An-Najah National University 

Faculty of Graduate Studies 

 

 

 

 

Characterization of Polyphenol-Containing 

Polar Extracts from Stachys Palaestina and 

Stachys Viticina and Evaluation of Their 

Pharmacological Properties 

 

 
By 

Laila Mohammed Abbas Badwan 

 

 

Supervisor 

Dr. Nawaf Al-Maharik 

 

Co- Supervisor 

Dr. Nidal Jaradat 

 

This Thesis Submitted in Partial Fulfillment of the Requirements for 

the Degree of Master of Chemistry, Faculty of Graduate Studies, at 

An-Najah National University, Nablus - Palestine.  

2021 





III 

Dedication 

To my parents 

To my sisters and brothers 

To my friends 

I dedicate this work  



IV 

Acknowledgments 

Praise to almighty Allah who has enabled me to finish this work. I 

would like to express my gratitude  to my supervisors, Dr. Nawaf Al-

Maharik and Dr. Nidal Jaradat, who always supported me with their 

knowledge and experiences. I would also like to thank Dr. Mohammed Al-

Qadi, Ms. Fatima Hussen and Mr. Nafith Dweikat.  

Specially thanks for my parents, sisters, brothers, and my friends 

who supported me during my study. 

Finally, thanks to chemistry department at my University.  

 

 

 

 

 

 

 

 

 

 

 

 





VI 

List of Contents 

Page Subject No. 

III Dedication  

IV Acknowledgements  

V Declaration  

VI List of Contents  

IX List of Figures  

X List of Tables  

XI List of Appendix  

XII Abstracts  

Chapter One: Introduction 

1 The use of plants in traditional medicine 1.1 

2 Use of plants in drug development 1.2 

3 A Palestinian perspective 1.3 

4 Lamiaceae family 1.4 

4 Stachys viticina 1.4.1 

6 Plant natural products 1.5 

7 Classes of secondary metabolites 1.5.1 

8 Phenols 1.5.1.1 

15 Terpenoids 1.5.1.2 

16 Alkaloids 1.5.1.3 

17 
An overview of techniques used in natural products 

chemistry 
1.6 

17 Extraction techniques 1.6.1 

17 Conventional extraction methods 1.6.1.1 

17 Soxhlet extraction 1.6.1.1.1 

18 Maceration 1.6.1.1.2 

18 Digestion 1.6.1.1.3 

18 Novel extraction methods  1.6.1.2 

19 Ultrasound assisted extraction (UAE) 1.6.1.2.1 



VII 

19 Microwave assisted extraction(MAE) 1.6.1.2.2 

19 Supercritical fluid Extraction (SFE) 1.6.1.2.3 

20 Chromatographic techniques 1.6.2 

20 Thin liquid chromatography (TLC) 1.6.2.1 

21 Open column chromatography 1.6.2.2 

22 High Performance Liquid Chromatography (HPLC) 1.6.2.3 

23 Structure elucidation technique 1.6.3 

23 Infrared spectroscopy (IR) 1.6.3.1 

24 Nuclear magnetic resonance spectroscopy (NMR) 1.6.3.2 

24 Mass spectrometry 1.6.3.3 

24 Biological activity 1.7 

24 anti-oxidant activity 1.7.1 

25 Anti-microbial activity (bacterial and fungi) 1.7.2 

26 Aims of the study 1.8 

Chapter Two: Experimental  

27 Chemicals and reagents 2.1 

27 Plant material 2.2 

27 Extraction 2.3 

28 Isolation 2.4 

30 Structure elucidation 2.5 

30 HPLC analysis 2.5.1 

30 IR analysis 2.5.2 

32 Pharmacological screening 2.6 

32 
Antioxidant activity 2, 2-diphenyl-1-picrylhydrazyl  

(DPPH assay) 
2.6.1 

32 antibacterial and antifungal activity tests 2.6.2 

32 Preparation of plant samples for testing 2.6.2.1 

33 Preparation of growth media 2.6.2.2 

33 Test microorganisms 2.6.2.3 

33 Preparation of bacterial and fungal suspension 2.6.2.4 



VIII 

34 Anti-microbial assay 2.6.2.5 

Chapter Three: Results 

38 
Phytochemical of Polyphenolic Composition of 

 S. viticina 
3.1 

40 Biological activity 3.2 

40 anti-oxidant Inhibitory Activity 3.2.1 

41 anti-microbial activity 3.2.2 

Chapter Four: Discussion and Conclusion 

43 The chemical composition 4.1 

43 Antioxidant activity 4.2 

44 Antimicrobial capacity 4.3 

45 Conclusion 4.4 

46 References  

65 Appendix  

  الملخص  ب

 

 

 

 

 

 

 

 

 

 



IX 

List of Figures 

Page Title Figure No. 

3 
Examples of pharmaceutical drugs developed 

from plants 
Figure (1.1) 

6 Major compounds of Stachys viticina essential oil Figure ( 1. 2) 

8 
Phenol, parent compound of all phenolic 

compounds 
Figure ( 1. 3) 

9 
Elementary chemical structures of flavonoids and 

their different class 
Figure (1.4) 

10 Structure of some flavanones Figure (1.5) 

10 Examples of some flavonols Figure (1.6) 

11 The major structures of flavones Figure (1.7) 

11 
Structures of the most well-known anthocyanins 

in plant 
Figure (1.8) 

12 Chemical structures of green tea catechins Figure (1.9) 

13 Chemical structures of major isoflavones Figure (1.10) 

14 
Chemical structures of the basic phenolic acids 

classes 
Figure (1.11) 

15 Hydrolysable and condensed tannins Figure (1.12) 

16 Examples of terpenoids classes Figure (1.13) 

17 Examples of alkaloids Figure (1.14) 

41 
Anti-oxidant % Inhibition concentration (µg/ml) 

of different extracts 
Figure (3.1) 

42 
Minimum Inhibitory concentration (µg/ml) of 

different plant extracts against different pathogens 

Figure (3.2) 

 

 

 

 

 

 

 

 



X 

List of Tables 

Page Subject Table No. 

40 IC50 (µg/ml) values for different extracts Table (3.1) 

42 

Minimum Inhibitory concentration values (µg/ml) 

for different Stachys viticina extracts against 

selected pathogens 

Table (3.2) 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



XI 

List of Appendices 

Page Appendix No. 

66 HPLC chromatogram for compound A Fig.a.1 

66 HPLC chromatogram for compound B Fig.a.2 

67 HPLC chromatogram for compound C Fig.a.3 

67 HPLC chromatogram for compound D Fig.a.4 

68 HPLC chromatogram for compound E Fig.a.5 

68 HPLC chromatogram for compound F Fig.a.6 

69 IR spectra for compound A Fig.a.7 

69 IR spectra for compound B Fig.a.8 

70 IR spectra for compound C Fig.a.9 

70 IR spectra for compound D Fig.a.10 

71 IR spectra for compound E Fig.a.11 

71 IR spectra for compound F Fig.a.12 

 

 

 

 

 

 

 

 

 

 

 

 

 



XII 

Characterisation of Polyphenol-Containing Polar Extracts from 
Stachys Palaestina and Stachys Viticina and Evaluation of Their 

Pharmacological Properties 

By 

Laila Mohammed Abbas Badwan 

Supervisor 

Dr. Nawaf Al-Maharik 

Co- Supervisor 

Dr. Nidal Jaradat 

Abstract 

The aromatic plant Stachys viticina (Lamiaceae) is a perennial herb 

growing in the Mediterranean countries including Palestine. And Like other 

Stachys species, it’s also used in folk therapy from ancient time. In view of 

this, the current work designed to isolate and characterize the chemical 

constituents and to assess the in-vitro, antioxidant, antimicrobial properties 

of the polar poly phenolic composition of Stachys viticina.  

Methanol extract of Stachys viticina was subjected to a sequence of silica 

gel column chromatography using different eluents with various polarities. 

The purity of the isolated fractions was conducted by thin layer 

chromatography (TLC) and confirmed using high performance liquid 

chromatography (HPLC). Moreover, functional groups of the pure fractions 

were detected using infrared spectroscopy. In addition, the radical 

scavenging capacity of plant methanolic extracts was assessed by the 

DPPH assay, and antimicrobial properties against seven microbial strains 

using the microdilution method were also screened. 

Seven extracts of Stachys viticina were separated (A-F and R). All of the 

extracts (A-D, F, and R) exhibited antioxidant activity with IC50values of 



XIII 

85.88 µg/mL, 54.37 µg/mL, 77.58 µg/mL, 68.36 µg/mL, 58.62 µg/mL, and 

18.58 µg/mL, respectively. Fraction C had the highest antibacterial activity 

at a minimum concentration (39 µg/mL) against methicillin-resistant 

staphylococcus aureus (MRSA) and Staphylococcus aureus. But all of the 

extracts had no activity against candida albicans fungi strain. 

 

 

 

 

 

 

 



1 

Chapter One 

Introduction 

1.1  Plants in Traditional Medicine 

From ancient time plants and herbs have been an essential provenance for     

treatment of various diseases ranging from minor illness to the more severe 

ones like malaria, cancer, tuberculosis, and even HIV/AIDS  ]1[.  

The Sumerians were the first civilization to use medicinal plants for drug 

preparation over 5000 years ago. They used more than 250 different plants, 

such as poppy, mandrake, and henbane. The Ebers Papyrus written by the 

Ancient Egyptians from around 1500 BCE listed over 800 herbal 

medicines, many of these herbs are still used nowadays ]2[. 

Traditional medicine system is deeply rooted in societal cultures that 

formed by people experiences and passed from generation to generation, 

for instance Chinese, Arabic and African folk medicine ]1[. 

 Seeds, flowers, leaves, fruits, roots, rhizomes, and oils are plant parts that 

are used for folk therapy in the form of powders, pills, creams, pastes, 

suppositories, and ointments, or sometimes combination of these ]3[. 

Recently, the request for herbal products is raising all over the world         

including herbal medicines, herbal pharmaceuticals, herbal health products, 

herbal cosmetics, nutraceuticals, and food supplements, etc. The main 

reason for this trend is that herbal products exhibit less side effects, and are 



2 

available at reasonable prices  ]4[. The World Health Organization (WHO) 

Statistics stated that ” approximately 80% of the world inhabitants reckon 

on folk medicine for their primary health care” ]5[. 

1.2  Use of Plants in Drug Development 

Initial used of medicinal plants taken a form of crude drug as herbal 

mixtures, teas, poultices, powders and tinctures ]6[. The isolation of 

morphine 1, used as an analgesic, in1806 as a pure natural product from the 

opium poppy Papaver somniferum was the introductory for drug 

development from plants ]7[. Since then several pure phytochemicals have 

been separated and used as drugs or analogues for the development of 

drugs. At the beginning of the 21st century, it was estimated that 11% of 

the 252 crucial drugs were of plant origin ]8[. 

Several examples of essential drugs in use today derived from plant are 

shown in Figure (1.1). Camptothecin 2 isolated from Camptotheca 

acuminata tree growing in China is used as antitumor agent ]9[, and 

Galantamine 3 (trade name Reminyl®) isolated from Galanthus woronowii 

Losinsk. (Amaryllidaceae) is currently used for the treatment of 

Alzheimer’s disease. The antimalarial drug Arteether 4 (trade name 

Artemotil®) was isolated from Artemisia annua L. (Asteraceae) ]10[. 



3 

 

 

Figure (1.1): Examples of pharmaceutical drugs of plant origin [7, 9, 10]. 

Despite drug development process from plants tends to be a long, boring 

and expensive, its value exceeds other routes because plants have an 

immense potential. With an estimated 250000 plant species dispersed 

across the world, where each plant can produce up to thousands of 

structurally different phytochemicals, the ability of plants as provenance of 

novel compounds cannot be matched. The rapid development in 

chromatographic and spectroscopic procedures employed in the isolation 

and identification of phytochemicals further advances their pharmaceutical 

potential  ]11[. 

1.3  A Palestinian Perspective  

Palestine with its special geographical location between three continents 

constitute of a wilderness in the south, a lot of mountains in the middle and 

the north in addition to the continental rift valley. Furthermore, it is 



4 

positioned at the coast of the Mediterranean. This geographical variation 

leads to the variety of soil and climate conditions and this in turns leads to 

biodiversity ]12[. Therefore, Palestine is renowned for its high abundance 

of medicinal plants that are used since a long period of time ]13[. However, 

the testing of pharmacologically active compounds in flora started in the 

late sixties ]14[. 

More than 2600 plant species from different families vegetate across the 

Palestinian mountains, valleys and desert, of which more than 700 are 

eminent for their usages as medicinal herbs or as botanical pesticides ]15[.  

1.4  Lamiaceae Family 

The Lamiaceae family (Labiatae) is an important medicinal plant family 

with a cosmopolitan distribution. It contains approximately 236 genera and 

more than 6000 species, of which Salvia is the largest genus with 900 

species followed by Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium, 

Vitex, Thymus, and Nepeta ]16[. Most of these species are aromatic and 

have essential oils that are valuable in different field, such as cosmetic, 

fragrance, flavouring, pesticide, and pharmaceutical industries ]17, 18[. 

This family includes several plants that are widely find applications in 

traditional medicine as a remedy for a wide range of disease ]17, 19[. 

1.4.1 Stachys Viticina 

Stachys L. (Lamiaceae, Lamioideae) is a large genus of herbs and shrubs 

that embraces approximately 450 species disseminated in the warm 

temperate and tropical regions worldwide such as the Mediterranean and 



5 

Southwest Asia ]20[. The common names of Stachys species are heal-all, 

self-heal, woundwort, betony, lamb’s ears, and in numerous native areas of 

the world as ‘mountain tea’.  They are employed in folk medicine for 

medication of genital tumors, inflammatory diseases, sclerosis of the 

spleen, fevers, cough and ulcers, sore mouth and throat, internal bleeding 

and weaknesses of the liver and heart, and diarrhea  ]21, 22[. 

Recently, examinations of the different extracts and constituents of Stachys 

species displayed different pharmacological activities, including among 

others anti-anxiety  ]23, 24[, antibacterial ]25, 26[, anti-inflammatory  ]27[, 

anticancer ]28[, and antioxidant activities ]26, 29[. They are used as 

antiaging and for curing other diseases related to radical scavenging 

mechanisms ]30[. 

From reported works, the key classes of plant secondary metabolites which 

have been recognized from Stachys species are flavonoids, phenolic acids, 

iridoids and fatty acids ]31[.  

Only 13 species of Stachys genus are growing in Palestine ]21[, of which 

Stachys viticina has been rather barely studied. Gӧren et al. reported the 

GC-MS analyses of the fatty acid composition of oil of S. viticina seeds in 

addition to 22 other Stachys species [32]. They found that linoleic acid 

(47.8%) and 6-octadecynoic acid (7.6%) are major acids isolated from S. 

viticina ]32[. N. Jaradat  and N. Al-Maharik  reported the identification of 

fifty two compounds of S. viticina essential oil using microwave-ultrasonic 

and GC-MS techniques, of which endo-borneol 5 was the major 



6 

component, followed by eucalyptol 6 and epizonarene 7 [33] (figure 1.2). 

To the best of our knowledge there are no published reports neither on the 

polyphenolic composition of Stachys viticina nor on its biological 

properties till now. 

 

Figure (1.2): Major compounds of Stachys viticina essential oil [34-36]. 

Stachys viticina   

1.5  Plant Natural Products 

Plants are continuously synthesizing natural products often called 

phytochemicals. These compounds categorized into two broad groups 

based on their functions in plants ]1[. Firstly, there are primary metabolites, 

they occur in all plants in sufficient amounts since they performing 



7 

essential metabolic roles in a plant, and they are directly engaged in plant 

growth and development ]37[. Examples on primary metabolites include 

carbohydrates, amino acids, lipids and nucleotides ]38[. Then there are 

secondary metabolites, which haven’t direct primary role in the growth and 

development of a plant, but they are crucial for the plants’ survival. Plants 

often synthesize secondary metabolites in response to attack by insects, 

microorganisms, herbivores and to suppress the growth of neighboring 

competitor plants. Some secondary metabolites are also produced in the form 

of aromas, flavors and colors to attract pollinators and seed dispersers ]39[. 

Secondary metabolites, which are the major active ingredients of medicinal 

plants, attract special interest due to their biological activity on other 

organisms especially animal cells [40]. Thus natural product chemistry 

field focuses mainly on isolation, structural elucidation, biological 

properties and preparation of secondary metabolites.  

1.5.1 Classes of Secondary Metabolites 

Classification of secondary metabolites into distinct groups is complicated, 

due to the wide structural diversity of them. Several classification 

approaches are used, of which the most use is the one based on the 

biosynthetic pathway of the compounds. Under this approach three major 

groups were isolated and identified: namely phenols, terpenoids, and 

alkaloids [41]. 



8 

1.5.1.1 Phenols 

Characterization of this group is referred to the occurrence of one or more 

hydroxy (OH) groups joined to an aromatic ring ]42[. Phenol can be 

assumed to be the parent compound as shown in Figure (1.3). The 

structures of phenolic compounds exist either as simple skeleton with one 

aromatic ring or as sophisticated polymers (polyphenols) having various 

functional groups attached. Major groups of phenols include the flavonoids, 

phenolic acids and tannins. This classification is established based on the 

number of carbon atoms in the elementary skeleton. Phenolic compounds 

accumulate in plants and related with flavor and color characteristics of 

fruits and vegetables ]43-45[.  

 

 

 

Figure (1.3): Phenol, parent compound of all phenolic compounds [44]. 

A) Flavonoids 

Flavonoids are the prevalent and highest diversified group of polyphenolic 

compounds. Their general skeleton structure is formed of two benzene rings 

(A and B) joined through a three-carbon bridge oxygenated heterocycle (C), 

which form the C6-C3-C6 structural backbone ]45–48[   (Figure (1.4)). Their 

main functions are protection against ultraviolet (UV) radiation and serving as 

signals to attract pollinators and seed dispersers ]49[. 



9 

Flavonoids have attracted special attention due to their pharmacological 

properties, which include antibacterial, anti-inflammatory, antimicrobial, 

estrogenic, anti-oxidant, cytotoxic and antitumor activity ]50-53[.  

Classification of flavonoids is based on the differences in their chemical 

structure (oxidation or saturation of the intermediate C ring)  ]46[, 

accordingly flavonoids can be categorized into six subclasses: flavanones 

9, flavonols 10, flavones 11, anthocyanins 12, flavanols 13, and 

isoflavonoides 14, and each group has particular characteristics ]53[. 

 

Figure (1.4): Elementary chemical structures of the flavonoids classes [53]. 

 

 



10 

Flavanones 9 

This type is found in aromatic plant (such as mint), citrus (especially 

grapefruit), and tomatoes ]45[. Figure (1.5) represents the most abundant 

simple flavanones (naringenin 15, eriodictyol 16, hesperetin 17). 

Flavanones were found to display a wide range of biological activities 

including among others radical scavenging, anticancer, anti-inflammatory, 

and antiviral activity ]54[.                               

 

Figure (1.5):  Structure of some flavanones [54]. 

Flavonols 10 

Flavonols are the most abundant flavonoids’ class in the plant kingdom 

]55[. Kaempferol 18, quercetin 19, and myricetin 20 (Figure (1.6)) are of 

the most important compounds that represent this group ]56[. Flavonols 

possess antioxidant activity that may protect against oxidative damage to 

cells, lipids or DNA. Additionally, they have anti-inflammatory and 

neuroprotective properties ]57[.     

                                 

Figure (1.6) : Examples of some flavonols [57]. 



11 

Flavones 11 

Flavones exist in all major land-plant lineages, and can be found in all parts 

of the plants (seeds, fruits, flowers, leaves, stem, buds, bark, roots, 

rhizomes). flavones were found in plant species belonging to over 70 

different families in the plant kingdom  ]58[. Apigenin 21 and luteolin 22 

are the main representative of the simple flavones  ]57[. 

                              

   Figure (1.7): The major structures of flavones [57]. 

Anthocyanins 12 

Anthocyanins are the chemicals that are accountable for plant tincture 

(such as blue, red, pink, and purple colour) ]59[. The most representative 

compounds of this subclass are pelargonidin 23, cyanidin 24, and 

delphinidin 25 (Figure (1.8)) ]45[. Anthocyanins play an essential role in 

preventing cardiovascular diseases, and have antioxidant properties ]60[.     

                                 

Figure (1.8): Structures of the most well-known anthocyanins in plant [57]. 



12 

Flavanols 13 

Flavanols (flavan-3-ols), also known as catechins, are categorized into free 

catechins and esterified catechins. The free catechins comprise catechin 10, 

gallocatechin, epicatechin 26, epigallocatechin 27, while the esterified 

catechins contain of epicatechin gallate 28, epigallocatechin gallate 29, 

gallocatechin gallate 30, and catechin gallate 31 (Figure (1.9)). The esterified 

catechins have bitter taste, while the free catechins have a slightly sweet taste. 

Compounds 26-29 are the four major catechins found in green tea ]61 [. 

Catechins represent the most complicated class of flavonoids due to their size, 

monomers (catechin), or polymeric forms (condensed tannins) ]57[. 

 

 

Figure (1.9): Chemical structures of some catechins [62]. 

 



13 

Isoflavonoids 14 

Isoflavonoids are another type of flavonoids, which have considerable 

estrogenic activity so they are referred to as phytoestrogens ]63[. These 

compounds are found predominantly in the Leguminosae family plants 

including among others soybeans, alfalfa sprouts, and red clover leaves. 

Isoflavones are an essential group that find use in medicinal, cosmetically, 

and nutritionally industry [64]. The major isoflavonoids that found in 

human diet are daidzein 32 and genistein 33 and their methyl ether 

formononetin 34 and biochanin A 35 (Figure (1.10)) ]57, 63]. 

 

 

Figure (1.10): Chemical structures of major isoflavones [63].  

B) Phenolic acids 

Phenolic acids represent the most copious class of phenolic compounds 

extracted from plants. They are found predominantly in plant seeds, fruits 

skins and vegetable leaves. Phenolic acids could be classified into two 

major groups: hydroxybenzoic acid 36 and hydroxycinnamic acid 37 

(Figure (1.11)) ]65, 66[. Phenolic acids possess anticancer, antioxidant 

activity, and are used as remedy for diabetes, cardiovascular diseases, and 

retard the development of Alzheimer’s disease ]67[. 



14 

 

 

Figure (1.11): Chemical structures of the basic phenolic acids classes [66]. 

C) Tanins 

Tannins are phenolic compounds having high molecular weight ranging 

from 500-3000 Da. They are found in many plants parts such as fruit, 

leaves, bark, wood and roots. They have been considered as plant defense 

mechanisms against herbivorous attacks because of their astringent taste. 

Based on their chemical structure and characteristics, tannins are 

categorized into two major groups: hydrolysable 38 and condensed tannins 

39 (Figure (1.12)). Tannins are used in industry field specially by the 

leather industry, and have found other applications like adhesives for wood 

industry, mineral industry, wine production industry, animal nutrition, oil 

industry, and protecting metal from corrosion ]68,69[. 

 

 

 

 

 



15 

 

 

Figure (1.12): Hydrolysable and condensed tannins [69]. 

1.5.1.2 Terpenoids 

Terpenoids are one of the most prevalent and diversified group of 

secondary metabolites. They are structurally characterized by a basic 

skeleton built from repeating isoprene building units, which are generally 

joined together in a head to tail fashion. Major groups of terpenoids are 

hemiterpenoid, monoterpenoid, sesquiterpenoid, diterpenoid, triterpenoid 

and tetraterpenoid  ]70, 71[. 

  



16 

 

   

Figure (1.13): Examples of terpenoids classes [72]. 

1.5.1.3 Alkaloids 

Alkaloids the third group of secondary metabolites. They contain at least 

one Nitrogen in their structure situated in some rings. About 20% of all 

flowering plants contain alkaloids ]37[. The two stimulants caffeine 46 

isolated from tea (mostly Camellia sinensis), coffee (Coffea arabica), and 

cacao (Theobroma cacao) plants, and nicotine 47 isolated from the tobacco 

plant Nicotina tabacum probably are two of the most common alkaloids. 

Alkaloids have been used for medication of illnesses related to the central 

nervous system (CNS), malaria and cancer ]73[. However, their toxic, 

narcotic and addictive nature, regulated or restricted their use and most are 

rarely used in their pure form but semi-synthetic analogues are used  ]73[. 

Examples of some of the most common alkaloids (46, 47, 48) are shown in 

Figure (1.14). 

  



17 
 

 

 Figure (1.14): Examples of alkaloids [37]. 

1.6  An Overview of Techniques Used in Natural Products Chemistry 

1.6.1 Extraction Techniques 

Extraction, the first step in the examination of medicinal plants, involves 

the transfer of phytochemicals from plant parts into organic solvents in 

standard extraction procedures. The obtained extracts from plants are 

almost impure and need further separation and characterization [74[. 

Several extraction methods have been used for the extraction of 

phytochemicals from plants. The most widely used extraction techniques 

will be briefly discussed. 

1.6.1.1 Conventional Extraction Methods  

Conventional extraction methods are centered on solid–liquid extraction 

with various solvents. These methods have considerable drawbacks, like 

the relatively large amounts of organic solvents required and long 

extraction time ]75[.  

1.6.1.1.1 Soxhlet Extraction 

In this process, a soxhlet extractor is used to transfer the soluble 

constituents of a solid matrix to the liquid phase. This method has some 



18 

advantages. This technique is simple, cheap, and can extract more sample 

mass than other conventional methods. The main drawbacks are the long 

extraction time (at least 6 h), the bulky volume of solvent used, and the 

target compound  may undergo thermal decomposition since the extraction 

usually occurs at the boiling point of the solvent for a long time ]76[. 

1.6.1.1.2  Maceration 

In this process, the coarse or powdered plant is submerged in the solvent in 

a sealed container, which was allowed to stir at room temperature for at 

least 3 days until most of the soluble matter has dissolved. Filtration or 

decantation after standing followed by removal of solvents gave a mixture 

of compounds. Maceration became popular extraction method for phenolic 

compounds because it is simple and inexpensive ]77[. 

1.6.1.1.3 Digestion 

Digestion is a maceration accompanied with gentle heat during the process. 

It's used when some of the plants' constituents can't be dissolved at room 

temperature, or when there are partially insoluble phytochemicals. [78]. 

1.6.1.2 Novel Extraction Methods  

Recently, modern environmentally friendly extraction techniques that 

are quicker and more reliable than conventional extraction methods have 

been developed. Many advantages in terms of extraction time, solvent 

used, extraction yields, and reproducibility have been achieved by using 

these methods in the extraction of active ingredients from plant [  79, 80[. 

These techniques include among others ultrasound assisted extraction 



19 

(UAE), microwave assisted extraction (MAE), and supercritical fluid 

extraction (SFE). 

1.6.1.2.1 Ultrasound Assisted Extraction (UAE) 

High ultrasound frequencies pulses ranging from 20 kHz to 2000 kHz are 

used in this technique. This causes cavitation, which destroys the plant's 

cell wall, allowing for better mass transfer [75]. UAE has a number of 

advantages. It takes less time and uses less solvent, and it improves 

extraction yield [81[. 

1.6.1.2.2 Microwave Assisted Extraction (MAE) 

Microwave Assisted Extraction is a technique that involves heating 

the solvent containing a sample matrix with microwave energy ranging 

from 300 MHz to 300 GHz in order to extract analytes from the matrix into 

the solvent. The rapid heating of the solvent and sample matrix is the main 

advantage of this technique. Microwave-assisted extraction takes only 15–

30 minutes and requires 10–30 mL of solvent. These volumes are roughly 

ten times smaller than those consumed by traditional extraction methods. 

Using MAE, the solute recovery and reproducibility are generally 

improved [75, 82[. 

1.6.1.2.3 Supercritical Fluid Extraction (SFE) 

It's a technique for isolating a specific component from a matrix with the 

aid of a supercritical fluid extraction solvent. In the fine chemical industry, 

supercritical carbon dioxide (CO2) is the most widely used extraction 



20 

procedure [83   [ . SFE under the right conditions, SFE yields purer extracts 

than traditional extraction with low or no organic solvents uses [75]. 

1.6.2 Chromatographic Techniques 

Chromatography is a physical technique used for separating chemical 

mixtures into their individual components [84[. In all chromatographic 

processes, the mobile phase (in which the sample is dissolved) is pushed 

into the stationary phase (which is settled in a column or on a solid 

surface). To different degrees, the sample components distributed 

themselves between the two phases [85[. 

Based on the form of mobile phase used, chromatography can be classified 

in three typed namely Gas chromatography (GC), liquid chromatography 

(LC), and supercritical fluid chromatography (SCF) [85]. Liquid 

chromatography is often classified into two types depending on the form of 

stationary material used for separation: normal phase and reversed phase 

silica gel. Since the normal phase consists of a polar stationary phase and a 

non-polar mobile phase, non-polar ingredients are eluted first. Reversed 

process, on the other hand, includes a non-polar stationary phase and a 

polar mobile phase, resulting in polar compounds eluting first. In most 

cases, though, reverse phase is used because many of the chemical drugs 

are polar in nature [86]. 

1.6.2.1 Thin Layer Chromatography (TLC) 

TLC is a form of chromatography in which a thin layer of adsorbent 

material (usually silica gel, aluminum oxide, or cellulose) is coated on a 



21 

solid support (aluminum sheet, glass, or plastic) and a liquid mobile phase 

is used [87].  

After the sample spot has been applied on TLC plate, the solvent system 

(mobile phase) drawn up the plate by capillary force. Different analytes 

moved on plate at different rates depending on their solubility and retention by 

the stationary phase. Each spot on plate equivalent to one analyte and it is 

characterized by its retention factor (Rf) value. This is calculated by dividing 

the analyte’s distance traveled by the solvent’s distance traveled  ]87]. 

TLC, in combination with UV detection and spraying reagents, provides 

knowledge about the number of mixture components and can be used to 

distinguish a compound in a mixture by comparing its Rf to the Rf of a 

known compound  ]87[. 

1.6.2.2 Open Column Chromatography 

It is a method of solid-liquid chromatography that is used to separate 

chemical compounds in a mixture. This technique utilizes a stationary 

phase, which is packed in a column, usually are silica gel (SiO2) and 

alumina (Al2O3), and a mobile phase that passes through the column, 

commonly volatile organic solvents. A solvent is used to elute the sample 

mixture from the top of the column. Analytes disperse as they travel down 

the column due to polarity variations, forming bands that are collected in 

small fractions [88]. TLC is used to track these processes on a regular 

basis, and related fractions are pooled and concentrated ]89[.  



22 

Eluting the column can be achieved in one of two ways, namely isocratic 

and gradient.  In isocratic elution, the column is eluted with a single solvent 

or solvent mixture, while in gradient elution, the column is eluted with a 

series of solvents of increasing polarity [88[. The best solvent flow rate for 

each column is determined by the form of mobile phase, analyte, and 

column size. The technique of open column chromatography is simple and 

inexpensive. However, it is time consuming, labor intensive, and boring. 

Since they spend too much time in the column, certain analytes are prone to 

decomposition ]89, 90[. 

1.6.2.3 High Performance Liquid Chromatography (HPLC) 

HPLC stands for high-performance liquid chromatography and is a type of 

column liquid chromatography. The HPLC instrument is an automated 

closed system consisting of a solvent delivery system containing a pump, a 

detector unit, injection port (manual or automated), data analysis unit, a 

column, and a reservoir for solution and waste deposit container ]91, 92[.  

The column is the main part of the system. There are various HPLC 

column sizes. The standard analytical HPLC column has a diameter of 4.6 

mm and a length of 10 to 25 cm. Larger columns are used in semi-

preparative and preparative HPLC (10 mm and 20 mm in diameter). The 

size of the columns is determined by the separation’s intent. Analytical 

columns are used to determine qualitative and quantitative information 

about a sample. Typically, once the components have been analyzed with 

an analytical column, they are discarded in a waste container.  To separate 



23 

and collect sample fractions, larger columns such as semipreparative and 

preparative columns are used, and more purification can be performed ]93[. 

Chemical partitioning with an HPLC system is possible because each 

compound migrates at a different rate in a certain mobile phase and 

column. The choice of stationary phase and mobile phase determines the 

degree of partitioning ]94[. This technique is currently more common than 

other techniques because it is the first option for studying fingerprinting to 

monitor herb quality [95[. 

1.6.3 Structure Elucidation Techniques 

Conventional spectroscopic techniques such as UV-visible, infrared (IR), 

mass spectrometry (MS), and nuclear magnetic resonance (NMR) 

spectroscopy are used in structure elucidation of organic compounds [96]. 

These techniques are always used to determine the number of atoms and 

bonds, type of bonds, as well as the structure and conformation of a pure 

compound. Improvements in technology have allowed the use of small 

quantities reached to milligram of sample, high resolution and minimized 

analysis times [97[. 

1.6.3.1 Infrared Spectroscopy (IR) 

Infrared spectroscopy is a technique used for identifying the functional 

groups in a compound that are recorded in a spectrum by the IR 

instrument.]98[. 

 

 



24 

1.6.3.2 Nuclear Magnetic Resonance Spectroscopy (NMR) 

NMR is an essential technique that provides detailed information 

concerning structural elucidation of organic compounds. Proton and 

Carbon -13  (1H ,13C) NMR are some of the most useful experiments for 

elucidation the compounds structures ]99, 100]. 

1.6.3.3 Mass Spectrometry 

The molecular weight of the compound and the fragmentation patterns of 

the origin compound are determined by analyzing the mass spectrum. 

Comprehensive study of the fragmentation patterns, in addition to 

information obtained from other techniques will lead to molecular 

structural elucidation of the studied compound [101[. 

1.7  Biological Activity  

Biological activity refers to ability of a certain molecule to obtain a defined 

biological effect on a target living tissue. It is determined by using 

biological assay ]102, 103]. 

1.7.1  Anti-oxidant Activity 

Free radicals are molecules with an odd number of electrons, such as 

reactive oxygen and nitrogen species. A chain reaction can be started when 

these very active unstable radicals are formed. These free radicals could 

be generated both internally, as normal cells create them during 

metabolism, and externally, as a result of contaminants in the air, exposure 

to harmful radiation, and chemicals created by industrial processes ]104[. 



25 

Oxidative stress is caused by an imbalance between the antioxidant defense 

mechanism and the production of oxidants. Oxidative stress has been 

linked to Alzheimer's disease, cancer, heart disease, Parkinson's disease, 

and death ]104, 105 [. 

An antioxidant is a substance that inhibits or prevents the oxidation of a 

substrate. Antioxidants come in two forms: natural antioxidants like 

ascorbic acid, glutathione, flavonoids, uric acid, melatonin, and vitamin E, 

and synthetic antioxidants like butylated hydroxytoluene and butylated 

hydroxyanisole. However synthetic antioxidants have greater hazard of side 

effects; therefore, searches on determining the natural antioxidants have 

become very important issue. Plants produce a huge amount of antioxidants 

and they can represent a potential source of new compounds having 

antioxidant properties ]106[. 

1.7.2  Anti-microbial Activity (Bacterial and Fungi) 

Microorganisms that cause disease for its host are called pathogenic 

organisms. These pathogens include:  bacteria, viruses, fungi, and parasites. 

They are infectious and transferred to human by animals, insects, and by 

taking contaminated water and food  ]107[  . 

Antibacterials are substances that inhibit the growth and reproduction of 

bacteria. All antibiotic drugs possess antibacterial properties ]108[. 

Antifungi is one of the antibiotic groups which kill or stop the growth of 

fungi. Candidiasis are one of the most popular type of fungi ]109[. 



26 

However, some pathogens such as Escherichia coli, Proteus sp., 

Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, 

gained resistance to antimicrobials ]110[, so finding new antimicrobials 

drugs a major challenge in global health care. 

  Researchers have recently attempted to use medicinal plants as an 

alternative source of medicines, since medicinal plants are abundant in 

antimicrobial compounds such as flavonoids, terpenoids, and alkaloids 

]111, 112]. 

1.8 Aims of the Study 

The objectives of this proposed study were: 

1. Isolate and identify the active components shown by Stachys viticina 

using various techniques. 

2. Exploring the antioxidant, antibacterial, and antifungal activities of 

Stachys viticina polar extracts. 

 

 

 

 

 

 

 



27 

Chapter Two 

Experimental Part 

2.1  Chemicals and Reagents 

The materials used in this research were of analytical grade and used  

without further purification.  

All solvents used (methanol, dichloromethane (DCM), ethyl acetate 

(EtOAc), hexane, and diethyl ether) were purchased from Sigma Aldrich 

(Germany). Silica gel (100–200 and 200–300 mesh) for column 

chromatography (CC), and GF254 silica gel for thin layer chromatography 

(TLC) were purchased from Sigma-Aldrich (USA), 2,2-diphenyl-1-

picrylhydrazyl (DPPH), and trolox were purchased from Sigma Aldrich 

(Germany). Finally, Mueller-hinton broth, Sabouraud dextrose agar, and 

dimethyl sulphoxide (DMSO) were obtained from Himedia (India). 

2.2  Plant Material 

 Stachys vicitina plant was collected on the end of May 2018 from the 

mountains of Nablus by Dr Nidal Jaradat from Al Najah University. The 

plant was washed with water and dried in well ventilated room at ambient 

temperature in a shaded area until it is completely dry. 

2.3  Extraction  

 The dried Stachys viticina leaves (585.53 g) were grinded, then macerated 

with sufficient amount of methanol (2L) and stirred using mechanical 

stirrer for 24 hours. The resulting extract was filtered, and the remaining 



28 

solid was subjected to extraction twice with methanol (0.5 L) under the 

same conditions. After filtration of the combined extract, methanol was 

removed under reduced pressure. Then concentrated extract was 

successively washed with pentane to remove the fats, waxes and the 

nonpolar compounds and with ethyl acetate to extract the flavonoids and 

other polyphenols present in the extract. 

2.4  Isolation  

Dried crude extract was dissolved in dichloromethane (DCM), the filtrate 

was concentrated under reduced pressure at 40 oC, afforded 3.42 g, and the 

residue precipitate (7 g) which didn’t dissolve in DCM was called R and  

was stored. 

The first step of separation of any extract started with determination of the 

best eluent that could be used in silica gel column chromatography, and 

which was achieved by TLC experiments. 

The crude extract was dissolved in small amount of (DCM), and subjected 

to a first silica gel column chromatography using dichloromethane (DCM) 

and ethyl acetate (EtOAc) (9:1 v/v) as eluent. During the chromatographic 

run, the polarity of the eluent was increased in order to consent the elution 

of more polar compounds by passing to a solution composed of 

dichloromethane and ethyl acetate (8:2, 7:3 v/v). A total of 22 test tubes 

were collected (100 mL each). The content of each test tube was analyzed 

with TLC using DCM/EtOAc in  (7:3) ratio as eluent. Compounds were 

visualized under UV light and similar tubes were pooled together to yield 



29 

four major fractions. Fraction 1 (F1) included tubes (1 and 2), Fraction 2 

(F2) included tubes (3-12), Fraction 3  (F3) included tubes (13-16), and 

Fraction 4 (F4) included tubes (17-22). Then the solvent from each fraction 

was evaporated under reduced pressure at 40 oC and saved for further 

purification.               

HPLC and TLC analysis of the isolated four fractions indicated the 

presence of a lot of compounds in F1, which proved to be very hard to 

separate.  On the other hand, fractions 2 and 3 were subjected to further 

chromatographic separation techniques.  

Fraction 2 was subjected to silica gel column chromatography using ether 

and hexane (7:3 v/v) as eluent. Five fractions namely, F21, F22, F23 , F24 

and F25 were isolated, from which the solvents were removed by rotary 

evaporator at 40 0C. From the five fractions F23 , F24 and F25 have been 

subjected to further purification. 

F23 fraction was subjected to the silica gel column chromatography using 

ether and hexane (8:2 v/v) as eluent gave three fractions. Based on results 

obtained from HPLC analysis two of the fractions were almost pure, and 

these fractions were called A (100.67mg, 97.82% purity) and B (85mg, 

82.81% purity). 

Further purification of F24 on the silica gel column chromatography 

employing ether and hexane (8:2 v/v) as eluent afforded two substances 

that were hard to separate by available simple techniques. They could be 

separated by preparative HPLC.  



30 

Fraction F25 was subjected to silica gel column chromatography using 

diethyl ether as eluent and resulted in the isolation of two fractions, among 

of them one was pure fraction C (102.7 mg, 90.82% purity) but the other 

one D (73.04 mg, 47.26% purity) need more purification. The purity of the 

two compounds was confirmed by HPLC analysis. 

Fraction F3 was chromatographed on silica gel column eluted with 

dichloromethane and ethyl acetate (7:3 v/v)  to obtain F31, F32, and F33. 

HPLC analysis indicated that F31 and F32 were pure fractions, they called E 

(45 mg, 96.8% purity) and F (91.4 mg, 94.13% purity).  

2.5  Structure Elucidation and Purity Determination of the Compounds 

 

2.5.1  HPLC Analysis 

To detect the purity of the extracted fractions  an analysis was conducted 

using HPLC-DAD Water 1525, with C18 column (5µm, 4.6×250 mm 

cartridge). The mobile phase consists of solvent A (water) and solvent B 

(methanol), HPLC separation was achieved using binary-solvent gradient 

elution that started with 100% of solvent A and 0% of solvent B till 0% of 

A and 100 % of B, with 0.7 ml/min flow rate. The detection of all extracts 

was at 254 nm and the injection volume was 20 µL. 

2.5.2  IR Analysis 

 Functional groups of the extracted fractions were characterized using FT-

IR spectrometer (NICOLIT iS5 from Thermo Fisher Scientific). 

 



31 

2.6 Pharmacological Screening 

2.6.1  Antioxidant Activity 2, 2-diphenyl-1-picrylhydrazyl (DPPH assay) 

Scavenging activity of Stachys viticina extracts were assessed using the 

method described in the literature [113, 114]. The anti-oxidant activities of 

the plants fractions (A, B, C, D, F, R) and Trolox (reference compound) 

were assessed by their ability to donate hydrogen atom or electron, which 

was recognized from the bleaching of deep violet color of the methanolic 

DPPH solution, as indicated in scheme 1.  

Stock solution of plant fractions and Trolox were prepared in methanol, at a 

concentration of 1 mg/mL. Each of these stock solutions were diluted in 

methanol to prepare 12 working solutions with the following 

concentrations: 1, 2, 3, 5, 7, 10, 20, 30, 40, 50, 80, 100 μg/mL.  

A freshly prepared DPPH solution (0.002% w/v) was mixed with both 

methanol and with each of the above-mentioned working solutions at 1:1:1 

ratio. A negative control solution was prepared by mixing the DPPH 

solution with methanol in 1:1 ratio. Then, all of these solutions were 

incubated at room temperature in a dark cabinet for 30 min. By the end of 

the incubation period, the absorbance of these solutions was measured by 

UV-Vis spectrophotometer at a wave length of 517 nm.  Methanol was 

used as the blank solution. 

 The antioxidant activity of the corresponding plant fractions and Trolox 

standard were determined in terms of inhibition percentage of DPPH 

activity using the following equation: 

𝑰𝒏% =
𝑨 𝒃𝒍𝒂𝒏𝒌−𝑨 𝒔𝒂𝒎𝒑𝒍𝒆

𝑨 𝒃𝒍𝒂𝒏𝒌 
 × 𝟏𝟎𝟎                                   Eq.1 



32 

Equation (1): Inhibition% of antioxidant activity [115] 

Where A blank and Asample represent the absorbance of the blank and the 

sample respectively. 

 

Scheme (1): Principle of DPPH radical scavenging capacity assay [116]. 

The antioxidant half-maximal inhibitory concentration (IC50) for the 

studied plant fractions and Trolox standard solution as well as their 

standard deviations, was calculated from the graph plotted of the inhibition 

percentage against fractions concentration, using Microsoft Office Excel 

2010. 

2.6.2 Antibacterial and Antifungal Activity Tests 

2.6.2.1 Preparation of Plant Samples for Testing 

10 mg of each of plant extracts (A, B, C, D, E, F, R) were dissolved in 0.5 

mL sterile dimethyl sulfoxide (DMSO) then diluted with 0.5 mL of 

distilled water to obtain a concentration of 10 mg/ml. All extracts solutions 

were kept in UV disinfection chamber for 20 minutes. 

 



33 

2.6.2.2 Preparation of Growth Media  

Mueller-Hinton Broth (MHB) media was prepared according to 

manufacturer's instructions labeled on the bottle. 8.4g of MHB powder was 

suspended in 400 ml distilled water, and heated with stirring to boiling until 

the medium was completely dissolved. The solution was autoclaved for 15 

minutes at 121 oC, and allowed to cool down to room temperature before 

use. 

2.6.2.3 Test Microorganisms  

a) Bacterial strains:  Six bacterial strains were used in this study. These 

strains consist of five references bacterial strains obtained from the 

American Type Culture Collection (ATCC): four gram-negative 

bacteria included Escherichia coli (ATCC 25922), Pseudomonas 

aeruginosa (ATCC 9027), Proteus vulgaris (ATCC 8427), klebsiella 

pneumonia (ATCC 13883), and two gram-positive bacteria included 

staphylococcus aureus (ATCC 6538P). In addition to clinical isolate of 

methicillin resistant staphylococcus aureus (MRSA). All the bacterial 

strains were sub-cultured on Mueller-Hinton Agar.  

b) Fungal strain: one fungal strain Candida Albicans (ATTC 90028) 

obtained from the American Type Culture Collection (ATCC) was used 

in this study. This strain was sub-cultured on Sabouraud Dextrose Agar. 

2.6.2.4 Preparation of Bacterial and Fungal Suspension 

All bacterial and fungal strains were cultured 24 hours before use. Then a 

sterile specimen was taken gently from the colony surface of each type of 



34 

cultured strains and transferred to a separable sterile tube containing 5 mL 

of sterile normal saline. The optical density of all solution was measured by 

spectrophotometer at ƛ = 620 nm, where normal saline was used as blank. 

The turbidity of the bacterial suspensions was adjusted into 0.5 McFarland 

turbidity standard (optical density 0.08 to 0.1), to obtain a bacterial 

suspension with a 1.5 x 10^8 colony forming units (CFU/mL). Also, the 

turbidity of the yeast Candida Albicans was adjusted to equal 0.5 

McFarland solution (optical density of 0.12 to 0.15) with concentration of 

1*106 – 5*106 CFU/mL. Finally, the stock solutions of each bacterial and 

fungal strains were prepared by transferring 100 µL strains suspension into 

10 mL of Mueller Hinton Broth media, these stock solutions were further 

used in experiment of the microdilution method. 

2.6.2.5 Anti-microbial Assay 

 The antimicrobial activity of the plant samples was assayed employing 

broth micro-dilution method defined by procedure as described in the 

literature with some modifications ]117, 118]. 

a. Anti-bacterial Assay 

In the sterile 96 micro-wells plate 50 µL from the Mueller Hinton Broth 

media were filled in all microplate wells except the last raw (H) using 

multichannel pipette. Then 50 µL from the first prepared solution of plant 

extract (A) was added to the wells of first column with excluding H raw. 

Thereafter, 50 µL of the solution from the wells number 1 was transferred 

by multichannel pipette to wells number 2, which were mixed to obtain (2-



35 

fold) serial dilution and so on till wells number 10. Then 50 µL from each 

type of the bacterial strain’s suspension was filled in its specific row for the 

wells 1-11. The wells number 11 contain Mueller Hinton Broth media and 

bacteria suspension and didn’t contain plant extract, served as positive 

growth control. Wells number 12 contain only the growth media and served 

as negative control. While G raw wells contain only the extract solution to 

ensure that there is no contamination and the resulted turbidity in the G raw 

wells was not due to the extract itself.  Same steps were performed for all 

plants extracts  (B-F, and R). Finally, all the inoculated plates were 

incubated at 35˚C for 24 hours. The resulting turbidity in the wells 

indicated the bacterial growth.  

The lowest concentration of plant extracts, at which no visible bacterial 

growth in that microwell was observed, considered as the minimum 

inhibitory concentration (MIC) of the examined plant extracts. All the 

established experiments were performed in triplicate, to control the 

sensitivity of the tested microorganisms. 

 

 

 

 

 

 

 

 

 



36 
 

     Serial dilution      1:2   1:4 1:8   1:16 1:32 ……………………Discard                  

b. Anti-fungal Assay 

The same procedure used for bacterial strains was used for the yeast C. 

albicans with some modifications. The plant extracts were added to 

microplate wells in duplicate; raw A and B for the first extract (A), C and 

D for the second extract (B), E and G for the third extract (C), G and H for 

the fourth extract (D). While the C. albicans suspension was added to raw 

A, C, E, and G. By the same steps experiment was carried out for the last 

three extracts (E, F, R). Finally, all the inoculated plates were incubated at 

35˚C for 48 hours. 

The lowest concentration of plant extracts, at which no visible candidal 

growth in that microwell was estimated as the minimum inhibitory 

  1 2 3 4 5 6 7 8 9 10 11 12 

MRSA A             

Proteus vulgaris B             

Klebsiella pneumonia C             

Escherichia coli D             

Staphylococcus 
Aureus 

E             

Pseudomonas 
aeruginosa   

F             

+ve extract control G             

  H             

           

+ 
ve

 c
o

n
tr

o
l 

- 
ve

 c
o

n
tr

o
l 

           



37 

concentration (MIC) of the examined plant extracts. All the established 

experiments were performed in triplicate, to control the sensitivity of the 

tested C. albicans. 

In parallel, a control experiment was run to study the impact of the solvent             

alone (without plant extracts) on growth of the seven test organisms. 

Dimethyl sulfoxide was diluted in a similar pattern with sterile MHB media 

followed by inoculation and incubation. 

 

 Serial dilution      1:2   1:4 1:8   1:16 1:32 …………………………Discard                  

 

 

 

 

  1 2 3 4 5 6 7 8 9 10 11 12 

Exract (A)+ C. albicans A             

Extract(A) only B             

Exract (B)+ C. albicans C             

Extract(B) only D             

Exract (C)+ C. albicans E             

Extract(C) only F             

Exract (D)+ C. albicans G             

Extract(D) only  H             

           

+ 
ve

 c
o

n
tr

o
l 

- 
ve

 c
o

n
tr

o
l 

           



38 

Chapter Three 

Results 

The prime target of this study was to isolate pure chemical compounds 

from phenolic extract of Stachys viticina plant and screen the potential 

pharmacological activities of these compounds. 

3.1 Phytochemical of Polyphenolic Composition of S. Viticina  

The isolation procedure was carried out using silica gel chromatography. 

Seven extracts were obtained. Among of them one was crude extract and 

was called R, and one with 47.26% of purity and was called (D), but others 

were almost pure (purity % < 80%) and were called (A, B, C, E, F). Purity 

of the obtained fractions was checked by TLC plates and confirmed using 

HPLC analysis. Unfortunately, due to the lack NMR techniques at An 

Najah National University as well as to the COVID-19 we were not able to 

send the fractions abroad in order to run 1H- and C-NMR and 2D-NMR 

that will enable us to identify the purified fractions. The fractions are stored 

in the fridge and they will be sent abroad as soon as the restriction imposed 

due the spread of COVID-19 will be lifted.  

 Fraction A: fraction A was isolated as yellow solid, UV active, Rf value = 

0.57 in eluent DCM and EtOAc (7:3 v/v), HPLC retention time = 4.234 

minutes (fig.a.1, appendix), IR analysis showed that fraction A had a band 

in (2361 cm−1) which could be a cyanide group, carbonyl group (1702 

cm−1), and carbon –carbon double bond group (1620 cm−1) (fig.a.7, 

appendix).    



39 

Fraction B: fraction B was isolated as green solid, UV active, Rf value = 

0.65 in eluent DCM and EtOAc (7:3 v/v), HPLC retention time = 4.320 

minutes (fig.a.2, appendix), IR analysis showed that fraction B had a band 

in (3200-3500cm−1) zoon which could be a hydroxyl group (OH), aromatic 

ring (2973 cm−1), conjugated carbonyl (1651 cm−1) and carbon –carbon 

double bond group (1596 cm−1) (fig.a.8, appendix).  

Fraction C: fraction C was isolated as green solid, UV active, Rf value = 

0.52 in ether, HPLC retention time = 4.323 minutes (fig.a.3, appendix), IR 

analysis showed that fraction C had aromatic ring (2900 cm−1), and 

carbonyl group (1651 cm−1), in addition to carbon –carbon double bond 

group (1600 cm−1) (fig.a.9, appendix).    

Fraction D: fraction D was isolated as dark green solid, UV active, Rf value 

= 0.66 in ether and ethyl acetate (7:3 v/v), HPLC retention time = 4.259 

minutes (fig.a.4, appendix), IR analysis showed that fraction D revealed 

just carbon –carbon double bond group (1600 cm−1) (fig.a.10, appendix).  

Fraction E: fraction E was isolated as green solid, UV active, HPLC 

retention time = 4.251 minutes (fig.a.5, appendix), IR analysis showed that 

fraction E had an aromatic ring (2919 cm−1), carbonyl (1650 cm−1) and 

carbon –carbon double bond (1605 cm−1) groups (fig.a.11, appendix). 

Fraction F: fraction F was isolated as green solid, UV active, Rf value 

=0.60 in ether, HPLC retention time = 4.246 minutes (fig.a.6, appendix), IR 

analysis showed that fraction F had a broad band in (3200-3500 cm−1) it 

seemed to be a hydroxyl (OH) group, and aromatic ring (2930 cm−1), and 



40 

carbonyl group (1710 cm−1), in addition to carbon –carbon double bond 

group (1603 cm−1) (fig.a.12, appendix). 

3.2 Biological Activity  

3.2.1 Anti-oxidant Inhibitory Activity 

Antioxidant activities of the obtained extracts of S.viticina plant were 

investigated using the DPPH assay as in vitro approach and are expressed 

as DPPH % inhibition activity (Eq.1) and IC50 values (the amount of 

extract that giving 50% inhibition of DPPH radical). Table 3.1 and figure 

3.1 present the DPPH inhibitory activity and the IC50 values of the extract 

R, the fractions A-D, F and Trolox. The higher % inhibition and Lower 

IC50 values reflect better anti-oxidant action of the extracts. The most 

powerful extract is the residue R with IC50 = 18.58 µg/mL, and % Inhibition 

= 93.55 % at concentration of 80 µg/mL.   

Table 3.1: IC50 (µg/ml) values for different extracts. 

 Trolox A B C D F R 

IC50 (µg/mL) ± 

Standard 

deviation 

2.70 

± 0.096 

 

85.88 

 ± 1.61 

 

54.37 

 ± 0.14 

 

77.58 

± 0.304 

68.36 

±. 0.94 

58.62 

± 0.74 

18.58 

± 0.99 

 

 

 



41 

 

Figure (3.1): Anti-oxidant % Inhibition concentration (µg/ml) of different extracts. 

3.2.2 Anti-microbial Activity 

The microdilution assay was used to evaluate the antimicrobial activity of 

the R extract, and fractions A-F against six bacterial strains included 

Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, klebsiella 

pneumonia, staphylococcus aureus and MRSA, inaddition to the yeast C. 

albicans. The results indicated in Table 3.2 revealed that only fractions C, 

B, and the extract R strongly inhibited the growth of MRSA and S.aureus. 

While, fractions D, E, F had moderate inhibitory effect against MRSA and 

S.aureus. And fraction A had poor inhibitory effect against all the 

microorganisms tested. However, C. albicans was not inhibited by the 

tested fractions.  

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Trolox

A

B

C

D

F

R

concentration (µg/mL)

%
 in

h
ib

it
io

n
 



42 

Table (3.2): Minimum inhibitory concentration values (µg/ml) for 

different Stachys viticina extracts against selected pathogens 

 Bacteria Fungus 

ATCC 
Number 

Clinical 

strain 

ATCC 

25923 

ATCC 

25922 

ATCC 

13883 

ATCC 

8427 
ATCC 9027 

ATCC 

90028 

Microbe 

/extract 

MIC 

(µg/ml )  

MRSA  S.aureus E. coli 
Klebsiella 

pneumonia 

Proteus 

vulgaris 

Pseudomonas 

aeruginosa 

Candida 

albicans  

A 1250 1250 0 1250 1250 0 0 

B 39 78.1 1250 1250 1250 1250 0 

C 39 39 0 0 0 1250 0 

D 156.3 156.3 0 1250 1250 0 0  

E 312.5 156.3 1250 1250 1250 0 0 

F 312.5 156.3 0 0 0 0 0 

R 78.1 78.1 0 1250 625 625 0 

 

 

Figure (3.2): Minimum inhibitory concentration (µg/ml) of different plant extracts 

against different pathogens. 

0

200

400

600

800

1000

1200

1400

A B C D E F R

Extract

MRSA

S.aureus

E. coli

Klebsiella
pneumonia

Proteus
vulgaris

Pseudomonas
aeruginosa

Candida
albicans

M
IC

  (
µ

g/
m

l )
 

tested microorganisim



43 

Chapter Four 

Discussion and Conclusions 

Scientific studies of plants secondary metabolites represent a global 

innovative strategy for the production of novel plant-derived 

pharmaceuticals drugs. Since plants contain an outstanding range of 

biological active compounds with intensive healing properties [119]. In this 

direction, characterization the profile of polyphenol-containing botanical 

extracts and their health-related properties have been a topic of the recent 

research. Since polyphenols appears as a prominent class of natural   

phytochemical compounds with enormous biological effects [120,121]. 

Thus, this investigation was designed in order to isolation and identification 

of the polar poly phenolic compositions of S. viticina plant, in addition to 

the antioxidant, and antimicrobial activity.  

4.1 The Chemical Composition 

In this study, seven extracts of S. viticina were obtained, one of them was 

crude R, another (D) was semipure fraction (47.26% purity), but the others 

(A, B, C, E, F) were almost pure fractions. Recently, Venditti et al. reported 

the identification of eight compounds from ethanolic extracts of S. palustris 

plant, of which verbascoside, echinacoside, and two of isoscutellarein 

derivatives were the major components  [122]. 

4.2 Antioxidant Activity 

The antioxidant capacity of the obtained extracts from S. viticina were 

measured using DPPH assay because it’s simple and highly sensitive 

method ]123[. The results of the current work showed that the phenolic 



44 

extracts of S.  viticina exhibited different antioxidant activity. R, B, F 

extracts had the highest antioxidant capacity with an IC50 value of 18.58 

µg/mL, 54.37 µg/mL, 58.62 µg/mL, respectively, less than that of the 

positive control (Trolox) which has an IC50 value of 2.70 µg/mL. A 

previous study performed by N.Jaradat  and N.Al-Maharik  revealed that 

the essential oil of S. viticina exhibited antioxidant activity with an IC50 

value of 19.95 µg/mL [33[, higher than that of the present study extracts 

except R. Furthermore, a study conducted by Kuki´c et al. reported that S. 

anisochila, S. beckeana, S. plumose, and S. 44lpine ssp. Dinarica have 

antioxidant activity with IC50 values of 17.9, 20.9, 101.61, and 26.14 

µg/mL, respectively ]124[. 

4.3 Antimicrobial Capacity 

The microdilution assay was used to screen the antimicrobial activity of 

phenolic S. viticina extracts against four gram-negative bacteria included 

Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, klebsiella 

pneumonia, and two gram-positive bacteria included Staphylococcus 

aureus, and MRSA, in addition to the yeast C. albicans. Some of S. viticina 

extracts inhibited the growth of the screened bacterial strains. In particular, 

the extracts B and C exhibited strong antibacterial activity against MRSA 

with MIC value of 39 µg/mL. Which is equal to inhibition activity of 

extract B against s.aureus and two folds more potential than the inhibition 

activity of extract R against s.aureus (MIC = 78 µg /mL). That is mean the 

obtained extracts revealed stronger activity against gram-positive strains 

compared to the gram-negative ones. Typically, gram-negative bacteria are 



45 

less sensitive because their cell walls have an outer membrane which 

composed of proteins and lipids that prevents easy penetration of 

compounds ]125[. However, all extract didn’t have inhibition activity 

against the fungal strain C. albicans. Recent study conducted by N.Jaradat  

and N.Al-Maharik  showed nearly matching result in the screening of the 

antibacterial activity of the S. viticina essential oil. And the essential oil of 

S. viticina inhibited the growth of the C. albicans with MIC value of 312.5 

µg /mL [33[. While C. albicans strain was resistance against all the extracts 

in the present study. 

4.4 Conclusions 

The HPLC analysis confirmed the purity of five fractions in the phenolic S. 

viticina extract (A, B, C, E, F), and one fraction (D) with only 47.26 % 

purity, in addition to one crude extract (R). All extracts showed DPPH 

scavenging activity in the following order: R< B< F< D< C> A. Some 

extracts also showed very strong antibacterial effects, of which the 

strongest were extracts B and C against MRSA, with MIC value of 39 

µg/mL.  But all extracts had no antifungal activity against the yeast C. 

albicans. Briefly, polyphenol-containing polar extracts from Stachys 

viticina contain pharmacologically active fractions which could be further 

confirmed and examined clinically for their therapeutic potential and for 

the design of new natural medicinal preparations. 

 

 



46 

References 

1- Gurib-Fakim, A. (2006). Medicinal plants: traditions of yesterday 

and drugs of tomorrow. Molecular aspects of Medicine, 27(1), 1-

93. 

2- Petrovska, B. B. (2012). Historical review of medicinal plants’ 

usage. Pharmacognosy reviews, 6(11), 1. 

3- Ntie-Kang, F., Njume, L. E., Malange, Y. I., Günther, S., Sippl, W., 

& Yong, J. N. (2016). The chemistry and biological activities of 

natural products from northern african plant families: From 

taccaceae to Zygophyllaceae. Natural products and 

bioprospecting, 6(2), 63-96. 

4- Sharma, A., Shanker, C., Tyagi, L. K., Singh, M., & Rao, C. V. 

(2008). Herbal medicine for market potential in India: an 

overview. Acad J Plant Sci, 1(2), 26-36. 

5- Farnsworth, N. R., Akerele, O., Bingel, A. S., Soejarto, D. D., & 

Guo, Z. (1985). Medicinal plants in therapy. Bulletin of the world 

health organization, 63(6), 965. 

6- Rashid, N., Paul, A. A., Islam, S., Sajib, S. A., Nasirujjaman, K., 

Hoque, K. M. F., & Reza, M. A. (2017). Studies on antioxidant 

potential, phytochemical properties and toxicity of four popular 

medicinal plants of Bangladesh. Journal of Bio-Science, 25, 27-37. 



47 

7- Rishton, G. M. (2008). Natural products as a robust source of new 

drugs and drug leads: Past successes and present-day issues. The 

American journal of cardiology, 101(10), S43-S49. 

8- Rates, S. M. K. (2001). Plants as source of drugs. Toxicon, 39(5), 

603-613. 

9- Singh, I., Kumaravadivel, N., Gnanam, R., & Vellaikumar, S. 

(2010). RP-HPLC analysis for camptothecin content in 

Nothapodytes nimmoniana, an endangered medicinal 

plant. Journal of Medicinal Plants Research, 4(3), 255-259.  

10- Balunas, M. J., & Kinghorn, A. D. (2005). Drug discovery from 

medicinal plants. Life sciences, 78(5), 431-441. 

11- Borris, R. P. (1996). Natural products research: perspectives from a 

major pharmaceutical company. Journal of ethnopharmacology, 

51(1-3), 29-38. 

12- Mendelssohn, H., & Yom-Tov, Y. (1999). Mammalia of Israel. 

Israel Academy of Sciences and Humanities. 

13- Crowfoot, G., and Baldensperger, L. (1932). From cedar to hyssob: 

A study in the folklore of plants in Palestine. London: Sheldon 

Press. 

14- Silva, F., & Abraham, A. (1981). Potentiality of the Israeli flora for 

medicinal purposes. Fitoterapia. 



48 

15- Jaradat, N. A., Ayesh, O. I., & Anderson, C. (2016). 

Ethnopharmacological survey about medicinal plants utilized by 

herbalists and traditional practitioner healers for treatments of diarrhea 

in the West Bank/Palestine. Journal of ethnopharmacology, 182, 57-66. 

16- Tamokou, J. D. D., Mbaveng, A. T., & Kuete, V. (2017). 

Antimicrobial activities of African medicinal spices and vegetables. 

In Medicinal spices and vegetables from Africa (pp. 207-237). 

Academic Press. 

17- Carović-StanKo, K., PeteK, M., Grdiša, M., Pintar, J., Bedeković, 

D., & Satovic, Z. (2016). Medicinal plants of the family Lamiaceae 

as functional foods–a review. Czech journal of food 

sciences, 34(5), 377-390. 

18- Özkan, M. (2008). Glandular and eglandular hairs of Salvia 

recognita Fisch. & Mey.(Lamiaceae) in Turkey. Bangladesh 

Journal of Botany, 37(1), 93-95. 

19- Carović-StanKo, K., PeteK, M., Martina, G., Pintar, J., Bedeković, 

D., Ćustić, M. H., & Šatović, Z. (2016). Medicinal plants of the 

family Lamiaceaeas functional foods–a review. Czech journal of 

food sciences, 34(5), 377. 

20- Venditti, A., Frezza, C., Lorenzetti, L. M., Maggi, F., Serafini, M., & 

Bianco, A. (2017). Reassessment of the polar fraction of Stachys 

alopecuros (L.) Benth. subsp. divulsa (Ten.) Grande (Lamiaceae) from 



49 

the Monti Sibillini National Park: A potential source of bioactive 

compounds. Journal of intercultural ethnopharmacology, 6(2), 144. 

21- Goren, A. C. (2014). Use of Stachys species (Mountain Tea) as 

herbal tea and food. Records of Natural Products, 8(2), 71. 

22- Conforti, F., Menichini, F., Formisano, C., Rigano, D., Senatore, F., 

Arnold, N. A., & Piozzi, F. (2009). Comparative chemical 

composition, free radical-scavenging and cytotoxic properties of 

essential oils of six Stachys species from different regions of the 

Mediterranean Area. Food Chemistry, 116(4), 898-905. 

23- Kumar, D., Bhat, Z. A., Kumar, V., Raja, W. Y., & Shah, M. Y. 

(2013). Anti-anxiety activity of Stachys tibetica Vatke. Chinese 

journal of natural medicines, 11(3), 240-244. 

24- Sajjadi, S. E., Ghanadian, S. M., Rabbani, M., & Tahmasbi, F. 

(2017). Isolation and Identification of Secondary Metabolites from 

the Aerial Parts of Stachys lavandulifolia Vahl. Iranian journal of 

pharmaceutical research: IJPR, 16(Suppl), 58. 

25- Grujic‐Jovanovic, S., Skaltsa, H. D., Marin, P., & Sokovic, M. 

(2004). Composition and antibacterial activity of the essential oil of 

six Stachys species from Serbia. Flavour and Fragrance 

Journal, 19(2), 139-144. 

26- Ferhat, M., Erol, E., Beladjila, K. A., Çetintaş, Y., Duru, M. E., 

Öztürk, M., ... & Kabouche, Z. (2017). Antioxidant, 



50 

anticholinesterase and antibacterial activities of Stachys guyoniana 

and Mentha aquatica. Pharmaceutical biology, 55(1), 324-329. 

27- Khanavi, M., Sharifzadeh, M., Hadjiakhoondi, A., & Shafiee, A. 

(2005). Phytochemical investigation and anti-inflammatory activity 

of aerial parts of Stachys byzanthina C. Koch. Journal of 

ethnopharmacology, 97(3), 463-468. 

28- Amirghofran, Z., Bahmani, M., Azadmehr, A., & Javidnia, K. 

(2006). Anticancer effects of various Iranian native medicinal 

plants on human tumor cell lines. Neoplasma, 53(5), 428. 

29- Grigorakis, S., & Makris, D. P. (2018). Characterisation of 

Polyphenol-Containing Extracts from Stachys mucronata and 

Evaluation of Their Antiradical Activity. Medicines, 5(1), 14. 

30- Tundis, R., Bonesi, M., Pugliese, A., Nadjafi, F., Menichini, F., & 

Loizzo, M. R. (2015). Tyrosinase, acetyl-and butyryl-cholinesterase 

inhibitory activity of Stachys lavandulifolia Vahl (Lamiaceae) and 

its major constituents. Records of Natural Products, 9(1), 81. 

31- Duru, M. E., Çakır, A., Harmandar, M., Izumi, S., & Hirata, T. 

(1999). The volatile constituents of Stachys athorecalyx C. Koch. 

from Turkey. Flavour and fragrance journal, 14(1), 12-14. 

32- Gören, A. C., Akçicek, E., Dirmenci, T., Kilic, T., Mozioğlu, E., & 

Yilmaz, H. (2012). Fatty acid composition and chemotaxonomic 

evaluation of species of Stachys. Natural product research, 26(1), 

84-90. 



51 

33- Jaradat, N., & Al-Maharik, N. (2019). Fingerprinting, 

Antimicrobial, Antioxidant, Anticancer, Cyclooxygenase and 

Metabolic Enzymes Inhibitory Characteristic Evaluations of 

Stachys viticina Boiss. Essential Oil. Molecules, 24(21), 3880 

34- Shareef, H. K., Muhammed, H. J., Hussein, H. M., & Hameed, I. H. 

(2016). Antibacterial effect of ginger (Zingiber officinale) roscoe 

and bioactive chemical analysis using gas chromatography mass 

spectrum. Oriental Journal of Chemistry, 32(2), 20-40. 

35- Naudé, Y., Makuwa, R., & Maharaj, V. (2016). Investigating 

volatile compounds in the vapour phase of (1) a hot water infusion 

of rhizomes, and of (2) rhizomes of Siphonochilus aethiopicus 

using head space solid phase microextraction and gas 

chromatography with time-of-flight mass spectrometry. South 

African Journal of Botany, 106, 144-148. 

36- Kilonzo, M., Ndakidemi, P. A., & Chacha, M. (2017). Mystroxylon 

aethiopicum chloroform root bark extracts phytochemical analysis 

using gas chromatography mass spectrometry. Journal of 

Pharmacognosy and Phytotherapy, 9(4), 44-50. 

37- Croteau, R., Kutchan, T. M., & Lewis, N. G. (2000). Natural 

products (secondary metabolites). Biochemistry and molecular 

biology of plants, 24, 1250-1319. 



52 

38- Abdel-Aziz, S. M., Elsoud, M. M. A., & Anise, A. A. (2017). 

Microbial Biosynthesis: A Repertory of Vital Natural Products. 

In Food Biosynthesis (pp. 25-54). Academic Press.   

39- Pichersky, E., & Gang, D. R. (2000). Genetics and biochemistry of 

secondary metabolites in plants: an evolutionary 

perspective. Trends in plant science, 5(10), 439-445. 

40-  Yadav, A. N., Kour, D., Rana, K. L., Yadav, N., Singh, B., 

Chauhan, V. S., ... & Gupta, V. K. (2019). Metabolic engineering to 

synthetic biology of secondary metabolites production. In New and 

Future Developments in Microbial Biotechnology and 

Bioengineering (pp. 279-320). Elsevier. 

41- Mera, I. F. G., Falconí, D. E. G., & Córdova, V. M. (2019). 

Secondary metabolites in plants: main classes, phytochemical 

analysis and pharmacological activities. Rev Bionatura, 4, 1000-9. 

42- Lone, R., Shuab, R., & Kamili, A. N. Plant Phenolics in 

Sustainable Agriculture. 

43- King, A. M. Y., & Young, G. (1999). Characteristics and 

occurrence of phenolic phytochemicals. Journal of the American 

Dietetic Association, 99(2), 213-218. 

44- Kabera, J. N., Semana, E., Mussa, A. R., & He, X. (2014). Plant 

secondary metabolites: biosynthesis, classification, function and 

pharmacological properties. J Pharm Pharmacol, 2, 377-392. 



53 

45- Zuiter, A. S. (2014). Proanthocyanidin: Chemistry and biology: 

From phenolic compounds to proanthocyanidins. 

46- Karabin, M., Hudcova, T., Jelinek, L., & Dostalek, P. (2015). 

Biotransformations and biological activities of hop 

flavonoids. Biotechnology advances, 33(6), 1063-1090. 

47- Sharma, V., & Janmeda, P. (2017). Extraction, isolation and 

identification of flavonoid from Euphorbia neriifolia 

leaves. Arabian Journal of Chemistry, 10(4), 509-514. 

48- Arora, S., & Itankar, P. (2018). Extraction, isolation and 

identification of flavonoid from Chenopodium album aerial 

parts. Journal of traditional and complementary medicine, 8(4), 

476-482. 

49- Jain, C., Khatana, S., & Vijayvergia, R. (2019). Bioactivity of 

secondary metabolites of various plants: a review. Int. J. Pharm. 

Sci, 10, 494-404. 

50- Nijveldt, R. J., Van Nood, E. L. S., Van Hoorn, D. E., Boelens, P. 

G., Van Norren, K., & Van Leeuwen, P. A. (2001). Flavonoids: a 

review of probable mechanisms of action and potential 

applications. The American journal of clinical nutrition, 74(4), 

418-425. 

51- Havsteen, B. H. (2002). The biochemistry and medical significance 

of the flavonoids. Pharmacology & therapeutics, 96(2-3), 67-202. 



54 

52- Peterson, J., & Dwyer, J. (1998). Flavonoids: dietary occurrence 

and biochemical activity. Nutrition research, 18(12), 1995-2018. 

53- Kale, A., Gawande, S., & Kotwal, S. (2008). Cancer 

phytotherapeutics: role for flavonoids at the cellular 

level. Phytotherapy Research: An International Journal Devoted to 

Pharmacological and Toxicological Evaluation of Natural Product 

Derivatives, 22(5), 567-577. 

54- Khan, M. K., & Dangles, O. (2014). A comprehensive review on 

flavanones, the major citrus polyphenols. Journal of Food 

Composition and Analysis, 33(1), 85-104. 

55- Rybarczyk-Plonska, A., Wold, A. B., Bengtsson, G. B., Borge, G. I. 

A., Hansen, M. K., & Hagen, S. F. (2016). Flavonols in broccoli 

(Brassica oleracea L. var. italica) flower buds as affected by 

postharvest temperature and radiation treatments. Postharvest 

Biology and Technology, 116, 105-114. 

56- Cui, B., Hu, Z., Zhang, Y., Hu, J., Yin, W., Feng, Y., ... & Chen, G. 

(2016). Anthocyanins and flavonols are responsible for purple 

color of Lablab purpureus (L.) sweet pods. Plant Physiology and 

Biochemistry, 103, 183-190. 

57- Brodowska, K. M. (2017). Natural flavonoids: classification, 

potential role, and application of flavonoid analogues. European 

Journal of Biological Research, 7(2), 108-123. 



55 

58- Martens, S., & Mithöfer, A. (2005). Flavones and flavone 

synthases. Phytochemistry, 66(20), 2399-2407. 

59- Xu, Y., Simon, J. E., Ferruzzi, M. G., Ho, L., Pasinetti, G. M., & 

Wu, Q. (2012). Quantification of anthocyanidins in the grapes and 

grape juice products with acid assisted hydrolysis using 

LC/MS. Journal of Functional Foods, 4(4), 710-717. 

60- Bagchi, D., Sen, C. K., Bagchi, M., & Atalay, M. (2004). Anti-

angiogenic, antioxidant, and anti-carcinogenic properties of a novel 

anthocyanin-rich berry extract formula. Biochemistry 

(Moscow), 69(1), 75-80. 

61- Vuong, Q. V., Golding, J. B., Nguyen, M., & Roach, P. D. (2010). 

Extraction and isolation of catechins from tea. Journal of 

separation science, 33(21), 3415-3428. 

62- Wein, S., Beyer, B., Gohlke, A., Blank, R., Metges, C. C., & 

Wolffram, S. (2016). Systemic absorption of catechins after 

intraruminal or intraduodenal application of a green tea extract in 

cows. PLoS One, 11(7), e0159428. 

63- Křížová, L., Dadáková, K., Kašparovská, J., & Kašparovský, T. 

(2019). Isoflavones. Molecules, 24(6), 1076. 

64- Chávez-González, M. L., Sepúlveda, L., Verma, D. K., Luna-García, 

H. A., Rodríguez-Durán, L. V., Ilina, A., & Aguilar, C. N. (2020). 

Conventional and Emerging Extraction Processes of 

Flavonoids. Processes, 8(4), 434. 



56 

65- Kumar, N., & Goel, N. (2019). Phenolic acids: Natural versatile 

molecules with promising therapeutic applications. Biotechnology 

Reports, 24, e00370. 

66- Khadem, S., & Marles, R. J. (2010). Monocyclic phenolic acids; 

hydroxy-and polyhydroxybenzoic acids: occurrence and recent 

bioactivity studies. Molecules, 15(11), 7985-8005. 

67- Kiokias, S., Proestos, C., & Oreopoulou, V. (2020). Phenolic Acids 

of Plant Origin—A Review on Their Antioxidant Activity In Vitro 

(O/W Emulsion Systems) Along with Their in Vivo Health 

Biochemical Properties. Foods, 9(4), 534. 

68- Chang, Z., Zhang, Q., Liang, W., Zhou, K., Jian, P., She, G., & 

Zhang, L. (2019). A Comprehensive Review of the Structure 

Elucidation of Tannins from Terminalia Linn. Evidence-Based 

Complementary and Alternative Medicine, 2019. 

69- Hassanpour, S., MaheriSis, N., & Eshratkhah, B. (2011). Plants and 

secondary metabolites (Tannins): A Review. 

70- Zwenger, S., & Basu, C. (2008). Plant terpenoids: applications and 

future potentials. Biotechnology and Molecular Biology 

Reviews, 3(1), 1 

71- Las Heras, B., Rodriguez, B., Bosca, L., & Villar, A. M. (2003). 

Terpenoids: sources, structure elucidation and therapeutic 

potential in inflammation. Current topics in medicinal 

chemistry, 3(2), 171-185. 



57 

72- Abdallah, I. I., & Quax, W. J. (2017). A Glimpse into the 

Biosynthesis of Terpenoids. KnE Life Sciences, 81-98. 

73- Matsuura, H. N., & Fett-Neto, A. G. (2015). Plant alkaloids: main 

features, toxicity, and mechanisms of action. Plant toxins, 1-15. 

74- Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K. M., & Latha, 

L. Y. (2011). Extraction, isolation and characterization of bioactive 

compounds from plants’ extracts. African Journal of Traditional, 

Complementary and Alternative Medicines, 8(1). 

75- Oreopoulou, A., Tsimogiannis, D., & Oreopoulou, V. (2019). 

Extraction of polyphenols from aromatic and medicinal plants: an 

overview of the methods and the effect of extraction parameters. 

In Polyphenols in Plants (pp. 243-259). Academic Press. 

76- De Castro, M. L., & Garcıa-Ayuso, L. E. (1998). Soxhlet extraction 

of solid materials: an outdated technique with a promising 

innovative future. Analytica chimica acta, 369(1-2), 1-10. 

77- United Nations Industrial Development Organization, Handa, S. S., 

Khanuja, S. P. S., Longo, G., & Rakesh, D. D. (2008). Extraction 

technologies for medicinal and aromatic plants. Earth, 

Environmental and Marine Sciences and Technologies 

78- Pandey, A., & Tripathi, S. (2014). Concept of standardization, 

extraction and pre phytochemical screening strategies for herbal 

drug. Journal of Pharmacognosy and Phytochemistry, 2(5). 



58 

79- Wang, L., & Weller, C. L. (2006). Recent advances in extraction of 

nutraceuticals from plants. Trends in Food Science & 

Technology, 17(6), 300-312. 

80- Kadam, S. U., Tiwari, B. K., & O’Donnell, C. P. (2013). Application 

of novel extraction technologies for bioactives from marine 

algae. Journal of agricultural and food chemistry, 61(20), 4667-

4675. 

81- Novak, I., Janeiro, P., Seruga, M., & Oliveira-Brett, A. M. (2008). 

Ultrasound extracted flavonoids from four varieties of Portuguese 

red grape skins determined by reverse-phase high-performance 

liquid chromatography with electrochemical detection. Analytica 

chimica acta, 630(2), 107-115. 

82- Vinatoru, M., Mason, T. J., & Calinescu, I. (2017). Ultrasonically 

assisted extraction (UAE) and microwave assisted extraction 

(MAE) of functional compounds from plant materials. TrAC 

Trends in Analytical Chemistry, 97, 159-178. 

83- Kaur, K. (2016). Functional nutraceuticals: Past, present, and 

future. Nutraceuticals; Elsevier: London, UK, 41-78. 

84- Anderson, J., Berthod, A., Pino, V., & Stalcup, A. M. 

(2016). Analytical separation science, 5 volume set. John Wiley & 

Sons. 

85- Schoen, H. (2015). Handbook of Purified Gases (Vol. 416). Berlin, 

Heidelberg: Springer. 



59 

86- Kondeti, R. R., Mulpuri, K. S., & Meruga, B. (2014). 

Advancements in column chromatography: a review. World J. 

Pharm. Sci., 2, 1375-1383. 

87- Cai, L. (2014). Thin layer chromatography. Current Protocols 

Essential Laboratory Techniques, 8(1), 6-3. 

88- Braithwaite, A., & Smith, J. F. (2012). Chromatographic methods. 

Springer Science & Business Media. 

89- Bhusal, R. D., Nahar, D. M., & Dalvi, P. B. (2017). Review on: flash 

column chromatography. Indo Am. J. Pharm. Res, 7(01). 

90- Heftmann, E. (Ed.). (2004). Chromatography: Fundamentals and 

applications of chromatography and related differential migration 

methods-Part B: Applications. Elsevier. 

91- Waksmundzka-Hajnos, M., & Sherma, J. (Eds.). (2010). High 

performance liquid chromatography in phytochemical analysis. 

CRC press. 

92- Thammana, M. (2016). A review on high performance liquid 

chromatography (HPLC). Res Rev J Pharm Anal RRJPA, 5(2), 

22-28. 

93- Lee, P. (2008). Development of a semi-preparative C₃₀ HPLC 

column for carotenoids separation. 

94- Shanmugam, S. (2009). Enzyme technology. IK International Pvt 

Ltd. 



60 

95- Thirumal, Y., & Laavu, S. (2017). HPLC profile of medicinal plant 

extracts and its application in aquaculture. J Aquacult Res 

Dev, 8(484), 2-6. 

96- Cordell, G. A. (1995). Changing strategies in natural products 

chemistry. Phytochemistry, 40(6), 1585-1612. 

97- Rodríguez, J., Crews, P., & Jaspars, M. (2012). Contemporary 

strategies in natural products structure elucidation. In Handbook 

of marine natural products (pp. 423-517). Springer Netherlands. 

98- Gautam, C., Yadav, A. K., & Singh, A. K. (2012). A review on 

infrared spectroscopy of borate glasses with effects of different 

additives. ISRN ceramics, 2012. 

99- Gunther, H., & Gunther, H. (1994). NMR spectroscopy: basic 

principles, concepts, and applications in chemistry. Chichester, 

UK: John Wiley & Sons. 

100- Gerothanassis, I. P., Troganis, A., Exarchou, V., & Barbarossou, K. 

(2002). Nuclear magnetic resonance (NMR) spectroscopy: basic 

principles and phenomena, and their applications to chemistry, 

biology and medicine. Chemistry Education Research and 

Practice, 3(2), 229-252. 

101- Field, L. D., Li, H. L., & Magill, A. M. (2020). Organic structures 

from spectra. John Wiley & Sons. 

102- Jackson, C. M., Esnouf, M. P., Winzor, D. J., & Duewer, D. L. 

(2007). Defining and measuring biological activity: applying the 



61 

principles of metrology. Accreditation and quality 

assurance, 12(6), 283-294. 

103- Pelikan, E. W. (1995). Glossary of Terms and Symbols Used in 

Pharmacology. Boston University School of Medicine, 

Pharmacology & Experimental Therapeutics. 

104- Phaniendra, A., Jestadi, D. B., & Periyasamy, L. (2015). Free 

radicals: properties, sources, targets, and their implication in 

various diseases. Indian journal of clinical biochemistry, 30(1), 

11-26. 

105- Ozcan, A., & Ogun, M. (2015). Biochemistry of reactive oxygen 

and nitrogen species. Basic principles and clinical significance of 

oxidative stress, 3, 37-58. 

106- Lobo, V., Patil, A., Phatak, A., & Chandra, N. (2010). Free radicals, 

antioxidants and functional foods: Impact on human 

health. Pharmacognosy reviews, 4(8), 118. 

107- National Institutes of Health. (2018). Biological Sciences 

Curriculum Study. NIH Curriculum Supplement Series [Internet]. 

Bethesda (MD): National Institutes of Health (US); 2007. 

Understanding Emerging and Re-emerging Infectious 

Diseases. Information about Mental Illness and the Brain. 

108- Ullah, H., & Ali, S. (2017). Classification of anti-bacterial agents 

and their functions. Antibacterial Agents, 1-16. 



62 

109- Dixon, D. M., & Walsh, T. J. (1996). Antifungal agents. In Medical 

Microbiology. 4th edition. University of Texas Medical Branch at 

Galveston. 

110- Barbour, E. K., Al Sharif, M., Sagherian, V. K., Habre, A. N., 

Talhouk, R. S., & Talhouk, S. N. (2004). Screening of selected 

indigenous plants of Lebanon for antimicrobial activity. Journal of 

ethnopharmacology, 93(1), 1-7. 

111- Van Der Watt, E., & Pretorius, J. C. (2001). Purification and 

identification of active antibacterial components in Carpobrotus 

edulis L. Journal of ethnopharmacology, 76(1), 87-91. 

112-  Iwu, M. W., Duncan, A. R., & Okunji, C. O. (1999). New 

antimicrobials of plant origin In: Perspectives on new Crops and 

new Uses, eds. J. Janick. 

113- Sonboli, A. (2015). Biological activity of various extracts and 

phenolic content of Micromeria persica and M. hedgei. Research 

Journal of Pharmacognosy, 2(4), 27-31. 

114- Jaradat, N., Adwan, L., K’aibni, S., Zaid, A. N., Shtaya, M. J., 

Shraim, N., & Assali, M. (2017). Variability of chemical 

compositions and antimicrobial and antioxidant activities of Ruta 

chalepensis leaf essential oils from three Palestinian 

regions. BioMed research international, 2017. 

115- Güllüce, M., Sökmen, M., Şahin, F., Sökmen, A., Adigüzel, A., & 

Özer, H. (2004). Biological activities of the essential oil and 



63 

methanolic extract of Micromeria fruticosa (L) Druce ssp 

serpyllifolia (Bieb) PH Davis plants from the eastern Anatolia 

region of Turkey. Journal of the Science of Food and 

Agriculture, 84(7), 735-741. 

116- Teixeira, J., Gaspar, A., Garrido, E. M., Garrido, J., & Borges, F. 

(2013). Hydroxycinnamic acid antioxidants: an electrochemical 

overview. BioMed research international, 2013. 

117- Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in 

vitro evaluating antimicrobial activity: A review. Journal of 

pharmaceutical analysis, 6(2), 71-79. 

118- Jani, N. A., Ibrahim, N., Hashim, S. E., & Sirat, H. M. (2015). 

Antimicrobial and Antioxidant Activities of Hornstedtia leonurus 

Retz. Extracts. Journal of Science and Technology, 7(2). 

119- Wang, Y. (2008). Needs for new plant-derived pharmaceuticals in 

the post-genome era: an industrial view in drug research and 

development. Phytochemistry Reviews, 7(3), 395-406. 

120- Li, A. N., Li, S., Zhang, Y. J., Xu, X. R., Chen, Y. M., & Li, H. B. 

(2014). Resources and biological activities of natural 

polyphenols. Nutrients, 6(12), 6020-6047. 

121- Dai, J., & Mumper, R. J. (2010). Plant phenolics: extraction, 

analysis and their antioxidant and anticancer 

properties. Molecules, 15(10), 7313-7352. 



64 

122- Venditti, A., Frezza, C., Bianco, A., Serafini, M., Cianfaglione, K., 

Nagy, D. U., ... & Maggi, F. (2017). Polar constituents, essential oil 

and antioxidant activity of marsh woundwort (Stachys palustris 

L.). Chemistry & biodiversity, 14(3), e1600401. 

123- Zheng, C. D., Li, G., Li, H. Q., Xu, X. J., Gao, J. M., & Zhang, A. L. 

(2010). DPPH-scavenging activities and structure-activity 

relationships of phenolic compounds. Natural product 

communications, 5(11), 1934578X1000501112. 

124- Kukić, J., Petrović, S., & Niketić, M. (2006). Antioxidant activity of 

four endemic Stachys taxa. Biological and Pharmaceutical 

Bulletin, 29(4), 725-729. 

125- Delcour A. H. (2009). Outer membrane permeability and antibiotic 

resistance. Biochimica et biophysica acta, 1794(5), 808–816. 

https://doi.org/10.1016/j.bbapap.2008.11.005 

 

 

 

 

 

 

 

 



65 

 

 

 

 

 

 

 

Appendix 

 

 

 

 

 

 

 

 

 

 

 



66 

Appendix 1 

 

Fig.a.1: HPLC chromatogram for fraction A. 

 

 

           Fig.a.2: HPLC chromatogram for fraction B. 

 



67 

 

               Fig.a.3: HPLC chromatogram for fraction C. 

 

Fig.a.4: HPLC chromatogram for fraction D. 

 



68 

 

Fig.a.5: HPLC chromatogram for fraction E. 

 

Fig.a.6: HPLC chromatogram for fraction F. 



69 

 

Fig.a.7: IR spectra for fraction A. 

 

Fig.a.8: IR spectra for fraction  B. 



70 

 

Fig.a.9: IR spectra fraction C. 

 

Fig.a.11: IR spectra for fraction D. 



71 

 

Fig.a.10: IR spectra for fraction E. 

 

Fig.a.12: IR spectra for fraction F. 



 جاح الوطنية جامعة الن  

راسات العليا ة كلي    الد 

 

 

 
 ونبتة  الفلسطينيالبطنج  الكشف عن محتويات نبتة  

من المركبات متعددة الفينوالت في   الكرميالبطنج 
 مستخلصهما القطبي وتقيم تأثيرها الدوائي 

 
 
 

 إعداد  
 ليلى محمد عباس بدوان

 
 إشراف  

 نواف المحاريق د. 
 نضال جرادات د. 

 
هذه   درجة    األطروحةقدمت  على  الحصول  لمتطلبات  الكيمياءاستكمااًل  في  كلية ب  ،الماجستير 

 . فلسطين –النجاح الوطنية، نابلس   في جامعة ،الدراسات العليا
2021 



 ب 

 الكرميالبطنج  ونبتة الفلسطيني البطنج الكشف عن محتويات نبتة 
 الدوائي من المركبات متعددة الفينوالت في مستخلصهما القطبي وتقيم تأثيرها 

 إعداد 
 ليلى محمد عباس بدوان

 إشراف 
 نواف المحاريقد. 

 نضال جراداتد. 

 الملخص 

ينها فلسطين, نبتة عطرية تنتشر في دول البحر األبيض المتوسط ومن ب  Stachys viticinaنبتة  
المكونات   عن  الكشف  إلى  الدراسة  هذه  تهدف  الشعبي.  الطب  في  القدم  منذ  استخدامها  وتم 
لنبتة   القطبي  المستخلص  في  الفينوالت  المتعددة  للمركبات  العالجية  والخصائص  الكيميائية 

Stachys viticina. 

نبتة   من  مستخلصات  سبعة  عزل     silica gel columnباستخدام    Stachys viticinaتم 
chromatography  وسميت المستخلصات كالتالي(A, B, C, D, E, F, R)  تم التحقق من .

. وتم الكشف عن المجموعات الوظيفية في المستخلصات  HPLCنقاوة المركبات باستخدام تحليل  
 .IR spectroscopyباستخدام تقنية 

  free radicalكمضاد لألكسدة من خالل تثبيط   (A-D, F, Rتم اختبار فاعلية المستخلصات )

picrylhydrazyl)-1-diphenyl-(2, 2      تراكيز    ت أظهر المستخلصات   كالتالي:  50ICهذه 
85.88 µg/mL, 54.37 µg/mL  77.58 µg/mL,  68.36 µg/mL  ,58.62 µg/mL ,
18.58 µg/mL بالترتيب ,. 

كمضادات   المستخلصات  فعالية  فحص  )تم  طريقة  باستخدام  وتم  microdilutionللميكروبات   )
المركب   أظهر  الفطريات.  من  واحدة  وساللة  البكتيريا  من  ستة سالالت  نشاط     (C)اختبار  أعلى 

 ضد نوعين من البكتيريا  , وذلكµg/mL 39يساوي  (MIC)بحد أدنى من التركيز

  methicillin-resistant staphylococcus aureus (MRSA), S. aureus.