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2018 In vivo, in vitro micropropagation and chemical characterisation of medicinal compounds in chamomile and yarrow species (Asteraceae)

Mahmood, Banaz http://hdl.handle.net/10026.1/10662

University of Plymouth

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In vivo, in vitro micropropagation and chemical characterisation of medicinal compounds in chamomile and yarrow species (Asteraceae)

by Banaz Mahmood

A thesis submitted to the University of Plymouth in partial fulfilment for the degree of

DOCTOR OF PHILOSOPHY

School of Marine and Biological Sciences Faculty of Science and Engineering

August 2017

Acknowledgements

I would like to thank God for allowing me to complete this thesis and for giving me this opportunity in life. I would not have been able to complete my thesis without the assistance and guidance of my supervisor team, help from my family and the unwavering support of my husband.

First and foremost, I would like to express my immense gratitude to the Director of my study, Dr.

Stephen Burchett, he has been a great source of moral support since I stared the project whenever I had needed it. Special thanks go to Dr. Richard Billington and Dr. Michael Foulkes for being my 2nd and 3rd supervisors, both of whom have encouraged me to stay and continue in the lab. Thank you to Dr. Michael Foulkes for his guidance in phytochemical analysis and Dr.

Richard Billington for giving me his experience and knowledge in bioassay work. They have all gave me unconditional support during the difficult times in progressing through the writing up of this thesis.

I will always be very thankful to my sponsor the Ministry of higher education, Kurdistan Regional

Government (KRG/ HCDP programme), for providing me with this opportunity and for giving me a chance and funding. I offer my gratitude and deepest appreciation to all the technicians on the ground, first, third and fifth floors of the Davy building/ Plymouth University, they have always been willing to give their support and best suggestions.

I would also like to extend my thanks to Michael Whiffen from the Skardon garden for his kindness and support; he has been an amazing source of support throughout my glasshouse work. Special acknowledgement goes to my husband; he always encouraged me to be honoured and to see everything positively. Also, my brother and sisters for their ongoing support, best wishes and encouragement at all times. Finally, I want to thank those who worked with me and supported me over the last four years.

Dedication

I dedicate this thesis to

My lovely husband, Dear brother and sisters, Also, to my wonderful little boy (Zad) I am really thankful for having all of you in my life……..

AUTHOR’S DECLARATION

At no time during the registration for the degree of Doctoral of Philosophy has the author been registered for any other University award without prior agreement of the Doctoral College Quality Sub- Committee.

Work submitted for this research degree at the University of Plymouth has not formed part of any other degree either at the University of Plymouth or at another establishment.

This study was financed with the aid of studentship from the Ministry of Higher Education/ Kurdistan Regional Government.

A programme of advanced study was undertaken, which included Postgraduate Research Skills and Methods (BIO5124) and Plant Biotechnology (BIOL301).

Publications:

 Ahmed, B., Jamal, B. (2013). Determination of Flavonoids in the Leaves of Hawthorn (Crataegus Azarolus) of Iraqi Kurdistan Region by HPLC Analysis. International Journal of Bioscience, Biochemistry and Bioinformatics (IJBBB), Vol. 3, No. 1.  Mahmood, B. (2016). Bioactive compounds identification and antibacterial activity of the essential oil of chamomile and yarrow species. Plant Physiol Pathol, Vol 4, Issue 4 (Suppl).  Mahmood, B. J., Rihan, H. Z., Foulkes, M., Burchett, S. (2017). The effect of drought on phytochemical active compounds content in chamomile and yarrow. Agricultural Research & Technology: Open Access Journal, Vol 12, Issue 4.

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Presentations at conferences:

1. Oral presentations

 Extraction and chemical characterisation of medicinal compounds found in chamomile and yarrow species. CARS symposium, Cornwall College Newquay, 12th Jun 2015.  Characterisation of phytochemical compounds found in Matricaria chamomilla and Achillea millefolium using HPLC and GC-FID analysis. The Postgraduate Society short conference, University of Plymouth, 2nd December 2015.  Evaluating the growth characteristics and production of medicinal compounds in chamomile and yarrow using HPLC and GC-FID technologies. Lunch meeting conference. University of Plymouth, 20th January 2016.

2. Poster presentations

 In vivo and in vitro micropropagation cultures, essential oil contents of chamomile and yarrow species. Iraqi day conference, University of Plymouth, 1st March 2014.  Micropropagation technique and bioactive chemical characterization of the essential oil of chamomile and yarrow species (Asteraceae). The postgraduate society annual conference, University of Plymouth, 24th March 2014.  In vitro seeds culture of chamomile and yarrow species using different plant hormones. The postgraduate society annual conference, University of Plymouth, 17th June 2014.  Study of chamomile and yarrow in vitro growth trials and, chemical extraction and characterization. 1st Student Symposium on Computational Biology and Life Sciences, University of South Wales, 8th October 2014.  Micropropagation protocols and HPLC chemical analysis of bioactive compounds found in chamomile and yarrow. 5th CARS Postgraduate Symposium at Eden Project. University of Plymouth, 19th November 2014.

 Extraction protocols and chemical identification of Matricaria chamomilla and Achillea millefolium medicinal compounds. The postgraduate society annual conference, University of Plymouth, 23th June 2015.  Bioactive compounds identification and antibacterial activity of the essential oil of chamomile and yarrow species (Asteraceae). 2nd Global Summit on Plant Science. London, UK. 6th -8th October 2016.

3. Exhibition

 The identification of bioactive compounds and antibacterial activity of the essential oil of chamomile and yarrow species. The postgraduate society annual conference, University of Plymouth, 5th December 2016. 1st place was awarded.

Word count of main body of thesis: 41,493 words, total: 49,547 words .

Signed ……………………………………….. Date ………………………………………….

Banaz Mahmood In vivo, in vitro micropropagation and chemical characterization of medicinal compounds in chamomile and yarrow species (Asteraceae) Abstract The Asteraceae family is frequently used to describe several medicinal plants which contain various phytochemical compounds including phenols, flavonoids and terpenoids. Among the Asteraceae family German chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) plants are extant species used in contemporary medicine. These phytochemical compounds have been traditionally used since ancient times in health care systems worldwide as a source of medicines. The use of micropropagation is essential to improve and increase these active compounds via plant tissue culture within a short period of time using the application of key plant growth regulators (PGRs). Furthermore, quantitative and qualitative analysis using high performance liquid chromatography- ultraviolet detector (HPLC-UV) and gas chromatography- flame ionisation detector (GC-FID) of potential medicinal compounds expressed by both chamomile and yarrow are important points. The protocol of in vitro shoots, roots and callus formation of chamomile and yarrow seeds culture were investigated using Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs). MS culture medium containing 0.5 mgL-1 IAA and 1.0 mgL-1 of GA3 were found to be the best culture medium for chamomile and yarrow seeds. In this project in vitro and in vivo growth rates of selected plant species were also investigated. In the earlier growth stages yarrow plants were found to grow much quicker than chamomile, while the yield of chamomile flowers was significantly (p ≤ 0.001) more than yarrow flowers. The phenolic, flavonoid and terpenoid compounds content of leaves and flowers of plants produced from both cultures were also studied. HPLC-UV analysis showed that chlorogenic acid, apigenin-7-O-glucoside and luteolin dominated as the main phenol and flavonoid compounds recovered in both in vitro and in vivo chamomile and yarrow cultures. However, GC-FID analysis indicated that farnesene and nerolidol were detected as the main terpenoid compounds present in the two culture conditions used to grow chamomile and yarrow plants. Moreover, this research examines how chamomile and yarrow plants can produce and improve their phytochemical compounds content not only under well-watered conditions but also under drought stress conditions. The main phenol and flavonoid compounds of chlorogenic acid, caffeic acid, apig-7-glucoside, umbelliferon and luteolin were found in chamomile and yarrow varieties grown under both well-watered and drought stress conditions using (HPLC-UV), however farnesene, nerolidol, chamazulene, α-(-)- bisabol and bisabolol oxide A were observed in the plant essential oils (EOs) using Soxhlet extraction and GC-FID analysis. The antibacterial activity of plant EOs was also investigated using disc diffusion and 96 well plates. In vivo chamomile EO showed the highest antibacterial activity against gram-positive and gram-negative bacteria strains. In addition, in vitro yarrow EO showed the greatest effect on the death of bacteria strains.

List of Contents

Acknowledgements ...... I Dedication ...... II Author’s Declaration ...... III Abstract ...... VI List of Contents ...... VIIII List of Tables ...... XIIII List of Figures ...... XIIIII List of Abbreviations ...... XIX Chapter One General introduction and literature review 1.1 General Introduction ...... 1 1.2 Literature review ...... 3 1.2.1 Historical use of medicinal plants in particular plants belonging to the Asteraceae family ...... 3 1.2.2 Plant taxonomy and botany...... 7 1.2.2.1 Chamomile (Matricaria chamomilla L.) ...... 8 1.2.2.2 Yarrow (Achillea millefolium L.) ...... 10 1.2.3 Molecular and Genetic Characterisation ...... 11 1.2.4 Commercial issues ...... 15 1.2.5 Medicinal plants and drought stress ...... 20 1.2.6 Plant tissue culture and micro-propagation technique ...... 23 1.2.7 Medicinal compounds or secondary metabolites identification from the Asteraceae family and their biochemical properties ...... 27 1.2.8 Anti-microbial activities ...... 34 1.3 Aims of study ...... 38 1.4 Objectives of study...... 39 Chapter two Chamomile and yarrow micro-propagation technique 2.1 Introduction ...... 40 2.2 Objectives ...... 42 2.3 General Materials and methods ...... 43

VII

2.3.1 Sample collection and preparation ...... 43 2.3.2 The effect of 6-Benzylaminopurine (BAP) and Kinetin on callus shoots and roots formation of in vitro chamomile and yarrow seeds culture ...... 43 2.3.3 Seed medium culture preparation using different PGRs ...... 44 2.3.4 Chamomile and yarrow plants production from nutrient agar ...... 45 2.3.5 Statistical analysis ...... 45 2.4 Results ...... 46 2.4.1 Callus, shoot and root formations ...... 46 2.4.2 In vitro chamomile and yarrow seeds culture using different plant hormones (PGRs) ...... 49 2.5 Discussion ...... 57 2.6 Conclusion ...... 61 Chapter three Characterisation of the growth and development of in vitro and in vivo seeds culture 3.1 Introduction ...... 62 3.2 Objectives ...... 63 3.3 Materials and methods ...... 64 3.3.1 The effect of light and temperature on the germination of chamomile and yarrow 64 3.3.2 In vivo culture of winter and summer planting seasons ...... 64 3.3.3 In vitro culture ...... 68 3.3.4 Statistical analysis ...... 68 3.4 Results ...... 70 3.4.1 The influence of light regimes and different temperatures on plant seed germination ...... 70 3.4.2 In vitro and in vivo winter and summer growth rate characterisations ...... 74 3.5 Discussion ...... 85 3.6 Conclusion ...... 88 Chapter four The extraction of bioactive phytochemicals from chamomile and yarrow plants with their separation and measurement using HPLC-UV and GC-FID 4.1 Introduction ...... 90 4.2 Objectives ...... 92 4.3 General Materials and methods ...... 93

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4.3.1 In vitro and in vivo leaves preparation and shaking extraction ...... 93 4.3.2 Standards and reagents ...... 95 4.3.3 HPLC analysis ...... 96 4.3.4 Calibration curves ...... 96 4.3.5 Preparation of flowers ...... 97 4.3.6 In vitro and in vivo flowers preparation and Soxhlet extraction ...... 97 4.3.7 Standards and reagents ...... 98 4.3.8 GC-FID analysis ...... 98 4.3.9 Calibration curves ...... 99 4.3.10 Statistical analysis ...... 99 4.4 Results ...... 100 4.4.1 Methanol and hexane extraction ...... 100 4.4.2 In vitro and in vivo chamomile and yarrow leaves HPLC analysis ...... 101 4.4.3 In vitro and in vivo chamomile and yarrow flowers GC-FID analysis ...... 107 4.5 Discussion ...... 112 4.6 Conclusion ...... 114 Chapter five A study of the phytochemical active compounds content of chamomile and yarrow varieties based on a drought stress regime 5.1 Introduction ...... 116 5.2 Objectives ...... 118 5.3 Materials and methods ...... 119 5.3.1 Characterization of the soil oven drying capacity in the presence of chamomile and yarrow plants...... 119 5.3.2 The measurement of soil moisture content ...... 120 5.3.3 Growth conditions ...... 122 5.3.4 Sample collection for final chemical analysis ...... 127 5.3.5 Sample extraction and identification ...... 128 5.3.6 Statistical analysis ...... 129 5.4 Results ...... 129 5.4.1 Soil oven drying method and the measurement of soil moisture content ...... 129 5.4.2 Chamomile and yarrow well-watered (WW) and droughted (D) HPLC-UV analysis..132

5.4.3 Chamomile and yarrow well-watered (WW) and droughted (D) GC-FID analysis…144 5.5 Discussion ...... 151 5.6 Conclusion ...... 154 Chapter six Antibacterial activity of the essential oil of chamomile and yarrow flower heads ...... 121 6.1 Introduction………………… ……………………………………………….……………..156 6.2 Objectives ...... 158 6.3 Materials and methods ...... 158 6.3.1 Plant samples ...... 158 6.3.2 Culture method ...... 158 6.3.3 Antibacterial activity ...... 159 6.3.3.1 Agar disc diffusion assay ...... 159 6.3.3.2 Measurement of growth curves and bacteria death using microplate reader 159 6.3.4 Statistical analysis ...... 160 6.4 Results ...... 160 6.4.1 The measurement of bacteria strains growth using disc diffusion method ...... 160 6.4.2 Determination of the effect of plant EO on the death of bacteria strains using microplate reader ...... 164 6.5 Discussion ...... 167 6.6 Conclusion ...... 170 Chapter seven General discussion and future work 7.1 General discussion ...... 171 7.2 Future work and recommendations ...... 177 7.2.1 Tissue culture protocols: impact of gene expression and medicinal compounds . 177 7.2.2 Molecular study and genetic characterization ...... 178 7.3 General conclusions……………………………………………………………………………………………………………178 References……………………………………………………………………………………….181 Appendices Appendix 1: Data related to chapter 2, African violet medium preparation ...... 196 Appendix 2: Data related to chapter 4 and 5 ...... 197 Appendix 3: Data related to chapter 4, HPLC-UV chromatograms using hexane extraction209

Appendix 4: Data related to chapter 5, Soil moisture content calibration curves ...... 210 Appendix 5: Data related to chapter 6 ...... 211 Appendix 6: Research publication ...... 213 Appendix 7: Professional membership ...... 213 Appendix 8: Conference attended ...... 213 Appendix 9: Training courses and workshops ...... 215

List of Tables

Table 1.1 Historical uses of medicinal plants……………………………………………………6

Table 1.2 Classification of chamomile species...... 8

Table 1.3 Classification of yarrow Species...... 10

Table 1.4 Type of molecular markers of some medicinal plants...... 15

Table 1.5 The effect of sowing dates on chamomile yields...... 20

Table 1.6 The role of plant secondary products of Asteracea family...... 31

Table 2.1 The percentages of callus, root and shoot formation of chamomile and yarrow seeds after 2-3 weeks of culture, data are presented as mean and standard error...... 46

Table 3.1 Growth stages description of the chamomile and yarrow plant growth cycles. .. 67

Table 3.2 Summary of the effect of the temperature and light regimes on the percentage of chamomile and yarrow germinated seeds...... 71

Table 4.1 Phenolic and flavonoid chemical standards detected by HPLC at different retention times, peak areas and the limit of detection (LOD) value...... 105

Table 4.2 Terpenoid chemical standards detected by GC-FID at different retention times, peak areas and the limit of detection (LOD) value...... 110

Table 6.1 The percentages of the number of bacteria strains killed under the effect of pure, 1:10 and 1:100 in vivo and in vitro chamomile and yarrow EOs within 1 hour...... 166

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List of Figures

Figure 1.1 Botanical comparison of German and Roman chamomile...... 9

Figure 1.2 Botanical descriptions of Achillea millefolium L. (Yarrow) plant...... 11

Figure 2.1 Experimental design of chamomile and yarrow in vitro seeds culture (five seeds per treatment) in MS medium containing different plant hormones...... 45

Figure 2.2 The effect of BAP and Kin (0.1mg L-1 BAP plus 0.2 mg L-1 Kin) on chamomile and yarrow callus development (LSD value for plant sp.= 4.27, days= 9.07 and for plant sp.*days= 12.82). Data are presented as mean ± SE of three replicates. (* = interaction between variables)………………………………………………………………………………..47

Figure 2.3 The effect of BAP and Kin (0.1mg L-1 BAP plus 0.2 mg L-1 Kin) on chamomile and yarrow roots formation (LSD value for plant sp. = 4.73, days= 10.04 and for plant sp.*days= 14.20). Data are presented as mean ± SE of three replicates...... 48

Figure 2.4 The effect of BAP and Kin (0.1mg L-1 BAP plus 0.2 mg L-1 Kin) on chamomile and yarrow shoots production (LSD value for plant sp. = 5.48 and for days= 11.61). Data are presented as mean ± SE of three replicates...... 49

Figure 2.5 Micropropagation developments of in vitro chamomile and yarrow seeds. A: Chamomile growth stages, B: Yarrow growth stages. A1 & B1: callus and multiple shoots production1-2 weeks after culture on MS medium containing 0.1mg L-1 BAP and 0.2 mg L-1 Kin. A2 & B2: root production from shoots and soil transfer 2-3 weeks after culture. A3 & B3: Plantlets potting up and growth developments in the glasshouse 2-4 months after culture.……51

Figure 2.6 Effect of BAP concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp. = 5.34, BAP conc. = 7.56, plant sp.*conc. of BAP= 10.69 and control = 8.86). Data are presented as means ± SE of three replicates for each concentration...... 52

Figure 2.7 Effect of Kin concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 5.24, Kin conc. = 7.41 and plant sp.*conc. of Kin=10.47). Data are presented as means±SE of three replicates for each concentration...... 52

Figure 2.8 Effect of IAA concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 6.21, IAA conc. = 8.78 and plant sp.*conc. of IAA= 12.42). Data are presented as means ± SE of three replicates for each concentration...... 54

Figure 2.9 Effect of NAA concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 9.40). Data are presented as means ± SE of three replicates for each concentration...... 54

Figure 2.10 Effect of 2, 4-D concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 5.79, 2,4-D conc. = 8.20 and plant sp.*conc. of 2,4-D = 11.59). Data are presented as means ± SE of three replicates for each concentration…………………………56

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Figure 2.11 Effect of GA3 concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 7.32 and GA3 conc.= 10.35). Data are presented as means ± SE of three replicates for each concentration...... …..56

Figure 3.1 Chamomile and yarrow plants production from in vitro seeds culture, callus, shoots and roots formations in MS medium supplemented with 0.5 mgL-1 of IAA (chamomile seeds) and 1.0 mgL-1 of GA3 (yarrow seeds)...... 69

Figure 3.2 The percentage of chamomile (RH/SS) germinated seeds at 18°C, 8h light: 16 h dark. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed sources= 6.78)...... 72

Figure 3.3 The percentage of chamomile (RH/SS) germinated seeds under 22°C and 24 h light. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed sources= 5.94)………………………………………………………………………..72

Figure 3.4 The percentage of yarrow (RH/SG) germinated seeds at 18°C, 8h light: 16 h dark. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed source= 7.05)...... 73

Figure 3.5 The percentage of yarrow (RH/SG) germinated seeds under 22°C and 24 h light. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed sources= 4.94)...... 73

Figure 3.6 Average temperature and relative humidity of in vivo chamomile and yarrow grown into the glasshouse...... 75

Figure 3.7 In vitro chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension)...... 76

Figure 3.8 In vivo winter chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension)...... 77

Figure 3.9 In vivo summer chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension)...... 78

Figure 3.10 in vitro chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation...... 79

Figure 3.11 In vivo winter chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation...... 80

Figure 3.12 In vivo summer chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation...... 81

Figure 3.13 In vitro chamomile (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering...... 82

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Figure 3.14 In vivo winter (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering...... 83

Figure 3.15 In vivo summer (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering...... 84

Figure 4.1 Methanol extraction procedures of in vitro and in vivo chamomile and yarrow leaves: (A) freeze drying (B) filtration process (C) rotary evaporation of samples (D) bulking samples (E) final extracted samples...... 95

Figure 4.2 Soxhlet extraction procedures of in vitro and in vivo chamomile and yarrow flowers...... 98

Figure 4.3 The effect of methanol and hexane solvent using Soxhlet and shaking extraction on the amount of final extract (LSD value for plant sp.= 0.04, extraction process= 0.05 and for plant sp.*extraction process= 0.07). Data are presented as mean ± SE of three replicates. .... 101

Figure 4.4 The HPLC-UV chromatogram of phenol and flavonoid compounds content of in vivo chamomile leaves detected at 256 nm...... 102

Figure 4.5 Standard calibration curve for chlorogenic acid polynomial regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent...... 102

Figure 4.6 HPLC-UV analyses of selected phenol and flavonoid compounds found in in vivo and in vitro chamomile leaves (LSD value for compounds= 3.52, culture conditions = 2.22 and for compounds *culture conditions = 4.97). Data are presented as mean ± SE of three replicates...... 106

Figure 4.7 HPLC- UV analyses of selected phenol and flavonoid compounds found in in vivo and in vitro yarrow leaves (LSD value for compounds = 2.10, and culture conditions = 1.71). Data are presented as mean ± SE of three replicates...... 106

Figure 4.8 Standard calibration curve for bisabolol oxide A linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent...... 107

Figure 4.9 GC-FID analyses of the selected essential oil compounds of in vivo chamomile flowers extracted by methanol using Soxhlet extraction...... 109

Figure 4.10 GC-FID analyses of selected terpenoid compounds found in in vivo and in vitro chamomile flowers essential oil (LSD value for compounds= 20.25, culture conditions = 16.53 and for compounds *culture conditions = 28.64). Data are presented as mean ± SE of three replicates...... 111

Figure 4.11 GC-FID analyses of selected terpenoid compounds found in in vivo and in vitro yarrow flowers essential oil (LSD value for compounds = 2.54 and compounds *culture conditions = 3.59). Data are presented as mean ± SE of three replicates...... 111

Figure 5.1 Soil drying process in the glasshouse and oven at 105°C containing chamomile and common yarrow plants...... 121

Figure 5.2 Chamomile and yarrow varieties growth stages and flowering under well-watered and drought stress...... 123

Figure 5.3 Average temperature and relative humidity in the glasshouse used for well watered and drought experiment...... 125

Figure 5.4 The final stage of vegetative growth under the effect of drought stress...... 126

Figure 5.5 Chamomile and yarrow leaves and flowers freeze drying samples for phytochemical bioactive compounds content analysis by High-performance liquid chromatography-ultraviolet (HPLC-UV) and Gas Chromatography-Flame Ionisation Detector (GC- FID)...... 127

Figure 5.6 Reduction in the soil dry mass in the presence of chamomile (Ch) and yarrow (Y) plants at 105 °C oven drying over time...... 130

Figure 5.7 Determination of soil moisture (MC) content of the pots containing chamomile (Ch) plants...... 131

Figure 5.8 Determination of soil moisture content (MC) of the pots containing common (Y1) and summer berries yarrow (Y2) plants...... 131

Figure 5.9 The main phenol and flavonoid compounds isolated from chamomile under well- watered (Ch WW) and drought stress (Ch D) conditions: chlorogenic acid LSD values (time=131.2 and growth conditions*time=185.5) caffeic acid LSD values (growth conditions= 2.8, time= 4.4 and growth conditions*time= 6.2) apigenin -7-glucoside LSD values (growth conditions= 24.4, time= 38.6 and growth conditions*time= 54.5) umbelliferone LSD values (growth conditions= 1.1, time= 1.7and growth conditions*time= 2.4). Data are presented as mean ± SE of three replicates...... 135

Figure 5.10 The HPLC- UV chromatogram of phenol and flavonoid compounds content of chamomile leaves detected at 256 nm under drought stress conditions...... 136

Figure 5.11 The main phenol and flavonoid compounds isolated from common yarrow under well- watered (Y1 WW) and drought stress (Y1 D) conditions: caffeic acid LSD values (growth conditions= 2.4, time= 3.8 and growth conditions*time= 5.4) apigenin -7-glucoside LSD values (growth conditions= 9.2, time= 14.6 and growth conditions*time= 20.6) umbelliferone LSD values (time= 6.9 and growth conditions*time= 9.8) luteolin LSD values (time= 3.8 and growth conditions*time = 5.3). Data are presented as mean ± SE of three replicates...... 139

Figure 5.12 The main phenol and flavonoid compounds isolated from summer berries yarrow under well- watered (Y2 WW) and drought stress (Y2 D) conditions: caffeic acid LSD values (time= 7.2) apigenin-7-glucoside LSD values (growth conditions= 1.4, time= 2.2 and growth conditions*time= 3.1) umbelliferone LSD values (time= 9.9 and growth conditions*time = 13.9) luteolin LSD values (growth conditions= 1.0 , time= 1.6 and growth conditions*time= 2.3). Data are presented as mean ± SE of three replicates...... 142

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Figure 5.13 The total yield of chlorogenic acid and apigenin-7-glucoside compounds content of chamomile and yarrow varieties under well-watered (WW) and drought stress (D) conditions...... 143

Figure 5.14 The total yield of caffeic acid, umbelliferone and luteolin compounds content of chamomile and yarrow varieties under well-watered (WW) and drought stress (D) conditions...... 143

Figure 5.15 The main terpenoid compounds content isolated from chamomile EO under well- watered (Ch WW) and drought stress (Ch D) conditions...... 146

Figure 5.16 The main terpenoid compounds content isolated from common yarrow EO under well-watered (Y1 WW) and drought stress (Y1 D) conditions...... 148

Figure 5.17 The main terpenoid compounds content isolated from summer berries yarrow EO under well-watered (Y2 WW) and drought stress (Y2 D) conditions...... 149

Figure 5.18 The total yield of terpenoid compounds content of chamomile and yarrow varieties under well-watered and drought stress conditions...... 150

Figure 5.19 GC-FID analyses of the essential oil of in vivo common yarrow flowers extracted by methanol using Soxhlet extraction under drought stress conditions...... 150

Figure 6.1 Radius of the inhibition zone (millilitres) at different concentrations of (A) 10-2 chamomile and (B) pure yarrow EOs against B. subtilis on (MC002) agar medium...... 161

Figure 6.2 The measurement of inhibition zones of E. coli, B. subtilis, E. faecalis, P. aeruginosa and P. syringae on (MC002) agar medium using pure MeOH. Data are presented as means ± SE of three replicates for each strain. (LSD value for bacteria strains= 0.60) ...... 162

Figure 6.3 The effect of in vivo chamomile extracted EOs activity (pure, 10-1, 10-2 and 10-3) against the inhibition zones of bacteria strains (E. coli, B. subtilis, E. faecalis, P. aeruginosa and P. syringae) cultured on (MC002) agar medium. Data are presented as means ± SE of three replicates for each strain. (LSD value for bacteria strains= 0.46, pure EO and dilutions = 0.41 and for bacteria strains* pure EO and dilutions. = 0.93)...... 163

Figure 6.4 The effect of in vivo yarrow extracted EOs activity (pure, 10-1, 10-2 and 10-3) against the inhibition zones of bacteria strains (E. coli, B. subtilis, E. faecalis, P. aeruginosa and P. syringae) cultured on (MC002) agar medium. Data are presented as means ± SE of three replicates for each strain. (LSD value for bacteria strains= 0.49, pure EO and dilutions = 0.44 and for bacteria strains* pure EO and dilutions= 0.97)……...….164

Figure 6.5 Growth curves of E. coli, B. subtilis, E. faecalis, P. aeruginosa and P. syringae, overnight cultured on (MHB2) at required temperatures 37°C, 25°C 37°C+CO2 based on the bacteria strain. Each well filled with 160 μl of overnight cultured bacteria. The microplate was read every 20 minutes for 24 hrs at 595 nm...... 165

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Figure 6.6 The effect of in vitro yarrow pure EO on the percentage of the number of E. coli living in the MHB2 culture at 37°C. The well filled with 160 μl of overnight cultured bacteria. The microplate was read every 20 minutes for 24 hrs at 595 nm...... 166

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List of Abbreviations

ANOVA Analysis of variance

AWC available water content

BA benzyl adenine

BAP Benzyl amino purine oC Celsius cm centimetre

D Drought

2, 4-D 2, 4- dichlorophenoxyacetic acid

DCM dichloromethane

DNA Deoxyribonucleic acid

DPPH 2, 2- diphenyl-1-picrylhydrazyl

EOs Essential oils

F variation within the samples g gram

GA3 Gibberellic acid

GC Gas chromatography

GC-FID Gas chromatography- flame ionisation detector

GC-MS Gas chromatography–mass spectrometry g L-1 gram per litre

GLC Gas liquid chromatography g/m2 grams per meter squared

GS Growth stage h hour ha hectare

XIX

HCl Hydrochloric acid

HPLC High-performance liquid chromatography

HPLC-EIMS High performance liquid chromatography-electrospray ionization mass spectrometry

HPLC-MS High-performance liquid chromatography- mass spectrometry

HPLC-UV High performance liquid chromatography- ultraviolet detector

IAA Indole-3 - acetic acid

IBA Indole-3-butyric acid

IPA Isopentenyl adenine

ISSR Inter-simple sequence repeat

ITS2 Internal transcribed spacer 2 kg ha-1 Kilogram per hectare

Kin kinetin

KRG Kurdistan regional government

LOD Limit of detection

LS Linsmaier and Skoog Medium

LSD Least significant difference mAU Milliabsorbance units

MC moisture content

MHB2 Mueller Hinton Broth 2

MIC Minimum inhibitory concentration mg g-1 milligram per gram mg Kg-1 milligram per kilogram mg L-1 milligram per litre

μl Microlitres ml millilitre ml min-1 millilitre per minute

XX mm millimetre

µm micrometre

MS Murashige and Skoog medium

MSMO Murashige and Skoog Medium with Minimal Organics

NAA Naphthalene acetic acid

NaOH Sodium hydroxide

P probability

p.a per annum

PCA Principal component analysis

PCR Polymerase chain reaction

PGRs Plant growth regulators

PPM Plant preservative mixture

RAPD Random amplified polymorphic DNA

RH Richters Herbs

RH Relative humidity

Rt Retention time

SCAR Sequence Characterized Amplified Regions

SE standard error

SG Skardon garden

SS stickysticky

TDR Time- Domain Reflectometry

TDZ Thidiazuron

WHO World health organization

WPM woody plant medium

WW Well-Watered

XXI

Chapter One

General introduction and literature review

1.1 General Introduction

German chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) are two main medicinal plants belonging to the Asteracea family (Hădărugăa et al., 2009).

The characterisation of the medicinal compounds produced in both plant parts in relation to the plant production system employed i.e., a) micropropagation using different plant growth regulators (PGRs) or b) traditional soil based culture, and mapping the production of medicinal compounds to the growth cycle, may elucidate new insights into the culture and production of key bioactive compounds in these key plants (Kindlovits et al., 2014, Sayadi et al., 2014).

Many of the plant natural products are phenol, flavonoid and terpenoid compounds which are generally used for their pharmaceutical properties; they have antiinflammatory, antioxidant and antibacterial properties. Additionally, they protect human bodies from numerous diseases such as cancer (Deng et al., 2014, Mahmoudi et al., 2016, Wink, 2015). The use of micropropagation techniques maybe a useful tool to enhance these active compounds via plant tissue culture within a short time

(Echeverrigaray et al., 2000a)

High performance liquid chromatography- ultraviolet detector (HPLC-UV) and gas chromatography-flame ionisation detector (GC-FID) techniques are considered as an efficient methods to determine the phytochemical compositions of phenols flavonoids and terpenoid compounds from dried chamomile and yarrow leaves and flowers

(Azwanida, 2015, Derwich et al., 2010, Dias et al., 2013, Szoke et al., 2003). Solvent extraction such as methanol and hexane extractions as well as methods of extraction including Soxhlet extraction are also used as a first step of the study of medicinal

1 plants to gain the highest yield of extracted plant material used for quantitative and qualitative analysis (Azwanida, 2015).

Researchers revealed that the change of environment and stress conditions predominantly affect the phytochemical compounds content especially essential oil composition, and the pharmacological properties of plants (Abdelmajeed et al., 2013,

Sharafzadeh and Alizadeh, 2011). Drought is one of the most common abiotic factors that limits plant production and, causes further implications for marketing of the final component of yield. Drought will impact on several parameters of plant growth and development, such as plant vigour and consequently plant height, with further implications on the production of side shoots and thus the final number of branches.

Drought will also influence final fresh and dry weight of flowers and the quantity of flowers produced. Evidence from the literature also illustrates abiotic factors such as drought will also influence the production of secondary metabolites including the total concentration of phenolic and flavonoid compounds. (Cicevan et al., 2016, Farooq et al., 2012, Razmjoo et al., 2008).

The phytochemical compounds of chamomile and yarrow essential oils (EOs) contain monoterpenes and sesquiterpenes that can act as an antimicrobials for example antibacterial activity against Gram-positive and Gram-negative bacteria such as

Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, and used to treat some infections caused by bacteria (Frey and Meyers, 2010, Solidônio et al., 2015).

1.2 Literature review

1.2.1 Historical use of medicinal plants in particular plants belonging to the Asteraceae family

Historically medicinal plants have played a big role in health care systems, for example plants belonging to the Asteraceae have been used since ancient times in Iraq, Egypt,

China, India and America. Traditionally they have been used to reduce cholesterol levels, malaria, skin and cancer diseases. Moreover, plants from other families including Rosaceae, Fabaceae, Asphodelaceae and Zingiberaceae have been used to treat eczema, kidney pain, fever and diarrhoea (Table 1.1). Globally, according to the

World Health Organization (WHO), more than 80% of people rely on medicinal plants for primary health care (Gurib-Fakim, 2006, Mazid et al., 2012, Uprety et al., 2010,

Edrah et al., 2016). A recent study by Gurib-Fakim (2006) indicated that globally the clinical use of drugs are based on 50% of compounds derived from medicinal plants and natural products for instance terpene lactones, flavonol glycoside and flavone glycoside. Additionally, Washimkar and Shende (2016) reported that the great important source of several diseases treatment in some traditional systems including

Siddha, Ayurveda and Unani in India is herbal medicine.

Moreover, González-Tejero et al. (2008) and Nedelcheva et al. (2007) concluded that in some Mediterranean regions (Egypt, Algeria, Spain, Cyprus and Albania), eastern, southeast and southern Europe (Macedonia, Bulgaria, Romania and Italy ) traditionally medicinal plants are not only used for medicine, but also in different areas such as foods, dyes, household and handicraft sectors. In Egypt medicinal plants are used to treat cough and abdominal colic however, in Italy they are used for detoxification and wounds. Among medicinal plant families, the Asteraceae, also known as Compositae

3 or Daisy family is the most commonly used group of plants in medicine worldwide

(Kumar and Ramaiah, 2011, Saeidnia et al., 2011). Common plant species that belong to this family are: Matricaria chamomilla L. (chamomile), Achillea sp. (yarrow),

Centaurea solstitialis L. (yellow star-thistle), Carthamus tinctorius L. (safflower) and

Echinacea purpurea L. (purple coneflower), (Asgarpanah and Kazemivash, 2013, Kilic,

2013, Kumar and Ramaiah, 2011, Saeidnia et al., 2011, Saeidnia et al., 2005,

Salamon et al., 2010). For instance, Moghaddasi Mohammad (2011) reported that medicinal plants belonging to the Asteraceae family are traditionally used to treat various diseases such as cancer in Iran.

Furthermore, Al-douri (2000) indicated that ninety seven medicinal plants from different Iraqi regions belonging to 43 families are used as a folk medicine. Asteraceae is reported to be the greatest family amongst them which includes beneficial plants such as Matricaria chamomilla L. (Arabic name: Babonage) which is used to treat depurative, cold, esophagitis and colpitis, and A. santolina L. (Arabic name: Kaisoom) which is commonly used as a restorative, anticolic, dysentery and carminative. Four main medicinal plants belonging to the Asteraceae family are also found in Erbil-

Kurdistan region Anthemis nobilis L. (Kurdish name: Gula Hajela), Artemisia campestris L. (Kurdish name: Sheeh), Calendula officinalis L. (Kurdish name: Hamish

Baha) and Cichorium intybus L. (Kurdish name: Jakjaka), these plants have been used for many years to treat hypercholesterolemia, sore throat, peptic ulcer, cleaning and opening of skin pores, blood purification and healing of wounds (Naqishbandi, 2014).

Moreover, twelve medicinal plants from different families were also investigated by

O'Hara et al. (1998) in the United States. From the Asteraceae family only Matricaria recutita L. was mentioned as an herbal remedy. These authors reported that Matricaria

4 recutita L. is cultivated for spasmolytic, wound healing (vulnerary), sedative and anti- inflammatory uses in the world.

Srivastava et al. (2010) report that chamomile is one of the most important medicinal plants belonging to the Asteraceae family, chamomile contains 1– 2% essential oil including terpenoids which are used in aromatherapy and cosmetic. The major components of the essential oil extracted from plant flowers are alphabisabolol oxides

A & B, alpha-bisabolol, and chamazulene. Furthermore, chamomile bioactive compounds have moderate antiphlogistic and anticancer properties. Additionally,

Srivastava et al. (2010) and Viola et al. (1995) mentioned that dried flower heads of chamomile have been used both internally and externally as a sedative and to treat spasmolytic rheumatic pain, hay fever, muscle spasms, insomnia, gastrointestinal disorders and ulcers. It is also used for skin problems like eczema, type one and type two diabetes anxiety and sedative (Amsterdam et al., 2009, Avallone et al., 1996,

Awad et al., 2007, Cemek et al., 2008, Eddouks et al., 2005, Kato et al., 2008, Patzelt-

Wenczler and Ponce-Pöschl, 2000).

Table 1.1 Historical uses of medicinal plants.

Countries Plant Species and Common Traditional References Families Uses Centaurea solstitialis Cancers and diabetes Gurib-Fakim (2006) (Asteraceae) Koc et al. (2015) Shanidar (North of Iraq) Ephedra altissima Various type of Lietava (1992) (Ephederaceae) infections Edrah et al. (2016)

Carthamus tinctorius Atherosclerosis Gurib-Fakim (2006) (Asteraceae) Reduce cholesterol Arpornsuwan et al. (2010)

Middle East& Egypt Prunus dulcis Cancer, Esfahlan et al. (2010) (Rosaceae) cardiovascular Mangalagiri Mandal disease, eczema and (2012) Pimples

Acacia senegal Stomach disorders Gurib-Fakim (2006) (Fabaceae) kidney pains, injury Eltahir et al. (2013) healing and cure

Africa Aloe vera Skin itching, Rajeswari et al. (Asphodelaceae) swellings, insect bites, (2012) cuts, skin cancer and Sahu et al. (2013) laxative.

Artemisia annua Malaria, fever and Gurib-Fakim (2006) (Asteraceae) cancer Räth et al. (2004)

China& India Centella asiatica Fever, diarrhoea, Ferreira et al. (2010) (Zingiberaceae) amenorrhea, lupus, Gohil et al. (2010) ieprosy and anxiety

Echinacea purpurea Infections, Gurib-Fakim (2006) (Asteraceae) autoimmune, wounds, Burgoyne and Bone and allergic (2004) North, central & Erythroxylum coca Hudson (2011) south America (Erythroxylaceae ) Diabetes, laryngeal Weil (1981) fatigue and motion sickness

Yarrow, is also widespread all over the world the same as chamomile and has been

used in traditional and folk herbal medicine thousands years ago to treat inflammation,

dyspepsia, flatulence, infants abdominal pain and diarrhoea (Honda et al., 1996,

Saeidnia et al., 2011). It is an astringent and aromatic plant and has been used

6 traditionally in Italy and India as a remedy for estrogenic activity, influenza, stimulant, tonic, gastroprotective, diaphoretic, carminative and antiseptic (Innocenti et al., 2007,

Lakshmi et al., 2011)

Furthermore, Radulović et al. (2012) demonstrated that Achillea sp. are used in modern medicine as an analgesic and antianxiety properties because it contains, alcoholic monoterpene fragranyl and benzoate as a major active compounds responsible for their medicinal properties. Rezaie et al. (2012) showed that Achillea millefolium L. herbal extract at the dose of 10% has greater wound healing properties compared to zinc oxide ointment, because of their terpenes, phenols and flavonoids content. In addition, Nemeth and Bernath (2008) reported that yarrow is used internally to treat gastric catarrh, loss of appetite and spastic discomfort, and externally for skin inflammation, fungal and bacterial infections and healing wounds. These effects might be related to sesquiterpenes content. Nowadays, the use of chamomile and yarrow are still popular in modern medicine and it will continue to be used in the future healthcare system because they contain numerous bioactive compounds such as essential oil, terpenoid and flavonoid which can work as a therapeutic agent.

1.2.2 Plant taxonomy and botany

In terms of medicinal plant taxonomy and botany, the Asteraceae is the largest family among flowering medicinal plants which includes more than 20,000 domesticated, weedy and wild species and consist of almost 10% of all flowering plants (angiosperms) such as trees, annual herbs, perennial herbs and shrubs (Kane et al., 2011).

Conversely, Gao et al. (2010) revealed that Asteraceae comprises 23,000 species and

1,600 genera, most of them are used in medicine, economic and ornamental purposes.

The selected species of this family are:

1.2.2.1 Chamomile (Matricaria chamomilla L.)

Chamomile belongs to the subfamily Asteroideae which is widely spread all over the world and grown in Europe, US, England and South America. The common name is from the Greek words chamos (ground) and melon (apple) which refers to the apple like scent of the plant flowers (Gardiner, 1999, Moghaddasi Mohammad, 2011). Two main species of chamomile are traditionally used in medicine as an herbal tea:

Matricaria chamomilla L. (German chamomile) and Anthemis nobilis L. (Roman chamomile), (Gardiner, 1999), Table 1.2.

Table 1.2 Classification of chamomile species.

German Chamomile

Family Asteraceae

Subfamily Asteroideae

Genus Matricaria

Species recutita, aurea, matricarioides, occidentalis, macrotis, tzvelevii, songaria

Roman Chamomile

Family Asteraceae

Subfamily Asteroideae

Genus Anthemis

Species nobilis, cotula, carpatica, cretica, austriaca, cinerea, austriaca, punctate, glaberrima, macedonica

German chamomile is an annual plant which grows 610 – 914 mm in height. It is also called, genuine, Hungarian, single, sweet false or wild chamomile. The roots are thin and spindle shaped. The erect branched stem grows 100 – 800 mm high. German chamomile leaves are narrow and long. The strongly aromatic flower heads are 25 mm

8 in diameter with a hollow centre which is covered with tiny golden- yellow florets between 1.5 - 2.5 mm in length, surrounded by white flowers which are 6 -11 mm long,

3.5 mm wide. In contrast, Roman chamomile is a perennial aromatic plant and also known as English, true, common, lawn, or garden chamomile. Plant height is only about 350 mm. The flower heads are approximately 25 mm in diameter, with a yellow conical disk bordered by some white flowers. It also has a number of hairs and branching stems and some small or thin separated leaves. (Gardiner, 1999), Figure

1.1.

Matricaria chamomilla L.

(German Chamomile)

Anthemis nobilis L. Roman Chamomile

Figure1.3.2.2 1.1 Yarrow Botanica l comparison of German and Roman chamomile.

http://essentialhealth.com/wp1.3.2.1 Yarrow -content/uploads/2013/04/RomanChamomile -drawing.jpeg

1.2.2.2 Yarrow (Achillea millefolium L.)

Yarrow (Achillea sp.) is one of the youngest perennial genera of the Asteraceae family worldwide which belongs to the same subfamily of chamomile (Asteroideae). It is also called common yarrow or milfoil. The Latin name is derived from the name of Achilles, who used Achillea plants to heal wounds in the Trojan War. It is commonly grows in

Europe, North America, Poland and central Asia (Nowak et al., 2010). The most common worldwide species are shown in Table 1.3.

Table 1.3 Classification of yarrow Species.

Yarrow

Family Asteraceae

Subfamily Asteroideae

Genus Achillea

Species millefolium, filipendulina, atrata, ptarmica, ageratifolia, aegyptiaca, ageratum, sibirica, asplenifolia

The height of Achillea sp.is about 800 mm and has erect stems. The strongly aromatic leaves are semi evergreen and are about 6 - 31 mm wide and 30 - 150 mm long. The inflorescence is 70 - 200 mm broad and 4.5 -7 mm in diameter. The ray flower numbers are 15 – 20, white to cream in colour, 0.8 - 2.5 mm in length. However, the numbers of the disk flowers are 15 – 60, yellow- coloured, (Figure 1.2).

Figure1.3.3 Molecular1.2 Botanical anddescriptions Genetic of AchilleaCharacterization millefolium L. (Yarrow) plant.

1.2.3 Molecular and Genetic Characterisation

The Asteraceae family provides a unique opportunity to learn about the genomic evolution of several phylogenetic scales diversification and the radiation of lineages with the use of bioinformatics tool to identify the selected genes in different plant species (Kane et al., 2011). In fact, since ancient the identification of the quality of medicinal plant species is traditionally completed by macroscopic morphological examination. This process needs to be achieved by a specially trained, expert; therefore, additional methods for medicinal plant identification of different plant species need to be studied (Zhao et al., 2011).

In modern times, the use of molecular biological techniques have helped to confirm the authentication of plant species including polymerase chain reaction (PCR) technique.

PCR is an in vitro molecular biology technique which generates numerous copies of specific selected sequences of DNA. Generally, the PCR technique consists of three important stages: DNA denaturation at 90°C – 97°C, annealing of primers to the template strands of DNA and to the extension of prime and occurrence of the annealed

11 primers extension at the end to generate a different copy of DNA (Joshi and

Deshpande, 2011). DNA-based markers have proved useful in various fields in plant science including embryology, taxonomy, genetic engineering, physiology, etc. These

DNA markers have been modified to bring the mechanism of genome analysis process to improve their efficiency. In this effort, the detection of PCR was found to be a single method to bring a new category of DNA marker (Joshi et al., 1999).

Moreover, medicinal plants have an important role in economy because of their use within medicinal, nutritonal and pharmacutical industries. Therfore, DNA extraction and purification techniques are required to get the noble PCR production from small amount of plant organs (Alatar et al., 2012). There are several ways to identify plant genetic sequences including ITS2 (internal transcribed spacer 2) found in four medicinal plants (Angelica biserrata, Aralia continentalis, Heracleum moellendorffii and

Levisticum officinale. ITS2 sequences were identified from each plant by designing primers and combined for sample analysis in a PCR technique by using Sequence

Characterized Amplified Region (SCAR) markers (Table 1.4), (Kim et al., 2016) .

Due to the important role of the chamomile plant in the world trade, drug and food industries, the evaluation of genetic diversity of chamomile based on molecular markers such as PCR was studied. PCR based molecular marker techniques like

Random Amplified Polymorphic DNA (RAPD) were used to evaluate different chamomile genetic diversity. The maximum variance coefficient of essential oil content, yield and number of flowers and minimum variance coefficient of height and flower diameter were noted. In terms of cluster analysis on both molecular markers and

12 morphology, in 5 clusters, 25 different populations were identified, 369 bands and 314 bands from 29 primers were detected polymorphic, respectively (Solouki et al., 2008).

Recent evidence by Okoń et al. (2013) confirmed that Inter-Simple Sequence Repeat

(ISSR) markers provide a practical method to the relationships and genetic similarity evaluation of the genotypes of Matricaria chamomilla L. In this study, five primers were repeatable fragments and polymorphic between twenty ISSR primers. Overall, fourty eight fragments were formed, out of this fourty one polymorphic were detected. The authors also suggested that it is important to observe a new source of chamomile diversity because the analysis of the genotypes of chamomile were detected by very low genetic variability.

In recent years, there has also been an increasing amount of literature on genetic diversity of Achillea sp. for instance (Ebrahimi et al., 2012b) reported recently, that there are a wide range of the studies on cultivatated Achillea sp. due to their bioactive compounds content, even though, the variation of the genes are still unknown.

Therefore, researchers decided to use ISSR and RAPD markers to study the genetic diversity of two native species in Iran (A. santolina L. and A. tenuifolia L.). As a result,

187 fragments were identified from Nine RAPD and seven ISSR primers, 159 of fragments were exactly polymorphic. Consequently, a minimum similarity between both species was noticed, which is consistent with their distribution. The low similarity between A. santolina (40- 84%) and A. tenuifolia (61- 86%,) were observed. Hence, the two species are totally separated and different from one another as a result of PCA

(Principal Component Analysis) and cluster analyses. In another major study, Ebrahimi et al. (2012a) considered the genetic relationship of the thirty seven accessions of

Achillea millefolium L. with the use of RAPD markers. To estimate the genetic diversity, nine RAPD primers were applied, all of them were appeared as polymorphic.

Furthermore, 118 fragments were generated from these nine RAPD primers and most of them were again polymorphic. In addition, the highest and the lowest values of genetic similarity were observed (Table 1.4).

In an earlier study, several researchers have analysed thirty seven Achillea millefolium

L. accessions using ISSR markers for their variability evaluation. Seventy two fragments were produced from seven ISSR primers. As a result of clustering, Achillea millefolium L. was indicated as an endemic to the North of Iran. In addition, the results showed that the ISSR markers, phonological and morphological traits selection could support further studies of domestication and plant breeding programs in Iran,

(Farajpour et al., 2012).

Table 1.4 Type of molecular markers of some medicinal plants.

Plant Type PCR Molecular Primer References Marker

An. biserrata Ab_F Ar. continentalis SCAR Ac_F1, Ac_F3 Kim et al. (2016) H. moellendorffii Hm_F L. officinale Lo_F ET42 M. chamomilla RAPD Solouki et al. (2008) IT36

Ch. recutita ISSR SR 14, SR 16, SR 33, Okoń et al. (2013) SR 36, SR 37

ISSR (1, 2, 3, 4, 5, 6, 7) A. santolina and ISSR and

Ebrahimi et al. (2012b) RAPD- (g11, m2, m3, a2, A. tenuifolia RAPD a10, e3, f19, b10, b11)

RAPD-( g11, m2, m3, A. milefolium RAPD Ebrahimi et al. (2012a) a10, e3, f19, b10, b11)

A. milefolium ISSR ISSR (1, 2, 3, 4, 5, 6, 7) Farajpour et al. (2012)

1.2.4 Commercial issues

Medicinal and aromatic plants from the Asteraceae family have been widely used as traditional medicine in several countries. These plants still play an important role within health care and their use will continue. Trade in these plants is rising in exports and in volume, which is globally estimated at US$800 million p.a. Moreover, the global market for plant chemical derivatives, colour, fragrances, pharmaceuticals and flavours ingredient were evaluated more than US$1billion p.a. With regard to the botanical markets of medicinal plants and herbs, China exports 120,000 tonnes p.a. and Europe imports approximately 400,000 tonnes annually (Hoareau and DaSilva, 1999).

Three ways of understanding healthcare systems from the research agenda come from the contribution of medicinal care quality in low income countries: firstly, the agenda showed that it was possible to measure medicinal advice quality using direct observation and vignettes. Secondly, it was emphasized that medicinal quality is perfectly measured to make a balance in market. Thirdly, it was presented that the quality of healthcare standard measurement relied on certain drug availability and physical infrastructure in low income countries (Das et al., 2008). Moreover, WHO has encouraged, and promoted the improvement of traditional and folk medicines for many decades, particularly for the least developed countries. For example WHO has supported some countries in Africa to start African traditional medicine (it is a socio economic and socio traditional culture, which services more than 80% of African populations) development (Elujoba et al., 2005).

A recent study by Mazid et al. (2012) state that in some Indian regions where people use medicinal plants for their treatment is because of the perception that these plants are free from side effects, safe and healthy. India is one of the most highly populated countries worldwide, cultivated most of the common traditional medicinal plants.

Therefore, it exports a large quantity of these plants as raw materials to other continents such as Asia. The authors also illustrated that farmers, village elders and tribal people have detailed knowledge about health issues and the use of medicinal plant and herbs as a folk remedies. This approach coincided with the origins of medicinal plants and can be traced back to ancient times and is still in continuous use today. The knowledge of village elders is still part of medical practice by several countries for instance Egypt, China, South America, Africa, and further world developing countries. Furthermore, farming in the area of study was established under:

16 family oriented, largely traditional, rain-fed condition, low income, diverse, small-scale, and characterised by farmer to farmer skills transfer. These points were play an important role in the genetic diversity of local crops management (Al-Rimawi et al.,

2004).

Only a few countries that dominate the international trade of pharmaceutical plants.

Europe and temperate Asia are among these countries; for global importation annually,

Europe is responsible for 33.3% and Asia for 42%. In terms of single countries, in

Germany the share of importation was 11 % and in USA was 13 %. Consequently,

Hong Kong was the main importer of these plants with 67,000 tonnes, annually. It was followed by Japan, the USA and Germany (4th place) with an importation average of around 51,350 tonnes, 49,600 tonnes, and 45,350 tonnes p.a. On the other hand,

China tops the list on around 12 export countries including India, Hong Kong, USA and

Germany, with an average of 147,000 tonnes in 1991-2000. Further exporter countries like Chile, Mexico, Bulgaria and Egypt were reported. As a result, Japan is a major country of pharmaceutical plants consumers. Germany, Hong Kong and USA were namely as the greatest botanical trade centres and presenting high export and import quantities (Lange, 2002).

Moreover, there is a growing demand for medicinal and aromatic plants, plant extracts and essential oils of these plants by flavour and food industries, as well as fragrance and cosmetic industries because of their beneficial use. The main consumers of these plant compounds across the global market were Japan, Western Europe and United

States (Govindasamy et al., 2011). A recent study has shown that the greatest essential oil markets from the South of Africa and Europe, where Asia, Americas and

Oceania were indicated as the lowest levels of essential oils export. Essential oil exportation between Europe and the South of Africa, Africa and the South of Africa in

2003 were started to rise about 572 tonnes and 245 tonnes, respectively. Nevertheless, essential oil exports between the South of Africa and Europe in 2012 was declined

100% compared to year 2003 (Marketing, 2013).

In the global market, chamomile is in great demand due to its pharmacological property and medicinal values. Also, the use of natural products from herbal medicines has superseded the use chemicals that are easy to obtain, create income, are free from side effects and are considered to be healthy (Singh et al., 2011). In addition,

South of India has become a good international market by cultivating a wide zone of chamomile as an industrial crop and commercial medicinal plant. This oil production was sold to the international markets at the amount of US$700 kg-1. Because of this high price of chamomile, it is essential to stimulate this plant as a main plant exports from the South of India (Nidagundi and Hegde, 2007).

In terms of plant production area, Iraq is considered to be a region in an excellent geographical position and is rich in plant biodiversity, genetic resources and several plants are also grown for medicinal, forestry and ornamental purposes such as cultivated and wild barley and wheat, dates, cauliflower, rice, onion, olive, lemons, etc.

North Iraq precipitation could be a perfect place to encourage winter plants cultivation such as wheat and barley, while in the south and the middle of Iraq, cultivation of different plants based on irrigation programme both in summer and winter seasons

(Leipzig, 1996). According to this fact, the government seeks to encourage and help people not only to increase the rate of crop production but also the production of

18 aromatic and medicinal plants. For instance, Kurdistan Regional Government (KRG) of

Iraq needs to support people continues farming by moving back to their villages instead of stay in the cities (RTI-International, 2008).

The environmental condition is one of the most important factors affecting the production of medicinal plants, particularly the yield of dry flowers and essential oil content (Ahmadian et al., 2011). A previous study reported by (Pirbalouti et al., 2011) indicated that agriculture land use potential was detected by evaluating agro ecological variables. The most important variables of components of agro ecology were topographical environmental, climate and soil. These variables were useful to identify suitable areas for crops and medicinal plant production such as chamomile. By using this approach, farmers could easily decide to select their crops pattern, which supports economical evaluation, production and marketing.

Chamomile and yarrow are two medicinal plants which are widely cultivated in the world for their dried flowers and essential oil content. Therefore, it is important to focus on the cultivation processes under controlled conditions to increase the yield of production and marketing issue. Naderidarbaghshahi et al. (2013) studied the effect of

German chamomile sowing date on the yield and growth properties in 2011-2012. The highest dry flower and biological yields of chamomile were observed in early October

(1602.00 kg ha-1 and 8880.2 kgha-1), respectively (Table 1.5). However (Nidagundi and

Hegde, 2007) investigated that a dry flower yield of German chamomile was 1.88 tonnes ha-1 and a fresh flower yield was 6.35 tonnes ha-1.

Table 1.5 The effect of sowing dates on chamomile yields.

Sowing date Early October Early November Early April Dry flower yield (kg ha-1) 1602 1167.23 893.3 Biological yield (kg ha-1) 8880.2 4640.2 4168.42

1.2.5 Medicinal plants and drought stress

Medicinal plants produce some plant secondary production which source for flavors, pharmaceuticals, biochemical industry and food additives. Accumulation of these plant products are affected by environmental stress conditions such as drought (Akula and

Ravishankar, 2011). In general, medicinal plants grown under moderate climate conditions produce secondary plant products less than those grown in semi-arid. In moderate climate conditions plants could be exposed to a lower level of drought than plants grown in semi-arid condition because of the lower level of light intensity and unlimited range of water supply. Hypothetically, secondary plant products, synthesis and accumulation of medicinal plants grown under drought condition are formed the same as plants grown under well-watered condition, but the concentration of these natural products are basically enhanced, caused by biomass decreasing (Selmar and

Kleinwächter, 2013). Chemical compounds and yield of secondary products in medicinal plants could be affected by several environmental factors starting from their first formation until the last stage of isolation in different plant parts.

One of the main issues among all environmental stresses is drought, which is an abiotic environmental factor that affects plant productivity, biochemistry, physiology and morphology. Many plants including chamomile and yarrow dehydrated and dried under uncontrolled drought conditions because of increasing solute concentration

20 outside of the plants which leads to poor growth and reduce yield. Root production, stem extension, leaves size and water use efficiency are reduced under drought stress.

In the environment, it is also causes a higher production of solute concentration and leads to an osmotic flow of water out of plant cells. In turn, inside the plant cells this causes and increase the solute concentration, and then dropping water potential and disrupting plant membranes, continue with some important process such as photosynthesis (Farahani et al., 2009b). Cicevan et al. (2016) also reported a significant difference of some medicinal plant cultivars between drought stressed and control plants. In general, drought stress among cultivars caused a clear decrease in fresh weight, stem length, carotenoid and chlorophyll photosynthetic pigments, total soluble sugars, total flavonoids and total phenolic compounds.

Moreover, the effect of the levels of water deficiency (25%, 50% and 100% control) on flavonoid and total phenolic content of two different varieties of Carthamus tinctorius

(eg, 104 and Jawhara safflower) were examined by Salem et al. (2014). The authors revealed that total phenolic compounds and flavonoids were significantly declined under 25% water deficiency of both varieties which was about 13.45 - 15.68%

(Jawhara) and 19.79-10.31% (104), respectively. In addition, the content of flower heads, phenolic acids were increased under drought condition, in particular gallic acid is significantly increased by 2.87 fold for Jawhara and 2.73 fold for 104 varieties.

Klunklin and Savage (2017) also indicated that phytochemical compounds content of tomato (Solanum lycopersicum) plants have been increased under water stress without affecting on the quality content.

Baghalian et al. (2011) studied the effect of drought on oil composition, oil content, flavone content (apigenin) and some morphological parameters including yield of dry flowers, weight of fresh flowers, roots and shoots weight of chamomile. They have observed that the oil content was not significantly affected by drought stress, however apigenin content, shoot weight, flower yield and plant height were dramatically decreased. Furthermore, high drought stress reduced the level of German chamomile chamazulene yield and essential oil content; however these variables have been increased in low drought stress. Plant height, number of main stem and number of flower heads per plant, were significantly decreased with the increase of drought stress level. In addition, the yield of fresh and dry flower, essential oil content and biomass of chamomile were significantly affected by drought stress. The highest yield of chamazulene and oil content were obtained in 70% field capacity, while the maximum biomass, yield of fresh and dry flower heads were noticed from 90% field capacity

(Ahmadian et al., 2011).

Conversely, Khalil et al. (2011) observed that relative water content, transpiration rate, leaf water potential, cell osmotic potential and stomatal conductance have been reduced in yarrow plants under drought condition (irrigated every 3, 6 and 9 days) compared to control (irrigated daily) in 2004 and 2005 under greenhouse conditions.

Relative growth rate and the ratio of root-to-shoot dry weight of plants irrigated every 9 days were lower than control. Furthermore, root weight, leaf weight and leaf areas of the control were higher than plants irrigated every 9 days in both year. Furthermore,

Fetri et al. (2014) revealed that percentage of germination, shoot length and rate of germination of common yarrow were significantly decreased under drought stress condition. There was not a significant differences observed for shoots and roots dry

22 weights of all treatments. However, significant differences were noticed for root length.

It was also found that the length of shoot was quite sensitive compared with other traits.

1.2.6 Plant tissue culture and micro-propagation technique

Medicinal plants have the greatest role in the world medicine due to their therapeutic agents which prevent and treat various diseases. Therefore, the study of plant biotechnology is important to conserve, multiply and select medicinal plant genotypes.

Regeneration (development of plant tissue and organ) of in vitro culture is also important to allow high quality production of medicinal plants active compounds

(Tripathi and Tripathi, 2003). Furthermore, medicinal plants genetic biodiversity is under threat because of unfavourable environmental conditions and the loss of habitat.

Thus, in vitro micropropagation is the greatest way to mass produce a range of traditional medicinal and aromatic plants. Recently, through this in vitro technique many plants and their secondary products were easily produced under controlled conditions (Ahsan et al., 2012).

Among all biotechnological techniques, plant tissue culture is a technique that provides a basic understanding of the chemical and physical requirements of the culture of tissue, cell and organ by using in vitro technique (micropropagation), this system provides ideal conditions to study the growth and development of test plants (chemical and physiological) under very controlled culture conditions (Dagla, 2012, Malik et al.,

2012, Victório et al., 2012). Tissue culture is also extensively used for pathogen free plant materials such as virus to develop a good quality of seed production and to improve effective, novel and safe products for customers, then producing bioactive compounds for both pharmaceutical and herbal industries (Debnath et al., 2006, Sidhu,

2010, Taşkın et al., 2013).

Micropropagation is the whole plant production from a small section of a plant such as embryo, internodes, nodes, hypocotyls or even a seed (Kintzios and Michaelakis, 1999,

Kulkarni et al., 2000, Sato et al., 2006) . It is considered that the multiplication of in vitro culture provides a great increase of health and successful plantlets established in the soil which had typical morphology and growth (Echeverrigaray et al., 2000a).

Furthermore, Debnath et al. (2006) and Malik et al. (2012) mentioned several micropropagation advantages for example, plants produce a large population from a small amount of explants within a short time, this technique could be commonly used to enhance the multiplication rate of vegetative part propagation, even so, it provides an economical and reliable process for producing a large number of healthy plants free from pathogens including bacteria and fungi, as well as producing virus free plants. In addition, improvement in in vitro culture method can provides a good means to produce exotic or rare plants commercially, chemical production and cost effective.

Malik et al. (2012) indicated that the general methods of plant regeneration through in vitro tissue culture are somatic embryogenesis and callus productions. Thereafter, the most common mediums used for in vitro culturing techniques are Gamborg B5 medium,

Anderson’s Rhododendron medium, McCown woody plant medium, Nitsch medium,

Che’e’ and pool medium, Murashige and Skoog medium (MS), chu medium, etc.

However, Murashige and Skoog medium has been successfully used for numerous medicinal plant species microropagation.

Four steps for optimization of tissue culture technique have been studied by

Ghasempour et al. (2012): firstly seeds were initially surface sterilized for 1 min using

70% ethanol, and for 8.0 mins in 1.5% sodium hypochlorite on a shaker and rinsed

24 three times with distilled water lastly, then seeds were placed in 6-Benzylaminopurine

(BAP) MS medium. After ten days of culture, young seedlings were formed and then transferred to MS medium containing different concentration of BAP (2.0, 5.0, and 10.0 mgL-1) after removing their roots. Callus and shoot were formed in both 5.0 and 10.0 mgL-1 BAP (with the highest amount at 5.0 mgL-1). Three characteristics have been investigated at this point of young seedlings (length of leaves, number of leaves and the tallest leaf). In third step, a free hormone of MS medium was used for seedlings.

Finally, young seedlings were moved to MS medium containing 2.0 mgL-1BAP.

The most important components of plant tissue culture are water, sugars, amino acids, vitamins, salts, plant growth regulators and agar or gelatin. Each ingredient has a special function in in vitro tissue culture process. Plant growth regulators (PGRs) have an important role among all these constituents, they encourage organ formation cell growth, cell division, differentiation of tissue and shoot formation and improve the yield of secondary products and culture (AbouZid, 2014, Malik et al., 2012, Rout et al., 2000,

Sato et al., 2006) The main plant growth hormones and their functions are:

 Auxins: cell division and elongation, callus formation, shoot elongation and

multiplication, root formation and inflorescence development. Examples: indole-

3- acetic acid (IAA), 2,4- dichlorophenoxyacetic (2,4-D) acid naphthalene acetic

acid (NAA), indole-3-butyric acid (IBA), (Gana, 2011, Keerthiga et al., 2012,

Pandey et al., 2011, Rout et al., 2000, Rout, 2004, Victório et al., 2012, Ziv¹ and

Naor, 2006)

 Gibberellins: elongation, growth and flowering, Examples: gibberellin A3 (GA3),

(Gana, 2011, Ziv¹ and Naor, 2006).

 Cytokinins: shoot induction, initiation of callus cell division, proliferation and

development. Examples: isopentenyl adenine (IPA or 2i-p), benzyl amino purine

BAP (benzyl adenine BA), 4- hydroxyl-3- methyl- trans-2-butenylaminopurine

(zeatin), furfurylamino purine (kinetin), (Gana, 2011, Keerthiga et al., 2012, Rout

et al., 2000, Victório et al., 2012, Ziv¹ and Naor, 2006).

Previous research has reported that micropropagation and somatic embryogenesis induction from Matricaria chamomilla L. flower explants represented a significant result for the regeneration of plantlets from embryos by using MS medium supplemented with

BA, NAA, Kin and 2,4-D. Consequently, results indicated that both types (ray and disk) of flowers reacted to the shoot and callus inductions but the globular somatic embryo formation was noticed only on the disk flowers on MS medium complemented either by

1.07 μM NAA and 8.87 μM BA or by 11.5 μm Kin and 26.8 μM NAA after 15-30 days of culture. Conversely, the best development of the stage of somatic embryos cotyledons could be enhanced on the kinetin and NAA medium after ten weeks of culture (Kintzios and Michaelakis, 1999) .

Moreover, a recent in vitro study by Sato et al. (2006) has shown that maximum shoot height (4.61 cm) and number of shoots per explant (3.31) of Matricaria chamomilla L. were observed on MS medium supplemented with 1.0 mgL-1 2,4-D. The increased concentration of 2, 4-D to 4.0 mgL-1, as well as decreased to 0.5 mgL-1, reduced shoot elongation and formation. Shoots were successfully improved to root formation with all different concentrations treatment, where the highest percentage of rooting (100%) was noticed with 2.0 mgL-1 GA3, however, the formation of callus was not observed with this treatment.

Moreover, Alvarenga et al. (2015b) studied the multiplication of Achillea millefolium L. in vitro culture with different concentrations of Thidiazuron TDZ (type of cytokinin).

Initially, explants (10 mm length of rhizome) were placed in 40 mL MS complemented by 1.00, 0.75, 0.50 and 0.25 mgL-1 TDZ. The best results of dry mass, length and number of shoots were observed with the use of 0.75 mgL-1 TDZ after six weeks of culture. In addition, Shatnawi (2012) revealed that a maximum number of shoots 5.9 of

Achillea millefolium L. per microshoots were discovered on 0.9 mg L-1 BAP MS medium. They also reported the influence of different auxin concentrations and types such as IAA, NAA and IBA. The highest number of root per explant (20.8 roots) was observed on 1. 2 mg L-1 o f I BA MS medium.

1.2.7 Medicinal compounds or secondary metabolites identification from the Asteraceae family and their biochemical properties

Medicinal plants contain some active substances which are known as secondary metabolites. Recent evidence suggested that among these plants the Asteraceae family contains numerous beneficial medicinal genera like Achillea millefolium L. and

Matricaria chamomilla L. Their chemical compositions are very different; many compounds are identified in all species, whereas some compounds are specific to a species (Kováčik et al., 2008, Trumbeckaite et al., 2011). In general, the first step of the medicinal plant study is an extraction of bioactive compounds from plant materials such as: roots, rhizomes, stems, leaves, barks, twigs, flowers, fruits and seeds.

Subsequently, the quantitative and qualitative testing is also an important step in the identifications of these compounds. Therefore, a number of studies demonstrated that hydrodistillation, hydroalcoholic and organic solvent extractions for instance methanol, dichloromethane, diethyl ether, ethanol and hexane and are more standard methods for extracting a high amount of medicinal plants bioactive compounds content (Candan

27 et al., 2003, Elouaddari et al., 2013, Johari et al., 2015, Kováčik et al., 2008, McCune and Johns, 2002, McCune and Johns, 2007, Mohammad Al‐Ismail and Aburjai, 2004,

Roby et al., 2013, Srivastava and Gupta, 2007, Srivastava and Gupta, 2009,

Trumbeckaite et al., 2011, Tuberoso et al., 2005, Tuberoso et al., 2009, Yucharoen et al., 2011).

Medicinal compounds are mainly responsible for organoleptic and pharmaceutical properties. Sesquiterpenes including bisabolol and chamazulene were found to be a main compound in chamomile essential oils with lower concentrated in the leaves compared to the flowers with the exception of chamazulene which was approximately

18.7% in leaves and 3.0-3.6% in flowers. However, in the leaves the concentration of

α-bisabolol (2.4-3.3%) and bisabolol-oxides (0.4-2.4%) were slightly lower. In terms of flower content, the authors revealed that the concentration of α-bisabolol (26 - 31% at

22.6), bisabolol-oxid A (12- 14% at 23.8) and bisabolol-oxid B (16 - 26% at 22.1 min) were much higher than the leaf content. Furthermore, low concentrations of these compounds were indicated in the extraction of chamomile roots and stems. In contrast, chamazulene (11.7-12.9% at 23.6 min) and α-bisabolol (0.7-2.6% at 22.6 min) were noted to be major volatile oil compounds found in the extraction of Achillea millefolium

L. flower heads. The concertation of α-bisabolol was decreased in the stem and root extraction, however, chamazulene content was increased (Hădărugăa et al., 2009).

Several studies have reported that the majority of plant natural products are phenols, terpenoids, sulphur and nitrogen containing compounds (Table 1.6). In addition, recent studies indicated that plants can protect themselves by the production of these bioactive compounds against some pathogens caused by microorganisms, abiotic

28 stresses (heat shock, water and drought stresses) as well as some herbivore varieties

(Edreva et al., 2008, Mazid et al., 2011). Conversely, Azmir et al. (2013) revealed that these compounds do not just act as a plant functions, but also a great source of protection for human against various illnesses and they are also used commercially as food products and within pharmaceutical and chemical industries.

Rahimi et al. (2011) published a paper in which they demonstrated that supercritical fluid extraction was also successfully used as a method for chamomile bioactive compounds extraction by using carbon dioxide. Eight compounds were identified in each sample including palmitic acid, bisabolol oxide A&B, cis-β-farnesene, linoleic acid,

7-methoxycoumarin, en-in-dicycloether and α-bisabolol. In contrast, a recent study by

Azmir et al. (2013) involved various new methods that have been investigated for bioactive compound extraction from plants, to date, however; there is no single method that is considered a standard method. They also mentioned that these methods of extraction are typically based on scientific skills, chemistry of the content of these active compounds and having a background of the plant matrix nature.

The principal terpenoid compounds of the flower heads of chamomile essential oil are

α-bisabolol and chamazulene. The primary flavonoids are quercetin, luteolin, apigenin and patuletin, which have found significant uses as antioxidant, anti-inflammatory, antimicrobial, antimutagenic activities, anxiolytic, antiplatelet and antispasmodic effects

(McKay and Blumberg, 2006). Additionally, a recent study has reported that the phytotheraputically useful part of chamomile is inflorescences due to the presence of the main bioactive compounds of their essential oil such as polyphenols, ascorbic acid and carotenoids and they possess as antioxidant activity (Telesinski et al., 2012).

Much research has been conducted into the terpene compounds of whole German chamomile plants, in particular ray and disk chamomile florets. The total terpenoids content of ray florets was approximately four times less than in the disk florets (ray florets, 308±46 μg/g, fresh weight and disk florets, 1098±180 μg/g, fresh weight).

However, both types of flowers produced bisabolol oxide A &B, α-bisabolol, (−) - germacrene D and (E) - β-farnesene as major sesquiterpene constituents and they act as antimicrobial, spasmolytic and antiphlogistic activities. In addition, some monoterpenes were found as minor constituents. In contrast, the major sesquiterpene components of chamomile leaves were β-selinene 4.8% , (E, E)-α-farnesene 54.1%, bicyclogermacrene 6.4% and (−)-germacrene D 22.4%), while monoterpene compounds were found in quite low levels. Bisabolol oxides and α-Bisabolol were not identified in the chamomile leaves. Moreover, the major terpene constituents detected in chamomile stems was (E)-β-farnesene 71.8% as sesquiterpene hydrocarbons.

However, in chamomile roots, (E)-β-farnesene 63.5%, geranyl valerate 10.7% and α- isocomene 10.8% were identified as major constituents (Irmisch et al., 2012).

Table 1.6 The role of plant secondary products of Asteracea family.

Secondary Products Bioactive Molecular Formula Activities Compounds Terpenoids Sesquiterpenes Bisabolol C15H26O Antimutagenic, Azulene C 10 H 8 Anixolytic. Chamzulene C 14 H 16 Antiinlamatory , Farnesene C 15 H 24 Antimicrobial Nerolidol C15H26O

Monoterpenes Tans- anethole C10H12O Antimicrobial, Limonene C10H16 Antioxidant, Antifungal 1,8 cineole C 10 H 18 O Antinociceptive, Antibiotic, Anti- hemorrhagic, Antidiaphoritic

Antioxidant, Phenols Chlorogenic Acid C 16 H 18 O 9 Antimicrobial, Free Ferolic Acid C 10 H 10 O 4 Radical Scavenging, Ascorbic Acid C6H8O6 Cytoprotective Activities

Pyrrolizidine C 7 H 13 N Antidiabetic, Nitrogen Containing Berberine C 20 H 18 NO 4 Antioxidant , Cytotoxic, Compounds Palmatine C 21 H 24 NO 4 Anticancer Demethylalangiside C 24 H 29 NO 10

Glutathione C 10 H 17 N 3 O 6 S Antioxidant, Sulphur Containing Taurine C 2 H 7 NO 3 S Antifungal , Compounds Cysteine C 3 H 7 NO 2 S Antibacterial

The essential oil content for chamomile flowers was established to be 0.73%. Gas

Chromatography Mass Spectrometry (GC-MS) analysis exposed the presence of the

15 main monoterpenoid compounds in the essential oil including limonene, trans- anethol, fenchone and estragole which were determined as antimicrobial and antioxidant activities. Moreover, some phenolic and flavonoid compounds for instance chloroginic acid, caffeic acid, quercetin, apigenin, apigenin-7-o-glycoside and luteolin

31 were determined by High Performance Liquid Chromatographic (HPLC), (Roby et al.,

2013). Accordingly, in a study which set out to determine volatile oil constituents with the use of GC-MS and Gas Chromatography Flame Ionisation Detector (GC-FID),

Orav et al. (2001) demonstrated that the main components were α-bisabolol 8–14%, bisabolol oxide A 20–33% & B 8–12%, en-yn-dicycloether 17–22%, (E)- β- farnesene

4–13%, bisabolon oxide A 7–14% and chamazulene 5–7%. They also reported that

70% of total essential oil consists of sesquiterpenoid constituents.

Srivastava and Gupta (2009) showed that dried flowers of German chamomile are mostly used for their pharmacological properties caused by the presence of some bioactive compounds. The major compound of chamomile methanolic extraction identified by HPLC-MS analysis was apigenin-7-O-glucoside, which prevented the growth of cancer cell and was used as an anticancer; some glycosidic compounds in chamomile essential oil were also detected. In addition, several researchers have examined the minor compounds of M. chamomilla essential oil characterized by Gas

Liquid Chromatography (GLC), were bisabolol oxide A and B, α-bisabolol, farnesene and chamazulene (El-Wahed et al., 2004).

In contrast, Tuberoso et al. (2009) indicated that the majority of flavonoid compounds of Achillea ligustica identified by High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-EIMS) were: apigenin-6-C-glucoside-8-C- arabinoside, apigenin, hydroxykaempferol-3,6,4- trimethyl ether and luteolin. They also found that ethanolic flavonoids extraction performed at higher antioxidant and antiradical activities using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) trial. Furthermore,

Trumbeckaite et al. (2011) established that an extract of Achillea millefolium L. was

32 possess significant antiradical and antioxidant activities due to their bioactive components content including phenols and flavonoids. They revealed that chlorogenic acid 19.3 ± 1.3 mg g-1 dominated among a mixture of secondary products. Regarding the flavonoid compounds, luteolin 10.2 ± 0.3 mg g-1 and apigenin 14.2 ± 0.4 mg g-1 dominated as major flavonoids; however their glucosides pattern was identified in lower levels, apigenin- 7-O-glucoside 2.0 ± 0.1 mg g-1 and luteolin-7-O-glucoside 0.67

± 0.01 mg g-1. In addition, rutin 3.2 ± 0.1 mg g-1 and vicenin-2, 1.8 ± 0.1 mg g-1 were detected to be the lowest quantities among all flavonoid compounds.

GC-MS analysis of the essential oil from Achillea sp. was studied by several authors

(Candan et al., 2003, Ghasempour et al., 2012). Thirty six monoterpenoid compounds were identified as principal constituents from the 90.8% total oil of Achillea millefolium

L. including, α-terpineol 10.2%, camphor 16.7%, borenol 4%, eucalyptol 24.6% and β- pinene 4.2%, which was including 60.7% of the essential oil. Furthermore, the result showed that these essential oil constituents possess antioxidant and antimicrobial activities (Candan et al., 2003).

In contrast, Ghasempour et al. (2012) determined Forty two major compositions which covering 98.0% of the Achillea bibersteinii oil compounds, and thirty seven minor compositions using GC and GC-MS analysis. The main monoterpenoid compositions were α-terpinene 9.6%, 1,8 cineole 30.9%, camphor 4.3%, cis-ascaridole 12.8% and

α-terpineole 6.3%. They also concluded that these compounds were used as, antifungal, antihemorrhagic, antibiotic, antioxidant, antidiaphoretic and antiinflammation activities. Furthermore, thirty major constituents of Achillea millefolium

L. were characterized by GC and GC-MS, which contain (0.70±0.05%) of the total

33 essential oil and comprising 93.43% of the total oil. The principal compounds were borneol 12.41%, sabinene 17.58%, chamazulene 5.28%, 1,8-cineole 13.04%, bornyl acetate 7.98%, and terpinine-4-ol 6.17%, (Nadim et al., 2011).

A total of ninety six constituents were also detected on Achillea ligustica L. with the use of GC-MS analysis. The majority of the essential oil compounds content were sabinol

2.1-15.5%, α- thujone 0.4-25.8%, sanatolina alcohol 6.7-21.8%, trans-sabinyl acetate

0.9-17.6%, borenol 3.4-20.8%, and viridiflorol 0.7-3.6% as a main sesquiterpenes constituent (Tuberoso et al., 2005).

1.2.8 Anti-microbial activities

At present, the resistance of drugs to pathogenic bacteria found within humans is giving rise to grave concern regarding the developing resistance in pathogens to antibiotics. Research is therefore important to identify new antimicrobial substances from different sources such as plants. Generally, there are thousands of active chemical compounds produced from plants that act as antimicrobials against some human and plant pathogenic micro-organisms (Amenu, 2014).

The antifungal and antibacterial activities of ethanolic and hot water extractions of six medicinal plants (Cinchorium intybus, Acacia Arabica, Sphareranthus hirtus, Cardus marianum, Emblica officinalis and Nymphaea lotus) for liver damage treatment were studied. Plant extracts were in vitro tested against two species of fungi (Aspergillus niger and Candida albicans) and seven species of bacteria (Proteus vulgaris,

Escherichia coli, Bacillus subtilus, Pseudomonas aeruginosa, Klebsiella pneumoniae,

Staphylcoccus aureus and Salmonella typhi) by microdilution and well diffusion methods. In total, the maximum inhibition zones of P. aeruginosa, S. typhi and E.coli were noticed. E. coli and P. aeruginosa were most affected by S. hirtus extract. In addition, S. hirtus hot water extraction showed a lower effect than the ethanolic extraction (Hassan et al., 2009).

Furthermore, the antibacterial activity of some medicinal plant extracts such as

Artemisia herba-alba, Ficus sycomorus, Withania somnifera, Nerium oleander,

Eucalyptus camaldulensis and Allium sativum with antibiotic drugs against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus has been studied by

Amenu (2014). As a result, aquatic and methanolic extractions indicated antimicrobial activity with antibiotics less than ethanolic extraction of all plant extracts. The highest effect of Ficus sycomorus (bark and leave extraction) was observed against E. coli when a mixture of plant extracts and Ofloxacin was used. The biggest effect of a mixture of A. sativumwith with Tetracyclin and Ofloxacin against S.aureus was detected. And the biggest effect against P. aeruginosa was determined when W. somnifera, A. herba-alba and N. oleander extracts were mixed with Amikacin, and when W. somnifera mixed with Neomycin, also when F. sycomorus extract was combined with Ceftazidime. Conversely, Ushimaru et al. (2007) demonstrated that

Caryophyllus aromaticus (Clove) methanolic extraction showed the strongest antibacterial effect against Staphylococcus aureus.

Researchers identified that natural plant products might exhibit human health benefits due to their anti-microbial activates against some bacteria, virus and fungi species.

Several in vitro studies reported that chamomile essential oil and extract are

35 responsible for antimicrobial activity and human health benefit due to their chemical bioactive compounds such as sesquiterpenes content (Petronilho et al., 2011, Saderi et al., 2003). Saderi et al. (2003) discovered that the use of chamomile essential oil and extract in different mouthwash reduced the growth of Porphyromonas gingivalis using disc diffusion method, and control periodontal disease, with the means of inhibition zone 13.33±3.4 and 20.5±0.5 mm, respectively. Moreover, ethanolic extraction of roots, leaves and flowers of chamomile were tested against

Pseudomonas syringae pv. maculicola (M Moricz et al., 2012). Coumarins and cis-, trans-spiroethers were observed as the strongest antibacterial activity compared with other compounds. They have also shown that coumarins like umbelliferone and herniarin can act as a plant defence against bacterial and fungal attack. Additionally,

Roby et al. (2013) found that estragole, limonene, fenchone and trans-anethole components found in the chamomile essential oil were significantly prevented the growth of Candida albicans, Staphylococcus aureus, Aspergillus flavus and Bacillus cereus depending on the applied dosage using disc diffusion method.

Furthermore, the volatile oil active compounds of Achillea clavennae L. were analysed against Gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Klebsiella pneumonia) and Gram positive bacteria (Streptococcus faecalis, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis,). Oil activity against Gram positive was fewer than Gram negative. As a result, the extracted oil was found to be antibacterial activity against all bacteria strain except Gram positive bacteria Bacillus cereus. However, the greatest activity against Gram negative was noticed in Pseudomonas aeruginosa (18 mm inhibition zone) and Klebsiella pneumoniae (26.4 mm inhibition zone), (Bezić et al., 2003)

Additionally, the antibacterial activity of the chemical compounds found in the essential oil of the flower tops of Achillea ligustica L. was investigated. Low activity (minimum inhibitory concentration (MIC) values more than 900 µg L-1) of the essential oil against

Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa was detected

(Tuberoso et al., 2005). In contrast, Candan et al. (2003) reported that the strongest activity of extracted oil of Achillea millefolium L. was detected against Candida albicans, Clostridium perfringens and Streptococcus pneumoniae, while the weakest activity was observed against Candida krusei, Acinetobacter lwoffii and Mycobacterium with the growth inhibition zones between 4.5 mg ml-1 and 4.5 mg ml-1.

Therefore, it was important to produce a protocol for antimicrobial activities including antibacterial activity against various strains for both chamomile and yarrow plants by using different methods such as disc diffusion and 96 well plate readers. The antimicrobial bioactive compounds content of medicinal plants is not only useful in treating some infections with the addition of some drugs as an antimicrobial agent.

There is also a use as active ingredients for food industries and preservatives (Kazemi,

2014, Mohamed et al., 2016).

According to previous work, both Matricaria chamomilla L. and Achillea millefolium L. were very important plants in medicine worldwide (Azmir et al., 2013, Hădărugăa et al.,

2009). Therefore, in vitro seeds culture, growth characterisation, drought stress and bioactive compounds identification really need to be investigated as a main direction by way of different experiments. Moreover, based on the agronomic issues, establishment of some pharmaceutical industries and commercial medicinal centres in

Kurdistan Regional Government (KRG) is an important issue with essential oil

37 production of medicinal plants, which is widely distributed in the region such as chamomile, yarrow, fennel, mint, rosemary, thyme, and there are several areas of interest for the development of oil production from the raw materials of these plant species. Collaboration between KRG producer, dealers and customers with their contact in the international market is another challenge for industries future development. Overall, due to the high price international market of these plants bioactive compounds, it is important to cultivate and promote these valuable medical plants as commercial plants predominantly for chamomile and yarrow essential oil export from KRG.

1.3 Aims of study

This thesis aims to:

1- Establish the optimization of in vitro micropropagation of chamomile and yarrow seeds in order to regenerate a mass production of whole plants through the optimum medium to study the phytochemical analysis, antibacterial activity of extracted essential oil and compared with in vivo culture.

2- Develop in vitro and in vivo plant growth stages grown in both inside and outside glasshouse conditions.

3- Study the mechanism of changing the quality and quantity of phytochemical compounds of plant leaves and flowers content under drought conditions compared to well-watered conditions.

4- Study the antibacterial activity of plant EOs produced from the flower heads of in vivo and in vitro cultures.

1.4 Objectives of study

The specific objectives of this thesis were to:

1- Establish a novel scheme for finding a high level of tissue differentiation in vitro culture using several plant growth regulators (PGRs) and compare how these tissue culture plants develop with plants grown in soil media.

2- Investigate the influence of temperature and light conditions on the percentage of germinated seeds.

3- Characterise the effect of in vitro and in vivo winter and summer culture conditions on the growth stages of plant height, number of branches and number of flowers of both plants.

4- Investigate the efficiency of methanol extraction for obtaining the highest amount of plant extracts using Soxhlet and shaking extractions.

5- Characterise and identify in vitro and in vivo phytochemical compounds using HPLC and GC-FID analysis.

6- Evaluate a comparative chemical analysis of drought and well-watered conditions by examining phytochemical changes of phenol, flavonoid and terpenoid compounds content.

7- Determine the antibacterial activity of plant essential oils against gram-negative and gram-positive bacteria strains placed on nutrient agar medium using disk diffusion and microplate methods.

Chapter two

Chamomile and yarrow micro-propagation technique

2.1 Introduction

Medicinal and aromatic plants are one of the greatest sources of medicine worldwide, for example, in Indian tradition most people depend on the use of these plants for their physiological condition and the treatment of several diseases such as diabetes, infections and skin diseases (Dhal et al., 2015, Washimkar and Shende, 2016).

Therefore, the application of plant biotechnological techniques is an important tool to select, improve, modify, conserve and multiply medicinal plant genotypes. In plant biotechnology one of the most conventional methods is micropropagation (in vitro tissue culture). This technique has been used for some plants, especially those plants used in pharmaceutical industries (Debnath et al., 2006).

Currently, micropropagation is the most common and commercial technique used for whole plant regeneration under controlled culture conditions in a specific medium such as Murashige and Skoog 1962 (MS) medium. MS medium is the best media for the in vitro regeneration (Madke et al., 2014). Moreover, micropropagation is an efficient method in plant breeding for transgenic plants production (Loberant and Altman, 2010).

It has been reported that a vast amount of medicinal and aromatic plants were in vitro regenerated from different parts of seeds, leaves and flowers (Chaturvedi et al., 2007).

The use of two types of hormones in in vitro tissue culture have been reviewed by

Gspar et al. (1996); new and classical hormones. These hormones interact together to achieve their final effects of cell divisions, initiation of callus and shoot elongation; however a few of them rarely act alone. The main classical plant hormones are gibberellins, ethylene, cytokinins, abscisic acid and auxins. However, sterols, turgorins, oligosaccharins, brassinosteroids, polyamines, myoinositol, jasmonates,

40 dehydrodiconiferyl alcohol glucosides and salicylates have recently been determined as phytohormones which act like growth regulators. It has been found that greater shoot and root induction were formed in free hormone MS medium and also with the use of different concentration of these plant regulators (Hanus-Fajerska et al., 2012,

Sato et al., 2006). Furthermore, cytokinins and auxins (BAP, Kin, IAA, NAA and 2, 4-D) are two important hormone classes that control shoot branching (Umehara et al., 2008).

Several points including multiple shoots production, somatic embryogenesis induction, callus production and root formation have been studied regarding the use of chamomile (Matricaia chamomilla L.) and yarrow (Achillea millefolium L.) in vitro (is a process of culture taking place in a Petri dish) micropropagation using different plant parts such as chamomile leaves, seeds and inflorescences (Ahmad and Misra, 2015,

Echeverrigaray et al., 2000b, Kintzios and Michaelakis, 1999, Sato et al., 2006), and yarrow seeds, meristems and rhizomes (Alvarenga et al., 2015a, Evenor and Reuveni,

2004, Shatnawi, 2012).

Because of the importance of the type and concentration of plant growth regulators

(PGRs) of chamomile and yarrow micropropagation technique, several studies have been conducted to increase the final production of callus, shoot and root formation

(Ghasempour et al., 2012, Sayadi et al., 2014). It has been shown that auxins such as naphthaleneacetic acid (0.5 mg L-1 NAA) and indole-3-butyric acid (1.0 or 0.5 mg L-1

IBA) had a positive effect on the production of yarrow shoots (Turker et al., 2009).

However, the best treatment for chamomile shoot and root generation were 2,4-

Dichlorophenoxyacetic acid (1.0- 0.5 mg L-1 2, 4-D) and gibberellin (2.0 mg L-1), (Sato et al., 2006).

Through the last decade researchers illustrated a greater understanding of the role of

PGRs (Gspar et al., 1996, Santner et al., 2009). It is known that Matricaria chamomilla

L. and Achillea millefolium L. are two important medicinal plants in herbal medicine because of their valuable compounds. Higher production of callus, shoots and roots stimulation from in vitro culture of chamomile and yarrow explants were therefore important to be determined as a main aim of this study using MS medium containing

PGRs like auxins, gibberellin and cytokinins. Many of these hormones including kinetin and NAA produce the best final effect on callus formation. It has been found that the callus formation from German chamomile explants (stems, leaves and axillary buds) was quicker on MS medium containing 1.0 mgL-1 kinetin and 1.0 mg L-1 NAA, and a maximum percentage of callus induction from leaves (93.26%) has been found by

(Sayadi et al., 2014). In addition, the greatest generation of callus from stems (80.75 %) and axillary buds (89.68%), and a maximum volume of callus have been observed with the same concentration of kinetin and NAA.

2.2 Objectives

1. To investigate in vitro chamomile and yarrow micropropagation.

2. To study the effect of different concentrations of PGRs on shoot and root formation and callus induction.

3. To maximize the number of germinated seeds under the optimum plant hormone concentration.

4. To optimize understanding of the culture and characterisation of chamomile and yarrow plants raised from seeds.

2.3 General Materials and methods

2.3.1 Sample collection and preparation

Dried chamomile seeds from Richters Herbs (Source for Everything Herbal, 357

Durham Regional Hwy 47, Goodwood, ON L0C 1A0, Canada) and yarrow seeds from

Skardon garden (Plymouth University) were surface sterilized after removing any obvious deleterious plant debris and placed into a sterilizing solution consisting of 10% household bleach (contains less than 5% Chlorine and 1.5% w/w Sodium Hypochlorite) for 3 min, and then the seeds were washed in sterile distilled water three times before culture.

2.3.2 The effect of 6-Benzylaminopurine (BAP) and Kinetin on callus shoots and roots formation of in vitro chamomile and yarrow seeds culture

Seeds were cultured as a preliminary test in order to achieve in vitro micropropagation technique using 0.1mg L-1 BAP (6- benzyl amino purine) + 0.2 mg L-1 kinetin (Kin) +

4.4 g L-1 MS + 30 g L-1 sucrose + 10 g L-1 bacteriological agar and 1.0 ml L-1 PPM

(plant preservative mixture) using a standard African violet (Saintpaulia ionantha L.) culture medium (Appendix1) previously described by Khan et al. (2007) as a efficacious medium for the growth and development of African violet ex-plants. Callus and multiple shoots were formed approximately 1-2 weeks after culture. Shoots that established good root systems (3-10 cm root length) on MS media were transferred to disposable pots containing John Innes compost No.1 and plantlets were then transported to the glasshouse approximately after 2-4 months of culture into 11 ×

11cm2 and 12 cm height pots (0.75 L) and soil were also changed to John Innes compost No.2.

2.3.3 Seed medium culture preparation using different PGRs

The seed culture process was to establish specific controlled chemical and physiological culture conditions of tissue, cell and organ culture. All instruments were sterilized by 10% household bleach and autoclaved (Boxer autoclave for 2 hours at

78°C) prior to use to reduce and prevent any microbial contamination. Forceps, needles and scalpels were sterilized by quartz glass bead sterilizer for 5-10 seconds at 240°c. Plant material was also previously surface sterilized (as described in section

2.3.1).

Dried seeds (5 seeds per plate) of both species were cultured in a petri dish which contained MS medium supplemented with 4.4 g L-1 MS + 30 g L-1 sucrose + 10 g L-1 bacteriological agar and 1.0 ml L-1 PPM, and then 0.5 mg L-1, 1.0 mg L-1, 1.5 mg L-1, and 2.0 mg L-1 of each BAP, Kin, IAA, NAA, GA3 and 2,4-D were used separately with triplicates for each PGR, respectively, (Figure 2.1). After adjusting pH to 5.8 (pH of optimal tissue culture medium) using 1M of HCl and 1M NaOH the culture media were poured onto the plate and placed into a SANYO plant growth chamber with controlled conditions (23.8°C: temperature and 8/16 h dark/ light: photoperiod. The effect of

PGRs on the percentage of callus induction, shoot and root formation were recorded throughout 20 days of culture.

Plant seeds MS growth medium

Figure 2.1 Experimental design of chamomile and yarrow in vitro seeds culture (five seeds per treatment) in MS medium containing different plant hormones.

2.3.4 Chamomile and yarrow plants production from nutrient agar

According to the results of preliminary test using different PGRs (Section 2.3.3), a new experiment was designed to produce a higher production of chamomile and yarrow leaves and flowers. The aim was to determine plant growth characterisation and identification of bioactive compounds using GC-FID and HPLC analysis. MS medium was used for both plant seeds supplemented with 1.0 mg L-1 of GA3 (used for yarrow seeds) and 0.5 mg L-1 of IAA (used for chamomile seeds) as the best concentrations noticed in different PGRs test for callus (section 2.3.3), shoots and root formation of both plant species. To do this experiment the same procedure has been used to prepare the MS nutrient medium as described in section 2.3.3.

2.3.5 Statistical analysis

All statistical testing were carried out using Minitab™ statistical software (Version17) in triplicate for three different samples. Analysis of variance (ANOVA) was performed for multiple group comparisons and data are presented as mean and standard error. Least

45 significant difference (LSD) was tested by comparing with means value and standard error, and significant differences were accepted at p < 0.05.

2.4 Results

2.4.1 Callus, shoot and root formations

Callus, shoots and roots were produced from chamomile plants seed culture; however shoots and roots were formed directly from yarrow plants seed culture without callus induction after 2-3 weeks of culture in MS medium supplemented with 0.1mg L-1 BAP and 0.2 mg L-1 Kin. Data relating to the percentage of chamomile and yarrow germinated seeds culture used in this experiment are summarised in Table 2.1. The highest percentage of callus induction was found in chamomile seeds (65.93± 5.32 %), while the greatest root and shoot were formed in yarrow seeds culture (36.30± 5.45 % and 60.74± 5.49%), respectively.

Table 2.1 The percentages of callus, root and shoot formation of chamomile and yarrow seeds after 2-3 weeks of culture, data are presented as mean and standard error.

Plant sp. Callus induction % Roots formation % Shoots formation%

Chamomile 65.93 ± 5.32 5.93 ± 2.57 23.70 ± 4.78

Yarrow 0.0 36.30 ± 5.45 60.74 ± 5.49

Results indicated that callus was only formed from chamomile culture. Chamomile seeds significantly produced the greatest callus growth compared to yarrow, where the

46 mean and standard error for chamomile callus formation were 65.9±5.32 %, (F 982.90

1, 34; p < 0.001), respectively (Table 2.1). Also, the interaction between plants and days was significant for chamomile callus production over time (p<0.001). Chamomile callus production was progressively increased over time; however time did not affect the improvement of yarrow callus production (Figure 2.2).

In terms of roots and shoots formation, yarrow produced more roots and shoots (36.30

± 5.45 % and 60.74 ± 5.49%) compared to chamomile seeds, respectively (Table 2.1) and significant differences were reported for both chamomile and yarrow plants (F

170.101, 34; p<0.001) and (F 188.891, 34; p<0.001), respectively (Figure 2.3 and 2.4).

Furthermore, the interaction between days and plant species of root production was significant at p<0.001, where yarrow seeds produced roots faster than chamomile seeds over time and started after 4 days of culture, however chamomile rooting started after 14 days of culture. Also, there was no significant difference between plant species and days of shoot formation (p= 0.323).

2.4.2 In vitro chamomile and yarrow seeds culture using different plant hormones (PGRs)

Effects of different PGRs on in vitro germinated seeds (seedlings) of both chamomile and yarrow plants were determined after 20 days of culture in MS medium. Plants were successfully established and adapted to the glasshouse conditions after a few months of culture (2-4 months), Figure 2.5. The results revealed that the effect of plant hormone concentrations (0.5 mg L-1, 1.0 mg L-1, 1.5 mg L-1, and 2.0 mg L-1 ) were significant for in vitro seeds culture. According to the number of germinated seeds and

49 the percentage of germination of both plant species most of the chamomile seeds and a few of yarrow seeds were germinated with the use of different PGRs.

Results of BAP concentrations showed that the maximum percentage of germinated seeds being observed in chamomile compared to yarrow and significant differences were found between in vitro chamomile and yarrow germinated seeds (F65.341, 72; p<

0.001). Moreover, free hormone use (control, 0.0) had a significant effect on the germination percentage of both plant seeds, where the highest germination percentage was obtained in chamomile (82.0 ± 3.98 %) compared to yarrow (24.67 ± 1.42%),

(F183.881, 18; p< 0.001), (Figure 2.6). There was also a significant difference observed in the interaction between plant species and BAP concentrations (p= 0.001); maximum percentages were obtained in chamomile using all four different concentrations with the highest value observed in 1.0 mg L-1 BAP, while 2.0 mg L-1 was optimal for yarrow seeds.

Figure 2.7 shows the effect of Kin on germinated seed. Chamomile seeds have a significantly higher percentage germination rate compared to yarrow culturing in MS medium supplemented by different concentrations of Kin, where the highest

-1 percentage was found by adding 0.5 mg L of Kin in to the medium (F 459.84 1, 72; p

≤ 0.001). Conversely, yarrow seeds were not affected by using 0.5 and 1.0 mg L-1 of

Kin and very low concentrations were observed in 1.5 and 2.0 mg L-1 compared to chamomile seeds. The interaction between plant species and Kin conc. was also significant (p = 0.001). Chamomile seeds were totally affected by all concentrations compared to yarrow seeds and the best concentration for yarrow seeds was 2.0 mg L-1.

A1 A2

B A3

B1 B2

Figure 2.5 Micropropagation developments of in vitro chamomile and yarrow seeds. A: Chamomile growth stages, B: Yarrow growth stages. A1 & B1: callus and multiple shoots production1-2 weeks after culture on MS medium containing 0.1mg L-1 BAP and 0.2 mg L-1 Kin. A2 & B2: root production from shoots51 and soil transfer 2-3 weeks after culture. A3 & B3: Plantlets potting up and growth developments in the glasshouse 2-4 months after culture.

The maximum values of chamomile seeds germination were observed on MS medium containing IAA concentrations. High significant differences were also found of the percentage of chamomile and yarrow germinated seeds under four different concentrations of IAA (F 94.821, 72; p< 0.001). In addition, the interaction of plant sp. and conc. of IAA had a significant (p< 0.001) effect on the percentage of germinated seeds (Figure 2.8). Concentrations of the selected PGRs at 0.5 mg L-1 and 2.0 mg L-1 were found to have the greatest influence on the percentage germination of seeds from both chamomile and yarrow.

In terms of NAA, results indicated that both plant species were affected by different concentrations. Interestingly, 0.5 mg L-1 was found as the best concentration for both chamomile and yarrow seeds germinations. Moreover, chamomile seeds culture was significantly more affected compared to yarrow (F 7.211, 72; p ≤ 0.001), (Figure 2.9). In contrast, the interaction of plant sp. and conc. of NAA had no significant effect on the percentage of germinated seeds (p= 0.661).

Figure 2.9 Effect of NAA concentrations on the chamomile and yarrow seeds germination (LSD value for plant sp.= 9.40). Data are presented as means ± SE of three replicates for each concentration.

A concentration of 1.5 mgL-1 of 2, 4-D was found as the best concentration for chamomile germinated seeds compared to other three 2, 4-D concentrations (Figure

2.10). However, yarrow seeds did not show any germination by using 0.5 mgL-1 and

1.0 mgL-1 (Figure 2.7). Chamomile and yarrow were significantly affected by 2, 4-D concentrations (F 103.021, 72; p ≤ 0.001). Moreover the interaction of plant sp. and conc. of 2, 4-D was also significant at p ≤ 0.001; chamomile seeds were totally affected by different concentrations, while yarrow seeds were only poorly germinated on MS medium containing 1.5 mgL-1 and 2.0 mgL-1 of 2,4-D.

Lastly, chamomile and yarrow seeds germination were also influenced by GA3 concentrations and chamomile seeds were highly germinated compared to yarrow. For both plant seeds 1.0 mgL-1 was found to be the best concentration. Significant differences were also observed between both plants (F75.99 1, 72; p ≤ 0.001), (Figure

2.11). Moreover, the interaction between plant species and GA3 concentrations was no significant (p= 0.640).

Overall, the use of 0.5 mgL-1 of IAA was considered to be the optimal concentration and PGRs among all hormones for the in vitro chamomile seed culture (81.33± 4.64%).

In terms of yarrow seed, the greatest PGRs and concentrations for giving the maximum percentage of germinated seeds was 1.0 mgL-1 GA3 (46.0± 6.77%).

Moreover, the maximum percentage of yarrow germinated seeds was observed in free hormone use compared to Kin and 2, 4-D (Figure 2.7 & 2.10).

2.5 Discussion

The micropropagation technique was successfully used to improve the growth of in vitro chamomile and yarrow seeds culture. BAP and Kin use in MS medium had the capacity to develop the callus, root and shoot formation as shown in Table 2.1. It was found that 0.1mg L-1 BAP and 0.2 mg L-1 Kin significantly increased the growth of chamomile callus production, as well as yarrow shoot and root formation. Moreover, the differences were also observed regarding the effect of BAP and Kin on the total percentage of germinated seeds over time. Both PGRs had a negative effect on yarrow callus production. However, more studies are required to discover the negative results of the use of 0.1 mg L-1 BAP and 0.2 mg L-1 Kin in in vitro yarrow seeds culture.

It might be that using other PGRs can aid and enhance the initiation of yarrow callus production by using a higher concentration, for example 3.0 mg L-1 of 2, 4-D found to be a good treatment for callus initiation reported by Gopitha et al. (2010), and selecting young leaf segments as an explants. Famiani et al. (1994) reported that young leaves show more regeneration than older leaves as an explant because young leaves have more active cells and less differentiation, therefore easily contact with a suitable hormone in the MS culture medium .

In this research, the second part aimed to find out the best MS media for both chamomile and yarrow in vitro seeds culture. The use of PPM was aimed to decrease the risk and controlling culture contamination, without causing a problem for the in vitro processing. Application of six different PGRs was used as a basic and sufficient medium for both chamomile and yarrow in vitro seeds culture. Significant differences were observed between different hormones and their concentration on the percentage of germinated seeds. Other reports indicated the effect of PGRs on chamomile and

57 yarrow seeds culture (Sato et al., 2006, Turker et al., 2009). The main findings in this study indicated that the greatest PGRs culture medium for chamomile and yarrow seeds germination was the culture with 0.5 mgL-1 of IAA and 1.0 mgL-1 GA3, respectively. However the lowest percentages of both chamomile and yarrow germinated seeds were observed in MS medium containing 0.5 mgL-1 and 2.0 mgL-1 of

2, 4-D, respectively (Figure 2.10). This suggested that 0.5 mgL-1 of IAA and 1.0 mgL-1

GA3, as well as chamomile free hormone use were sufficient to use for the next experiment of the highest production of in vitro leaves and flowers as mentioned in section 2.3.4. Moreover, one of the advantages of the use of different PGRs here indicated that the technique is an easy and stable way to manipulate the level of seed germination, and to select the best concentration and suitable hormone for in vitro explants culture. Yang and Xiong (2012) demonstrated that the use of different hormones is essential to maintain different type of cells function. For example the combination of BAP and 2, 4-D can cause embryo development and differentiation from primary cell culture (Haensch, 2007). These hormone combinations could be useful to germinate more yarrow seeds, callus production, shoot and root development from explants.

Several studies have been reported to examine the role of different concentrations of

PGRs on in vitro micropropagation trial. Khalafalla et al. (2007) showed that the optimal root productivity from shoot (95%) and the best numbers of root/shoot were observed only on the medium culture supplemented with 0.5 mgL-1 and 1.0 mgL-1 of

IAA, respectively. However, our study found that roots were also able to form from micro shoots in the absence of PGRs in the MS medium (control), and then adapted easily into the soil. This may related to the chamomile and yarrow seeds content of

58 micro shoot endogenous hormone that enhances and enables rooting process on MS medium without PGRs.

Results showed that yarrow seeds did not germinate in 0.5 mgL-1 and 1.0 mgL-1 of both Kin and 2, 4-D. Also, 2, 4-D lacked capacity to increase the percentage of chamomile germinated seeds compared to other PGRs. This could be because of either adding a small amount of PGRs in culture media not being sufficient enough to complete the germination process or possibly due to the harmful effects of high levels or concentrations of some macro and micro elements on plant seeds, which could reduce the germination rate (Ghasempour et al., 2012). This might then lead to the use of IAA, GA3, NAA or BAP instead of Kin and 2, 4-D for improving in vitro chamomile and yarrow germinated seeds.

The results also showed that seeds of both plants can germinate and continue to grow and produce callus, shoot and roots at different concentration of PGRs especially those cultured in the studied media containing NAA, IAA and GA3. This finding appears to agree with the study done by (Turker et al., 2009) who discovered that the highest roots and shoots were obtained from the seed culture on MS media supplemented by IAA, NAA, 2, 4-D and GA3. Therefore, the most sufficient culture medium for in vitro micropropagation was essential to produce the maximum number of both plant seeds germination.

Ghasempour et al. (2012) have also previously found that the number of leaves of the in vitro seed culture of Achillia bibersteinii L. were formed on the different concentrations of BAP (0.0, 2.0, 5.0, 10.0 mg L-1) and no significant differences in leaf

59 formation between 5.0 mg L-1 and 10 mg L-1 BAP. Subsequently, both shoots and callus were formed on MS media complemented with 5.0 and 10.0 mg L-1 BAP, while the ratio of callus was much higher than shoot induction on 5 mg L-1 BAP. Furthermore, the best effective MS medium for Achillea bibersteinii L. micropropagation was 2.0 and

5.0 mg L-1BAP, however, the 2.0 mg L-1 BAP was more economic and optimal. In contrast, the optimal concentration for Achillea millefolium L. was 1.0 mgL-1 GA3 in the present study compared to BAP.

In addition, the use of different PGRs for regenerated shoots of Achillea millefolium L. was reported by Turker et al. (2009). They discovered that good shoots and root segments were formed on MSMO (Murashige and Skoog Medium with Minimal

Organics) medium comprising different concentrations of NAA, IAA, 2,4-D or IBA

(Indole-3-butyric acid) , however, IBA at 0.5 or 1.0 mg L-1 or NAA at 0.5 mg L-1 were the most productive. Thus, the higher numbers of shoots were gained when shoot-tips were cultured on MSMO medium complemented with 1.0 mg L-1 IBA and 5.0 mg L-1

Kin, or 0.5 mg L-1 IAA and mg L-1 BA, producing 17.0 and 17.3 shoots per explant at

100% frequency, respectively. Therefore, using IBA is suggested for in vitro yarrow seeds culture as an effective auxin for reducing germinated seeds problem as observed in the present finding. According to the data collected in this present study it is clear that the production of shoots and roots were faster in in vitro cultured seeds of chamomile and yarrow plants than in glasshouse plants. Due to the importance of in vitro micropropagation protocols more work will be required such as shoots, roots and callus bioactive compounds identification using PGRs to gain high chamomile and yarrow regeneration rates and to provide maximum levels of plant secondary metabolites such as terpenoid, phenol, and flavonoid compounds (Rout et al., 2000).

2.6 Conclusion

The current study was focused on the micropropagation technique as a successful procedure for in vitro chamomile and yarrow seeds culture by the effect of different concentration of PGRs on the number of germinated seeds, shoots, roots and callus formation. IAA and NAA were found to be the best hormones. Furthermore, the use of

PGRs appeared to be able to modify cell growth in the culture medium and they will become the main plant hormones to improve growth and organ differentiation in tissue culture process. Seeds of both plant species were successfully germinated by the use of PGRs. It was demonstrated that tissue culture technique provides a large amount of plants via root and shoot multiplication from a parent plant under sterilized conditions.

Overall, it is considered that both plants were affected by PGRs and free hormone within the MS culture medium. This could be a focus for future work to establish the best concentration to enhance in vitro medicinal plant growth and development.

Chapter three

Characterisation of the growth and development of in vitro and in vivo seeds culture

3.1 Introduction

Chamomile and yarrow are both medicinal plants cultivated all over the world in an effort to determine the main active compounds from plant materials. The main factors that influence plant growth and development to increase the bioactive compounds are temperature, light, and planting date or growing seasons. Extensive studies have been done to explore the effect of controlled conditions on the growth of in vitro and in vivo chamomile (Matricaia chamomilla L.) and yarrow (Achillea millefolium L.) cultures

(Giorgi et al., 2013, Mohammad et al., 2010). Homogeneity, time, and rate of germination are factors which could be measured during germination process (Ranal and Santana, 2006).

Mohammad et al. (2010) reported that date of planting and age of seedling had significant effects on chamomile morphological characterizations grown in field conditions. The maximum values for plant height, number of branches and the diameter of flower heads were gradually decreased with a late planting date (30th May).

In terms of yarrow plants, yield and plant growth have been affected by seasons and weather conditions including hot and cold treatments. It has been illustrated that cold weather can increase leaf size and plant height. Empirical manipulation of culture temperatures from a high diurnal temperature range of 27.0 D/14.0 N °C to a lower diurnal temperature range of 12.5 D /7.5 N °C raised larger leaves by 35% and plant height by 50% and additional biomass was observed, approximately 0.117 kgL-1 plant compared to the warmer temperatures. In the cold chamber the first flower bud was detected after approximately three months, whilst in warmer conditions they appeared after two months (Kindlovits et al., 2014).

Conversely, temperature was an important factor, strongly affecting all growth parameters including plant height, vegetative growth and flowering of yarrow during different growing seasons. Plants grown at warmer temperatures were much taller than those grown at lower temperatures. In terms of flower bud formation, the time of flowering was delayed by about 20 days at low temperatures (Zhang et al., 1996).

Additionally, the measurement of plant height, number of branches and flowers per plant are also important characteristics which need to be considered by plant physiologists before any experimental manipulation can be fully evaluated. Adaptability of plant species into a wide range of soils and climates are also essential issues

(Razmjoo et al., 2008).

The different culture room temperature and light regimes of 18°C in 8 h light and 16 h dark and at 22°C in 24 h light used in this study was important as a preliminary work to determine the percentage of germinated seeds before growth in different seasons.

Growth parameters like plant height, branch numbers and flower numbers were observed to compare the growth rate trial of both in vitro and in vivo cultures (winter and summer seasons) under different environment. A further aim was to examine the best controlled conditions to get a final result of plant growth characterization.

3.2 Objectives

The main purposes of these present studies were:

1. To determine the ideal culture conditions of selected plant species.

2. To investigate in vitro and in vivo winter and summer growth stages under different environmental conditions.

3. To investigate a protocol for bringing plants from tissue culture to the soil in the glasshouse and compare how these plants improve with plants grown in soil media.

4. To determine the effect of temperature and light on plant seeds germination under controlled conditions.

3.3 Materials and methods

3.3.1 The effect of light and temperature on the germination of chamomile and yarrow

Two different temperatures (18°C and 22°C) and two different light periods (24h light,

8h light: 16h dark) were used. The source of chamomile seeds used in this test was

Richters Herbs (RH) and stickysticky.com (SS). Yarrow seeds were obtained from

Skardon garden (SG), (University of Plymouth) and Richters Herbs (RH). The temperature ranges were between 18 °C in 8 h light and 16h dark in the SANYO plant growth chamber and at 22°C in 24 h light at culture room temperature. In this test 20 seeds were selected for each species. No.1 Whatman filter papers were used and placed on the petri dishes (Benvenuti et al., 2001). Number of germinated seeds were counted after 2 days of germination for all chamomile and yarrow sources under 18°C and 22°C for a period of (20 days).

3.3.2 In vivo culture of winter and summer planting seasons

Initially, John Innes compost (seed sowing compost, No.1 and No.2 potting compost) were used for all following experiments in both seasons. In 2013- 2014, this work was carried out in the glasshouse (Cropley commercial glasshouses, controlled by a

Tomtech T200 environmental computer) of the Skardon garden, Plymouth University

UK. RH chamomile and SG yarrow seeds were used in this experiment. Seeds were sown in two different planting years as follows:

 On the 2nd December 2013. These winter plants were grown under controlled

conditions of temperature relative humidity, and normal sunlight by using the

automatic shading system on hot days during plant growth and development

(Figure 3.6).

 The 2nd June 2014. The summer plants were grown under normal

environmental condition outside the glasshouse.

 For both seasons, seeds were sown using the John Innes seed sowing compost

placed into the plastic seed trays (220×160mm) with triplicates for each species.

Growth stages (GS) including seeds germination (GS0), leaf production (GS1),

stem extension (GS2), branch elongation (GS3), start flowering (GS4) and

complete flowering (GS5) were recorded and classified in both seasons (Table

3.1).

Plant height (cm), branching of side shoots, number of flowers per plant were also measured as an indication of growth and development. Data was recorded every 3 days, starting at the beginning of December through to the middle of July for winter plants and starting from the beginning of June until the end of September for summer plants. Temperature and relative humidity for winter season were also recorded every

3 days by data logger (Tiny Tag explorer version 4.7) placed between plants into the glasshouse, (Figure 3.7).

In terms of winter season, after two weeks of sowing, plants were transferred into the small black plastic pots (88.9 mm2) when they were (5-7cm) tall (one plant per pot), with the mixture of John Innes compost No.1. Then, after five weeks of sowing the

65 small pots were changed to new, larger pots (127 mm2) containing John Innes potting compost No.2 when plants were (12-15 cm) tall. Subsequently, in summer (five months after sowing) plants with their own pots were transported outside the glasshouse to prevent harm from whiteflies, aphids and mildew. Conversely, six weeks after sowing summer plants were transferred directly into the normal soil outside the glasshouse in the middle of July, where they improved well over the summer.

Winter and summer plants were sprayed twice against whiteflies, bugs and aphids with

Bayer Garden Provado Ultimate Bug Killer (14.8 ml L-1) and once with Miracle-Gro

(3.33 ml L-1) as a plant food to keep them free from pests and disease. All plants were watered every 2-3 days.

Table 3.1 Growth stages description of the chamomile and yarrow plant growth cycles.

Growth stages Growth descrip tion Chamomile Yarrow

GS0 Seed germination

GS1 Leaf production

Stem extension GS2 (Plant height)

GS3 Branch elongation

GS4 Start flowering

GS5 Complete flowering

3.3.3 In vitro culture

In terms of in vitro culture, yarrow seeds were cultured in MS medium complemented with 1.0 mgL-1 of GA3 from the beginning of culture until transferring plantlets to soil.

Chamomile MS medium was supplemented with 0.5 mgL-1 of IAA in the first culture until callus formation. After two weeks of seed culture, callus formations from chamomile seeds were transferred to the free plant growth regulator MS medium and shoots production from yarrow seeds were transferred to the 1.0 mgL-1GA3 medium culture. Explants from both plant species were sub-cultured on MS medium every 4-6 weeks. Plantlets were removed from the MS medium culture whenever roots appeared; they were then transported into disposable plastic pots containing John Innes No.1 compost. Plantlets were incubated for a further two weeks to allow adaptation to the soil; they were irrigated with sterilised water every 2-3 days. They were then transplanted in to the small seed pots containing John Innes No.2 compost and kept under glasshouse conditions until flowering began. Plants developed and grew well in the glasshouse until (GS4), they were then transported outside the glasshouse to develop and complete their growth with in vivo plants (Figure 3.1).

3.3.4 Statistical analysis

Data from this study was statistically analysed using Minitab™ statistical software

(Version17). ANOVA test was applied to determine the significant difference among groups for all parametric data. Least significant difference (LSD) was also tested. All data are presented as mean and standard error, and significance differences were accepted at p ≤ 0.05.

In vitro chamomile seeds culture process

In vitro yarrow seeds culture process

3.4 Results

3.4.1 The influence of light regimes and different temperatures on plant seed germination

The effect of light and temperature on chamomile and yarrow germination was investigated. Results in this test indicated that temperature ranges and light had a significant effect on the percentage of seed germination. Seeds from different sources were germinated gradually starting from seeds embryo and continued to produce roots and shoots under different light and temperature regimes. After 20 days of trial, results revealed that the highest and lowest percentages of germination rates were observed at 18°C and 8 h light: 16 h dark of chamomile/ RH and SS seeds (87.71 ± 3.26 % and

39.58 ± 2.44 %) respectively. However, the highest and lowest germination rate for yarrow/SG and RH seeds were noticed at 22°C and 24 h light (94.17±1.85 % and

11.88 ±1.80 %), respectively which is shown in Table 3.2.

Results were also showed that the highest germination rates of chamomile seeds were found in chamomile/ RH compared to chamomile/ SS in both different temperature and light regimes. The statistical analysis of chamomile seeds showed higher significant differences of both chamomile (RH and SS ) germinated seeds under 18°C and 22 °C

(F 204.231, 44; p< 0.001) and (F 229.191, 44; p < 0.001), respectively, (Figure 3.2 and

3.3).

Table 3.2 Summary of the effect of the temperature and light regimes on the percentage of chamomile and yarrow germinated seeds.

Plant Sp.& Sources Temperature °C and Light Germinated Seeds %

Chamomile/ RH 18 °C 8 h light: 16 h dark 87.71± 3.26

22 °C 24 h light 86.25± 1.91

Chamomile/SS 18 °C 8 h light: 16 h dark 39.58± 2.44

22 °C 24 h light 41.67± 2.34

Yarrow/ RH 18 °C 8 h light: 16 h dark 12.08± 1.9

22 °C 24 h light 11.88± 1.8

Yarrow/ SG 18 °C 8 h light: 16 h dark 87.92± 3.11

22 °C 24 h light 94.17± 1.85

Both temperature and light regimes also showed a great positive effect on yarrow germinated seeds. The highest germination was observed in yarrow/ SG compared to yarrow/ RH in both temperature and light regimes. The test of analysis of variance showed that the percentages of germinated seeds were significantly affected by different temperatures at 18°C and 22°C (F 467.961, 44; p< 0.001) and (F 1128.021, 44; p< 0.001), respectively (Figure 3.4 & 3.5). In total, high differences were not observed between two temperature regimes for each seed source. Additionally, the best conditions to obtain a maximum percentage of Ch/ RH and Y/ RH germinated seed was 18 °C 8 h light: 16 h dark, however 22°C 24 h light was found to be the best conditions for both Ch/ SS and Y/ SG (Table 3.2).

Figure 3.4 The percentage of yarrow (RH/SG) germinated seeds at 18°C, 8h light: 16 h dark. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed source= 7.05).

Figure 3.5 The percentage of yarrow (RH/SG) germinated seeds under 22°C and 24 h light. Data are presented as means ± SE of three replicates for each variable. (LSD value for seed sources= 4.94).

3.4.2 In vitro and in vivo winter and summer growth rate characterisations

The results of growth rate parameters (plant height, branch numbers and flower numbers) of in vitro and in vivo chamomile and yarrow cultures revealed that the plant growth and development of both cultures were extremely different under controlled environmental conditions of temperature and relative humidity (Figure 3.6). In vitro plant species started growing slowly after being transplanted into the soil. In terms of plant height, it was found that in vitro yarrow started growing better and taller (30.64 cm) with lower rate than in vitro chamomile (22 cm), Figure 3.7. However, in vivo winter sown chamomile plants grew better than yarrow plants regarding the plant height. It was found that chamomile had stronger growth and was taller (26.63 cm) than yarrow (20.67 cm), Figures 3.8. Similar to in vivo winter plants, in vivo summer chamomile grew and developed more quickly and higher (23.93 cm) than yarrow

(13.52 cm). Both in vivo summer plants started growing rapidly in the soil (Figures 3.9).

Overall, highly differences were found between date, in vitro and in vivo winter and summer plants for plant height. Moreover, the growth rate of plant height was progressively increased with time throughout stem elongation (growth stage GS2).

Figure 3.6 Average temperature and relative humidity of in vivo chamomile and yarrow grown into the glasshouse.

Figure 3.7 In vitro chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension).

Figure 3.8 In vivo winter chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension).

77 l;.#’

GS1 GS2

Figure 3.9 In vivo summer chamomile (Ch) and yarrow (Y) plant height developments. GS1= Leaf production GS2= Plant height (Stem extension).

Regarding the vegetative growth, it was observed that in vitro and in vivo winter chamomile and yarrow plants start branching at almost identical times with maximum rates observed in yarrow (5.26 and 5.85) compared to chamomile (3.64 and 4.68), respectively (Figures 3.10 and 3.11). Different from in vitro and in vivo winter plants, the highest rate and production of in vivo summer branches noted in chamomile (4.65) compared to yarrow (1.55), Figures 3.12. Plants from both in vitro and in vivo (winter and summer) cultures were well branched over time of growth stage (GS3).

GS1 GS2 GS3

Figure 3.10 in vitro chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation.

GS1 GS2 GS3

Figure 3.11 In vivo winter chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation.

GS1 GS2 GS3

Figure 3.12 In vivo summer chamomile (Ch) and yarrow (Y) vegetative branch developments. GS1= Leaf production GS2= Plant height (Stem extension) GS3= Branch elongation.

In terms of chamomile flower and yarrow florets, it is clear that for all in vitro and in vivo

(summer and winter) plants chamomile produced more flowers and at a faster rate

(19.19, 16.78 and 28.85) than yarrow plants (0.68, 1.61 and 1.26), respectively (Figure

3.13, 3.14 and 3.15). The results showed that all in vitro and in vivo plants were flowering well over time, where chamomile plants started and completed flowering

(growth stage GS4 and GS5) before yarrow plants.

GS3 GS4 GS5

Figure 3.13 In vitro chamomile (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering.

Figure 3.14 In vivo winter (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering.

Figure 3.15 In vivo summer (Ch) and yarrow (Y) flowering developments. GS3= Branch elongation GS4= Start flowering GS5= Complete flowering.

Overall, it was found that summer plants grew and developed much faster for all three parameters of plant height, branching and flowering compared with in vivo winter plants. Moreover, a highly differences was also found in growth between in vitro and in vivo winter and summer plants at the time of flowering

As a result, in vitro plants growth parameters (GS2, GS3, GS4 and GS5) were taken at approximately five and half months, which started from the bigeninig of February until late July. In vivo winter plants growth parameters took the longest time at almost seven months starting in the middle of December through to the bigenning of July.

Additionally, in vivo summer plants growth parameters were shorter than both in vitro and in vivo winter growth parameters, which took around three and half months, starting from the beginning of June through to late September.

3.5 Discussion

In this experiment the effect of light and temperature on chamomile and yarrow seeds and the effect of two different culture conditions on plant growth characterization were described. This series of experiments were designed to characterise and evaluate the growth and development of both chamomile and yarrow species that were either grown during the summer months or during the winter, and temperature ranges and light had also a significant effect on the percentage of seed germination. According to these investigations, the researchers must select the best conditions of temperature, light and growing season to provide the highest percentage of seed germination and plants final production based on their aims. The characterisation of seed germination under

85 different light and temperature regimes is also important for the study of seed ecology

(Ranal and Santana, 2006).

The current results showed significant differences at p≤0.001 between both plants under two different temperature regimes. Chamomile seeds from RH and yarrow seeds from SG have much better germination rates than chamomile and yarrow seeds from SS and RH, respectively. This could be related to the storage conditions and plant seeds age. In this case a viability test is suggested to determine the activity of seeds before germination. These results confirm the important role of temperature and light to encourage seed germination. Our result are similar to those described by El-

Keblawy and Al-Rawai (2005), who reported that the highest percentage of germinated seeds occurred at 25°C compared to 40°C and germination in the dark was significantly lower than in the light at high temperature. It seems that these plant seeds are germinated and adapted well under controlled light and temperature conditions.

In this study the most important finding is a measurement of plant height, branch numbers and flower numbers during two different seasons, which are recommended for the study of the medicinal and aromatic plants production such as flower numbers to provide a high level of essential oil content. Also, it is quite interesting that these parameters were compared between in vitro and in vivo cultures. It seems that in vitro and in vivo winter yarrow is taller and grows guicker than chamomile for vegetative growth. This might be due to the glasshouse temperature range as a main factor affecting plant growth, it is probable winter temperature was better suited for yarrow plant heights compared to hot summer temperatures. However flowering in chamomile occurs earlier than that of yarrow in both cultures and seasons. This result suggests

86 that both plants adapt to a wide range of different cultures and seasons very easily however, as a result plant habit and morphology might change significantly, where they adapt to grow best and faster in the summer season outside the greenhouse compared to the winter season (Salim et al., 2014).

It was observed that during a period of three to six months in vivo plants were successfully grown and developed well both inside and outside the glasshouse.

Moreover, at six to seven months plants that were successfully grown in tissue culture media and soil were getting a higher number of flowers of both plant species. Three months after transferring winter plants into the large pots, plants began flowering and progressed well over the summer outside the glasshouse Nidagundi & Hegde, (2007).

Different culture conditions had a statistically significant effect on plant growth parameters, conforming with Ebrahimi et al. (2011). Depending on the environmental conditions, plants start flowering about two to three months after transplantation.

It was clear that growth parameters were different for both plant species in winter glasshouse controlled conditions and summer plants grown outside under normal seasonal conditions. As a result, the summer season was found to be best suited for optimal plant height and branching. These results were probably due to the net photosynthesis of plants being higher in summer time than winter time which encourage plants to start growing fast. A higher number of flowers were also obtained in plants grown in summer months compared to plants grown in winter months. This is caused by the fact that photoperiod and long day season have a great role in the formation and improvement of flower (Heide and Sønsteby, 2011).

Our current results can be supported by a previous study reported by Zhang et al.

(1996), who revealed that yarrow flowering and plant height is variable and responds

87 positively to warmer temperatures normally seen during the summer months. Flower buds formed quickly at 18°C compared to 10°C; plants were also taller than those grown at 18°C. This indicates that the net photosynthesis of yarrow plants was high at higher temperatures than those grown at cooler temperatures. This point provides a very beneficial guideline to promote flowering palnts growth in the summer season and avoid growing during cold weather.

In case of the flowering period, Nidagundi and Hegde (2007) indicated that depending on environmental conditions, plants start flowering about two to three months after transplanting. This is in agreement with the present study in terms of chamomile flowering stage. The maximum plant height (111.8 cm) and the yield of flower (270.4 g m-2) were also observed as a final result of chamomile plant under 12°C by Ebrahimi et al. (2011). These results demonstrate that plant height and flower yield were significantly affected by temperature. Regarding the growth characterisation, more studies might be needed to investigate the effect of phosphorous, nitrogen, micro- nutrient and potassium on plant growth stages of Matricaria recutita L. (Upadhyay and

Patra, 2011). After these observations of growth stage parameters like GS2, GS3 and

GS4 in this work as the most important parameters, further studies of herbal plants and economical crops grow in different seasons may be required to elucidate the role of these parameters in order to increase the oil yield and crop production.

3.6 Conclusion

In this chapter the studies indicated that number of germinated seeds of both chamomile and yarrow seeds under different temperature and light conditions, and the in vitro and in vivo winter and summer plant growths were significantly different. Both plant species provide good examples of medicinal plants which are affected by the

88 date of planting, temperature and relative humidity which significantly affects chamomile and yarrow growth stages. The use of in vivo cultivation seasons had a significant effect on growth parameters. Depending on the date of in vitro an in vivo cultivation different growth parameters of plant height, branching and flowering developments were informed for both plant species.

It was also confirmed that flowering stage appears to be more affected by temperature than plant height and vegetative growth, and summer season is the best time for growing chamomile and yarrow plants. These results might be used as a good way to encourage farmers to start cultivating these plant species under full control conditions using different seasons of culture.

Chapter four

The extraction of bioactive phytochemicals from chamomile and yarrow plants with their separation and measurement using HPLC-UV and GC-FID

4.1 Introduction

Medicinal plants produce some beneficial secondary products such as phenols, flavonoids and terpenoids which make up one of the most diverse and largest groups of chemical compounds (Lin et al., 2016, Pichersky and Gang, 2000). Chamomile

(Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) are medicinal plants that contain phytochemical compounds such as chlorogenic acid, caffeic acid, apigenin-7-glucoside, luteolin, chamazulene and nerolidol (Haghi et al., 2014,

Judzentiene and Mockute, 2010). These plants can provide phytochemical compounds for use in food and pharmaceutical industries, aromatherapy and cosmetics. Additionally, they have anti-inflammatory and antioxidant properties (Dias et al., 2013, Nowak et al., 2010, Srivastava et al., 2010). To date, several methods have been used to investigate the extraction and identification of the phytochemical compounds found in chamomile and yarrow plants such as Soxhlet and shaking extractions using methanol as a solvent (Ahmad et al., 2009, Złotek et al., 2016).

Methanol extraction, HPLC-UV and GC apparatus were found to be more efficient for quantitative and qualitative analysis of these bioactive compounds (Hădărugăa et al.,

2009, Nováková et al., 2010, Roby et al., 2013).

Several solvents have been used based on their polarity in order to extract a maximum amount of active compounds with the greatest biological activity (Doughari,

2012). It has been demonstrated that selecting polar solvents such as methanol and non-polar solvents for instance hexane are key to extracting a high range of plant active compounds (Green, 2004). Soxhlet (hot) and shaking extractions using several solvents have been widely used for plant parts extraction (Khan et al., 2012, Sultana et al., 2009). Soxhlet extraction is an approved method used on semi-volatile and

90 volatile phytochemical compounds from plant material (Schwab et al., 1999).

However, shaking extraction is a conventional method usually used for phenol and flavonoid compounds of plant material such as leaves (Dent et al., 2013). Ncube et al.

(2008) have reported the yield of extraction is affected by the origin of sample tissue such as leaves, stems and/or floral tissue, the type of solvent and extraction methods.

The authors have also found that type and time of extraction, concentration of solvent and temperature will affect plant secondary products and the yield of extraction.

Various studies have been explored to identify potential bioactive compounds and their medicinal properties (Srivastava and Gupta, 2009, Tuberoso et al., 2009).

Phenols and flavonoids have been found in the methanol extraction of chamomile

(Guimarães et al., 2013). Apigenin-7-O-glucoside has been also found to be the main chamomile flavonoid compound using shaking extraction (Srivastava and Gupta,

2009). However, apigenin, luteolin, quercetin and 6-hydroxykaempferol-3, 6, 4- trimethylether from shaking extraction have been found to be major flavonoid compounds of in vivo Achillea sp (Tuberoso et al., 2009). Moreover, it has been demonstrated that chlorogenic acid and apigenin- 7-O-glucoside were found in wild yarrow flowers as major compounds (Benetis et al., 2008).

Alvarenga et al. (2015a) suggested that the important issue for in vitro medicinal plants study is a variation of essential oil components under controlled conditions.

With regard to in vitro chamomile and yarrow plants, leaf and flower secondary products such as monoterpene (cis-ascaridole, 1,8 cineole, camphor) and sesquiterpene (alpha-bisabolol, chamazulene, beta-eudesmol) have been isolated from seeds, and were analysed by GC and GC-MS (Ghasempour et al., 2012, Szőke et al., 2004). It has also been demonstrated that the essential oil content of German

91 chamomile are mainly responsible for the pharmacological properties such as azulenes and α-bisabolol (Sharafzadeh and Alizadeh, 2011). Chamazulene and α- bisabolol were also found to be the most important compounds in the flowers and leaves of yarrow essential oil (Costescu et al., 2014).

Identification of these phytochemical compounds from chamomile and yarrow leaves and flowers indicate that plant species of the Asteraceae family are a natural source of phytochemical active compounds. These compounds appear to be important in prevention of several human diseases including cardiovascular, neurodegenerative, cancers and diabetes diseases (Ganesan and Xu, 2017, Lin et al., 2016).

Thus, to explore this issue, two approaches were used. Firstly, a preliminary test was carried out of in vitro (a process of culturing seed taking place in a Petri dish within a nutrient medium) and in vivo (a process of growing seed in the glasshouse within a soil) seed culture aimed at developing primary shoot and root formation using

Murashige and Skoog (MS) medium and John Innes compost to get the whole plant for chemical analysis. Following this, plant leaves and flowers from in vitro tissue culture and in vivo culture were developed as described in Chapter 3.3.2 and 3.3.3, and then extracted by soxhlet and shaking extractions using methanol and hexane as solvents with the aim of analyse and identify any phytochemical compounds present by HPLC-UV and GC- FID

4.2 Objectives

The objectives of this study were to:

1- Assess the method of shaking extraction at room temperature using methanol for extracting phenol and flavonoid phytochemical

92 compounds content from in vitro and in vivo chamomile and yarrow leaves.

2- Determine the efficiency of non-polar (hexane) and polar (methanol) solvents using Soxhlet (hot) extraction in order to obtain the highest amount of extracted samples from the flower of both subject plants and culture.

3- Investigate quantitative and qualitative analysis of chamomile and yarrow phytochemical compounds content using HPLC- UV and GC-FID.

4.3 General Materials and methods

Extraction protocols for vegetative material (leaves)

4.3.1 In vitro and in vivo leaves preparation and shaking extraction

This trial was carried out to evaluate the efficacy of methanol extraction on the recovery of phytochemical compounds from both chamomile and yarrow leaves grown under in vitro and in vivo culture conditions. Extraction was carried out by the use of 99.9% HPLC grade methanol. Chamomile and yarrow leaves from in vivo and in vitro seeds culture were washed in distilled water and placed into a zip lock freezer storage bags. Prepared samples were then placed into the freezer (Indesit freezer) at

-20°C for one day and subsequently freeze dried in freeze drier, Edwards Modulyo at

-50°C under 10-1 mbar vacuum for 2 days. The principle of freeze drying or lyophilization method is sublimation (a process of removing 95%-99.5% water of the frozen samples and passing from solid (ice) phase to vapour (gas) phase directly).

Freeze drying is also known as a process to preserve important medicinal compounds such as phenols, flavonoids and volatiles among most drying process and consist of freezing, primary drying and secondary drying stages (Abascal et al.,

2005, Nireesha et al., 2013).

After freeze drying, 2 g of chamomile leaves were weighed into 250 ml conical flask and powdered before extraction using a porcelain pestle and mortar (Srivastava and

Gupta, 2009), all samples were prepared in triplicate. Then 60ml of methanol was added to the samples and placed on a shaker (KS 501 digital, IKA labortechnik,

Germany) for 30 min (200 rpm) at room temperature, the samples were then covered with parafilm and aluminium foil to avoid degradation of compounds and the loss of solvent, and then left in darkness for 5 days at room temperature to stand. After 5 days of dark, storage samples were adjusted to 100 ml of methanol and filtered through filter paper Whatman No.1 in a filter funnel (Nováková et al., 2010), (Figure

4.1). All three extracted samples were combined together, and then evaporated with a rotary evaporator (Buchi, Rotavapor R-3) at 40°C and further dried with a Nitrogen blow down apparatus (Dias et al., 2013). Samples were finally stored at 3 to 5°C until

HPLC analysis use (Figure 4.1). The same process was adopted for yarrow leaves.

`A B

C D

4.3.2 Standards and reagents

The standard compounds of chlorogenic acid, caffeic acid, umbelliferone, luteolin and apigenin-7-O-glucoside were supplied by Sigma Aldrich Ltd (Gillingham, Dorset, UK).

All standards were listed as HPLC grade purity. The solvent methanol and acetonitrile HPLC grade were purchased from Sigma Aldrich Ltd (Gillingham, Dorset,

UK). Acetic acid solution for HPLC grade was also purchased from Sigma Aldrich Ltd

(Gillingham, Dorset, UK). Ultra-pure (Elga) water was previously prepared on the 4th floor and 2nd floor Davy building/ Plymouth University by Water Purification System.

4.3.3 HPLC analysis

In vitro and in vivo leaves of both plant species were prepared and extracted using methanol as a solvent. The extracted samples were then analysed by Hewlett-

Packard UltiMate 3000 chromatograph (ground floor Davy building, Plymouth

University, UK) with the UltiMate 3000 pump, injection loop volume (20 μL) and a diode array detector (DAD) using 256 nm wavelength. A reversed phase cartridge guard column (Thermo Scientific, BDS Hypersil C18, 4 × 3.0 mm, AJO-4287, particle size= 5µm) was used at 25 °C. Two Analytical gradients were created as a solvent:

(A) 1% citric acid in ultrapure water (Elga) water and (B) HPLC grade acetonitrile.

The extracted samples were prepared for analysis by making two different dilutions for each sample: in the first step add 1ml methanol (99.9% HPLC grade) to the known mass of each final extracted sample return to the fridge and stand for 24 hours. The second step was preparing 10 fold dilutions from the first step samples.

1.5 ml of each prepared dilution was then placed into the HPLC vials separately and each sample was run for 20 minutes. The phenolic and flavonoid compounds were identified according to their retention times, peak area, with the injection volume 20μL and flow rate 1ml min-1. The detected samples were then compared with the standards.

4.3.4 Calibration curves

Standard calibration curves were determined from known standards (chlorogenic acid, caffeic acid, apigenin-7-glucoside, umbelliferon and luteolin) and are the average of three repeat HPLC-UV runs. Five different dilutions were prepared for each standard (5, 10, 25, 50, 100 mgL-1), by using methanol 99.9% HPLC grade as solvent. Linear and polynomial regressions were carried out on the extraction curves

96 and subsequently used to develop a calibration to determine the concentration curves. All phenolic and flavonoid compounds found in the samples were compared with standards according to their retention times and peak area. The results are presented as mean and standard error of three different analyses in mg kg-1 of plant leaves dry weight (Barros et al., 2012).

Extraction protocols for reproductive material (flowers)

4.3.5 Preparation of flowers

Freeze dried chamomile and yarrow flowers were powdered with a porcelain pestle and mortar, and then sieved through a 250 µm nylon mesh. 2 g of each prepared sample was weighed into the 250 ml flask.

4.3.6 In vitro and in vivo flowers preparation and Soxhlet extraction

Extraction was carried out by a Soxhlet extraction method using polar solvent

(methanol) in the 5th floor/ Davy building, Plymouth University, UK, and non-polar solvent (hexane), in the ground floor/ Davy building Plymouth University, UK. The process was conducted to compare the extraction quality between both solvents.

Samples were prepared by adding 150 ml of hexane or methanol (methanol 99.9% and hexane 97.0%) to 2g freeze- dried flower heads separately (samples were prepared in triplicate). After that, all samples were placed on the Soxhlet, and then the thimbles containing plant materials were situated into the Soxhlet extraction chamber (Kothari et al., 2012). After 3h of extraction the obtained samples from each replicate were collected and evaporated with a rotary evaporator (Buchi, Rotavapor

R-3) at 40°C, (Figure 4.2). The remaining essential oils were finally weighed and stored at -10°C until GC-FID analysis.

Flower extraction process

Soxhlet extraction

Figure 4.2 Soxhlet extraction procedures of in vitro and in vivo chamomile and yarrow flowers.

4.3.7 Standards and reagents

All standard compounds of α-(−)-bisabolol, bisabolol oxide A, chamazulene, limonene, nerolidol and farnesene were supplied by Sigma Aldrich Ltd (Gillingham,

Dorset, UK). Methanol, hexane and dichloromethane (DCM) HPLC grade were also purchased from Sigma Aldrich Ltd (Gillingham, Dorset, UK). Deionized water was previously prepared on the 5th floor Davy building/ Plymouth University by Water

Purification System.

4.3.8 GC-FID analysis

The essential oil analysis was carried out with Agilent Gas Chromatograph 7890A equipped (5th floor Davy building, Plymouth University, UK) with a 7683 series autosampler and 7683B series autoinjector/ Flame Ionisation Detector (FID). The GC detector temperature was 260°C with 40 ml min-1, hydrogen flow rate and 400 ml

98 min-1 air flow rate. The carrier gas was nitrogen, set to 1.0 ml min-1 flow rate. The capillary column (HP- 5 Column, 5% phenyl – 95% dimethylsiloxane, low polarity column), with (30 m × 320 µm ×0.25 µm film thickness) was used for all fractions; injection volume was 1.0 μl at 240°C.

The extracted samples were prepared for analysis by adding 250 mg of sample into

10 ml DCM for each sample. 1 ml of each sample was then placed into the GC vials and run for 40 minutes. The terpenoid compounds were identified according to their retention times and peak area. The detected samples were then compared with actual standards.

4.3.9 Calibration curves

Calibration curves were developed for different chemical standards as a quantitative analysis for the essential oils bioactive compounds. The stock solution of essential oil concentrations were determined by dissolving from quantities of limonene, farnesene, nerolidol, α-(−)-bisabolol, chamazulene and bisabolol oxide A (1, 2, 5, 10, 25, 50 mgL-1) in 10ml dichloromethane. Logistical regression, linear trendline on extraction curves were then used to develop a concentration calibration curve.

4.3.10 Statistical analysis

In this experiment the data analysis was carried out with the use of

Minitab™ statistical software (Version17). All data were analysed by balanced

ANOVA test. Means significant differences were determined by using Least

Significant Difference (LSD) test at p < 0.001.

4.4 Results

4.4.1 Methanol and hexane extraction

Results (Figure 4.3) show that methanol extraction was more efficient than hexane in both Soxhlet and shaking extractions. The highest yield of extract from 2 g dried leaves was noted in yarrow methanol extraction compared to chamomile in both

Soxhlet (0.4 ± 0.03 g) and shaking extraction (0.5 ± 0.06 g), respectively. In contrast, the greatest amount of hexane extraction was in chamomile (0.1 ± 0.01g) compared to yarrow (0.03 ± 0.01g).

Analysis of variance indicated significantly differences between both plants from

Soxhlet and shaking extraction using methanol and hexane (F 48.811, 10; p< 0.001).

Moreover, the interaction between plant species and extraction process was also significant at p< 0.001, where both Soxhlet and shaking extractions using methanol as a solvent were found to be the most efficient methods for extracting compounds from chamomile and yarrow leaves and flowers, however hexane solvent was less effective for both plant species (Figure 4.3).

100

4.4.2 In vitro and in vivo chamomile and yarrow leaves HPLC analysis

The absorbance of bioactive compounds was measured at 256 nm (Figure 4.4). In this present experiment standard calibration curves were constructed for phenolic and flavonoid compounds at different retention times. The straight lines were characterised for caffeic acid and apigenin 7-glucoside standard calibration curves from the equation (y=mx +c), however polynomial regression for chlorogenic acid, umbelliferon and luteolin were calculated from the quadratic equation solver (y = ax2

+ bx +c). All curve points are the result of three injection analyses, for example chlorogenic acid standard curve (Figure 4.5), and for the remaining standard curves

(see Appendix 2).

101

Time (min)

Figure 4.4 The HPLC-UV chromatogram of phenol and flavonoid compounds content of in vivo chamomile leaves detected at 256 nm.

Figure 4.5 Standard calibration curve for chlorogenic acid polynomial regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent.

102

Table 4.1 shows all phenol and flavonoid standard compounds detected by HPLC-

UV at different retention times and area. In terms of samples analysis, all subject compounds (chlorogenic acid, apigenin 7-glucoside, caffeic acid, umbelliferone and luteolin) were detected in chamomile leaves, however only three of them (caffeic acid, umbelliferone and luteolin) were found in yarrow leaves (Figure 4.6 and Figure 4.7).

Chlorogenic acid was detected as a main compound found in both chamomile culture conditions. Chlorogenic acid and apigenin 7-glucoside were also the most abundant compounds isolated from chamomile leaves from the in vivo (96.38 ± 0.53 mg kg-1,

72.96 ± 0.42 mg kg-1) and in vitro (48.80 ± 4.70 mg kg-1, 13.44 ± 0.75 mg kg-1) cultures, respectively, (Figure 4.6). Results showed higher significant differences for both culture conditions of chamomile bioactive compounds content (F 648.331, 18; p<

0.001). Moreover, the interaction between bioactive compounds content and culture conditions was also significant (p< 0.001), where the maximum values of both phenolic (chlorogenic acid and caffeic acid) and flavonoid (apigenin 7-glucoside, umbelliferone and luteolin) compounds were found in in vivo chamomile leaves compared to in vitro leaves (Figure 4.6).

Caffeic acid, umbelliferone and luteolin were the only main compounds found in in vivo and in vitro yarrow leaves. Luteolin dominated among two other compounds in both in vivo (9.51± 2.29 mg kg-1) and in vitro (6.35± 0.04 mg kg-1) yarrow leaves, respectively, (Figure 4.7). Analysis of variance showed that yarrow bioactive compounds content was also significantly affected by culture conditions (F 41.891, 10; p≤0.001). Furthermore, there was no significant interaction between bioactive compounds content and culture conditions (p= 0.073).

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In addition, hexane extraction from in vivo chamomile and yarrow leaves was also analysed by HPLC-UV. Chlorogenic acid was only found as a main compound in chamomile leaves however some unknown peaks were also detected in yarrow leaves, this might be caused by keeping and storing extracted samples in the fridge for long periods of time. (Appendix 3) The results indicated that methanol extraction positively affected the bioactive compounds content in comparison to hexane.

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Table 4.1 Phenolic and flavonoid chemical standards detected by HPLC at different retention times, peak areas and the limit of detection (LOD) value.

Standard Name Chemical Structure Formula Rt Peak LOD -1 (min) Area* (mg L ) (mAU*min)

Chlorogenic acid C16H18O9 6.09 33.18 0.46

Caffeic acid C9H8O4 6.81 80.07 1.49

Apigenin 7- glucoside C21H20O10 7.75 28.28 3.42

Umbelliferone C9H6O3 7.91 32.80 0.11

Luteolin C15H10O6 8.87 168.21 1.06

* To calculate the amount of chemical compounds see figures 4.6 & 4.7.

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4.4.3 In vitro and in vivo chamomile and yarrow flowers GC-FID analysis

In this present work standard calibration curves were constructed for terpenoid compounds found in the flower essential oil at different retention times. The straight lines were characterized for all standard calibration curves from the equation (y=mx

+c). All curve points are the result of three injection analyses and they indicated good linearity with a regression coefficient (R2) between 0.9998 and 0.9999, for example bisabolol oxide A standard curve as shown in Figure 4.8 (for the remaining standard curves see Appendix 2).The results were presented as mean and standard Error of three different analyses in mg kg-1 of plant flowers dry weight.

Figure 4.8 Standard calibration curve for bisabolol oxide A linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

Two different cultures were carried out on the chamomile and yarrow flowers extraction to identify tepenoid compounds content and to compare terpenoids content between both in vitro and in vivo cultures. Table 4.2 shows all terpenoid standard compounds were detected by GC-FID at different retention times and peak areas.

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Some sesquiterpene compounds such as farnesene, chamazulene and bisabolol oxide A were found as main terpenoids in both chamomile culture conditions (Figure

4.9). Farnesene dominated both cultures with the highest production in in vivo

(165.44± 21.97 mg kg-1) chamomile flowers compared with in vitro (42.80 ± 2.09 mg kg-1), respectively. However, chamazulene and bisabolol oxide A were identified in lower concentrations. Results from the chamomile plants showed significant differences between in vitro and in vivo leaves bioactive compounds content (F

51.512, 10; p< 0.001). Moreover, the interaction between bioactive compounds content and culture conditions was also significant at p= 0.001, where the maximum values of all terpenoid compounds were found in in vivo chamomile essential oil compared to in vitro essential oil (Figure 4.10).

Results from the yarrow plants indicated that in vivo and in vitro cultures positively affected flower essential oil content compared to phenols and flavonoids content. The total terpenoids (sesquiterpenoids) content of in vitro culture was significantly higher than in vivo culture regarding the flowers compounds content, where limonene was only found as a main monoterpene in in vivo yarrow flowers (F 65.69 3, 14; p< 0.001).

Nerolidol dominated among other terpenoid compounds found in both culture conditions with the minimum value found in in vivo (16.09± 0.75 mg kg-1) flowers compared to in vitro (23.51±1.96 mg kg-1) flowers, respectively (Figure 4.11).

Additionally, the interaction between bioactive compounds content and culture conditions was also significant (p< 0.001), where the maximum values of all terpenoid compounds were found in in vitro yarrow essential oil compared with in vivo essential oil and limonene was not detected in in vitro culture.

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Figure 4.9 GC-FID analyses of the selected essential oil compounds of in vivo chamomile flowers extracted by methanol using Soxhlet extraction.

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Table 4.2 Terpenoid chemical standards detected by GC-FID at different retention times, peak areas and the limit of detection (LOD) value.

Standard Name Chemical Structure Formula Rt Peak LOD -1 (min) Area* (mg L ) (mAU*min)

Limonene C10H16 9.02 289.0 0.02

16.67 13.37 0.19 trans-β- Farnesene C15H24 17.10 15.50 0.08

17.97 24.16 0.10

Nerolidol C15H26O 18.90 247.48 0.02

α-(−)-bisabolol C15H26O 20.88 214.89 0.02

Chamazulene C14H16 21.74 124.38 0.02

Bisabolol oxide A C15H26O2 21.91 186.83 0.02

* To calculate the amount of chemical compounds see figures 4.10 & 4.11.

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4.5 Discussion

Methanol extraction was found to be more efficient than hexane for phytochemical compounds from both leaf and flower parts. The different percentages of extracted amounts with hexane and methanol might be influenced by solvent polarities. This is in agreement with those reported by Ahmad et al. (2009) who previously indicated that their study showed methanol was a superior solvent in comparison to hexane with regard to the percentage of extraction yield using Soxhlet extraction, where the yield of methanol extraction was approximately 50% more than the percentage of hexane extraction.

The methanol extraction, HPLC-UV and GC-FID methods used here have been adapted from previous studies, which are based on the use of these methods for the same reason of the identification of phenol, flavonoid and terpene compounds content (Costescu et al., 2014, Dias et al., 2013, Srivastava and Gupta, 2009, Szoke et al., 2003). In both methods preparation of stock and dilutions of extracted samples were slightly difficult to complete, possibly because of the presence of some solvent in the extracted samples after rotary evaporation process especially in the Soxhlet extraction of the essential oil, where samples were not blowing down to evaporate the solvent because of the volatile compounds content.

According to the current results, HPLC-UV analysis indicated that a maximum phytochemical compounds of in vivo compounds content were significantly (p <

0.001) higher than in vitro leaves content (Figure 4.7 and 4.8), this may be due to the environmental conditions such as light and temperature, as well as plant growing period, different growth media and soil vs nutrient medium (Ghasemzadeh et al.,

2010). Chlorogenic acid and apigenin 7-glucoside were not detected in yarrow leaves,

112 however chlorogenic acid was found as a main phenolic compound in chamomile leaves, this could be caused by degradation of these two compounds in yarrow leaves during the process of shaking extraction under room temperature and light.

This indicates that in vitro growing was not stable for phenols and flavonoids total yield found in yarrow leaves.

In support of these current findings, previous studies revealed that in chamomile methanolic extraction, chlorogenic acid and apigenin- 7-O-glucoside are a major phenol flavonoid compounds (Kováčik et al., 2008, Srivastava and Gupta, 2009), however apigenin-6-C-glucoside-8-C-arabinoside (1.41 mg g-1) and luteolin (1.70 mg g-1) were the main flavonoids detected in yarrow (Tuberoso et al., 2009). Moreover,

Benetis et al. (2008) in their study identified eight major phenolic and flavonoid compounds from yarrow extraction using ethanol by HPLC analysis including chlorogenic acid, apigenin-7-O-glucoside, vicenin-2, luteolin-7-O-glucoside, rutin, luteolin-3, 7-di-O-glucoside, apigenin and luteolin.

It appears that minimum values of terpenoid compounds found in in vitro chamomile and in vivo yarrow, which could be related to disadvantages of the Soxhlet extraction such as a long extraction process period and which may have led to loss of some volatile compounds content. In addition, limonene was not found in in vitro yarrow flowers content (Figure 4.11), this could be due to the relative insolubility of some essential oil compounds in dichloromethane. Similar observation was reported in

Santalum album L. by Misra and Dey (2012). They investigated that in vivo sesquiterpenoids content (8- 37.4 mg g-1) was found to be less than that of in vitro extract (17.3- 51.4 mg g-1).

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Nerolidol was detected as a main terpenoid compound found in yarrow flowers essential oil compared to in vivo. As reported previously, Judzentiene and Mockute

(2010) similarly demonstrated that (cis and trans) nerolidol, 1,8-cineole and β-pinene were mainly detected in the oils extracted from yarrow flowers.

According to the current results, further research will be established to identify these active compounds found in different plant parts of in vivo grown plants; including root, stem, leaf and seed, and also in in vitro callus production. Interestingly, all evidence in the present work indicated that in vitro micropropagation technique used in current investigation was suitable for producing perfect, healthy plants like in vivo plants. In addition, a process of phenol, flavonoid and terpenoid identification by HPLC-UUV and GC-FID and using methanol as a solvent for extraction process may be considered a successful method for determining these compounds found in chamomile and yarrow plant parts at different growth stages. Furthermore, to obtain the highest amount of chamomile and yarrow phytochemical compounds content, in vivo growing appears to be suitable for most of the compounds content.

4.6 Conclusion

Phenols, flavonoids and terpenoids content of chamomile and yarrow leaves and flowers are considered to be the main phytochemical compounds. Chlorogenic acid and luteolin were found to be major phenol and flavonoid compounds identified in in vitro and in vivo chamomile and yarrow leaves, respectively. Farnesene and nerolidol were detected as a main terpenoid compounds found in in vitro and in vivo chamomile and yarrow flower essential oil, respectively.

Exploration of the chemical characterisation of in vitro and in vivo plant species cultures might be useful for the pharmaceutical and medicinal industries by

114 enhancing the production these phytochemical compounds as plant secondary products. Furthermore, both plant species could be a source of these beneficial compounds which act as anti-inflammatories and antioxidants. They may also be used in different food and medicine industries.

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Chapter five

A study of the phytochemical active compounds content of chamomile and yarrow varieties based on a drought stress regime

5.1 Introduction

Drought stress or low availability of water is considered to be an important abiotic factor that influences plant growth, development and biochemical processes such as the production of secondary metabolites and aromatic compounds (Tátrai et al.,

2016, Salem et al., 2014). Soil oven drying and the measurement of soil moisture content are therefore initially important as a first step of drought study using Time-

Domain Reflectometry (TDR) technique (O'Kelly, 2005). The measurement of TDR is an accurate method used to determine the soil water content (Jones et al., 2002,

Olszewska and Nowicka, 2015, Topp et al., 1980). Moreover, Theta Probe technique has been used by some researchers for the accurate measurement of soil moisture content and volumetric soil water content which is important to understand plant watering system (Kaleita et al., 2005, Van Bavel and Nichols, 2002)

The soil water content might be expressed by volume, which is a ratio between the volume of water and soil total volume, or by weight as a ratio between a mass of water and the soil sample dry weight. These ratios have been previously determined by measuring the constant soil weight from soil drying, and by measuring the mass of soil before and after oven drying process. Additionally, the oven drying method was used as a basic to determine the calibration of the soil moisture content at temperature ranges between 100-110°C (typically is 105°C), which is based on the boiling point of water (DeAngelis, 2007).

In the controlling and management of agriculture production, drought stress is one of the most important environmental factors that determine plant growth worldwide.

Plant phytochemical compounds content including total phenols and essential oil

116 constituents such as limonene, terpinolene, myrcene and sabinene are mainly influenced by drought stress (Gnanasekaran and Kalavathy, 2017, Saeidnejad et al.,

2013). Stress factors can damage plant mechanisms for example, an abnormal plant metabolism may occur and cause reduced growth and development and subsequently influence crop yield, or can lead to complete plant/crop death (Fathi and Tari, 2016). It has been reported that drought stress affects plant growth and the production of secondary products when grown under greenhouse conditions, for example plant vegetative growth has been reduced under stress however, some essential oil compounds content such as geraniol and carvacrol were increased

(Tátrai et al., 2016). Riaz et al. (2013) have informed that plant height and leaf numbers were reduced under drought stress conditions. Flower numbers decreased by approximately 50% under stress compared with control (Nuñez Barrios et al.,

2005).

To date chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) are cultivated around the world mainly for secondary products content such as essential oil. These natural compounds are affected by environmental stress factors such as drought (Saeedfar et al., 2015). Jeshni et al. (2015) have successfully reported that percentage of essential oil percentage and final yield of chamazulene, bisabolol oxide A and B content of German chamomile were affected by drought stress. They suggested that irrigation, less than 50% field capacity, could increase the content of medicinal components and the yield of essential oil. It has been found that drought stress levels not only effect the phytochemical compounds content of chamomile plant, but also significantly influence the fresh and dry flower yield

(Ahmadian et al., 2011). Drought stress level (50% field capacity) was also found to

117 be optimal for total phenol and flavonoid compound content of yarrow compared to

100%, 75% and 25% field capacity (Gharibi et al., 2016).

Several studies have reported on the effect of drought stress on quality, yield and growth of field crops (Basu et al., 2016, Blum, 2005, Farooq et al., 2012); however only few studies linked to the effect of environmental stress on medicinal plants phytochemical compounds content have been investigated (Abdelmajeed et al., 2013,

Kirakosyan et al., 2004). The current study aimed to determine a standard protocol for the application of soil oven drying and soil moisture content, to examine how chamomile and yarrow respond to the drought stress regime. And to evaluate the impact of drought and well-watered regimes on the leaf and flower bioactive compounds content.

5.2 Objectives

1- To assess available water content (AWC) of the selected loam-based compost and construct calibration curves based on a TDR Theta Probe in:

a) The presence of plants, and

b) To determine the water use in irrigation during the study of drought.

2- The main objective of this work was to determine if the quantity and quality of phenol, flavonoid and essential oils in the leaves and flowers of German chamomile and yarrow varieties would change under drought stress, compared with well- watered conditions. In addition, the compare and contrast the suite of phytochemical active compounds between drought stressed plants and well-watered plants.

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5.3 Materials and methods

Experiment 1: Soil characterization

5.3.1 Characterization of the soil oven drying capacity in the presence of chamomile and yarrow plants.

This experiment was carried out in the glasshouse of Skardon gardon, Skardon

Place, Plymouth University, UK. The soil used for all experiments was a loam-based topsoil (LBS Horticulture Ltd production, England). Soil was free of stones and had a pH of 5.65. Chamomile and common yarrow seeds were firstly sown in small trays

(220×160 mm, four trays per plant species) in early December 2014. Two weeks from sowing, when the young plants were (10-15 cm) tall, plants (one plant per pot) were transferred into small plastic pots (88.9 mm2). Then, after five weeks from sowing, when plants were (15-25 cm) tall, the small pots were changed to new, larger pots (127 mm2). All plants were left to grow until early April 2015. Subsequently, four plants from each variety were transferred directly in to the larger pots (15 litres).

Three plastic pots (15 litre) were prepared for each chamomile and yarrow plants

(common yarrow and summer berries yarrow). Pots were filled with topsoil to a final weight of approximately 14 kg. To get the final claibration curves, the measurement of gravimetric moisture content is essential using portable TDR Theta Probe (type

ML2x, Delta-T Devices Ltd Cambridge, UK) based on the followeing procedure reported by Jones (2007):

Step 1 Firstly, all loam topsoil pots needed to be saturated to the maximum water content, this was done by adding water until drainage happened, the pots were then left in saucers overnight to ensure full water saturation through capillary action. Full

119 saturation and 100% field capacity was expected when the pots stopped draining at this point the pots were weighed.

Step 2 When the pots reached full saturation they were not watered again. The fresh weight of the pots was now approximately 17 kg meaning they had taken up nearly 3 litres (or 3 kg) of water. Pots containing plants were then left to dry gradually in the glasshouse and Theta Probe measurements were completed every two days on the top of topsoils.

Step 3 The soil drying characteristics was measured by TDR Theta probe. The TDR rods were pressed into the soil surface of pots and then measurements were recorded for each pot. Subsequently, the pots were weighed on digital top-pan balance (Toledo Model 47141, UK). These weight measurements began in early April

2015 and were recorded every two days continuing until the readings stabilized.

Step 4 A small amount of water remained in the pots, even after the readings of

Theta Probe had stabilized. In this case, soil from the pots was poured and spread out onto metal, then left to air dry for six days and weighed every two days. To get the final true weight, the soil was then put in the oven at 105 °C and weighed again every two days until a constant weight occurred. After approximately 50 days the soils were fully dried (Figure 5.1 and 5.2).

5.3.2 The measurement of soil moisture content

Soil moisture content (MC%) was measured using the portable battery-powered TDR

Theta probe contained a three parallel steel rods (Topp et al., 1984). For data

120 recording in each chamomile and yarrow varietes well-watered and droughted pots, the rods were placed into the soil surface to a depth of 15 cm. To get the soil water content (MC%) from TDR readings value, calibration equations (below) were used

(Topp et al., 1980), Appendix 4.

y= -153.08x2 + 265.25x - 21.216 (Chamomile pots) y = -194.27x2 + 306.71x - 23.768 (Yarrow pots)

Where; x = Theta o readings.

Figure 5.1 Soil drying process in the glasshouse and oven at 105°C containing chamomile and common yarrow plants.

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Experiment 2: drought study, plant material extraction and

identification

5.3.3 Growth conditions

The experimental design of this study consisted of a randomized complete block design (RCBD) at Skarden Garden glasshouse. Two varieties of common yarrow seeds were collected from the previous year’s project in summer 2015 and summer berries yarrow; seeds were purchased from Chiltern seed (Wallingford, OX10 6SL,

England), as well as one variety of German chamomile; seeds were collected from the previous year’s project in summer 2015 with two treatments well-watered (WW) and drought stress (D) in triplicate were carried out. Varieties were randomly allocated into the pots (Fisher and Yates, 1938). Seeds of all varieties were germinated in plastic seed trays (220×160 mm, three trays per variety), 11th October

2016, with the loam based topsoil. The plants were then transferred into small plastic pots (88.9 mm2) when they were 7-10 cm tall (two plants per pot with forty pots per variety), 12th November 2015. Three months after germination, plantlets (20-25 cm tall) were transplanted in early January 2016 into 15 litre plastic pots (four plants per pot with 18 pots per variety); the pots were filled with the same amount of loam based topsoil (Figure 5.2). Plants were only sprayed twice (28th January 2016 and

30th March 2016) against whiteflies with bug clear ultra (Bayer Provado

Ultimate Bug Killer).

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Vegetative growth stages

Uniform growth stages of all three varieties

Seeds sowing Transplanting into Transplanting to larger pots intermediate pot size

Flowering Stages

Summer berries yarrow

German chamomile Common yarrow Figure 5.2 Chamomile and yarrow varieties growth stages and flowering under well-watered and drought stress.

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All small plants were well-watered until they were two months old (leaf production growth stage GS1). Six months after germination when plants were well developed, two watering regimes were applied well-watered (WW) and droughted (D) in early

April 2016 (04th April 2016). Based on the first small experiment results, the well- watered pots (control) were allowed to drop to 70% of available water capacity, then pots were brought up to fully watered again (100%) by adding 2 litres of water. The droughted pots were allowed to drop to 50% available water capacity, and then brought up to 70% available water capacity again by adding 1 litre of water. The control pots were labelled with white coloured labels and the droughted pots were labelled with red coloured labels. Blue labels were used for chamomile pots, yellow coloured labels were used for common yarrow (y1) and purple coloured labels were used for summer berries yarrow (y2) in order to be easily noticed during irrigation.

At least 2 litres of water were added to the control pots for all varieties in order to keep them between 70% to 100% moisture content (MC) and maintain them under well-watered condition. To retain the droughted pots for all varieties between 50% and 70% MC, the pots were kept under water stress condition by adding 1 litre of water to the pots. The water content was measured by Theta probe (Delta-T Devices

Ltd) for all treatments. Data was recorded at the soil surface of the pots daily until the end of experiment when plants started wilting and stopped growing (Figure 5.4). The temperature (oC) and relative humidity (RH%) were also measured by data loggers

(Tiny Tag explorer version 4.7) during the period of experiment. Data is shown in

Figure 5.3.

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Figure 5.3 Average temperature and relative humidity in the glasshouse used for well watered and drought experiment.

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Figure 5.4 The final stage of vegetative growth under the effect of drought stress.

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5.3.4 Sample collection for final chemical analysis

Leaves and flowers of all varieties were collected separately in this study to identify the total phytochemical active compounds of phenols, flavonoids and essential oil accumulated by plant materials during the well-watered and droughted stress growing period. Only plant leaves of chamomile and yarrow varietes have been taken in order to identify phenolic and falivonoid bioactive compounds analysed using

HPLC-UV. Samples were generally collected five times every 10 days from the end of April to the biginning of June (22nd April, 2nd May, 12th May, 22nd May and 2nd June

2016). Flower samples were also collected three times between June and July (15th

June, 25th June and 15th July 2016) during blooming and flowering to determine the quantity and qualtiy of essential oil compounds analysed using GC-FID (Figure 5.5).

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5.3.5 Sample extraction and identification

Samples were freeze dried at -50°C for 2 days before extraction and analysis.

Methanol shaking extraction was carried out for leaf extraction (Chapter 4.3.1).

Soxhlet extraction using methanol as solvent was used for flowers extraction

(Chapter 4.3.6). To identify phenol and flavonoid compounds dried leaves were crushed and powdered using a porcelain pestle and mortar. Dried flowers were also crushed using a coffee grinder (Andrew James, QF-3001HY) and sieved to the 250

µm nylon mesh to determine terpenoid compounds. 2.0 g of each sample was weighed in triplicate. Final extraction of each triplicate were combined together because of the lower concentration level of boiactive compounds content. Freeze dried leaves were extracted by shaking and stored at 3 to 5°C. Flower essential oil was extracted by soxhlet and stored for a short period at -10°C until a time of analysis (as described in Chapter 4).

All extracted samples were analysed using HPLC-UV or GC-FID (Zygmunt and

Namieśnik, 2003, Wadood et al., 2014) under the same conditions as previously described (Chapter 4.3.3 and 4.3.8.). A set of chemical standards, and a large number of chamomile, common yarrow and summer berries yarrow samples were prepared and run at the same time over 72h to provide comparison. One sample from each plant condition was run there times among all standards and samples.

Standards and reagents were purchased from Sigma Aldrich Ltd (Gillingham, Dorset,

UK), (Chapter 4.3.2 and 4.3.7). All calibration curves were also set up same as in vitro and in vivo chamomile and yarrow analysis (Chapter 4.3.4 and 4.3.9).

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5.3.6 Statistical analysis

The statistical analysis and significant differences between the mean values of phenol, flavonoid and terpenoid bioactive compounds content were tested by balanced ANOVA and Minitab™ statistical software (Version17). Data are presented as mean ± SE mean. All samples preparation was carried out in triplicate. Microsoft

Excel 2010 was also used to calculate the final concentration of compounds from the equation of calibration curves.

5.4 Results

Experiment 1

5.4.1 Soil oven drying method and the measurement of soil moisture content

Figure 5.6 illustrates soil drying and final true weights, in the presence of plants, by declining progressively through a period of approximately 22 days at 105°C oven drying. Results indicated that soil containing chamomile plants dried faster than that containg yarrow plants.

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Figure 5.6 Reduction in the soil dry mass in the presence of chamomile (Ch) and yarrow (Y) plants at 105 °C oven drying over time.

Soil moisture content of pots containing chamomile and yarrow plants was measured and calculated from the equation of Theta Probe calibration curves as previously illustrated in section 5.3.2. Results clearly report differences between the moisture content of well-waterd and droughted pots. In both chamomile and yarrow varieties the values of MC% were lower in drought pots compared to well-watered pots (Figure

5.7 and 5.8).

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Figure 5.7 Determination of soil moisture (MC) content of the pots containing chamomile (Ch) plants.

Figure 5.8 Determination of soil moisture content (MC) of the pots containing common (Y1) and summer berries yarrow (Y2) plants.

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Experiment 2

5.4.2 Chamomile and yarrow well-watered (WW) and droughted (D)

HPLC-UV analysis.

All data obtained from samples were firstly compared with the standard peak areas and retention times, and then calculated by linear and polynomial equations of standard calibration curves to determine the final concentration (mg kg-1) of each compound found in the plant species. Five samples of plant leaves at five different times were taken in 2016 during the vegitative growth (22nd April, 2nd May, 12th May,

22nd May and 4th June). Data under well-watered and drought stress conditions were compared for each plant and variety. Five major phenolic and flavonoid compounds were identified in chamomile well-watered (Ch WW) and droughted (Ch D) including chlorogenic acid, caffeic acid, apigenin-7-glucoside, umbelliferon and luteolin.

The levels of caffeic acid, apiginin-7-glucoside and luteolin increased during vegetative growth (April to June) in chamomile well-watered (Ch WW), however chlorogenic acid production increased in the first time of analysis and declined at the beginning of the second time, it then increased again during the vegitative growth as shown in Figure 5.9. Umbelliferone levels was increased from the beginning of vegitative growth, and then reduced. The results of chamomile under drought conditions (Ch D) revealed that chlorogenic and umbelliferone were the only two compounds reduced over time however, some increasing levels were observed particularly at late of vegetative growth in caffeic acid, apigenin-7-glucoside and luteolin.

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Analysis of variance shows a significant difference between the time of analysis during the vegetative growth of chlorogenic acid under both Ch WW and Ch D growth conditions (F34.174, 20; p< 0.001). Moreover, the interaction between growth conditions and time also produced a statistically significant effect (F12.344, 20; p<

0.001) on the chlorogenic acid content, where it was progressively increased during a vegetative growth phase of the plants life- cycle under drought conditions compared to well-watered conditions (Figure 5.9). Caffeic acid was significantly affected under both growth conditions during the time of vegetative growth (F44.321,

20; p< 0.001) and (F7.464, 20; p= 0.001), respectively. Also, the interaction between growth conditions and time was significant (F58.224, 20; p< 0.001), where caffeic acid was gradually increased during a vegetative growth under well-watered conditions compared to drought conditions.

In terms of apig-7-glucoside, growth conditions and time were two factors significantly affected the content of apigenin-7-glucoside (F20.501, 20; p< 0.001) and

(F4.474, 20; p≤ 0.001). Additionally, the interaction between growth conditions and time was also significant (F11.834, 20; p< 0.001), where apigenin-7-glucoside was regularly increased and decreased during a vegetative growth under well-watered and drought stress conditions, respectively. Statistical analysis was also showed a significant differences between the growth conditios during the time of vegetative growth regarding the chamomile content of umbelliferone (F93.111, 20; p< 0.001) and

(F149.704, 20; p< 0.001) respectively. In addition, the interaction between growth conditions and time was also significant (F41.034, 20; p< 0.001), where umbelliferone was continually decreased during a vegetative growth under drought stress conditions. Under both conditions chamomile luteolin content was not significantly affected over the time of vegetative growth (p= 0.09 and p= 0.07) respectively.

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Moreover, the interaction between growth conditions and time was not significant (p=

0.77) as shown in Figure 5.9.

Results showed a signifficant decrease for most of the chamomile leaves phytochemical active compounds content under drought stress conditions. In total, the highest amount of chlorogenic acid was observed in both well-watered and drought conditions with the highest production found in Ch WW compared to Ch D

(513.80 ± 43.80 mg kg-1 and 444.20 ± 98.60 mg kg-1), respectively (Figure 5.13).

Moreover, results showed that caffeic acid, apig-7-glucoside, umbelliferone and luteolin were more drought resistant, where their maximum production were observed in Ch D compared to Ch WW (Figure 5.13 and 5.14). The HPLC- UV chromatogram of chamomile drought conditions is shown in Figure 5.10.

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Time (min)

Figure 5.10 The HPLC- UV chromatogram of phenol and flavonoid compounds content of chamomile leaves detected at 256 nm under drought stress conditions.

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HPLC-UV results of common yarrow (Y1) showed that four main compounds of both well-watered and droughted conditions were identified as follows; caffeic acid, apigenin-7-glucoside and luteolin were increased over time however, in (Y1 WW), decreasing levels of umbelliferon were observed from the middle to the end of vegetative growth (Figure 5.11). No detection of chlorogenic acid was observed during this experiment. Moreover, luteolin and apigenin-7-glucoside detected in common yarrow were more affected under drought conditions compared to the other detected compunds. Caffeic acid and umbelliferone were also affected by drought from the behinning to late of vegetative growth.

Statistical analysis showed significant differences between compounds during vegetative growth. Different growth conditions and time showed significant effect on caffeic acid content (F40.991, 20; p< 0.001) and (F12.514, 20; p< 0.001) respectively.

Moreover, the interaction between growth conditions and time also showed a significant effect (F20.684, 20; p< 0.001), where caffeic acid was progressively increased during a vegetative growth under well-watered conditions compared to drought stress. Analysis of variance was also showed that well-watered and drought conditions had significant effect on apigenin-7-glucoside compounds content upon time (F4.931, 20; p≤ 0.001) and (F101.604, 20; p< 0.001) respectively (Figure 5.11). In addition, the interaction between growth conditions and time was also significant

(F351.19, 20; p< 0.001) and showed that apigenin-7-glucoside was the only compound that continually decreased and reduced under well-watered and drought conditions, respectively.

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Time of vegetative growth was significantly affected the content of Y1 umbelliferone content (F13.454, 20; p< 0.001) however, growth conditions were not significantly affected (p= 0.17). Additionally, the interaction between growth conditions and time was also significant (F12.384, 20; p< 0.001), where umbelliferone was regularly increased and decreased until late of vegetative growth under both growth conditions.

Luteolin content was not significantly affected under both growth conditions (p= 0.09) however, statistical analysis showed that luteolin content was changed over the time of vegetative growth (F3.734, 20; p≤ 0.001). Moreover, the interaction between growth conditions and time was also significant (F4.584, 20; p≤ 0.001) as shown in Figure

5.11.

Apigenin-7-glucoside was found as the major phytochemically active compound in both grwoing conditions based on the total yield during vegetative growth. (165.30 ±

22.10 mg kg-1 and 155.50± 27.90 mg kg-1), respectively (Figure 5.13).

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Similar to the common yarrow, four main phenolic and flavonoid compounds were also found in summer berries yarrow (Y2). Also, chlorogenic acid was not detected however, umbelliferon and luteolin increased over time. The concentration of apigenin-7-glucoside increased at the beginning of vegetative growth and then started to decline however, caffeic acid increased from the beginning to late of the time of vegetative growth and started declining only at the end of growth life cycle

(Figure 5.12). Among the phenolic and flavonoid compounds content of Y2 under drought stress conditions, apigenin-7-glucoside was the only flavonoid compound that decresed over time.

The results showed that most of the phenol and flavonoid compounds found in Y2

WW well-watered were significantly increased and affected during plant vegetative growth compared to Y2 D. Caffeic acid was significantly affected over time (F5.394,

20; p≤ 0.001) however, it was not significantly affected by growth conditions (P= 0.3 ).

Moreover, results of the interaction between growth conditions and time was also no significant p=0.33 (Figure 5.12). Growth conditions and time were significantly affected the content of summer berries yarrow apigenin-7-glucoside (F1015.111, 20; p<0.001) and (F1889.854, 20; p<0.001) respectively. The interaction between growth conditions and time also had a signifficant effect on the apigenin-7-glucoside content

(F284.764, 20; p<0.001), where it gradually declined under drought conditions.

In terms of umbelliferone content, it was found that growth conditions were not significantly affect the Y2 umbelliferone content (P= 0.06) however, it was changed over time under both conditions (F19.864, 20; p<0.001). Furthermore, the interaction between growth conditions showed a significant difference (F3.944, 20; p≤ 0.001), where it progressively increased under weill-watered conditions. Statistical analysis of Y2 luteolin content showed that both growth conditions and time were significantly

140 affect the content of luteolin (F31.901, 20; p<0.001) and (F20.944, 20; p<0.001) respectively. Highly significant differences were also found in the interaction between growth conditions and time (F3.99, 50; p≤ 0.001), where it gradually increased under weill-watered and drought conditions. Overall, apig-7-glucoside (81.44± 7.90 mg kg-1) and umbelliferone (18.91± 3.08 mg kg-1) were found as main compounds in both (Y2

WW) and (Y2 D), respectively (Figure 5.13 and 5.14).

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Figure 5.12 The main phenol and flavonoid compounds isolated from summer berries yarrow under well- watered (Y2 WW) and drought stress (Y2 D) conditions: caffeic acid LSD values

(time= 7.2) apigenin-7-glucoside LSD values (growth conditions= 1.4, time= 2.2 and growth conditions*time= 3.1) umbelliferone LSD values (time= 9.9 and growth conditions*time = 13.9) luteolin LSD values (growth conditions= 1.0 , time= 1.6 and growth conditions*time= 2.3). Data are presented as mean ± SE of three replicates.

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Figure 5.13 The total yield of chlorogenic acid and apigenin-7-glucoside compounds content of chamomile and yarrow varieties under well-watered (WW) and drought stress (D) conditions.

Figure 5.14 The total yield of caffeic acid, umbelliferone and luteolin compounds content of chamomile and yarrow varieties under well-watered (WW) and drought stress (D) conditions.

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5.4.3 Chamomile and yarrow well-watered (WW) and droughted (D) GC-

FID analysis

The impact of the well-watered and drought stress conditions on the level of EO phytochemical compounds content of both chamomile and yarrow flowers was carried out. Three samples were taken at three different times in 2016 of the flower heads of chamomile, common yarrow and summer berries yarrow from well-watered and droughted plants at the following growth stages (GS4 and GS5) blooming and flowering stages (15th June, 25th June and 15th July). The extraction and analysis of different varieties of flowers were examined by Soxhlet extraction and GC-FID analysis (as described in chapter 4). Data from each plant was firstly compared with the standard peak areas and retention times, and then calculated by linear regression from the standard calibration curves (Appendix 4.2) to establish the final concentrations of extracted phytochemically active compounds (mgkg-1). Running a set of standards and samples together at the same time and for more than 72 hrs was not detrimental for the GC detector therfore, the statistical analysis for this experiment was not carried out due to the lack of replication of samples analysed using GC-FID.

Results of this study showed that chamomile and yarrow EO phytochemical compounds content were affected by their growing environment (well-watered and droughted). Five main terpenoid compounds were identified in the chamomile well- watered (Ch WW) and droughted (Ch D) containing two isomers of farnesene however, farnesene (1) and nerolidol were not detected. Increasing numbers were positively observed for all mono- and sesquiterpenes under well-watered conditions over time as shown in Figure 5.14. A maximum total yield of farnesene (2) at (17.1 min) were found among other compounds in both Ch WW (847.51 mg kg-1) and Ch D

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(840.97 mg kg-1), respectively (Figure 5.17). Chamazulene and bisabolol oxide A were the only two sesquiterpenes compounds highly affected by drought stress, where both compounds progressively decreased over time compared with other compounds (Figure 5.14).

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Figure 5.15 The main terpenoid compounds content isolated from chamomile EO under well-watered (Ch WW) and drought stress (Ch D) conditions. 146

In terms of common yarrow (Y1) and summer berries yarrow (Y2) plants, five main terpenoids were also found containg three different isomers of farnesene.

Chamazulene was not detected. All compounds found in (Y1 WW) were reduced over time except farnesene 2, α-(-)- bisabol and bisabolol oxide A (Figure 5.15).

Interestingly, results of (Y1 D) showed some differences with reducing amount of all terpenoid compounds content over time. Farnesene (1) at (16.6 min) was dominant among other compounds in both Y1 WW(280.55 mg kg-1) and Y1D(222.70 mg kg-1),

Figure 5.17 and 5.18. Moreover, the total yields of limonene, α-(-)- bisabol and bisabolol oxide A were higher in (D) conditions compared with (WW) conditions.

Conversely, results of (Y2 WW) showed that the terpenoid compounds content increased regularly over time, however farnesene isomers were decreased in late

June then began to rise (Figure 5.16). Moreover, α-(-)- bisabol was the only compound that had a good response under drought stress among other compounds observed in (Y2 D). Limonene, farnesene (1) and farnesene (2) did not show any drought effect, whereas the rest of compounds showed a medium drought response

(Figure 5.16). In both (Y2 WW) and (Y2 D), total yields of nerolidol were dominant

(219.94 mg kg-1 and 241.41 mg kg-1), respectively. In addition, a total yield of all compounds were increased under drought conditions except farnesene (1) and bisabolol oxide A (Figure 5.17).

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Figure 5.16 The main terpenoid compounds content isolated from common yarrow EO under well-watered (Y1 WW) and drought stress (Y1 D) conditions.

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Figure 5.17 The main terpenoid compounds content isolated from summer berries yarrow EO under well-watered (Y2 WW) and drought stress (Y2 D) conditions.

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Figure 5.18 The total yield of terpenoid compounds content of chamomile and yarrow varieties under well-watered and drought stress conditions.

1. Farnesene1 (16.69 min) 4- Nerolidol (18.89 min)

2. Farnesene2 (17.10 min) 5- α-(-)- bisabol (20.86 min) 3. Farnesene2 (17.74 min) 6- Bisabolol oxide A (21.91 min)

4 6 1 2

Figure 5.19 GC-FID analyses of the essential oil of in vivo common yarrow flowers extracted by methanol using Soxhlet extraction under drought stress conditions.

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5.5 Discussion

The results of the first experiment showed that TDR measurement and oven drying technique were successfully used to determine soil moisture content in the presense of chamomile and yarrow plants. TDR measurement was also previously used as a precise technique to measure soil water content compared to others such as gravimetric method, which requires along time and a great physical effort to collect the samples and determine the moisture contet (Menziani et al., 1996, Olszewska and Nowicka, 2015, Reynolds, 1970, Topp and Davis, 1985, Topp et al., 1984). For example Topp et al. (1984) and Topp and Davis (1985) indicated that the values of soil surface water content by TDR technique and gravimetric measurement were not different, where Topp et al. (1984) revealed that TDR measurement was rapid and more accurate than those measured by gravimetric method. These findings support what has been found in the present study.

The current results also show that soil water content (MC%) in well-watered pots was higher than that in drought pots, which is in agreement with the finding reported by

Puértolas et al. (2017), where the soil water content of WW pots was 50% higher.

Also, they indicated that maintaining soil water content in pots containing plants might be related to the leaf area and root distribution, which are two factors affect the gradient of soil moisture content.

The results of the second experiment indicated that drought conditions had a significant effect on the levels of phenol, flavonoid and terpenoid compounds in both chamomile and yarrow leaves and flowers. Results also showed a significant difference between the two analyses p< 0.001. Regarding the HPLC-UV analysis, more phytochemically active compounds were identified in chamomile compared with

151 yarrow however, GC-FID analysis showed that most of the terpenoid compounds were found in yarrow compared with chamomile. Using and selecting different instrumental analyses for different plant parts is considered to be one of the main protocols to identify maximum numbers of beneficial active compounds content especially for commercial medicinal plants.

Despite the successful methods used in this work, some difficulties for the detection of some compounds were observed, for example chlorogenic acid and chamazulene were not observed in yarrow plants, which perhaps, whether due to the storage of samples in a fridge or freezer for long periods of time. This is particularly problematic with some volatile oils as they can be lost over long storage periods. Another problem can arise in detection, as very small concentrations of target compounds from extraction processes may lead to poor absorption by the detector.

The study of drought stress conditions has been successful for most of the phenol and flavonoid compounds content found in chamomile, this could be due to the accumulation of phenolic acid compounds usually increasing under abiotic stress conditions (Hale et al., 2005). In addition, some abnormalities were also observed under both well-watered and drought conditions. This might be caused by sampling during analysis, particularly at the time of dilution method (Chapter 4.3.3). The results obtained in this study are in agreement with those indicated by Salem et al. (2014) who reported a significant increase of total phenolic acids content of Carthamus tinctorius L. (safflower) under water stress. Conversely, Król et al. (2014) reported that all the levels of phenolic acid such as caffeic acid found in Vitis vinifera L.

(grapevine) leaves were significantly decreased under drought stress conditions.

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Moreover, Saeidnejad et al. (2013) confirmed that applied drought treatments reduced yield components of Bunium persicum L. (Black Cumin). Some differences were observed in yield components when plants were exposed to drought stress at different growth stages. They also reported that the concentration of phenolic compounds improved under drought conditions.

It has been previously confirmed that the total phenolic compounds content of Tridax procumbens L. (tridax daisy) leaves and flowers significantly increased under drought stress (Gnanasekaran and Kalavathy, 2017), this result was supported in the current study that the total yield of caffeic acid detected in (Y2 D) increased under drought stress however in (Y1 D) it was decreased compared to well-watered conditions

(Figure 5.14). The main reason for these differences may be linked to the variation of the mechanism of compounds biosynthesis of two yarrow varieties, for example the reduction of the activity of some enzymes under drought stress such as 2, 2- diphenyl-1-picrylhydrazyl (DPPH), particularly those connected to the phenols and flavonoids biosynthesis (Farooq et al., 2012, Król et al., 2014).

Conversely, in the current research drought stress did not affect only the leaf phenolic and flavonoid compounds content, but it also strongly affected the flower EO compounds content of chamomile and of the two yarrow varieties. The use of drought stress trial for chamomile and common yarrow was also previously tested by some authors; Ahmadian et al. (2011), Baghalian et al. (2011), Gharibi et al. (2016),

Houshmand et al. (2011), Jeshni et al. (2015), Razmjoo et al. (2008), however nothing was reported about summer berries yarrow. Most of the compounds found in

Y2 EO were increased under drought conditions, this might be because the production of metabolites under stress conditions could be more leading to high

153 production of substances (Farahani et al., 2009a) or, because yarrow plants have a rhizome root system and feathery type leaves which might make plants drought tolerant (Khalil et al., 2011). Therefore, growing summer berries yarrow plants under drought conditions is required to obtain the highest amount of terpenoid compounds content. The same trend was observed by Farhoudi (2013) in Rosmarinus officinalis

L. (Rosemary) essential oil. The author indicated that drought stress conditions increased the main essential oil compounds such as α- bisabolol, α- bisabololoxide A,

1, 8-cineole, α-pinene and β-pinene compared with control.

The findings in this current study are also in agreement with a previous study reported by Jeshni et al. (2015), who discovered that drought stress significantly affected the essential oil yield, chamazulene, bisabolol oxide A and bisabolol oxide B content. Otherwise, Baghalian et al. (2011) indicated that drought stress had no significant effect on oil compounds content, however apigenin content and flower yield decreased. Moreover, Houshmand et al. (2011) explored that drought stress also had a significant reduction of flower diameter, number of flower, as well as fresh and dry weight of chamomile flower per plant. These parameters might be important to investigate besides our work, because all parameters related to the flower part which contains the main essential oil bioactive compounds.

5.6 Conclusion

In summary, total moisture content of the topsoil used in all the experiments in this chapter was firstly determined and measured by TDR and oven drying methods at

105°C. The total values of MC% of chamomile and yarrow plants were higher in well- watered pots compared with drought pots. In irrigation system the application of

154 these methods provides a mechanism where the level of water supply maybe manipulated to enhance the production of secondary metabolites and might be extended for further, useful, studies relating to water stress condition.

Regarding the phytochemical bioactive compounds content: the identification of the main phenol, flavonoid and terpenoid compounds found in plants, analysed using

HPLC- UV and GC-FID, were significantly affected by well-watered and drought stress conditions. Total yields of some compounds significantly increased under drought stress. However, most of the compounds were reduced under stress.

Therefore, manipulating enough water supplies for the chamomile and yarrow plants might be one of the most important points to focus on during growth stages.

The total yield of chlorogenic acid, apigenin-7-glucoside, umbelliferone, farnesene (1), farnesene (2) and nerolidol were dominant among other compounds as main bioactive components found in chamomile and yarrow varieties grown under both well-watered and drought stress conditions. Results of this study showed that chamomile was highly drought resistant regarding the total yields of phenolic and flavonoid compounds content compared with yarrow plants, however summer berries yarrow was found to be highly drought resistant and common yarrow moderate drought resistant compared with chamomile plants regarding the total yield of terpenoids content. These findings of current study might be support future studies about drought programs for growing and improving plants in some soils that availability of water is not enough, especially for economical medicinal and aromatic plants. Sandy soils can also result in periods of drought, even in moderate climates.

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Chapter six

Antibacterial activity of the essential oil of chamomile and yarrow flower heads

6.1 Introduction

Chamomile (Matricaria chamomilla L.) and yarrow (Achillea millefolium L.) produce a range of phytochemical active compounds from secondary metabolites such as essential oils (EOs) compounds which possess antibacterial activity (Issabeagloo and Abri, 2012). These EO contain an important array of monoterpenes such as limonene, estragole, trans-anethole and fenchone with antibacterial activity and have been used in the treatment of bacterial infections such as urinary tract infection

(Mittal et al., 2009, Roby et al., 2013). These compounds have been extracted by different methods including Soxhlet and shaking extractions using methanol and hexane solvents (as described and discussed in Chapter 4).

It has been widely focused on the use of EO compounds against Gram-positive and

Gram-negative bacteria using several methods for example agar disc diffusion

(Alireza, 2012, El, 2014, Nascimento et al., 2000, Shahbazi and Zadeh, 2008) and 96 well plate test (Frey and Meyers, 2010, Kazemian et al., 2015). Because medicinal plants are great sources of compounds with antimicrobial activities, several studies have been reported the development of new antimicrobial agents from different sources that inhibit the growth of microorganism such as bacteria strains (Monte et al., 2014). The antibacterial activity has been studied against two main types of bacteria, Gram-positive and Gram-negative bacteria, (Alireza, 2012, El-Kalamouni et al., 2017). Both types of bacteria have different sensitivity toward antibiotics

(Shahbazi and Zadeh, 2008). Gram-positive bacteria contain a single membrane however, Gram-negative bacteria cell walls contains a high level of lipids which protect the strains from antibiotics (Lambert, 2002).

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It has been reported that 1:2 dilutions of chamomile EO showed 73% and 62% inhibition zones against the gram-negative bacteria Escherichia coli (E. coli) and the

Gram-positive bacteria Bacillus subtilis (B. subtilis), respectively (Alireza, 2012).

Alkuraishy et al. (2015) reported that chamomile disc diffusion method showed a significant antibacterial activity against E. coli more than Pseudomonas aeruginosa

(P. aeruginosa). Moreover, it has been demonstrated that the use of 96 well plate test is also an important method mainly used in microbiology testing especially for in vitro evaluation of antimicrobial activity (Balouiri et al., 2016). Vila et al. (2010) reported that trans-nerolidol, bisabolol oxide B and α-bisabolol isolated from Plinia cerrocampanensis, have the ability to inhibit the growth of P. aerginosa. However, α- bisabolol has been found to be the main content of Eremanthus erythropappus

(Asteraceae) EO compounds possess antibacterial activity against E. coli by giving the highest value of inhibition zone (Silvério et al., 2013)

Shahbazi and Zadeh (2008) have been demonstrated that EO extract from yarrow leaves and flowers containing eugenol, 1, 8-cineole and camphor have an antibacterial activity against E. coli using disc diffusion assay. Therefore, this study has been carried out to investigate the antibacterial activity of pure essential oil and different dilutions of two different cultures of in vitro and in vivo chamomile and yarrow plants (both cultures and extraction processes have previously been described in chapter four) against some gram-negative bacteria: E. coli,

Pseudomonas aerginosa and Pseudomonas syringae, as well as gram-positive bacteria: B. subtilis and Enterococcus faecalis (E. faecalis). Two different assays named the disc diffusion and 96 well plates method were also examined. The main aim was to study the effect of extracted plant EOs grown under different culture conditions on the death of some selected bacteria strains.

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6.2 Objectives

1- To determine chamomile and yarrow EOs antibacterial activity based on their in vivo and in vitro cultures.

2- To evaluate a maximum inhibitory zone of bacteria strains using disc diffusion methods.

3- To investigate the different levels of bacteria death under pure and different dilutions of EOs by 96 wells plate method.

6.3 Materials and methods

6.3.1 Plant samples

Samples were collected at the flowering stage of both plant species from June to July

2016. Flowers were freeze dried and powdered. The extraction was completed in triplicate using Soxhlet extraction using methanol for 3h per sample from each of the test plants (Chapter 4.3.6). 2.0 g of dried flowers were then added to a round bottom flask containing 150ml of methanol. After 3h of extraction the samples were collected and evaporated using a rotary evaporator (Buchi, Rotavapor R-3) at 40°C and stored at -10°C until bioassay work (this section has previously been described in details in

Chapter 4.3).

6.3.2 Culture method

The culture was performed by adding 12 g L-1 of bacteriological agar No.1 (MC002) and 22 g L-1 of Mueller Hinton Broth 2 (MHB2, pH= 7.3 ± 0.2). Agar culture was then autoclaved for 2.5 hrs and poured into the plate (10 ml each) and stored at 4-6°C until use. In addition, 22 g L-1 of MHB2 was prepared separately for bacteria strains overnight culture and dilution process of strains, and then stored at room temperature.

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6.3.3 Antibacterial activity

6.3.3.1 Agar disc diffusion assay

Bacteria strains (E. coli K12, B. subtilis 40P, P. aeruginosa 10817, P. syringae A6, and E. faecalis 40P) were obtained from the microbiology laboratory in the Plymouth

University culture collection. All bacteria strains were grown in agar media as prepared previously and incubated at different temperatures depending on the type of strains. E. coli, B. subtilis and P. aeruginosa were incubated at 37°C, P. syringae at 25°C and E. faecalis at 37°C+CO2. The diffusion method was used to determine the antibacterial activity of flower essential oil of plant species with pure and three different dilutions (10-1, 10-2 and 10-3) in methanol. The pure essential oil was already containing some unknown volume of methanol which remained after rotary evaporator process. In this case, the pure methanol 99.9% HPLC grade was plated separately as a control for each bacteria strain in triplicate and the inhibition zone was measured and taking out from the measurement of the inhibition zone for each sample. All sample plates were in triplicate and covered uniformly with strain dilutions

(1/10 dilution of E. coli and B. subtilis, 1/100 dilution of P.aeruginosa, P. syringae and

E. faecalis), and then 20 μl from each oil dilution series was added into each well

(they made by cork borer). The inhibitory zones (mm) were measured by Vernier caliper after 24hrs (Dezfooli et al., 2012).

6.3.3.2 Measurement of growth curves and bacteria death using microplate reader

Bacteria strains were sub-cultured in the bacteriological agar and cultured overnight in MHB2 at required temperatures (section 6.3.3.1). Wells of 96 well plates (Corning,

NY 14831/ USA) were filled with 160 μl of a mixture of different solutions in triplicate

(Figure 6.1). In each well the absorbance was measured by microplate reader

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(TECAN Infinite F200) at 595 nm and 37°C every 20 min for 24h. In this study pure

EO, 1:10 and 1:100 dilutions of extracted essential oil of in vitro and in vivo chamomile and yarrow flowers were prepared in methanol. Also, to compare both results of bacteria strain growth curves untreated and treated with extracted EOs, microplate wells were filled with 160 μl of overnight cultured bacteria strains untreated with EOs and placed on the microplate reader, and growth curves were measured every 20 min for 24hrs, at 595 nm and 37°C, and then the results were compared with plates treated with EOs (Appendix 5).

6.3.4 Statistical analysis

All data were completed in triplicate and were analysed in Minitab™ statistical software (Version17) using a balanced analysis of variance (ANOVA) test, a significant difference between means of each variable was accepted at p < 0.05.

Data are presented as mean ± standard error mean (SE).

6.4 Results

6.4.1 The measurement of bacteria strains growth using disc diffusion method

Antibacterial activity of pure and the three different dilutions (10-1, 10-2 and 10-3) of in vivo chamomile and yarrow essential oils (EOs) with methanol solvent were tested in bacteriological agar containing MHB 2. The inhibition zones or halo zones were measured as a radius of bacteria death in millimetre (mm), Figure 6.1.

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1.0 mm 2.8 mm

A B

Figure 6.1 Radius of the inhibition zone (millilitres) at different concentrations of (A) 10-2 chamomile and (B) pure yarrow EOs against B. subtilis on (MC002) agar medium.

Results of methanol preliminary test indicated the highest inhibition zone (1.83 ± 0.2 mm) against B. subtilis, however E. coli and E. faecalis showed negative results, where both strains were totally active and were not affected by methanol (Figure 6.2).

As a result, the use of pure methanol showed a significant effect on the death of bacteria strains (F 22.68 4, 8; p< 0.001).

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Occurrence of inhibition zones of bacterial death was observed in both chamomile and yarrow EOs, (Figure 6.3 and 6.4). Pure chamomile EO gave the greatest level of inhibition zones of 9.83 ± 1.09 mm, 2.70 ± 0.29 mm, 4.67 ± 0.44 mm and 0.70 ± 0.09 mm against E. coli, B. subtilis, E. faecalis and P. aeruginosa, respectively (Figure 6.3).

However, P. syringae was more sensitive to 10-1 dilution with 1.93 ± 0.17 mm inhibition zone compared to pure and two other dilutions.

The 10-1 dilution of yarrow EO showed a maximum value of inhibition zones of 3.13

± 0.19 mm, 3.20 ± 0.43 mm and 1.23 ± 0.29 mm against E. coli, B. subtilis and P. aeruginosa, as well as the lowest value (1.06 ± 0.27 mm) which was observed against P. syringae, respectively, (Figure 6.4). However, a great inhibition zone of both E. faecalis (2.20 ± 0.83 mm) and P. syringae (1.33 ± 0.32 mm) were found in pure EO. Unexpectedly, the activity of pure, 10-2 and 10-3 dilutions of yarrow EO

162 compounds did not show any effect against E. coli, E. faecalis or P. aeruginosa. In total, the disc diffusion assay showed that the death of all bacteria strains was significantly affected by the activity of chamomile and yarrow EOs compounds content with the highest antibacterial activity observed in chamomile compared to yarrow (F 63.854, 38; p< 0.001), (F 19.094, 38; p< 0.001), respectively. E. col, B. subtilis and E. faecalis were to be more sensitive of the chamomile and yarrow EOs, where the mean and standard error mean values were 3.59 ± 1.12 mm and 2.46 ± 0.28 mm, respectively. Also, the lowest EOs antibacterial activity was found in P. aeruginosa for both plants. Moreover, the interaction between bacteria strains and pure EOs& dilutions had a significant effect at p< 0.001. The greatest antibacterial activity was observed in chamomile pure EO against E. coli (Figure 6.3), while 10-1 dilution of yarrow EO showed the highest effect on B. subtilis as shown in Figure 6.4.

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6.4.2 Determination of the effect of plant EO on the death of bacteria strains using microplate reader

Results of bacteria strain growth curves using 96 well plates filled with overnight cultured bacteria and untreated with extracted EOs showed a different growth of each strain at 595 nm and 37°C based on the differentiations of the nature of absorbance (Figure 6.5). Both P. aeruginosa and P. syringae grew slightly over time however, no growth of E. coli, B. subtilis, E. faecalis was noted in fact, E. faecalis was completely stopped.

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The results of microplate reader (Table 6.1) showed that most of the bacteria strains were killed under the effect of pure in vitro and in vivo chamomile and yarrow EOs compared with 1:10 and 1:100 dilutions. Results from a random selection of data showed that 64.63% of E. coli strain was killed under the effect of in vitro yarrow pure

EO within 1 hour compared with other strains (Figure 6.6). The lowest and highest dilutions (1:10 and 1:100) of plant EOs except in vitro yarrow dilutions were found to be less effective. Moreover, the lowest dilution of in vitro yarrow also showed a maximum effect against E. coli (47.75%) however, 1:100 dilution killed 46.07% of P. aeruginosa. Also, the results of chamomile in vivo pure EO is shown in Appendix 5.

Overall, results indicated that all bacteria strains were affected by in vivo and in vitro chamomile and yarrow EOs, where in vitro yarrow EO was more effective than other plant cultures.

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Table 6.1 The percentages of the number of bacteria strains killed under the effect of pure, 1:10 and 1:100 in vivo and in vitro chamomile and yarrow EOs within 1 hour.

In vivo Ch In vitro Ch In vivo Y In vitro Y Bacteria strains Bacteria death % Bacteria death % Bacteria death % Bacteria death %

Pure 1:10 1:100 Pure 1:10 1:100 Pure 1:10 1:100 Pure 1:10 1:100

E. coli 17.83 -20.26 -2.85 10.49 -14.41 -4.99 11.17 -3.34 -0.43 64.63 47.75 42.54

B. subtilis 12.34 -15.32 -3.14 13.62 -11.32 -5.5 3.83 -3.61 -0.87 50.40 46.06 28.22

E. faecalis 10.76 -23.86 -3.48 10.6 -15.74 -7.09 5.33 -8.23 -0.34 20.85 44.46 44.62

P. aeruginosa 11.86 -20.97 -3.05 11.24 -12.35 -4.47 4.03 -3.7 0.76 53.26 30.03 46.07

P. syringae 8.86 -26.53 -2.83 8.85 -13.63 -5.79 -0.62 -4.05 -0.71 57.44 27.45 39.14

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6.5 Discussion

Essential oil components are greatly active against microorganism cytoplasm and membrane, and they also morphologically change the cells in different cases

(Nazzaro et al., 2013). The results of the present study therefore showed the important role of extracted EO from selected plant flowers when tested against a range of bacteria strains which are harmful to our health. From the results of chamomile and yarrow EOs antibacterial activity, it is clear that different culture conditions produce a range of essential oil compounds which possess different levels of antibacterial activity despite being from the same plants. Only in vivo culture was studied by disc diffusion method as a preliminary test to determine the role of antibacterial activity of EO compounds content, therefore the study of in vitro culture may be required to compare with current results for example study the antibacterial activity of callus, plantlets, shoots and leaves against E. faecalis and E.coli (Kumari et al., 2016). However for 96 wells plate method the in vitro culture was also studied as a new investigation in order to obtain different results compared to in vivo culture.

The current study demonstrated that the EO compounds of chamomile and yarrow flowers significantly inhibited the growth of selected bacteria by acting to change the hydrophobic property on the bacteria surface and simply connecting with the strain

(Lopez-Romero et al., 2015). Figure 6.3 and 6.4 show that Gram-negative bacteria P. aeruginosa, through disc diffusion method, illustrated a reduced effect when exposed to EOs compared to gram-positive bacteria, this could be relating to the sensitivity of bacteria cell wall, it was therefore decided to select microplate reader to investigate better results regarding the gram-negative bacteria.

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It was found that the inhibition zone of bacteria growth might be based on the dilution of the EOs. Regarding the disc diffusion method, the high antibacterial activity of chamomile may be because of chamomile EO ingredients able to interrupt the bacteria strains permeability of wall and easily inhibited the activity of membrane enzymes (Alkuraishy et al., 2015). In both methods, pure EO was found to be the most effective against bacteria strains with the superior against E coli (Figure 6.3 and

Table 6.1). This might be due to the content of higher levels of phytochemically active compounds such as farnesene, chamazulen and bisabolol oxide A found in in in vivo chamomile, as well as nerolido and α-(−)-bisabolol found in in vitro yarrow (Chapter

4). In addition, the effect of pure EOs on some bacteria strains using both methods were less active than EO dilutions, this might be because of the high loss of active compounds and evaporation during the process of microplate reader (Cuéllar and

Hussein, 2009).

In terms of chamomile antibacterial activity, this finding is different from those illustrated by (Solidônio et al., 2015) who reported that chamomile EO had no activity against Gram-negative bacteria of E. coli and P. aeruginosa. However, our study shows lower activity of plant EOs against P. aeruginosa, this is probably due to the presence of some phytochemically active compounds in our plant species which possess a great activity against bacteria. Regarding this observation, Aliheidari et al.

(2013) proposed that due to the presence of a double membrane in gram-negative bacteria it is more difficult linking to these strains compared to Gram-positive bacteria which consist of single membrane. The use of bacteria type and bioassay methods therefore are important to be requirements of the study of antibacterial activity based on the activity of compounds content in plant EOs.

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The use of plant EOs dilution is therefore required to compare the sensitivity of different strains of plants antibacterial activity. The sensitivity between both types of bacteria to plant EO is also supported by Shahbazi and Zadeh (2008). Generally, the

Gram-negative bacteria appear less sensitive than the Gram-positive bacteria toward plant EOs (Bosnić et al., 2006, Trombetta et al., 2005). In contrast the result from this study was observed here, regarding Gram-negative bacteria, E. coli was found to be more sensitive than the other strains of bacteria in the test in the presence of pure

EOs in both in vitro chamomile and yarrow culture (Figure 6.3 and Table 6.1). Based on these results, both methods showed a great affect against bacteria strains, but disc diffusion was found to be more acceptable due to the logic effect of EO levels.

This is probably due to the well diffusion effect of EO in the agar medium and thus directly attacking the bacterial membrane. In fact, it is not clear why Gram-negative bacteria would be more resistant than Gram-positive bacteria to plant extracts.

Nikaido (2003) suggested that it might be linked to the outer membrane, where the outside of Gram-negative bacteria have an extra cell membrane layer which ability to protect the cell membrane as a barrier from the harmful components such as terpenoid compounds including limonene, chamazulen and nerolidole found in the current study.

According to the current observations and results from the previous chapter of in vivo and in vitro chamomile and yarrow cultures phytochemical active compounds identification, the use of pure EO of in vivo chamomile using disc diffusion and in vitro yarrow using 96 well plates method which provide maximum levels of terpenoids content and their highest activity against Gram-negative bacteria mostly against E. coli bacteria are mainly required in the study of plant antibacterial activity.

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6.6 Conclusion

Based on all the observations of plant EOs antibacterial activity it is important to conclude that the growth of bacteria strains was affected by chamomile and yarrow

EOs in both methods. The highest antibacterial activity of in vivo chamomile pure EO against E. coli (9.83 ± 1.09 mm) and yarrow 10-1 EO against B. subtilis (3.20 ± 0.43 mm) were found in disc diffusion method. However, microplate reader showed that E. coli and P. syringae were more sensitive when subjected with pure in vitro yarrow EO.

Despite this, more studies must be conducted in the future to examine this activity not only for chamomile and yarrow flowers but also for root, leaf and seed against other bacteria strains using microplate reader. Plant extracts should also be tested against more microbials with the ability to cause human diseases. Moreover, further studies are suggested about the molecular levels of plant EO phytochemical compounds to examine their total mechanisms of action.

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Chapter seven

General discussion and future work

7.1 General discussion

This study was carried out with the aim of developing a protocol for the culture and chemical characterisation of chamomile (Matricaria chamomilla L.) and yarrow

(Achillea millefolium L.) using plants raised from in vitro and in vivo seeds culture. An additional aim was to develop a protocol for the micropropagation of the subject plants, in order to increase the rate and the quality of the plant phytochemical active compounds from tissue, organ and cell culture under specific controlled conditions

(Malik et al., 2012). The project also aimed to study the impact of in vitro and in vivo seed culture on the growth and developmental characteristics of the selected plants and subsequently characterise the phytochemical compounds content of both chamomile and yarrow plants.

The use of different concentrations of plant growth regulators (PGRs) during micropropagation is a challenge that needs to be characterised to determine which type and which concentration of hormone will substantially influence seed germination and produce the highest degree of callus, shoot and root formation. The observations from this study proposed that, the use of 0.5 mgL-1 of IAA for chamomile and 1.0 mgL-1 GA3 for yarrow is suggested in future studies to obtain the maximum production of callus, roots and shoots. Similarly, Siwach and Gill (2011) reported that the peak number of shoots of Ficus religiosa L. was observed in the woody plant medium (WPM) supplemented with 0.5 mgL-1 of IAA however, Ribeiro et al. (2009) indicated that the greatest rate of Annona crassiflora roots was obtained in

MS medium containing 2.0 mgL-1 of IAA, these differences could be due to the use of different medium, environmental conditions and genotype of mother plants.

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In this study, the effect of light regimes and temperature on the percentage of germinated seeds demonstrated that the required temperature and light regimes were different for each seed lot and is based on the source of seed supply. This could be related to the regulation and activity of photochromic genes which play the important role in the process of seed germination (Shinomura, 1997). This seems to be important in aiding researches to select the optimal conditions either in a greenhouse or in a field for the germination of seeds.

Due to the significant impact plant growth and development can have on experimental design it is necessary to characterise the growth and development of any potential test plants. Parameters that need to be described are the rate of growth and development of floral tissue, this leads to the requirement to enumerate plant height, date of side branching and the development of floral initials and final flowering.

Several scientists have tried to study the effect of the date of planting, temperature and light conditions on all growth characteristics (Fausey et al., 2005, Nidagundi and

Hegde, 2007). For example, Fausey et al. (2005) summarised the effect of daily light integrals on yarrow flowering time in a 22°C glasshouse and 16h photoperiod and they indicated that 15-20 mol.m-2 .d-1 of daily light integrals has been considered an acceptable range for high quality production. Similarly, in the current study, it was clear that external light and temperature were the two main factors that effected the growth and development of chamomile and yarrow during in vitro culture, as well as the in vivo winter and summer greenhouse growth trails. This indicates that temperature and light environments have a significant role to play in producing uniform development of a population of test plants. This concept is an essential parameter to characterise for chamomile and yarrow plants and before designing future experimental manipulation of these subject species.

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Moreover, results from this study indicated that summer plants grew faster and were more vigorous than winter grown plants (see Chapter 3 for more details), this is thought to be due to the consistently higher temperatures and quality of light in the summer season, which is especially important for the development of the flowering stage. It is also suggested that poor sensitivity of these naturally occurring summer season plants to the variable temperature and light spectrum of the winter months.

(i.e. these plant species have an innate dormancy mechanism that renders these species quiescent during the cool and dark winter months). This observation is an agreement with those described by Lee et al. (1999) who mentioned that the low activity of winter plant growth stages may be due to the the lower resistance of plants to the cooler winter environmental conditions. This evidence demonstrates to growers and researchers that growing chamomile and yarrow plants during the summer season, specifically in some countries that have short or highly variable summers like the United Kingdom. Such a strategy could improve the production of leaves, flowers and seeds rapidly. Another approach would be the application of in vitro seed culture and propagation of explants to obtain quality plantlets which can then be transferred to a temperature controlled greenhouse until flowering.

Alternatively it would be possible to transplant young plants to the field environment to establish a good population of plants for field scale cropping to maximise the production of flowers and seeds, which are main issues of commercial components of yield for the pharmaceutical industries (Papadopoulos et al., 2000). The controlled environmental conditions is also one of the main factors that should be considered during the cultivation of medicinal and aromatic plants to improve the levels and concentration of phytochemical active substances (Giurgiu et al., 2014).

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To achieve a more detailed understanding of the extraction process and identification of phytochemical compounds of plant materials, a study of Soxhlet and shaking extractions was carried out. This work was also completed by the analysis phytochemical compounds content in leaves and flowers under two different conditions as a main objective of this part of the project a) in vitro and in vivo culture conditions and b) well-watered and drought conditions as a comparative process for understanding the impact of changing abiotic environment has on the suite of active compounds contents of the test plants and furthermore to understand how drought impacts on the concentration and productivity of beneficial compounds (Chapter 4 and 5). It is believed that the final results of these current experiments will affect and improve an economic issue through the application of abiotic stress such as drought stress, which has the ability to increase the biosynthesis of plant natural products such as phenols, alkaloid and isoprenoids, thereby the amount of these natural products could be improved and increased in the stressed plants through the reduction of plant growth and the biomass (Al-Gabbiesh et al., 2015).

Results from this study has also demonstrated that most of the phenolic, flavonoid and terpenoid compounds found in in vivo leaves and flowers were significantly higher than in vitro content. This is probably due to the greenhouse environmental conditions such as temperature and light which may further enhance the final production of phytochemical active compounds content. However, a few compounds from the in vitro cultured plants had a higher concentration than in vivo cultured plants; such as nerolidol found in yarrow EO. The explanation for this observation may be proposed that in vitro cultured explants formed multiple shoots and this subsequently increased the concentration of nerolidol as a consequence of increased

174 production sites (i.e. more side shoots = more potential sites for secondary metabolites), this does appear to agree with results reported by Ghasemi et al.

(2014). This is a significant factor that shows micropropagation seems suitable for yarrow plants and could be scaled up to an industrial process that could be useful for the mass production of secondary metabolites such as essential oil.

The effect of drought stress and well-watered conditions on the phytochemical compounds content of chamomile and two varieties of yarrow was investigated

(chapter 5). It is proposed that this present study is the first work that reports the effect of drought stress on summer berries yarrow. Results confirmed that these two plants can produce a good mass production of phytochemical compounds content even when there is insufficient water in the soil. It is important to mention that this drought resistant may be due to the fact that both plants have small leaf structures which reduce water loss through transpiration system or it may be related to the deep root structures found in these plants (Kouressy et al., 2008). It is suggested that this factor is important for growing chamomile and summer berries yarrow in some dry areas such as our Kurdistan region (north of Iraq) which has inconsistent seasonal rainfall. Moreover, this work still needs further investigation to improve and identify more phytochemical compounds which can increase under drought stress conditions through analysing these active compounds from different plant parts such as root, stem and seeds.

Based on the various studies indicating the antimicrobial activity of some medicinal plant species (Gakuubi et al., 2017, Millezi et al., 2012, Si et al., 2005), the use of plant EOs showed good antibacterial activity. During the current study (disc diffusion

175 and 96 well plates methods) have been used to characterise the activity of EOs against some selected gram-positive and gram-negative bacteria strains. It is suggested that the activity of chamomile and yarrow EOs is mainly either due to the presence of high levels of some compounds produced for example in the greenhouse including farnesene and bisabolol oxide A found in in vivo chamomile and yarrow

EOs (Chapter 4), or having some compounds that are more active than others based on their structures. This result supports that informed by Lu et al. (2013). This study also showed that disc diffusion test was an easy way to show the great role of pure

EOs as an antibacterial activity especially against E. coli and B. subtilis. However, due to some negative results observed in the use of both methods in this bioassay work, these methods could possibly be further developed by using a range of different dilutions and by using the pure essential oil without any residual solvent in the extracted EO. Moreover, this work needs further development and future study to enable a more detailed analysis of the antibacterial activity of phenol and flavonoid compounds found in plant leaves in order to compare with flower EO antibacterial activity. It is also proposed that the use of more bioassay methods such as resazurin test (Mann and Markham, 1998) are investigated in future work to find out the efficiency of all three methods.

This project demonstrates that micropropagation technique is a viable option using different PGRs to produce complete plants containing variable phytochemical active compounds which act as an antibacterial activity therefore, in vitro micropropagation might possibly be used for those plants that cannot germinate easily in vivo. This technique however, is not economical and the cost is higher than in vivo propagation.

The summer season is the optimal season to grow both chamomile and yarrow

176 plants (due to the very fact they are occur naturally in the summer months in temperate countries) for obtaining the optimal production of leaf and flower parts. ln addition the application of drought stress appears to produce a maximum amount of some phytochemical active compounds content such as terpenoids which are less affected by stress, this could be important particularly in the area of marketing by producing the highest yield of essential oil from medicinal and aromatic plants.

7.2 Future work and recommendations

7.2.1 Tissue culture protocols: impact of gene expression and medicinal compounds

The results of this study indicated that plant leaves and flowers can regenerate from in vitro seed culture after being transplanted into the soil, and containing some beneficial phytochemical active compounds can act as an antibacterial activity.

Therefore, further work is required to identify these compounds such as quercetin and luteolin from callus, embryos, shoots and root formation (Agarwal and Kamal,

2007).

The study did not elucidate any of the molecular mechanisms that underpin the production of secondary metabolites, including those described above, therefore a recommendation would be to conduct a range of molecular studies to detect the gene/or genes responsible of secondary products including phenol, flavonoid and terpenoid compounds in higher quantities (Pazouki et al., 2015). Moreover, there are some difficulties in the use of different hormones for yarrow callus production using

MS medium. Therefore, future work could explore the possibility of using different

177 medium culture for example Linsmaier and Skoog Medium (LS) or Gamborg B5 medium, as well as different explants (Saad and Elshahed, 2012).

Furthermore, future work could be conducted to investigate the use of deoxyribonucleic acid (DNA) microarray applications as an easy and powerful way in a genomic tool. This approach provides some possible information which might be useful to find out the drug mechanism of action, discover gene production and novel molecular targets, as well as identifying the cause of diseases (Youns et al., 2009).

7.2.2 Molecular study and genetic characterization

Because of the role of chamomile and yarrow plants as a commercial trait, cultivation of different varieties of both plants in different seasons and under different abiotic stresses are important to be investigated. Also, to keep varieties of both plants, the study of genetic diversity needs to be achieved by the use of molecular biological methods for the authentication of plant species including Polymerase chain reaction

(PCR) technique using different markers such as Inter-simple sequence repeats

(ISSR), Random Amplified Polymorphic DNA (RAPD) and Sequence Characterized

Amplified Region (SCAR). This process also needs to investigate chamomile and yarrow genotypes characterization and discover more genotypes different from wild genotypes (Okoń et al., 2013).

7.3 General conclusions

This investigation proved that growth stages especially flowering stage were more affected by date of planting in different seasons. The application of in vitro and in vivo culture methods had also a great role in plant growth rate characteristics. This would support in vitro activities to enhance better management practices for plant species

178 biodiversity with the use of the micropropagation technique using different PGRs.

Moreover, this study provides at least some evidence, of how micropropagation can regenerate chamomile and yarrow plants from seed culture whilst producing a good quality of phytochemical compounds which possess great antibacterial activity.

Improvements in plant tissue culture could also provide a new means for producing exotic or rare plants and commercial phytochemical production.

In order to maintain the highest production of phytochemical active compounds content the process of drought stress in chamomile and yarrow should provide a good opportunity to produce some new commercial compounds able to cope with different environmental conditions. Results showed that the phytochemical compounds content was significantly affected by drought stress. The application of drought conditions is therefore essential and needed into farming systems, with regard to the land areas and varieties, local knowledge of species, cultural management and farmers' practices which promote and preserve plant diversity and minimize dangers, and increase the local use of wild herbs and plants of economic value.

Date of planting management and watering regimes are two main points required for the Kurdistan region where, water is a limiting and important environmental factor for plant growth development and total yield. This approach is possible to recommend for establishing the key growth stages of any new crop in order to maximize the yield of these active compounds.

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Moreover, chamomile and yarrow showed drought tolerance, this should provide a good opportunity to produce some new commercial compounds able to cope with different environmental conditions.

Finally, it has been demonstrated that selecting the best methods and solvents of plant extractions, as well as methods of antibacterial activity are needed to provide the effect of extracted EOs against Gram-positive and Gram-negative bacteria strains.

Consequently, it was confirmed that the methods of analysis and selecting a sufficient solvent of extraction needs to be considered to determine the phytochemical analysis with a high efficiency within the shortest run time. Therefore, one of the most interesting suggestions for future work will be to focus on the role of the phytochemical compounds content of chamomile and yarrow EOs as antiviral, antifungal and antiprotozoal agents.

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Appendices

Appendix 1: Data related to chapter 2, African violet medium preparation

This culture medium provides all compounds required for plant growth development such as organic compound (Sugar), mineral nutrients (MS) and plant hormones (BAP and Kin). Agar is also added as a solid agent. For preparing one litre of medium we added:

Step 1. 800 ml of distilled water to 1000 ml beaker.

Step 2. 4.4 gm of MS.

Step 3. 0.1 mg of BAP plus 0.2 mg kinetin as a PGR.

Step 4. 30 gm of sucrose.

Step 5. 10 gm of Agar.

Step 6. 1 ml plant preservative mixture (PPM).

Step 7. Adjust the pH to 5.8 using HCl and NaOH with a pH meter.

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Appendix 2: Data related to chapter 4 and 5

HPLC- UV chromatograms using methanol extraction

Figure 1 The HPLC-UV chromatogram of phenol and flavonoid compounds content of in vitro chamomile leaves detected at 256 nm.

Figure 2 The HPLC-UV chromatogram of phenol and flavonoid compounds content of in vivo yarrow leaves detected at 256 nm.

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Figure 3 The HPLC-UV chromatogram of phenol and flavonoid compounds content of chamomile leaves under well-watered conditions detected at 256 nm.

Figure 4 The HPLC-UV chromatogram of phenol and flavonoid compounds content of chamomile leaves under drought conditions detected at 256 nm.

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Figure 5 The HPLC-UV chromatogram of phenol and flavonoid compounds content of common yarrow leaves under well-watered conditions detected at 256 nm.

Figure 6 The HPLC-UV chromatogram of phenol and flavonoid compounds content of summer berries yarrow leaves under drought conditions detected at 256 nm.

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Phenol and flavonoid calibration curves

Figure 7 Standard calibration curve for caffeic acid linear regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent.

Figure 8 Standard calibration curve for apig-7-glucoside linear regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent.

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Figure 9 Standard calibration curve for polynomial umbelliferone polynomial regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent.

Figure 10 Standard calibration curve for polynomial luteolin polynomial regression by preparing (5.0, 10.0, 25.0, 50.0 and 100.0 mg L-1) using methanol as solvent.

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GC- FID analysis

1. Farnesene 2 (17.12 min) 2. Chamazulene (21.76 min) 3. Bisabolol oxide A (21.92 min)

2 3

Figure 11 GC-FID analyses of the selected essential oil compounds of in vitro chamomile flowers extracted by methanol using Soxhlet extraction.

1. Limonene (9.10 min) 6 2. Farnesene 2 (17.10 min) 3. Farnesene 3 (17.99 min) 4- α-(-)-bisabol (20.90 min) 5- Chamazulene (21.74 min) 6- Bisabolol oxide A (21.93 min)

2 5

1 3

Figure 12 GC-FID analyses of the selected essential oil compounds of chamomile flowers under drought conditions extracted by methanol using Soxhlet extraction.

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1. Limonene (9.10 min) 2. Farnesene 1 (16.69 min) 3. Farnesene 2 (17.10 min) 4. Farnesene 3 (17.97 min) 5- Nerolidol (18.89 min) 6- α-(-)- bisabol (20.86 min) 7- Bisabolol oxide A (21.91 min)

4 1 6 2 3 5 7

Figure 13 GC-FID analyses of the selected essential oil compounds of common yarrow flowers under drought conditions extracted by methanol using Soxhlet extraction.

1. Limonene (9.10 min) 2. Farnesene 1 (16.69 min) 3. Farnesene 2 (17.10 min) 4. Farnesene 3 (17.97 min) 5- Nerolidol (18.81 min) 6- α-(-)- bisabol (20.86 min) 5 7- Bisabolol oxide A (21.91 min)

6 4 7

1 2 3

Figure 14 GC-FID analyses of the selected essential oil compounds of summer berries yarrow flowers under well-watered conditions extracted by methanol using Soxhlet extraction.

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1. Limonene (9.09 min) 2. Farnesene 1 (16.68 min) 3. Farnesene 2 (17.10 min) 4. Farnesene 3 (17.97 min) 5- Nerolidol (18.89 min) 6- α-(-)- bisabol (20.86 min) 7- Bisabolol oxide A (21.91 min)

6 5

1 2 3 7

Figure 15 GC-FID analyses of the selected essential oil compounds of summer berries yarrow flowers under drought conditions extracted by methanol using Soxhlet extraction.

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Terpenoid calibration curves

Figure 16 Standard calibration curve for limonene linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

Figure 17 Standard calibration curve for farnesene 1 linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

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Figure 18 Standard calibration curve for farnesene 2 linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

Figure 19 Standard calibration curve for farnesene 3 linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

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Figure 20 Standard calibration curve for nerolidol linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

Figure 21 Standard calibration curve for α-(-)- bisabol linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

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Figure 22 Standard calibration curve for chamazulene linear regression by preparing (1.0, 2.0, 5.0, 10.0, 25.0 and 50.0 mg L-1) using methanol as solvent.

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Appendix 3: Data related to chapter 4, HPLC-UV chromatograms using hexane extraction

Figure 23 The HPLC-UV chromatogram of phenol and flavonoid compounds content of chamomile leaves using hexane extraction detected at 275 nm.

Figure 24 The HPLC-UV chromatogram of phenol and flavonoid compounds content of yarrow leaves using hexane extraction detected at 275 nm.

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Appendix 4: Data related to chapter 5, Soil moisture content calibration curves

Chamomile

Figure 25 Calibration curve of Theta probe measured the top of soil vs soil moisture content in pots with chamomile plants.

Yarrow

Figure 26 Calibration curve of Theta probe measured the top of soil vs soil moisture content in pots with yarrow plants.

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Appendix 5: Data related to chapter 6

C1 T1 T2

T3 C2 T1

T2 T3

Figure 27 Microplate cultures of in vivo and in vitro chamomile methanol extraction against five studied bacteria strains (n=3): C1 = control (1) without adding plant extract (10 μl sterilized distilled water+ 100 μl MHB2+ 50 μl culture (of bacteria strains separately)); C2= control (2) without any plant extract (10 μl MeOH+ 100 μl MB+ 50 μl culture ). T1, T2 and T3 were extracted essential oil of in vivo chamomile flowers; T1, T2 and T3 were extracted essential oil of in vitro chamomile flowers. T1 and T1= 10 μl pure extract + 100 μl MHB2+ 50 μl culture; T2 and T2= 10 μl 1:10 oil dilution+ 100 μl MB+ 50 μl culture; T3 and T3= 10 μl 1:100 oil dilution + 100 μl MB+ 50 μl culture (Mishra et al., 2015). The same process was approved for extracted essential oil of in vivo and in vitro yarrow flowers.

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In vivo Ch pure EO

Figure 28 The effect of in vivo chamomile pure EO on the percentage of the number of E. coli, B. subtilis, P. aeruginosa, P. syringae, and E. faecalis living in the MHB2 culture. The well filled with 160 μl of overnight cultured bacteria. The microplate was read every 20 minutes for 24 hrs at 595 nm.

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Appendix 6: Research publication

 Ahmed, B., Jamal, B. (2013). Determination of Flavonoids in the Leaves of Hawthorn (Crataegus Azarolus ) of Iraqi Kurdistan Region by HPLC Analysis. International Journal of Bioscience, Biochemistry and Bioinformatics (IJBBB), Vol. 3, No. 1.

 Mahmood, B. (2016). Bioactive compounds identification and antibacterial activity of the essential oil of chamomile and yarrow species. Plant Physiol Pathol, Vol 4, Issue 4 (Suppl).

Appendix 7: Professional membership

 Kurdish Postgraduate Society, University of Plymouth 2013.  Member of Society of Chemical Industry (SCI- BioResources Young Research) 2014.

Appendix 8: Conference attended Oral Presentations

 Extraction and chemical characterisation of medicinal compounds found in chamomile and yarrow species. CARS symposium, Cornwall College Newquay, 12th Jun 2015.  Characterisation of phytochemical compounds found in Matricaria chamomilla and Achillea millefolium using HPLC and GC-FID analysis. The Postgraduate Society short conference, University of Plymouth, 2nd December 2015.  Evaluating the growth characteristics and production of medicinal compounds in chamomile and yarrow using HPLC and GC-FID technologies. Lunch meeting conference. University of Plymouth, 20th January 2016.

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Poster presentations

 In vivo and in vitro micropropagation cultures, essential oil contents of chamomile and yarrow species. Iraqi day conference, University of Plymouth, 1st March 2014.  Micropropagation technique and bioactive chemical characterization of the essential oil of chamomile and yarrow species (Asteraceae). The postgraduate society annual conference, University of Plymouth, 24th March 2014.  In vitro seeds culture of chamomile and yarrow species using different plant hormones. The postgraduate society annual conference, University of Plymouth, 17th June 2014.  Study of chamomile and yarrow in vitro growth trials and, chemical extraction and characterization. 1st Student Symposium on Computational Biology and Life Sciences, University of South Wales, 8th October 2014.  Micropropagation protocols and HPLC chemical analysis of bioactive compounds found in chamomile and yarrow. 5th CARS Postgraduate Symposium at Eden Project. University of Plymouth, 19th November 2014.  Extraction protocols and chemical identification of Matricaria chamomilla and Achillea millefolium medicinal compounds. The postgraduate society annual conference, University of Plymouth, 23th June 2015.  Bioactive compounds identification and antibacterial activity of the essential oil of chamomile and yarrow species (Asteraceae). 2nd Global Summit on Plant Science. London, UK. 6th -8th October 2016.

Exhibition

 The identification of bioactive compounds and antibacterial activity of the essential oil of chamomile and yarrow species. The postgraduate society annual conference, University of Plymouth, 5th December 2016. 1st place was awarded.

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Appendix 9: Training courses and workshops

 Follow up- structuring your thesis/ Course of Microsoft Word 2010, University of Plymouth, 5th June 2013.  IEEE workshop/ Writing Workshop, University of Plymouth,13th June 2013.  Follow up- structuring your thesis/ Course of Microsoft Excel word 2010, University of Plymouth, 20th June 2013.  Biotechnology Module, University of Plymouth, 27th September 2013.  BIO5124 Module, University of Plymouth, 2nd October 2013.  Research skills (Using Excel as a Database), University of Plymouth, 30th October 2013.  SPSS workshop, University of Plymouth, 5 th February 2014.  The transfer process course, University of Plymouth, 16th June 2014.  Presenting to an audience, part 1, University of Plymouth, 3rd November 2014.  Critical thinking, University of Plymouth, 12th November 2014.  Overview to accessing and searching information resources, University of Plymouth, 18th November 2014.  Immersive writing workshop, University of Plymouth, 24th January 2017.

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