing - - - - Fatigability - - - - Itching - - - - Diarrhea - - - - Lacrimation - - - - Red tears - - - - Sedation light - - - - Sedation heavy - - - - Convulsion - - - - Push back legs - - - -

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Table (A28): Pharmacological behavioral screening of Pistacia lentiscus methanol fruits extract treated rats.

Zero time During 1st During 2nd During 3rd Notes Behavioral hour hour hour parameters No. of total No. of total No. of total No. of 6 rats 6 rats 6 rats total 6 rats Excitation - + (6) + (3) - Convulsion - - - - Grasp Normal(6) Normal(6) Normal(6) Normal(6) Aggressiveness - - - - Sapasm - + (all rats) +(all rats) - Expanded in 3 expanded 3rd hr. *Strong spasm every 10min.

Writhing - - + (3/6 rats) +(3/6 rats) Fatigability - - - - Itching - - - - Diarrhea - - - - Lacrimation - - - - Red tears - - - - Sedation light - - - - Sedation heavy - - - - Convulsion - - - - Push back legs - + (2/6 rats) + (2/6 rats) -

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Table (A29): Constituents of Krebs solution, Tyrode solution, D Jalon’s solution and Frog solution

Solution / Composition Krebs Tyrode De Jalon’s Frog solution solution solution solution Sodium chloride Nacl 43.5gm 40gm 45gm 32.5gm Sodium bicarbonate 10.5gm 5.0gm 2.5gm 1.0gm NaHCO3 D Glucose 10gm 5.0gm 2.5gm - Mono potassium 0.8gm - - - phoaphate KH2PO

Mono sodium - 0.25gm - - phosphate NaH2PO4 Potassium chloride KCl 1.8gm 1.0gm 2.1gm 0.7gm Magnesium chloride - 0.5gm - - MgCl2 Calcium chloride 1.85gm 1.32gm 0.4gm 0.79gm dihydrate CaCl2.2H2O

( Ian Kitchen, 1984)

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Some Preparations

Ferric chloride:

3 g of ferric chloride dissolved in 100 ml Methanol.

Gelatin salt reagent:

10 g of Gelatin powder (BDH chemical LTD England) dissolved in 100 ml hot distilled water.

Mayer’ reagent:

1. 36 g of mercuric chloride (BDH chemical LTD England) dissolved in 60 ml of distilled water. 2. 5g of potassium iodide (Loba Chemia India) dissolved in 10ml of distilled water. Solution (1) and (2) mixed and diluted to 100 ml with distilled water.

Valser' reagent:

10 g of potassium iodide (dissolved in 100 ml of distilled water. Mercuric iodide (BDH chemical LTD England) was added til the solution became saturated. Excess of mercuric iodide was removed out.

1% Potassium hydroxide in Methanol

1 g of Potassium hydroxide dissolved in 100 ml Methanol.

0.5N KOH:

0.89 g of potassium hydroxide dissolved in 100 ml distilled water.

Sodium carboxy methyl cellulose 3%:

Slowly, 3g of Sodium carboxy methyl cellulose was added to 100 ml hot distilled water (65 C0) and continuous vigorously mixed till came to room temperature.

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Materials

Chemicals

Acetic anhydride(Sharlu Spain)

Acetylcholine (Sigma Aldrich)

Anhydrous sodium sulphate (Sigma Aldrich)

Atropine (Sigma Aldrich)

Barium chloride dehydrate (Himedia)

Calcium chloride dehydrate (Himedia)

Chloroform (S D Fine India)

Crystal violet (British Drug House, England)

D Glucose(SDFCI, India)

Dimethyl sulfoxide (SDFCI, India)

DPPH (Sigma Aldrich)

Emulsifying wax (Medex, England)

Ethanol (S D Fine India)

Ferric chloride (Sharlu Spain)

Formaldehyde solution (Central drug house, India)

Gelatin powder (BDH chemical LTD England)

5-hydroxy tryptamine(Sigma Aldrich)

Halothane (ICI Ltd., India)

HPLC Methanol (Sigma Aldrich)

Hydrochloric acid (British Drug House, England)

Hydrogen peroxide (Sigma Aldrich)

Hydroxide solution (S D Fine India)

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Iodine (Hopkins and William, England)

Immersion oil (British Drug House, England)

Isopropanolol (Sigma Aldrich)

Kovac’s reagent (Sigma Aldrich)

Lead acetate(BDH chemical LTD England)

Liquid paraffin BP (Bell, sons & Co., England)

Magnesium chloride (Loba Chemia India)

Methanol (S D Fine India)

Mono potassium phoaphate (Loba Chemia India)

Mono sodium phosphate (Loba Chemia India)

Nor epeniphrine (Sigma Aldrich)

Normal saline (Ain Sudan company, Sudan)

Oxidase ( John baker Inc., USA)

Petroleum ether (SDFCI, India)

Phentolamine (Sigma aldrich)

Potassium hydroxide(Loba Chemia India)

Propranolol HCL (Sigma Aldrich)

Propyl Gallate (Sigma Aldrich)

Safranin red (British Drug House, England)

Sodium bicarbonate( Scharlau, Spain)

Sodium carboxy methyl cellulose salt (Sigma aldrich)

Sodium chloride (Scharlau, Spain)

Sulfo Rhodamine B stain (Sigma Aldrich)

Sulfuric acid (Sigma Aldrich)

Trichloroacetic acid (Sigma Aldrich)

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Chemotherapeutic Agents

Antibiotic Discs

Amoxicillin 20µg + Clavulanic acid 10µg (AMC) Disc

Cefotaxime 30µg (CTX) Disc

Ceftazidime 30 µg (CAZ) Disc

Ciprofloxacin 30 µg (CIP) Disc

Gentamicin 10 µg (CN) Disc

Vancomycin 30 µg (VA) Disc

All have been bought from Bioanalyse@ YSE Tibbi Malzemeler San. Ve Tic.Ltd. Expire dates of all reference drugs were 10 - 18 months valid after the date of the assay.

Antibiotic Ointments

Tetracycline ointment 3% ointment (Private Pharmacy)

Fucidin ointment 2% (Private Pharmacy)

Culture Media

DNAse media (Himedia)

Motility Indol Urea medium (Himedia)

Mueller Hinton agar (Himedia)

Nutrient agar (Himedia)

RPMI 1640 with glutamine (Plain media) (Gibco-Brl, Life technology)

Simmon citrate agar medium (Himedia)

Sabouraud dextrose agar (Micro Mister)

Triple sugar Iron agar medium (Himedia)

Serum

Fetal bovine serum (Bio West. France)

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Equipment and Instruments

American digital EazyLyte Na/ K analyzer instrument (American)

Autoclave (Griffen and George Ltd, England)

Automatic adjustable pipette (Avis-El-Reooba)

Balance (Shuangquan, China)

BD Phoenix™ automated identification and susceptibility testing system (Phoenix 100) (UK)

Centrifuge (Braun, Centrifuge PLC series)

Freeze Drier (Edwards 2507) (England)

GC/MS-QP2010-Ultra (Japans ’Simadzu Company) with serial number 020525101565SA and capillary column (Rtx-5ms-30m×0.25 mm×0.25µm).

Glass ware (Rasotherm, GDR)

Harvard Universal Oscillograph recorder(Harvard Apparatus Limited UK).

Hot Air Oven (Nuve, Turkey)

Incubator (Heraeus, Germany)

Manadary BC-6800 (Hematology analyser) (Chnia)

Mandary BS-480 (Biochemistry analyzer) (China)

Microscop (Olympus, Japan)

Microwave Histo-Processor System (Germany)

Tested Organisms

Bacillus subtilis NCTC 8236 Staphylococcus aureus ATCC 25923 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853

Aspergillus niger ATCC 9763 Candida albicans (ATCC 7596

The standard organisms were obtained from the National Collection of Type Culture (NCTC), Colindale, England and the American Type Culture Collection (ATCC), Rockville, Maryland, USA.

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