BIOLOGICAL EVALUATION OF PHOSPHOLIPID COMPLEXES
FROM EXTRACTS OF BISTORTA AMPLEXICAULIS
SALMA BATOOL 06-Arid-771
Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2019
BIOLOGICAL EVALUATION OF PHOSPHOLIPID COMPLEXES
FROM EXTRACTS OF BISTORTA AMPLEXICAULIS
by
SALMA BATOOL (06-Arid-771)
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Biochemistry
Department of Biochemistry Faculty of Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2019
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TO
ABBU
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CONTENTS
Page
LIST OF FIGURES x
LISTOF TABLES xii
LIST OF ABBREVIATIONS xv
ACKNOWLEDGEMENTS xvi
ABSTRACT Xviii
1. INTRODUCTION 1
2. REVIEW OF LITERATURE 8
2.1 CANCER STATISTICS WORLDWIDE 8
2.1.1 Types of Cancer 8
2.1.2 Liver Cancer 8
2.1.2.1 Available treatments for liver cancer 8
2.2 PLANTS AS HERBAL MEDICINES 8
2.2.1 Pharmacological Potentials of Plant Based Medicine 10
2.3 INTRODUCTION TO FAMILY POLYGONACEAE 10
2.4 BISTORTA AMPLEXICAULIS 11
2.5 CONSTRAINTS OF PLANT BASED MEDICINES 14
2.6 NANOTECHNOLOGY AND HERBAL MEDICINES 15
2.7 LIPOSOMES 16
2.7.1 Types of Liposome 16
2.7.2 Long Circulating Liposomes i-e Stealth Liposomes 17
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2.8 BENEFITS OF USING LIPOSOMES 18
2.9 DRUG TARGETING USING LIPOSOMES 18
2.9.1 Passive Targeting 18
2.9.2 Active Targeting 19
2.9.2.1 Antibody mediated liposomes targeting 19
2.9.2.2 Targeting though folate 20
2.9.2.3 Targeting through transferrin 20
2.9.2.4 Other ligands that could be used 21
2.10 PREPARATION METHODS 21
2.11 FDA APPROVED LIPOSOME BASED DRUGS 21
2.12 APPLICATIONS OF LIPOSOMES 22
2.12.1 Liposomes in Cancer Therapy 22
2.13 PLANT BASED LIPOSOMES 23
3. MATERIALS AND METHODS 29
3.1 PLANT COLLECTION 29
3.1.1 Preparation of Extract 29
3.2 INITIAL SCREENING OF PLANT EXTRACTS 32
3.2.1 Sample Preparation 32
3.2.2 Cell Seeding 32
3.2.3 Treatments 32
3.2.4 SRB Assay 33
3.3 SCREENING FOR HEPG2 AND MCF-7 CANCER CELL 33 LINES
3.3.1 Cell Culture for Hepg2 and MCF-7 Cancer Cell Lines 34
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3.3.2 Cytotoxicity 34
3.4 UPLC ANALYSIS OF RHIZOME EXTRACT ETHANOLIC 35 OF B. AMPLEXICAULIS
3.5 PREPARATION OF LIPOSOMES 37
3.5.1 Encapsulation of Rhizome Extract Ethanolic in to Liposomes 37
3.5.1.1 Thin film method 37
3.5.1.2 Fusion method 37
3.5.1.3 Preparation of stealth liposomes 40
3.5.2 Characterization of Liposomes 40
3.5.2.1 Visualization 40
3.5.2.1.1 Scanning electron microscopy of liposomes 40
3.5.2.1.2 Transmission electron microcopy 41
3.5.2.2 Size determination 41
3.5.2.3 Zeta potential 41
3.5.2.4 Encapsulation efficiency 44
3.6 CELL CULTURE FOR HEPG2 AND MCF-7 CANCER 45 CELL LINES
3.6.1 Cytotoxicity 46
3.7 CELL CULTURE OF HUVEC ENDOTHELIAL CELLS 47
3.7.1 Cytotoxicity 48
3.8 POLARITY EXTRACTS/FRACTIONS 49
3.8.1 Preparation of Different Polarity Extracts 49
3.8.2 UPLC Profiling of Polarity Extracts 49
3.8.3 Cytotoxicity of Polarity Extracts against HepG2 50
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3.8.3.1 Cytotoxicity 51
3.9 DETERMINATION OF FLUORESCENCE OF DIFFERENT 52 POLARITY EXTRACTS
3.9.1 Sample Preparation 52
4 RESULTS AND DISCUSSION 53
4.1 PREPARATION OF EXTRACT 53
4.1.1 Extraction Yield 53
4.2 INITIAL SCREENING AGAINST HCT-116 56
4.2.1 Anticancer Activity Against HCT-116 56
4.2.2 Cell Culture for Hepg2 and MCF-7 Cancer Cell Lines 57
4.3.1 UPLC Profiling of Rhizome Extract Ethanolic 59
4.3.2 Determination of Stability of Extracts with Different Concentrations 59
4.4 PREPARATION OF LIPOSOMES 59
4.4.1 Thin Film Method 59
4.4.1.2 Scanning electron microscopy of prepared liposomes 60
4.4.2 Fusion Method 60
4.5 PREPARATION OF SECOND GENERATION; STEALTH LIPOSOMES 70
4.5.1 Surface Morphology 70
4.5.1.1 Scanning Electron Microscopy (SEM) 70
4.5.1.2 Transmission Electron Microscopy (TEM) 73
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4.5.2 Zeta Potential 73
4.5.3 Determination of Size using Dynamic Light Scattering (DLS) 74
4.5.4 Estimation of Encapsulation Efficiency 83
4.5.4.1 Using UPLC 83
4.5.4.2 Using spectrophotometer 81
4.6 IN VITRO ANTICANCER ACTIVITY OF ENCAPSULATED EXTRACT VS RAW EXTRACT ON 82 HEPG2 CELLS
4.7 EFFECTS OF ENCAPSULATED EXTRACT VS RAW 83 EXTRACT ON HUVEC ENDOTHELIAL CELLS IN VITRO
4.8 PREPARATION OF POLARITY EXTRACTS/FRACTIONS 90 AND IN VITRO CYTOTOXICITY ASSAY
4.8.1 UPLC Profiling of Extracts/Fractions 91
4.9 DETERMINATION OF FLUORESCENCE FOR 91 POLARITY EXTRACTS/FRACTIONS OF REE
SUMMARY 104
LITERATURE CITED 108
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List of Figures
Figure No. Page
1 Collection and shade drying of plant samples 30
2 Voucher specimen of Bistorta amplexicaulis 31
3 SRB assay 36
4 Ultra-performance liquid chromatography(UPLC) 38
5 Loading of sample on copper grid for Transmission electron 42
microscopy
6 Transmission electron microscope 43
7 Graphical representation of preliminary screening of Shoot, 62
Rhizome and Leaf methanolic extracts with HCT-116
8 Graphical representation of preliminary screening of Shoot, 63
Rhizome and Leaf ethanolic extracts with HCT-116
9 Percentage viability and IC50 of Rhizome extract ethanolic 65
(REE) and Gallic acid against HepG2
10 Percentage viability and IC50 of Rhizome extract ethanolic 66
(REE) and Gallic acid against MCF-7 cell lines
11 UPLC profile of ethanolic extract of rhizome of B. 67
amplexicaulis
12 UPLC profile of ethanolic extract of rhizome of B. 69
amplexicaulis
13 Scanning electron microscope image of liposomes 71
14 SEM images of freeze dried liposomes 75
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15 Measurement of Zeta Potential of latex beads 77
16 Measurement of Zeta Potential of empty liposomes 78
17 Measurement of Zeta potential of 4mg/mL formulation of 79
liposomes
18 Measurement of Zeta Potential of 2mg/mL formulation of 80
liposomes
19 Calibration curve from the data obtained from UPLC for 85
supernatant
20 Calculations for encapsulation efficiency obtained from 87
Spectrophotometer
21 Graphical representation of cytotoxicity of encapsulated and 93
un-encapsulated extract against HepG2 cell lines
22 Graphical representation of cytotoxicity of encapsulated and 94
un-encapsulated extract against HepG2 cell lines
23 Graphical representation of cytotoxicity of encapsulated and 96
un-encapsulated extract against HUVEC cell lines
24 Graphical representation of percentage viability and IC50 of 98
fractions of crude extract of B. amplexicaulis
25 UPLC profile of ethanolic extract of rhizome of B. 99
amplexicaulis
26 Fluorescence intensity of Rhizome extract ethanolic fraction 100
27 Fluorescence intensity of acetone fraction of rhizome 101
28 Fluorescence intensity of 80% methanol fraction of rhizome 102
29 Fluorescence intensity of 80% ethanol fraction of rhizome 103
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List of Tables
Table No. Page
1 Program for UPLC 39
2 Percentage yield of the extract obtained extract of B. 55
amplexicaulis
3 Preliminary screening of Shoot, Rhizome and Leaf ethanolic 61
and methanolic extracts with HCT-116
4 Percentage viability and IC50 of Rhizome extract ethanolic 64
(REE) and Gallic acid against HepG2 and MCF-7 cell lines
5 Qualitative analysis of Rhizome extract ethanolic of B. 68
amplexicaulis through UPLC profiling
6 Screening the method of preparation of liposomes 72
7 Characterization of stealth liposomal formulations 76
8 Standard calibration curve obtained from UPLC for supernatant 84
9 Data represents calculations for the encapsulation efficiency 86
through UPLC
10 Data represents absorbance readings against standard 88
concentrations for spectrophotometer
11 Data represents calculations for the encapsulation efficiency 89
through Spectrophotometer
12 Cytotoxicity of encapsulated and un-encapsulated extract 92
against HepG2 cell lines
13 Cytotoxicity of encapsulated and un-encapsulated extract 95
against HUVEC cell lines
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14 Percentage viability and IC50 of fractions of crude extract of B. 97
Amplexicaulis
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List of Abbreviations
REE Rhizome extract ethanolic RAC Rhizome extract acetone fraction 80RE Rhizome extract 80% ethanol fraction 80RM Rhizome extract 80% methanol fraction SEM Shoot extract methanolic REM Rhizome extract methanolic LEM Leaf extract methanolic SEE Shoot extract ethanolic LEE Leaf extract ethanolic TFM Thin film method FM Fusion method UPLC Ultra-performance liquid chromatography MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium SEM Scanning electron microscope TEM Transmission electron microscope PDI Poly dispersity index SRB Sulphorhodamine B REE Rhizome extract ethanolic RAC Rhizome extract acetone fraction 80RE Rhizome extract 80% ethanol fraction 80RM Rhizome extract 80% methanol fraction SEM Shoot extract methanolic REM Rhizome extract methanolic LEM Leaf extract methanolic
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ACKNOWLEDGEMENTS
and His Holy ﷺ Foremost, I am truly grateful to Allah, Beloved Prophet
Progeny, those who are the head spring of all knowledge bestowed to mankind, for blessing me with this extent of enlightenment.
I would like to extend my profound etiquette to Dr. M. Sheeraz Ahmad, my supervisor for his patience, tolerance, guidance positive criticism, kindness and manifold support during the study tenure. Presence of Dr. Rahman Shah Zaib
Saleem along as a co. supervisor was truly a blessing. I feel indebted of his kind- heartedness and intellectual approach which paved the way towards the accomplishment of this degree.
I would like to express my sincerest gratitude to my committee members
Dr. Muhammad Javaid Asad and Prof. Dr. Muhammad Arshad (Dean Faculty of
Sciences), for their valuable suggestions, immense support, guidance and caring attitude during period of study. My special thanks also go to Dr. S.M.S Naqvi, for his selfless help and support.
I feel privileged to have great company of my intimate friends Dr. Umme
Hina and Safia Janjua. I would like to express my deepest thanks to Liu Yanna,
Gomes Alves, A.G. (Georgina), Rodriguez, L. (Lucia), Aida Moreira and Umara
Afzal, Bushra Javaid, Maria Mushtaq, Madiha Khalid, Dr. Shumaila Naz, Dr.
Farah Deeba, Kanwal Batool, Sana Nayab, Nosheen Saleem and all my batch fellows and friends for making me stand again in the times of huge depression, for their company made the time of the degree delightful. Special thanks are reserved for all members of Plant Biotechnology Lab II and for Ph.D squad at LUMS for the interesting discussions and care in the time of need.
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I would like to express my gratitude towards Nabiha Shahroz, Anoosha
Haseeb, Fatima Anjum and Maheen Lateef for being a sole source of encouragement to me always.
Words fail me in expressing my overwhelming sense of affection and gratitude for my parents Abbu, Amme and Papa, Mama and to my siblings, Danish,
Cyra and Hassan, who always encouraged me and are source of immense emotional and moral support to me in my life. . I will always regret Abbu’s extremely unfortunate dismissal just before 67 days of my final viva. I will always miss the spark in his eyes upon completion of my degree. I will forever miss him.
I would like to extend the most innocent thanks to my beloved son Haider
Ali who has sacrificed his mommy`s time when he needed me the most. It would be extremely unjust not to mention the man behind the success story, my kindred soul Ali Ziaa, without whom I would never accomplish my dreams. Colossal thanks to him for his divine love, interminable support and limitless forgoes for me to become a learned person of the society.
May Allah bequeathed them all with the best in all pursuits of their lives
(Ameen)
Salma Batool
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ABSTRACT
Medicinal plants cater healthcare needs of about 80% of world population particularly in developing countries. Bistorta amplexicaulis belongs to the genus
Polygonum (Polygonaceae), and is found in temperate regions of the world. The plant is extensively used as herbal medicine in North Pakistan, India and China and has proven antioxidant and antitumor activities with high phenolic contents. Plant phenolics being quantitatively highest in the extract and are most bioactive, suffer from low cellular uptake due to their hydrophilic nature. The in vivo activity of phenolics is often limited due to stability, low bioavailability and cellular uptake.
This translates to using a higher dosage of the extract for effective intracellular concentrations which in turn limits the affectivity and increases side effects. So the researchers are focusing on approaches that can improve the cellular uptake of these compounds to enhance their activity. Keeping in view, the current study was designed for nano-liposomal encapsulation of B. amplexicaulis extracts for increased anti-cancer activity due to increased cellular uptake. Plant extract was prepared using maceration technique, analyzed for its phytochemical constituents using Ultra performance liquid chromatography (UPLC) with C18 columns and screened for its anticancer activity against HCT-116 Human Colon cancer cell lines. Liposomes were prepared using thin film method with DPPC,
PEG2000DSPE and Cholesterol in a ratio of 1.85:0.15:1 with 2mg/mL and
4mg/mL ethanolic extract of B. amplexicaulis respectively. Prepared liposomes were characterized using Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) and Size was determined using dynamic light scattering (DLS) whereas charge was determined using Zeta sizer. Encapsulation
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efficiency was estimated using spectrophotometer and UPLC. A cytotoxicity comparison was established between encapsulated and un-encapsulated extract through in-vitro MTS cell proliferation assay using Hepatocellular carcinoma cell lines (HepG2) and breast cancer cell lines (MCF-7). Furthermore, encapsulated and un-encapsulated extract was also tested for cytotoxicity to normal Human
Umbilical Vein Endothelial Cells. The UPLC analysis identified gallic acid, caffeic acid, chlorogenic acid, catechin and epicatechin in ethanolic rhizome extract.
Liposomes encapsulating rhizome extract were characterized having zeta potential of -19.8 mV and -16.9mV, size of 155nm and 143nm with PDI of 0.09 and 0.16 for
2mg/mL and 4mg/mL ethanolic extract formulations respectively. Encapsulation efficiency was in a range of 71-81% for both formulations. The HepG2 cells were found most sensitive against free extract with IC50 of 27g/mL as compared to
MCF-7 with IC50 of 67g/mL. The rhizome extract loaded liposomes as compared to free extract has shown to improve anticancer activity by 30% against HepG2 cells. However, in normal Human Umbilical Vein Endothelial Cells (HUVEC) the
LD50 value of free extract was 13 g/mL compared to liposomes encapsulated extract having LD50 54.0 g/mL and LD50 65.9 g/mL for 2mg/mL and 4mg/mL ethanolic extract formulations respectively. The increased LD50 values of liposomes encapsulated extract against HUVEC cells indicates that its toxicity is decreased to the normal cell whereas liposomal encapsulation of extract has enhanced its anticancer activity. Hence, Nano-liposomal encapsulation could be used as a mean of targeting the B. amplexicaulis rhizome extract to the cancer cells to enhance its anticancer potential avoiding cytotoxicity to the normal cells. To our knowledge
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this is the first report of the liposomal encapsulation of an extract, enhanced uptake of extract and an improved activity against hepatocellular carcinoma (HepG2).
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Chapter 1
INTRODUCTION
All the known drugs somehow hold an integral portion in the scaffolds of natural products either being inspired from or being derived from them. In last three decades a keen insight of all the novel drugs introduced and approved by the
US Food and Drug Administration (FDA) have proven 34% of all the novel medicines were originated from natural products or natural products inspired or a derivative of natural products which includes the statins, tubulin-binding anticancer drugs and immunosuppressant (Newman and Cragg, 2007). Even the successfully commercialized medicines have been known to be adopted from the original idea of naturally produced secondary metabolites though the biologically active compounds obtained from nature (Hert et al., 2009). Another important benefit of plant based medicine is that they can also act as substrates for several compounds for their transportation to the required action site intracellularly (Schenone et al.,
2013; Eggert 2013).
The leads obtained from the nature i-e from plants and microbes have wide pharmacological applications because of their unique stereochemistry. The stereo- chemical profiles of these compounds pose medicinal attributes to natural products, particularly in protein interactions and targeting for medicinal activities i-e antitumor, antioxidant and anti-diabetic activities (Drewry and Macarron, 2010).
Among the most generously distributed 30000 bioactive natural products, a simplified division could be the production of about 13% leads from animals, 33%
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from bacteria, 26% from fungi and 27% from plants (Henkel et al., 1999; Kinghorn
2001).
Bistorta amplexicaulis (D. Don) Green synonym Polygonum amplexicaule
D. Don belongs to family Polygonaceae of plant kingdom and is known as
Anjabar/Maslun to layman in Pakistan. Rhizome of the plant has known Medicinal properties like curing ulcer; other uses off the plant include its use thatching, fodder, ethnoveternary. Dried roots are used in making tea which is very effective in flue, fever and joints pain (Qureshi et al., 2007; Jammu et al., 2014). Rhizome of plant is also used in fever and diarrhea as traditional medicine.
It grows in Semi shade to full sun/partially moist, shady edges. Flowering period of plant starts from June and stays till September in a year (Gilani et al.,
2014). It is sold as $1.50 per kg. Bistorta amplexicaulis (D. Don) Green is known to act as antiulcer and have the properties of purifying blood by local experts
(Hamayun et al., 2006; Adnan and Hölscher 2010).
It is reported that plant contains alkaloids, saponins, cardiac glycosides, steroids, fixed oils and fats, phenolics, gums and mucilages and tannins and terpenoids and anthraquinones. The rhizome was found potent for antitumor activities and cytotoxicity assay with IC50 of 13.57μg/mL. Rhizome was also found active for tyrosinase inhibition with suitability of 64.6% tyrosinase inhibition at 10μg/mL treatment (Ahmad et al., 2013).
The proven in vitro and in vivo antioxidant activities and anti-cancer
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activities of the Polygonum amplexicaule synonym Polygonum bistorta lead us to the further investigation of the plant for its pharmaceutical uses. As there is always a void to fill as far as the chemo-preventive drugs are concerned. Out of total
250,000 plants species, it is considered that above one thousand plants have noteworthy anticancer properties. This figure does not limit the huge potential of plant against cancer but it encourages the need to investigate even deep in to the vast sea of potential biological leads that could be obtained from plants. Among all the major anticancer drugs obtained from plants is Taxo, which is one of the most stupendous compounds, which is found active in dealing with, breast, refractory, ovarian and many other cancers. Another protuberant molecule is Podophyllotoxin.
Synthetic alteration of this molecule is Etoposide, which is known to be operative for small cell cancers of the lungs and testes. Camptothecin is obtained from
Camptotheca acuminata. Other significant molecules include Vincristine,
Vinblastine, Colchicine, Ellipticine and Lepachol along with Flavopiridol, a semi- synthetic analogue of the chromone alkaloid Rohitukine from India, a pyridoindole alkaloid from leaves of Ochrosia species and many more. Along with these are the lesser-known plants of sub-Himalayan region, which needs to be explored
(Mukherjee et al., 2001).
The triumph of pharmaceutical industry is now relying on the competent utilization of all existing data and generation of new data on the therapeutic potential of medicinal plants for frantically required products for healthcare.
Lately, several well reputed pharmaceutical companies have indulged themselves in production of herbal preparations which has increased the need for eminence of
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raw materials. This has increased the growth of medicinal plant industry at the rate of 7-15 % yearly. In 1991 herbal drugs market in Germany was $3.0 billion, in
France it was $1.6 billion and in Italy it was $0.6 billion respectively. Herbal extracts are vended as prescription drugs used in Germany and France. In 1996, the herbal medicine market was $ 4 billion in USA and for Europe it was about $ 10 billion. The local Indian market in 1996 was about $ 1 billion, but the export crude extract was only $ 0.08 billion. In the year 2001 the raw material export has surpassed a billion US dollars. So, far China is ranked the first among raw material exporters and India being the second. Germany has been the most active country among all other developed nations on herbal drug research. About 300 plants has been Published for their individual monographs on the therapeutic benefits. In developing countries, China has created adequate data on 800 or more medicinal plants and is a large exporter of herbal drugs. Only few articles have been prepared by India with minimal exports (Subramoniam, 2014).
Right now, despite of having huge potential herbal drug industry is facing certain challenges. Occurrence of numerous pharmacological properties is a very common attribute of a single medicinally important plant that makes the scenario complicated. Another major drawback is toxicity along with useful chattels. Only if the toxic molecules are different from the active molecules we can utilize the technique of fractionation using apt isolation practices. Most important challenge in phytomedicine is development of ecotype and genotype variants in effectiveness and safety and it depends on various factors like soil characteristics, agroclimatic conditions of the habitat, genotype, plant portions, stage of development, nitrogen-
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use efficiency, and association of microorganisms and the level of ecological contamination. This requires extensive research. Standardization of procedures for farming of medicinal plants is very critical in this regard. Many plants with broadly fluctuating degrees of the similar healing action are befalling. In such cases, a comparison for efficiency and safety must be established before any further development. Development of suitable dosage forms or drug delivery systems for extracts and fractions is a stoppage. In some cases, one of the important aspects is lack of enough good quality plant materials. Once found active in the preliminary screening, target plant collection in bulk quantity may be a problem due to limited availability of plant-mass, scattered distribution, etc. Authentication of plant species is difficult in some cases; this is particularly true and most crucial in the case of lower plants such as bryophytes, fungi and algae. As far as cancer is concerned, despite of having huge repertoire of already existing drugs a need for the development of unique chemopreventive and therapeutic agents with minimal side effects has never ended. Plant-derived phytochemicals raise high expectations, since undesirable side effects are comparatively low (Subramoniam, 2014).
For all the positive effects of Herbal drugs they have become more famous in the current era for potential to antidote variety of illnesses with reduced toxicity and extremely useful pharmacological effects. Despite of having all the good reasons there are few restraints of herbal extracts/plant actives, which are wavering stability profiles in highly acidic pH, liver metabolism etc has restricted the drug concentrations lower therapeutic dose in the blood causing minimal or no healing effect. Along with it plants based medicine suffers from low bioavailability inside
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the body. So, far invention of novel drug delivery technology has provided solution to this constraint to plant based drugs. It helps minimize pre-systemic metabolism and the drug degradation and severe side effects. Novel drug delivery system has superiority over conventional dosage forms being able to release the required drug at the specific site minimizing the severe side effects to undesirable sites. It also aids in increasing pharmacological activity with improved macrophage distribution
(Dhiman et al., 2012).
Liposomes has been widely studied for their use as drug delivery vehicles for anti-neoplastic drugs by accruing the amount of drug in target site and lessening the contact or buildup of drug in healthy cells or tissues hence foiling tissue toxicity profiles.
Liposomes are colloidal carriers, usually ranging in size from 0.05 to 5.0µm in diameter with spontaneous formation when specific lipids encounter aqueous media. Liposomes are circular vesicles which are made up of amphiphilic lipids which organize themselves to concentric rings like membranous structures upon contact with water (Dhiman et al., 2012).
As plant extracts are mixtures of hydrophilic and hydrophobic compounds so the major benefit using liposomes as drug carrier for plant extracts is that they can encapsulate both type of compounds. Hydrophilic substances are ensnared in the aqueous portion, while lipophilic substances are adsorbed in membrane. They are composed of phospholipids that are either synthetically made or obtained from
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natural origins, sterols, and an antioxidant. There are three types of classifications exist for liposomes based on their size, number of lamellae, and surface charge.
Liposomes are classified into three categories i.e anionic, cationic or neutral based on surface charge. Based upon the dimension and number of concentric rings formed, liposomes may be oligo, uni or multilamellar and small, large or giant.
Unilamellar liposomes contains only bilayer and categorised into small, large and giant unilamellar liposomes with diameters of approximately 25–100 nm, 100 nm to 1 µm and diameters greater than 1 µm upto tens of microns. Multilamellar liposomes which are also abbreviated as MLVs are composed of many concentric lamellae that resembles the structure of an onion. Dilute surfactant solutions contain ULVs whereas MLVs are present in more concentrated solutions of surfactants.
So, the current study was intended to inspect the enhancement of delivery of bioactive components of B. amplexicaulis by encapsulating extract of plant into liposomes. The main objectives of the study were
1. Preparation of phytophospholipid complexes with phosphatidylcholine
2. Characterization of prepared phospholipid complexes
3. Evaluation of transmembrane delivery of phospholipid complexes using
human cell lines.
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Chapter 2 REVIEW OF LITERATURE
2.1 CANCER STATISTICS WORLDWIDE
Mankind has been on the endeavor to seek the understanding of the reality and improve the life. The ubiquity behind all the knowledge attained so far is to protect, secure and facilitate the life. Despite of the discovery of number of therapeutics that have eradicated or cured number of ailments, we are still faced with the lack of prognosis and treatment of many lethal ailments.
Cancer is the prominent cause of mortality in the world. In 2012 about 14 million cases and 8.2 million cancer related deaths were reported and the numbers are expected to increase by 70% in 2032 (WHO).
2.1.1 Types of Cancer
Cancer is losing the homeostatic control of cell growth, which could be benign or malignant causing spread to various organs of the body becoming fatal.
Cancer is second major cause of mortality worldwide and have many subtypes depending upon the anatomic and molecular basis. Lung, colorectal and stomach cancer are the most common types of cancer hitting male and female equally whereas prostrate and liver cancer are specifically targeting men and breast and cervical cancers are major causes of death in women. Cancer is treated through surgery, radio-, chemo- or gene therapy. Various therapies often lack the specificity and show high toxicities. Gene therapy is still at budding stage and is yet not introduced as standard treatment of cancer. Targeted delivery of the anti-cancer
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drugs can enhance the specificity and lower the toxicities and side effects on the normal cells (WHO).
2.1.2 Liver Cancer
Liver cancer among all others is the third major cause of death being fifth major cause of death among men and eight most common cause of death among women. Liver cancer is much more common in Asia as compare to other parts of the world whereas in some countries it is the most usual cause of death. People who are diagnosed early live almost 5 years after diagnosis whereas after spread the survival rate is even decreased by 3% only. There are three major types of liver cancer i-e hepatocellular carcinoma, angiosarcoma and cholangiocarcinoma
(Pazgan-Simon et al.,2015).
2.1.2.1 Available Treatments for Liver Cancer
Depending upon the size and spread of liver cancer there are various therapies available which include surgery, thermal ablation, percutaneous ethanol injection, radiotherapy, chemotherapy, immunotherapy and targeted therapies.
Adverse side effects, multidrug resistance and being unaffordable are the major limitations to all these treatments particularly for the developing countries of the world (Li and Martin 2011).
2.2 Plants as Herbal Medicines
Plants have long been used as curatives and their healing properties have widely been exploited traditionally. Use of plants as medicines date back to 5000
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years ago or even earlier than that. The strongest evidence of the relevance of plants and human health was found in 1897 when salicylic acid from willow bark served the basis for synthetic acetyl salicylic acid used as antipyretic. Among all the known drugs nowadays most of them are either plant based or have their structures derived from some natural compound. Among 250000 angiospermic plant species there are a huge reservoir of bioactive moieties that could be used as synthetic analogues or as such against ailments serving humanity through nature
(Raskin et al., 2002).
2.2.1 Pharmacological Potentials of Plant Based Medicine
Out of 1.25 lakhs of angiosperms only 1-2% are explored for their pharmacological potentials. Anti-inflammatory activities of chlorophyll a is a proof that almost all plants on the face of earth could have some medicinal characteristics which needs to be explored for the benefit of mankind (Subramoniam, 2014).
2.3 INTRODUCTION TO FAMILY POLYGONACEAE
Family Polygonaceace is known for its medicinal properties traditionally and scientifically as well which makes it the need of time to get more powerful insight in to the benefits of its family members.
Family Polygonaceae is known for its antioxidant, anti-inflammatory, antibacterial, antifungal, antiviral, anticancer, neuroprotective, and lipid regulating and estrogenic effects. Many of its members contain valuable antioxidants that incur above mentioned medicinal activities against various ailments. Some of their
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cardioprotective and hepatoprotective activities are also reported. It has also been seen that some of the plants help boosts the proliferation of osteoblastic cells in vitro. Tyrosinase inhibitory activities are also known from some of the members of this family which makes the use of plant credible for cosmetics.
2.4 BISTORTA AMPLEXICAULIS
Bistorta amplexicaulis which was formerly known as Polygonum amplexicaule is known for antioxidant, anticancer, anti-bacterial, anti-fungal, cardio-protective activities. Its activities against atherosclerosis and promotion of cell proliferation in osteoblastic cells in vitro were also reported. These activities could be attributed to high phenolic contents of plant, out of which gallic acid, catechin, kaempferol, caffeic acid, quercetin and rutin was found in the plant.
Along with these formerly unknown compounds were also identified which were named as 5,6-dihydropyranobenzopyronean 5,6-dihydro- pyranobenzopyrone and
Emodin-8-O-β-D-Glucoside, amplexicine, khellactone, Vanillin, methyl cafeate,
5,7-dihydroxychromone, p- hydroxyphenethyl alcohol, ethyl cafeate, isovanillic acid and dihydro-kaempferol (Xiang et al., 2011; Tantry et al., 2012; Batool et al.,
2015).
GC-MS was used to identify the 81 compounds from Polygonum bistorta
L., which was obtained from three different Asian origins two from china and one from Pakistan. Out of 81 compounds 77 were identified successfully with percentage yield of 0.11 to 0.29% with a huge difference in their chemical composition validated through chemometric analysis. A remarkable difference in
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antibacterial activity was also found between Pakistani and Chinese origin plants.
The major chemical constituents were Furfural, linoleic acid, methyl ester, palmitic acid, 5-methyl furfural, oleic acid and cosanes (Intisar et al., 2012).
Crude extract of Polygonum bistorta roots of Turkish origin was obtained by with70% methanol and 70% Acetone by solid liquid extraction. Highest phenolic content was obtained through acetone extraction and liquid chromatography-electro spray ionization-mass spectrometry revealed three major antioxidants i-e chlorogenic acid, procatechuic acid and catechin. A low antioxidant activity as compared to other Turkish medicinal herbs was obtained from roots extract of Polygonum bistorta (Joshi, 2014).
Another study was done to investigate the cytotoxicity of methanol water extract of Polygonum bistorta L. Thirteen different fraction were obtained from the extract by preparative HPLC and components were identified using GC-MS and
LC-DAD-ESI-MS. Anticancer compounds of phenolic origin were identified i-e gallic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, protocatechuic acid, syringic acid, catechol, 4-methyl catechol, syringol and pyrogallol and fatty acids were identified from different fractions. Cytotoxicity was assessed using human hepatocellular carcinoma cell line (HCCLM3). Three fractions showed highest anticancer activity which contained highest phenolic compounds. Though two other fractions also contained good phenolic content but didn’t show anticancer activity this proves that the unique chemistry of plant based extract enables them to work synergistically to show dose dependent anticancer potential
13
(Intisar et al., 2012). Polygonum amplexicaule, rhizome, shoot and leaf extracts were analyzed after making their crude methanolic extracts, which were further extracted using solvent extraction with following solvents n-butanolic, ethanolic, ethyl acetate and aqueous fractions for DPPH assay. Crude methanolic extract obtained from leaf having IC50 1.03μg/mL showed highest activity. DNA protection properties were seen with the 10ppm and 100ppm concentrations of crude methanolic extracts of rhizome and leaf, and ethanolic fraction of rhizome extract. Gallic acid, quercetin, catechin, Caffeic acid were the antioxidants found using HPLC based Identification (Batool et al., 2015).
Another study reinforced the presence of Catechin, gallic acid and caffeic acid in the plant Polygonum amplexicaule whereas 5, 6-dihydropyrano- benzopyrone and amplexicine were two novel compounds identified with other already known compounds rutin , quercetin-3-O- -D-galactopyranoside , chlorogenic acid, galloyl glucose)and scopletin. All compounds showed considerable antioxidant activities (Tantry et al., 2012).
Polygonum amplexicaule D. Don has known traditional used in china against cardiovascular and cerebrovascular diseases, fractures, pain, etc. Twelve novel compounds of phenolic origin were identified which include Vanillin, p- hydroxyphenethyl alcohol, isovanillic acid, 5,7-dihydroxychromone and dihydro- kaempferol (Xiang et al., 2011).
Polygonum amplexicaule D. Don var. sinense Forb (P. amplexicaule) is
14
used as a treatment of hepatocellular carcinoma (HCC). To understand the underlying compounds and the mechanism of action involved in such activity a study was designed in which total flavonoids from P. amplexicaule was investigated. Nine compounds were isolated and identified as major flavonoids in the plant. It was shown that cell apoptosis was induced by flavonoid extract in
HepG2, H22 HCC and Huh-7 in dose dependent manner. In HCC cells, transcriptional activity of signal transducer and activator of transcription 3
(STAT3) was greatly inhibited. Along with it an increase in expression of SHP-1, a protein tyrosine phosphatase which catalyzes STAT3 dephosphorylation in cancer cells was seen. These compounds in combination have shown that no toxic effect was seen in tumor bearing mice while doing Animal studies. This study provides a valuable foundation for further studies in vitro and in vivo for anti-tumor activities of plant (Xiang et al., 2015).
2.5 CONSTRAINTS OF PLANT BASED MEDICINES
There is a long history of plant based natural products to be used as medicines. About ternary part of currently used pharmaceuticals are obtained from plants of synthetic analogues of plant based active compounds. Despite of this long history of benefits to human race and somehow due attention was not paid to the plant based medicine due to various reasons. There is a misconception about natural products that they can only be used as antibiotics that roots in to the era of
World War II but with the advances in the technology and research now various other biological activities have been reported for plants i-e anti-inflammatory, anti- fungal, anti-bacterial, anti-cancer etc. A mountainous constraint in the use of
15
natural products as drugs is their low bioavailability e.g. curcumin is known for its valuable bio-activities in the human body but suffers from low bioavailability to as low as 11.1nmol/L after administration of an oral dose of 3.6g per day. This phenomenon is universal for polyphenols and flavonoids obtained from plants
(Watkins et al , 2015).
Plant based compounds are either hydrophilic or lipophilic. In the case of lipophilic compounds large quantities of the compounds or the mixture of compounds are required to achieve therapeutic effects in the cell and due to excessive amount of dosage of plant based active compounds cell toxicity is suspected response. Encapsulation of such molecules in biodegradable nano- particles can help mask their lipophilic nature and increase their water solubility and hence a better therapeutic effect can be aimed. Whereas transmembrane delivery is a major constraint in the case of hydrophilic compounds. Hydrophilic compounds can get their bioavailability improved by incorporation in to nano- vehicles. Hence a therapeutic effect can be achieved with reduced amount of dosage with high sensitivity and accuracy for better efficiency.
2.6 NANOTECHNOLOGY AND HERBAL MEDICINES
Nanotechnology is beneficial to the delivery of natural products to the body in various ways. It helps boost the bioavailability, can help achieve targeting of the natural products to disease site e.g. in cancer therapy and controlled release of the drug may help reduce various cytotoxic effects associated with the natural products. Nanotechnology can also help reduce the amount of drug administered.
16
(Bonifacio et al., 2013).
2.7 LIPOSOMES
Liposomes are bilayers composed of amphiphilic molecules surround an aqueous internal compartment. Liposomes were first identified by Alec D
Bangham in last century. Liposomes are formed upon hydration of lipid films which happened upon their discovery when Bangham and Colleagues were trying to hydrate dry phospholipids with negative stain. Upon hydration lipid layers dissociate and form a vesicle like microscopic structures. Liposomes are lipid based concentric bilayer vesicles having a hydrophilic compartment for storing drugs for later release in the targeted areas.
2.7.1 Types of Liposomes
Liposomes are classified into several groups based upon lamellarity and chemical composition. i) the Giant Multilamellar vesicles (size 500nm-5000nm ii)
Giant unilamellar vesicles (size > 1000nm) iii) Large unilamellar vesicles (size:
>100nm) iv) Small unilamellar vesicles (size: 20nm-100nm) v) Oligolamellar vesicles (size: 100-500nm) vi) Multilamellar vesicles (size: >500nm). Smaller the vesicle size higher will be the uptake rate by the cancer cells and it will be rapidly eliminated from the blood circulation. To minimize stability and delivery issues, various strategies have been developed to produce liposomes by altering the chemical composition of liposomes which can be classified as i) conventional liposomes ii) Immunoliposomes iii) cationic liposomes iv) pH sensitive liposomes and v) long circulating liposomes. Various types of lipids, procedure of formation
17
of liposomes and the compounds that need to be entrapped, a huge variety of liposomes can be made. Depending upon the nature of the drug that needs to be incorporated in to liposomes hydrophilic drugs will be entrapped inside the aqueous part. The concentration of the drug entrapped will depend upon the volume of water taken and the size of liposome and its lamellarity (Tripathi et al.,
2013).
Whereas in case a drug that needs to be carried in to liposomes is lipophilic, it will most likely bind to the membrane bilayer. This needs to be dissolved in to the organic phase during the formation of liposomes. As far as the lipophilic compounds are concerned they could be entrapped with better encapsulation efficiency. Immuno-liposomes provided a big step forward in attaining these two objectives. Some site-specific ligand from IgG class of immunoglobulins attached to the surface of liposome can help target the cancer cell and drug accumulation.
2.7.2 Long Circulating Liposomes i-e Stealth Liposomes
Due to less circulation time and accumulation of liposomes in the liver rather than the targeted area, concept of long circulating liposomes has been introduced. Different types of biologically suitable polymers can be attached to the surface of liposomes to mask the liposomes from opsonization and elimination later in liver for instance PEG is considered worthy in this regard.
To cater low specificity of drugs, vehicle based drug delivery system approaches have long been exploited. Various vehicles include nanoparticles,
18
liposomes, polymers, SiRNA etc. Being a xenobiotic to the system, a delivery agent itself can be toxic to the body. Liposomes are shown to be least toxic delivery system and there are various methods available for improving their systemic circulation e.g. PEGylation (Immordino et al., 2006).
2.8 BENEFITS OF USING LIPOSOMES
Liposomes mask the characteristics of anticancer drugs and offers pharmacokinetics of liposomes rather than drug in to the blood stream. So, any alteration due to enzymatic degradation in the digestive tract in case of oral administration of drug encapsulated in liposomes is eliminated. They are less immunogenic, biodegradable and easily taken up by the cancerous cells due to
EPR-effect. Further, the liposomes are flexible enough to be surface modified according to the target tumor cells (Allen and Cullis 2013).
2.9 DRUG TARGETING USING LIPOSOMES
There are two methods of liposome based drug targeting to the tumor sites i-e passive targeting and active targeting. It has been investigated that the minimal cutoff size required by the liposomes to enter the leaky vasculature of tumors ranges from 200nm-1.5µm and depends on the type of tumor targeted and the location of tumor. In case of most of available tumor models pore size ranges from
200nm-800nm (Tripathi et al., 2013).
2.9.1 Passive Targeting
In passive targeting the leaky vasculature and the poor lymphatic drainage is exploited to get the encapsulated drug accumulated at the tumor site. Certain
19
type of biocompatible polymers e.g. PEG is being used to protect the liposomes from recognition by the serum proteins which leads to MPS recognition and finally phagocytosis. Small liposome size and optimization of amount of dosage, sterical stabilization and optimizing the composition of liposomes can help mask the liposomes from opsonins and ultimately accumulation in tumor tissues (Yadav et al., 2011).
2.9.2 Active Targeting
In active targeting, the tumor marker proteins that are expressed on the surface of tumor cells are used to locate and target the tumor cells. Molecular recognition is carried out through the ligand attached on the surface of the liposomes which can eventually bind to the tumor specific receptor proteins on the surface of tumor cells. Folate receptor is the most widely studied in this regard.
Due to its low presence in normal cells except in kidneys, high expression in cancer cells and easy binding to lipids makes it best suitable for the active targeting by liposomes. The most important feature of folate receptor based targeting is receptor mediated endocytosis of the liposome containing this ligand (Immordino et al., 2006).
2.9.2.1 Antibody mediated liposome targeting
Various types of cancers are mainly targeted through antibody mediated targeting, which utilizes the tagging if monoclonal antibodies on the surface of liposomes.CD-19 are used as a epitope for internalization of antibodies to get better efficacy. Some of the tumors which over-express HER-2 can be targeted well using
20
anti HER-2 liposomes. Another antibody CC51 was reported to improve the accumulation of related liposomes in the rat colon adenocarcinoma cells in vitro.
Immuno-liposomes can also be used in combination with the endosome destroying peptides for enhanced cytosolic delivery. Ovarian carcinomas are better targeted using diINF-7 peptide.
2.9.2.2 Targeting through Folate
A very well-known technique for targeting is folate mediated targeting of liposomes due to over expression of these receptors in broad range of tumor tissues. It can also be used to overcome multidrug resistance, which makes it even more fascinating technique. Among the famous marketed drugs are daunorubicin and doxorubicin which utilized the folate targeting methods to improve the efficacy of drugs.
2.9.2.3 Targeting through transferrin
Another targeting approach is transferrin mediated targeting, which is over expressed on majority of tumors which can help targeting the drug to and inside the tumors. Newly published data has shown that coupling of transferrin to PEG can help achieve the long circulation and targeting to enhance the delivery of nanoparticles to the tumor sites. This method was utilized for the delivery of cisplatin to gastric cancer and for photodynamic therapy containing hypericin.
Doxorubicin was studied against brain cancer cells after binding with Transferrin and has proven improved cytotoxicity. Discovery of over expression of transferrin in post ischemic cerebral endothelium has led us to develop transferrin mediated
21
liposomes for drug targeting to this area of brain.
2.9.2.4 Other ligands that could be used
The central dogma of targeting the liposome based drugs is to search for new receptors that are highly expressed on the surface of target cells. Most commonly known are the vitamins and growth factor receptors. Against breast cancer in rats, Vasoactive intestinal peptide i-e VIP is targeted to the VIP receptors.
Epidermal growth factor receptors are also one of those targets which are exploited in targeting the liposomes to specific cells which over express EGFR receptors
(Torchilin, 2005).
2.10 PREPARATION METHODS
Liposomes are very economically prepared by using different conventional techniques. Liposomes can be prepared by i) Hydration of thin film method also known as Bangham method ii) Reverse phase evaporation technique ii) Detergent dialysis and iii) Solvent injection technique. Large scale production of liposomes requires certain scale up methods. Important methods used are heating method, spray drying, Freeze drying, supra critical reverse phase evaporation modified ethanol injection method, the cross flow injection technique, micro fluidization and membrane contractor (Gregoriadis, 1984).
2.11 FDA APPROVED LIPOSOMES BASED DRUGS
There are many FDA approved liposomal anticancer drugs viz; Doxil,
Myocet and LipoDox in market against breast cancer and Kaposi sarcoma. Myocet
22
is PEGylated liposomal formulation. Along with these, there is a long list of the liposomal formulations under clinical trials at present which is an encouraging sign in the development of liposomal formulations for the treatment of cancer.
2.12 APPLICATIONS OF LIPOSOMES
Liposomes have fifty years of history with key roles as drug delivery agents. They have wide range of applications in Food and cosmetic industry. They could be a nano-drug carrier, SiRNA could be attached to change their surface morphology and polymer coating of liposomes can be achieved for efficient, targeted and optimal anticancer drug delivery and cell penetration. The major challenges to the liposome biotechnology are the stability of liposomes and large scale production of liposomes with optimal sterility as liposomes are sensitive to heat, chemical sterilizers and radiations.
2.12.1 Liposomes in Cancer Therapy
Liposomal formulations of many anticancer drugs have proven benefits being less toxic than the drug itself. A good example is of anthracycline which is cytotoxic to the normal cells equally well as to cancer cells so to reduce its toxicity liposomal formulations could be used. Liposomal formulations of anticancer drugs can be administered locally to get improved therapeutic index and this strategy can be used to monitor the slow release of the drug and tumor targeting can also be
23
achieved through liposomal encapsulation and engineering the surface of liposomes with several cancer cell specific ligands (Tripathi et al., 2013).
2.13 PLANT BASED LIPOSOMES
Among all known precious plant species with known biological activities is
Silybum marianum whose activity profile led it to be in to the clinical studies sooner than expected. Despite of the fact that it contained highly valuable silybin i- e flavonoid it was poorly absorbed in the digestive tract. In order to improve its bioavailability liposomal encapsulation was exploited (El-samaligy et al., 2006).
Essential oils obtained from Citrus extracts are highly promising against cancer but there are various constraints like low bioavailability and less stability which pose limitations to their usage as anticancer agents. To improve the anticancer efficacy essential oils from citrus extracts were encapsulated in liposomes and their anticancer potential was checked against human SH-SY5Y cell lines which are neuroblastoma cells (Celia et al., 2013).
Aisha et al reported the formation of Orthosiphon stamineus (Lamiaceae) liposomes improved water solubility with 66% entrapment efficiency. Though some interactions between soy phospholipids and extract has been shown through
FTIR. Particle size was 152nm whereas zeta potential was -49.8 which lies in the good stability range. Improved antioxidant activities are also reported from this formulation which will result in better pharmacological activities (Aisha et al.,
2014).
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Aqueous extracts of Ecballium elaterium (Cucurbitaceae) (Karimi and Bohlooli,
2016) was encapsulated in nano liposomal vehicles through thin film method with the size of 218nm and PDI of 0.3. Formulation was tested against AGS cell lines and about two to three fold increase in cytotoxity was seen with nano liposomal formulation as compared to the raw extract (Karimi and Bohlooli, 2016).
Aloe vera is widely known for its pharmaceutical use specifically its leaf gel extract. Encapsulation of leaf gel extract was done and evaluated for its activities. Liposomes were prepared using Bangham protocol with soybean lecithin. Table liposomes were prepared for which the size attained was less than
200nm for unilamelar vesicles with good encapsulation efficiency. Prepared liposomes were tested against NB1RGB human skin fibroblasts cells and better results for proliferation was obtained with liposomal encapsulated extract as compared to raw extract with an increase in collagen synthesis. Liposomal encapsulated AGE was also assessed for the outcome on proliferation in NHEK (F) human epidermal keratinocytes in vitro. The proliferation rate was found to increase by 77% and 101%, respectively with 4 and 20mg/mL formulations in comparison with raw extract. Concluding, liposomal encapsulation enhanced the bioavailability of the extract and can be used for other skin care products as well
(Takahashi et al., 2009).
Based upon already reported antioxidant and tryosinase inhibition activities
Artocarpus lakoocha (Moraceae) (Teeranachaideekul et al., 2013) heartwood
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hydroglycolic extract was encapsulated in liposomes to achieve prolonged release and improved skin whitening effects were seen (Teeranachaideekul et al., 2013).
A comparative study of encapsulated and un-encapsulated extracts of four different thymus species for their antioxidant and antimicrobial activities were done in comparison with standard tocopherol. Almost comparable results with tocopherol were obtained with the un-encapsulated extracts but for the entrapped extract in liposomes better activities were reported (Gortzi et al., 2006).
Delphinium denudatum Wall (Zaidi et al., 2016) is a species of
Ranunculaceae and its folkloric use is commonly known as a therapy for epilepsy and other diseases. Aqueous fraction of the extract was taken and encapsulated in to phospholipid complex which have shown better survival rate in neurological studies as compared to raw extract (Zaidi et al., 2016).
Bacopaephospholipid complex (BPC) was prepared to check it’s antiamnesic and. A higher plasma level was obtained with the phospholipid complex as compared to the extract which means that the bioavailability was enhanced which could be due to enhanced absorption of the extract due to phospholipid complex (Habbu et al., 2013).
Quercetin phospholipids has been developed for enhance therapeutic efficiency against the acute liver damage of the rats. Bioavailability of quercetin was assessed based upon the criteria of enzymatic activity of the super oxide
26
dismutase glutathione reductase and oxidase system and catalase. Lipid peroxidation profiles were done using thiobarbituric acid substances. The quercetin phospholipids took lead on the pure compound administered in protecting the rat liver damage (Maiti et al., 2005).
Anticancer activities of different fractions of extracts from Ginkgo biloba L.
(Ginkgoaceae) (Yamamoto et al., 2002) leaves in different solvents and hot water are well known against B-16 mouse melanoma and human lung adenocarcinoma
(RERF-LC-OK). Best activities were reported by the petroleum ether extract once encapsulated in to the hybrid liposomes against b-16 melanoma cell lines. This was further fractionated through preparative layer chromatography to obtain further four fractions. Among which one fraction was found highly potent against both cell lines used with 80% inhibition of cell proliferation. This indicates the existence of some hydrophobic molecules inside the extracts of G. biloba L. leaves which is promising in liposomal formulation against cancer (Yamamoto et al., 2002).
Oxidative stress induced liver damage and curative effects of curcumin were studied by Maiti and coworkers. A novel formulation of the antioxidant was made with liposomes and was tested in rats against acute liver damage. Free curcumin and phospholipid complex in a dosage of about 100 and 200mg/kg body weight was administered in a way that their effects were analyzed against various enzymes induced by the oxidative stress. The results obtained by the measurement of serum level of curcumin concentration have proven that due to the better
27
bioavailability of phospholipid complex it has better hepatoprotective activity then free curcumin when administered at similar dosage (Maiti et al., 2007).
In a study and silybin- N-methylglucamine was formulated in to liposomal complexes and was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), differential scanning calorimetry (DSC), dissolution and solubility. Its pharmacokinetic and enhanced oral bioavailability was tested in rats by RP-HPLC. Solubility of the complexes was enhanced in water and octane. Due to high solubility of liposomal complexes and in turn increased bioavailability has led to the conclusion that silybin-phospholipid complex has an impressive pharmacokinetic value as compared to free silybin- N-methylglucamine when checked for their plasma concentrations in rats though oral administration
(Yanyu et al., 2006).
Cratylia mollis (Fabaceae) (Andrade et al., 2004) lectin is obtained from seeds of Camaratu bean. Antitumor activity of the lectin was reported. To enhance its antitumor activity against sarcoma 180 in mice, it was encapsulated in to liposomes through thin film method. About 84% encapsulation efficiency was achieved and 71% inhibition in the tumor was seen reported. Along with it loaded liposomes protected liver and kidneys of the treated mice from toxicity (Andrade et al., 2004).
Silybym marianum belongs to the family compositae and its extract is widely known as Silymarin which has been standardized and is used against liver
28
diseases but suffers from low oral bioavailability. Therefore, El-Samaligy et al studied silymarin hybrid liposomes for buccal administration. Silymarin was encapsulated in hybrid liposomes made up of lecithin, stearylamine, cholesterol and Tween 20 (molar ratio 9:1:1:0.5). Liver studies were conducted using male albino rats weighing 180–220 g. Prepared liposomes were tested for their activity against CCl4 induced liver oxidative stress. Better hepatoprotection was seen with the hybrid liposomes encapsulating the extract than the raw extract (El-samaligy et al., 2006).
Breviscapine is a flavonoid isolated from Erigeron breviscapus (vant.)
(Zhong et al., 2005) which belongs to family Compositae. Its activities for protection against brain deterioration were reported through uninvestigated mechanism. To achieve longer systemic circulation DepoFoam® formulation with multilamellar vesicles was developed. Formulation was made through double emulsification process. A longer systemic circulation of four days was achieved using liposomes (Zhong et al., 2005).
Liposomal formulation encapsulating doxorubicin was made with lectoferrin the ligand on the surface for active targeting to HCC cells with 97% encapsulation efficiency and 100nm size. Lectoferrin targets asialoglycoprotein receptor (ASGPR) and a very high uptake was seen in asialoglycoprotein receptor
(ASGPR) positive cells with very considerable cytotoxic activities (Wei et al.,
2015).
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Chapter 3
MATERIALS AND METHODS
3.1 PLANT COLLECTION
Plant was collected from Meeran Jani Tract, Nathiya Galli, Abbottabad and
Murree Hills Rawalpindi Pakistan, in July 2014, identified by Dr. Rahmatullah
Qureshi (Assistant Professor, Pir Mehr Ali Shah Arid Agriculture University
Rawalpindi). A voucher specimen of the plant after identification was submitted to the ISL herbarium Islamabad figure 1 and figure 2. The plant leaves, shoots and rhizome were separated and were shade dried at room temperature. After drying each of the dried plant part was ground in to fine powder respectively.
3.1.1 Preparation of Extract
Each ground plant part was added in to methanol and absolute ethanol in
1:3 respectively and put on shaker incubator overnight (amounts given in table 2).
The mixture was then filtered and the cycles were repeated thrice to obtain enough of the extract out of aerial and underground parts of plant. Filtrate hence obtained was dried first through rotary evaporator according to the boiling point of ethanol and methanol and the final highly concentrated filtrates were dried in oven at 37ºC
(Batool et al., 2015).
The extract obtained was stored at 4ºC till further use and were named as
Rhizome extract methanolic (REM), Shoot extract methanolic (SEM), Leaf extract methanolic (LEM), Rhizome extract ethanolic (REE), Shoot extract ethanolic
(SEE) and Leaf extract ethanolic (LEE) respectively.
29
30
a b
c d
Figure 1. Collection and shade drying of plant samples; a) collection of plant
sample, b) whole plant sample, c) Shade drying of aerial parts, d)
Shade drying of rhizomes
31
Figure 2. Voucher specimen of Bistorta amplexicaulis
32
3.2 INITIAL SCREENING OF PLANT EXTRACTS
3.2.1 Sample Preparation
The stock solution was prepared as 200mg/ml of each extract in respective solvents i-e Ethanol and methanol and were named as REE, SEE, LEE and REM,
SEM and LEM respectively. Along with it 200mg/ml gallic acid stock solution was prepared in both ethanol and methanol to run as positive control.
3.2.2 Cell Seeding
All extracts were checked for their cytotoxicity with HCT 116 human colon cancer cell lines. For a screening experiment, the cells were inoculated into 96-well microtiter plates having 100µL DMEM media with cells at plating densities 20000 cells/well. After cell inoculation, the microtiter plates were incubated at 37°C, 5%
CO2, 95% air and 100% relative humidity for 24h prior to addition of samples.
Wells having only DMEM with 0% and 1% solvent were considered as negative control and gallic acid was used as positive control.
3.2.3 Treatments
After 24h, for treatments dilutions were made in dilution plate to achieve
1mg/mL 0.5mg/mL, 0.25mg/mL, 0.125mg/mL and 0mg/mL final concentrations of extract and positive control (gallic acid) in the already seeded HCT-116 96 well microtiter plate. Then 25µL of each concentration was added to each well respectively in triplicates.
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3.2.4 SRB Assay
After addition of extracts dilutions, the cells were incubated for 72h at 37ºC with 95% air, 5% CO2 and 100% relative humidity. After incubation, an aliquot of
41.3 µl of TCA was added in each well to fix the cells. After 2h incubation at 4ºC, the plates were washed with water and dried. An aliquot of 100µL of 0.06% SRB dye was added in each well followed by 15min incubation at room temperature.
Unbound dye was removed with 2% v/v solution of acetic acid in water. The plates were air dried and bound dye was solubilized in Tris base solution with 5 minutes shaking. Absorbance of solubilized dye was taken at 540nm figure 3. The growth of cell lines was calculated as:
While:
ODs, ODc and ODn were optical density of wells containing sample, control and negative control respectively. The tests were performed in duplicate and average value was calculated. IC50 was calculated by using graph pad prism (Houghton et al. , 2007).
3.3 SCREENING FOR HEPG2 AND MCF-7 CANCER CELL LINES
The stock solution of 200mg/ml rhizome extract ethanol (REE) was further diluted to 500g/mL, 250g/mL, 125g/mL, 62.5g/mL, 31g/mL, 15.6 g/mL and used for anticancer screening against Hepg2 and MCF-7 Cancer Cell Lines.
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3.3.1 Cell Culture for Hepg2 and MCF-7 Cancer Cell Lines
Fresh vials of HepG2 epithelial cells (ATCC HB-8065) (passage #12) were thawed and taken into culture. The cells were collected at 80% confluency after washing with 12 mL PBS (Sigma) and this step was followed by detaching the cells from the 75cm2 flask with 1 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (Sigma). After the cells were detached from the flask cells were resuspended in 10 mL high glucose DMEM (Sigma) containing 10% FBS (Sigma) medium. Cell density was determined by Biorad cell counter with trypan blue.
The cell suspension was made and was then reduced in culture medium to the requisite concentrations. Hundred microlitre of the cell suspension was introduced into each well of a 96-well cell culture plate. The plates were placed in the incubator at (37°C, 5% CO2, humidified) overnight. After overnight incubation
25 L of each concentration of extract was then added to each well to obtain the final concentration of 500g/mL, 250g/mL, 125g/mL, 62.5g/mL, 31g/mL,
15.6g/mL with four controls which were PBS only, cells and Medium only, SDS detergent and no cells only medium in final volume of 125 microliters per well.
The plates were then put at incubation (37°C/5% CO2/humidified) for 72hrs.
Similar method was repeated with MCF-7 cell lines.
3.3.2 Cytotoxicity
® Cell viability was evaluated with a CellTiter 96 AQueous One Solution
(Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
35
2-(4-sulfophenyl)-2H-tetrazolium (MTS). The principle behind using MTS is that it went digested by enzymes once taken up by the living cells to form formazan. The quantity of formazan produced is directly proportional to the amount of living cells and is easily detected by measuring absorbance at 490 nm.
In short, 5000cells/well HepG2 and MCF-7 cancer cells were seeded in 96 well plates and incubated overnight in 100µL high glucose DMEM medium containing 10% FBS for each well. After overnight incubation at 37˚C and 5%
CO2 the medium was aspirated and refreshed with high glucose DMEM medium containing 10% FBS and extracts only in HBS. The incubation time for the uptake of the extracts was 72h.
After incubation times the medium was aspirated and replaced with 100µL high glucose DMEM medium containing 10% FBS for each well and 10µL of
® CellTiter 96 AQueous One Solution. Subsequently, the well plate was incubated up to 4 hours and the absorbance at 490 nm was measured with a 96 well plate reader
(Biochrom EZ Read 400 Microplate reader, Biochrom, U.K.) (Lieberman et al.,
2001).
3.4 UPLC ANALYSIS OF RHIZOME EXTRACT ETHANOLIC OF B.
AMPLEXICAULIS
The extract solution 1mg/mL was made in Solvent A (94.9% water:
5%ACN: 0.1 of Formic Acid) of UPLC mobile phase was taken and sample was injected to UPLC for further investigation. The HPLC column was BEH C18
Column, 130Å, 3.5µm, 2.1mm X 100mm waters Ireland Figure 4 and Table 1. The
36
Figure 3. SRB assay
37
mobile phase comprised of two solvents solvent A and solvent B Solvent A was
94.9% water: 5%ACN: 0.1% of Formic Acid whereas solvent B was 98.9% ACN:
1% water: 0.1 % formic Acid. The flow rate was 0.45mL/min. The detection wavelength and the detection limit of extract adopted were 273nm and 324nm respectively.
3.5 PREPARATION OF LIPOSOMES
3.5.1 Encapsulation of Rhizome Extract Ethanolic (REE) into Liposomes
3.5.1.1 Thin film method
Phosphatidylcholine and cholesterol in 8:1 were taken and dissolved in to chloroform and ethanol. Solvents were evaporated using rotary evaporator at 60°C and thin film was obtained. About 8mL of PBS buffer was placed at 60°C and
10mg/mL and 4mg/mL of extract was dissolved in it to get hydration medium and added to thin film (Asili et al., 2012). Huge aggregations were seen in case of
10mg/mL as compared to 4mg/mL.
3.5.1.2 Fusion method
Using method of Asili et al., 2012 with some modifications phosphatidylcholine and cholesterol were taken in 8:1 and fused at 60°C in water bath. Rhizome Ethanolic extract (REE) of B. amplexicaulis was dissolved in acetone (10mg/mL and 4mg/mL), added to the fused lipids and evaporated in the water bath at 60°C. After that PBS was added as hydration medium and mixture was agitated to get proper mixing (Asili et al., 2012).
38
Figure 4. Ultra-performance liquid chromatography (UPLC)
39
Table 1. Program for UPLC
Sr No Time Concentration
1 0-1 mins 0%B -100%A
2 1-16 mins 30%B
3 16-20 mins 100%B
4 21-25 mins 0%B
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3.5.1.3 Preparation of stealth liposomes
Liposomal complexes were made following the method of Chen et al.,
2012. Lipid phase containing DPPC, PEG2000DSPE and cholesterol in the ratio of
1.85:0.15:1 was dissolved in organic solvent and dried to thin film by rotary evaporator. After that aqueous phase containing the extract was made in HEPES buffer pH 7.4 for hydration and was added to the lipid phase. Polycarbonate membrane filter with the pore size minimum 100nm was used for the size extrusion of liposomes.
3.5.2 Characterization of Liposomes
Prepared phospholipid complexes were analyzed for the shape and morphology, size determination, encapsulation efficiency and drug content.
3.5.2.1 Visualization
3.5.2.1.1 Scanning electron microscopy of liposomes
The Surface morphology of liposomes was studied by scanning electron microscopy (SEM) (Phenom, FEI, the Netherlands). For the visualization, under scanning electron microscope minimal amount of freeze-dried liposomes was mounted on the grid (aluminum specimen stubs with the diameter 12-mm Agar
Scientific Ltd., England) with the dual-sided adhesive tape. Before analyzing the liposomes into the microscope, liposomes were coated with 6mm platinum sputter coater for imaging following the protocol of Rahimian et al. (Rahimian et al.,
2015).
41
3.5.2.1.2 Transmission electron microscopy
FEI Tecnai™ transmission electron microscope (TEM) was used to study the morphology of prepared liposomal formulations (Figure5,6). Samples were diluted with HEPES to obtain opaque color dispersion. Copper grids with formvar film were glow discharged. 15uL of sample was dropped on parafilm, the grid incubated on top for 1min. The grid was blotted on filter paper for 5sec. Then the grid was incubated on a 15uL drop of 1% Uranyl acetate in double Bidest water for
1min and was blotted using filter paper and it was further air-dried for 5min (Yang et al., 2007).
3.5.2.2 Size determination
Malvern ALV CGS-3 was used to measure sizes of liposomes by dynamic light scattering (DLS) under standard conditions: - Measurement temperature 25
°C; Aqueous dispersants (viscosity 0.89 cP, refractive index 1.333); Measurement angle 90°;Calculation of z-average radius (ALV software) or size (DTS software) and polydispersity index. Sample was prepared by diluting the formulation 10 times with HBS (Bartneck et al., 2014).
3.5.2.3 Zeta Potential
Malvern Zetasizer Nano-Z was used for zeta potential measurements of samples. Measurements were done using Universal DIP cell ZEN 1002. Prior to the measurement Instrument’s accuracy was checked by measuring Latex Standard
42
Figure 5. Loading of sample on copper grid for Transmission electron microscopy
43
Figure 6. Transmission electron Microscope
44
beads (Zeta Potential Transfer Standard, Malvern). About 1 mL suspension of non-liposomes was diluted to 1/10 and was put into the DIP cell (minimum 0.7 mL). The DIP cell was mounted into the zeta sizer and the zeta potential was measured. (Bartneck et al., 2014).
3.5.2.4 Encapsulation efficiency
After size extrusion, the liposomal suspension (5 mL) was centrifuged at
64,000 × g at 4–8 °C for 60 min (Beckman LE-80K Ultracentrifuge UU.NL).
Supernatant was separated and stored for testing free drug content. Pellet was suspended again in 5mL of HBS and liposomal suspension was taken and destroyed using (chloroform: Methanol Chl: MeOH (1:1) the upper phase which was methanol was taken and was diluted 1:1 with solvent A (94.9% water:
5%ACN: 0.1 of Formic Acid) of UPLC mobile phase and sample was hence injected to UPLC for further investigation. The UPLC column was BEH C18
Column, 130Å, 3.5 µm, 2.1 mm X 100 mm waters Ireland. The mobile phase comprised of two solvents solvent A and solvent B. Solvent A was 94.9% water:
5%ACN:0. 1 of Formic Acid whereas solvent B was 98.9% ACN: 1% water:0.1 % formic Acid. The flow rate was 0.45 mL/min. The detection wavelength and the detection limit of extract adopted were 273nm and 325nm respectively. The destroyed liposomes containing drug and the supernatant were also tested at 279nm using spectrophotometer (Mullauer et al., 2011).
The destroyed nano-liposomes containing drug and the supernatant were also tested at 273nm using a spectrophotometer. The liposomes, containing the drug, were ultra-centrifuged at 64000 × g at 4-88 °C for 60 min. Pellet was washed
45
twice with HBS and the supernatant was collected. Supernatant was hence used to estimate the amount of un-entrapped extract at 273 nm against the standard concentrations of REE i-e 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 mg/mL respectively.
3.6 CELL CULTURE FOR HEPG2 AND MCF-7 CANCER CELL LINES
Fresh vials of HepG2 epithelial cells (ATCC HB-8065) (passage #12) were thawed and taken into culture. The cells were collected at 80% confluency after washing with 12 mL PBS (Sigma) and this step was followed by detaching the cells from the 75cm2 flask with 1 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (Sigma). After the cells were detached from the flask, cells were resuspended in 10 mL high glucose DMEM (Sigma) containing 10% FBS (Sigma) medium. Finally, the cell density and viability were determined by counting the cells with a Biorad TC20 cell counter after staining with trypan blue.
The cell suspension was made and was then reduced in culture medium to the requisite concentrations. Hundred microlitre of the cell suspension was introduced into each well of a 96-well cell culture plate. The plates were placed in the incubator at (37°C/5% CO2/humidified) overnight. After overnight incubation
25L of each concentration of extract and liposomes was then added to each well to obtain the final concentration of 500g/mL, 250g/mL, 125g/mL, 62.5g/mL,
31g/mL, 15.6g/mL with four controls which were PBS only, cells and Medium only, SDS detergent and no cells only medium in final volume of 125 microliters per well. The plates were then put at incubation (37°C/5% CO2/humidified) for
72hrs. Empty liposomes were used as the negative control whereas un-
46
encapsulated extract was used as positive control in cell culture experiments
(Mullauer et al., 2011).
Similar method was repeated with MCF-7 cell lines (Cat. No. :
ATTCC/EATCC HTB-22- Passage number12). The protocol was repeated with encapsulated extract as well.
3.6.1 Cytotoxicity
® Cell viability was evaluated with a CellTiter 96 AQueous One Solution
(Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS). The principle behind using MTS is that it went digested by enzymes once taken up by the living cells to form formazan. The quantity of formazan produced is directly proportional to the amount of living cells and is easily detected by measuring absorbance at 490 nm.
In short, 5000 cells/well HUVEC endothelial cells, HepG2 and MCF-7 cancer cells were seeded in 96 well plates and incubated overnight in 100µL high glucose DMEM medium containing 10% FBS for each well. After overnight incubation at 37˚C and 5% CO2 the medium was aspirated and refreshed with high glucose DMEM medium containing 10% FBS and prepared liposomal formulations or extracts only in HBS. The incubation time for the uptake of the
Liposomal formulation and un-encapsulated extracts was 72h. Empty liposomes were used as the negative control whereas un-encapsulated extract was used as positive control in cell culture experiments.
47
After incubation time, medium was aspirated and replaced with 100µL high glucose DMEM medium containing 10% FBS for each well and 10µL of CellTiter
® 96 AQueous One Solution. Subsequently, the well plate was incubated to 4 hours and absorbance at 490 nm was measured with 96 well plate reader (Biochrom EZ
Read 400 Microplate reader, Biochrom, U.K.) (Lieberman et al., 2001).
3.7 CELL CULTURE OF HUVEC ENDOTHELIAL CELLS
Fresh vials of HUVEC pooled epithelial cells (Lonza # C2519A) (passage
3) were thawed and taken into culture. The cells were picked at 80% confluency by washing with 12mL PBS which was followed by detaching the cells from the
75cm2 flask with 1mL of 0.25% (w/v) Trypsin / 0.53mM EDTA solution. After detachment of cells, they were re-suspended in 10mL EGM-2 medium (Lonza).
Finally, the cell density and viability were determined by counting the cells with a
Biorad TC20 cell counter after staining with trypan blue. The cells were either used in experiments or taken back into culture. The cell suspension hence obtained was then diluted in culture medium to the required concentrations. Hundred microliters of the diluted suspension were then added into each well of a 96-well cell culture plate. The plates were incubated at (37°C/5% CO2/humidified) overnight. After overnight incubation 25L of each concentration of extract and liposomes was then added to each well to obtain the final concentration of 128g/mL, 64g/mL,
32g/mL and 16g/mL with four controls which were PBS only, cells and Medium only, SDS detergent and no cells only medium in final volume of 125L per well.
The plates were then put at incubation (37°C/5% CO2/humidified) for 72h. This
48
protocol was used for un-encapsulated and encapsulated extracts (Mailloux et al.,
2001a).
3.7.1 Cytotoxicity
® Cell viability was evaluated with a CellTiter 96 AQueous One Solution
(Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS). The principle behind using MTS is that it went digested by enzymes once taken up by the living cells to form formazan. The quantity of formazan produced is directly proportional to the amount of living cells and is easily detected by measuring absorbance at 490nm.
In short, 5000cells/well HUVEC endothelial cells, HepG2 and MCF-7 cancer cells were seeded in 96 well plates and incubated overnight in 100µL high glucose DMEM medium containing 10% FBS for each well. After overnight incubation at 37˚C and 5% CO2 the medium was aspirated and refreshed with high glucose DMEM medium containing 10% FBS and prepared liposomal formulations or extracts only in HBS. The incubation time for the uptake of the
Liposomal formulation and un-encapsulated extracts was 72h. Empty liposomes were used as the negative control whereas un-encapsulated extract was used as positive control in cell culture experiments.
After incubation times the medium was aspirated and replaced with 100µL high glucose DMEM medium containing 10% FBS for each well and 10µL of
49
® CellTiter 96 AQueous One Solution. Subsequently, the well plate was incubated up to 4 hours and the absorbance at 490 nm was measured with a 96 well plate reader
(Biochrom EZ Read 400 Microplate reader, Biochrom, U.K.) (Lieberman et al.,
2001). It is important to mention here that any interference due to the colour of extract with the dye was nullified by several washing steps in separate experiments.
3.8 POLARITY EXTRACTS/FRACTIONS
3.8.1 Preparation of Different Polarity Extracts
About 100mg of the extract was weighed accurately and dissolved in
300mL of absolute ethanol, absolute acetone and 80% ethanol and 80% methanol in 1:3 ratio for overnight in three cycles to obtained different polarity based extracts of Rhizome. The extracts hence prepared were named as REE for absolute ethanolic extract, 80%RE for 80% ethanolic extract, 80%RM for 80% methanolic extract and RAC for acetone extract respectively (Batool et al., 2015).
3.8.2 UPLC Profiling of Polarity Extracts
About 1mg/mL of extract solution made in Solvent A(94.9% water:
5%ACN:0.1% of Formic Acid) of UPLC mobile phase was taken and sample was injected to UPLC for further investigation. The HPLC column was BEH C18
Column, 130Å, 3.5µm, 2.1mm X 100 mm waters Ireland. The mobile phase comprised of two solvents solvent A and solvent B. Solvent A was 94.9% water:
5%ACN:0.1% of Formic Acid whereas solvent B was 98.9% ACN: 1% water:0.1
% formic Acid. The flow rate was 0.45 mL/min. The detection wavelength and the
50
detection limit of extract adopted were 273nm and 325nm respectively (Eugster et al., 2011).
3.8.3 Cytotoxicity of Polarity Extracts Against Hepg2
Fresh vials of HepG2 epithelial cells (ATCC HB-8065) (passage #12) were thawed and taken into culture. The cells were collected at 80% confluency after washing with 12mL PBS (Sigma) and this step was followed by detaching the cells from the 75cm2 flask with 1mL of 0.25% (w/v) Trypsin / 0.53mM EDTA solution
(Sigma). After the cells were detached from the flask cells were resuspended in 10 mL high glucose DMEM (Sigma) containing 10% FBS (Sigma) medium. Finally, the cell density and viability were determined by counting the cells with a Biorad
TC20 cell counter after staining with trypan blue.
The cell suspension was made and was then reduced in culture medium to the requisite concentrations. Hundred microlitre of the cell suspension was introduced into each well of a 96-well cell culture plate. The plates were placed in the incubator at (37°C/5% CO2/humidified) overnight. After overnight incubation
25L of each concentration of extract and liposomes was then added to each well to obtain the final concentration of 4000g/mL, 2000g/mL, 1000g/mL,
500g/mL, 250g/mL, 125g/mL, 62.5g/mL, 31g/mL, 15.6g/mL with four controls which were PBS only, cells and Medium only, SDS detergent and no cells only medium in final volume of 125 microliters per well. The plates were then put at incubation (37°C/5% CO2/humidified) for 72h. (Mullauer et al., 2011).
51
3.8.3.1 Cytotoxicity
® Cell viability was evaluated with a CellTiter 96 AQueous One Solution
(Promega) containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS). The principle behind using MTS is that it went digested by enzymes once taken up by the living cells to form formazan. The quantity of formazan produced is directly proportional to the amount of living cells and is easily detected by measuring absorbance at 490nm.
In short, 5000cells/well HUVEC endothelial cells, HepG2 and MCF-7 cancer cells were seeded in 96 well plates and incubated overnight in 100µL high glucose DMEM medium containing 10% FBS for each well. After overnight incubation at 37˚C and 5% CO2 the medium was aspirated and refreshed with high glucose DMEM medium containing 10% FBS and prepared liposomal formulations or extracts only in HBS. The incubation time for the uptake of the
Liposomal formulation and un-encapsulated extracts was 72h.
After incubation times the medium was aspirated and replaced with 100µL high glucose DMEM medium containing 10% FBS for each well and 10µL of
® CellTiter 96 AQueous One Solution. Subsequently, the well plate was incubated up to 4 hours and the absorbance at 490 nm was measured with a 96 well plate reader
(Biochrom EZ Read 400 Microplate reader, Biochrom, U.K.).
52
3.9 DETERMINATION OF FLUORESCENCE OF DIFFERENT
POLARITY EXTRACTS
The Fluorolog fluorometer (FLUOstar OPTIMA fluorometer) was the instrument used to estimate the fluorescence, or a scatter intensity of liquid samples in cuvettes which is equipped xenon flash lamp with very high intensity and liquid light guide technology. It is used to cover a range of wavelength from 240 to
740nm with in build incubator which can incubate the samples from +8°C to
+45°C.
3.9.1 Sample Preparation
From each polarity extract i-e REE, 80%RE, 80%RM and RAC 1mg sample was taken and dissolved in to their respective solvents per mL. Samples were placed in the FLUOstar OPTIMA fluorometer plate reader and fluorescence intensity was determined (Drabent et al., 1999).
53
Chapter 4
RESULTS AND DISCUSSIONS
4.1 PREPARATION OF EXTRACT
4.1.1 Extraction Yield
The extraction yield (%) obtained from ethanolic and methanolic extraction is shown in the table 2. The best yield obtained was with the Rhizome extract in methanol and ethanol with no significant difference which was 19% and 20% respectively. Whereas in the case of leaf extract about 12% yield was obtained from methanolic extraction and 10% yield was obtained from ethanolic extraction procedures which again is a non-significant difference. In the case of shoot extract about 10% yield was obtained from methanolic extract and 11% yield was obtained from ethanolic extract which was not strikingly different. Among shoot rhizome and leaf extract significant difference was seen between yield of rhizome extract as compared to shoot and leaf extract and similar trend was observed in ethanolic and methanolic extraction. Whereas no significant difference in extraction yield was seen among leaf and shoot extracts in methanolic and ethanolic extraction.
Extraction was quite difficult with leaf and shoot because of the sticky nature and the minimal amount of the extract obtained out of the total powdered sample used initially for the extraction. As far as the toxicity profiles of the extraction solvents are concerned methanol has been proven highly toxic for the human consumption causing necrosis in nervous system (Gupta et al., 2013). Whereas minimal consumption of ethanol is considered safe comparatively since despite of evaporation small traces of solvents remains in the extracts. The reason behind choosing methanol and ethanol was to investigate the extraction yield and better
secondary metabolite extraction although literature supported the fact that despite
53
54
of high percentage yield of extract obtained with methanol better phenolic contents were extracted using ethanol i-e in the case of Limnophila aromatic Total phenolic content obtained from ethanolic extraction was 40.50 mg GAE/g and total flavonoid content obtained was 31.11 QCE/g which clearly indicates highest antioxidant potential but low extract yield was obtained (Do et al., 2014). The presence of polyphenols is directly proportional to the polarity of solvent used for the extraction as reported in a study conducted to investigate the total phenolic content and antioxidant activities from different solvents from three different plants namely Thymus vulgaris L., Salvia officinalis L., and Origanum majorana L. extracts. Though methanolic showed slightly better phenolic content but authors strongly recommended the use of ethanol for being more appropriate because there were non-significant differences between the antioxidant profiles of both solvents from all three plants mentioned above (Roby et al., 2013). In another study a comparison was generated between the extraction using different solvents namely ethyl acetate, benzene, pet-ether, chloroform and ethanol. Better flavonoid content was obtained from ethanolic extraction i-e 6.5g which was later confirmed by the identification and isolation of a novel compound from the leaves of Euphorbia neriifolia which is 2-(3,4-dihydroxy-5-methoxy-phenyl)- 3,5-dihydroxy-6,7- dimethoxychromen-4-one (Sharma and Janmeda, 2017).
As far as the previous studies done on the plant of interest are concerned ethanolic extract of Polygonum amplexicaule synonym to Bistorta amplexicaulis was reported to contain a novel compound i-e 5, 6-dihydropyranobenzopyrone.
55
Table 2. Percentage yield of the extract obtained extract of B. amplexicaulis
Amount of powdered Amount Yield S# Solvent Extract plant material obtained (g) (%) (g) Rhizome 1 33.33 6.3 19 Extract(REM) Leaf Extract 2 Methanol 16.66 1.9 12 (LEM) Shoot 3 16.66 1.67 10 Extract(SEM) Rhizome 4 100 20.2 20 Extract(REE) Leaf 5 Ethanol 100 9.8 10 Extract(LEE) Shoot 6 50 5.7 11 Extract(SEE)
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4.2 INITIAL SCREENING AGAINST HCT-116
Extracts were made at room temperature for the Rhizome, shoot and leaf and were named as Rhizome extract ethanolic (REE), Shoot extract ethanolic
(SEE), Leaf Extract Ethanolic (LEE), Rhizome extract methanolic (REM), Shoot extract methanolic (SEM) and Leaf extract methanolic (LEM) respectively. All extracts were tested against HCT-116 human colon cancer cell line i-e Homo sapiens colorectal carcinoma.
4.2.1 Anticancer Activity against HCT-116
The results of anticancer screening against HCT-116 human colon cancer cell line showed that LEE was most active with IC50 of 569µg/mL as compared to
REE and SEE which were 800µg/mL and 900µg/mL respectively. The Gallic acid was taken as positive control with IC50 of 1120µg/mL for Gallic acid dissolved in methanol and 806µg/mL when dissolved in ethanol with statistically insignificant difference due to solvent. Whereas in case of REM, SEM and LEM, best activities were obtained with LEM with IC50 of 1480µg/mL as compared to REM whose IC50 was 3210µg/mL and SEM with IC50 of 6030µg/mL. A significant difference was seen among all methanolic extracts whereas there was no significant difference seen among ethanolic extracts as compared to gallic acid (positive control) whose
IC50 was 1080µg/mL in the case of methanolic extract and 800µg/mL in ethanolic extract although the effects of solvents were minimized to least effective levels shown in table 3 and figure 7 and 8. This directs us towards the chemistry of gallic acid which might have reacted with the solvent before applied as treatment to the cells and resulted in the change of pH of the media which resulted in difference among results. As far as the difference between ethanolic and methanolic extracts
57
cytotoxicity profile is concerned, a significant difference was seen. Although there is a slight difference between the polarity of both solvents i-e 5.2 is the polarity index for ethanol and 6.6 is the polarity index of methanol but the different in cytotoxicity profile indicates that the target molecules may get binding with each other and their extract is influenced with the polarity of solvents (Franco et al.,
2008). We suspect that because novel compounds like amplexicine and 5, 6- dihydropyranobenzopyrone were identified from the ethanolic extracts of the B. amplexicaulis, this gives us a clue to the better cytotoxicity of ethanolic extracts because ethanolic extracts and novel compounds isolated have shown best antioxidant activities though this does not validate that there would be no or may be less extraction of these compounds with methanol and we suggest a study to investigate it (Xiang et al., 2011; Tantry et al., 2012).
4.2.2 Cell Culture for Hepg2 and MCF-7 Cancer Cell Lines
As we concluded from the previous experiment that Rhizome extract ethanolic would be used for the further experiments for more investigations so we further screened rhizome extract ethanolic along with gallic acid as standard against human hepatocellular carcinoma i-e HepG2 cell lines and breast cancer i-e
MCF-7 cell lines shown in table 4 and figure 9 and 10. Rhizome extract ethanolic was found most promising against HepG2 cell lines with IC50 of 30µg/mL which was found comparable with the standard gallic acid with IC50 of 32µg/mL, whereas with MCF-7 the IC50 value was of 67µg/mL for REE and 53µg/mL for gallic acid shown in table 4 and figure 9 and 10. Results of REE for HepG2 cell lines synchronizes with the results of the study published in 2015 which indicated the inhibition of STAT proteins in apoptotic pathways as molecular target of the polyphenolics and flavonoids of the rhizome extract ethanolic of B. amplexicaulis
58
synonym P. amplexicaule (Xiang et al., 2015). MCF-7 was another chosen cell line for initial screening because it is well characterized and due to its Multi drug resistance properties.
Despite of multidrug resistance it has become an ideal cell line to study the novel anticancer drugs against it (Metha et al., 2002, Comşa et al., 2015). resistance properties of MCF-7 IC50 value 67µg/mL is considerably important and warrants further research on this cell line with REE in future. Another plant from the same family, the Polygonum avicularis showed cytotoxicity against MCF-7 at higher concentrations than 300ng/mL which is very promising. High cytotoxicity of the plant extracts is attributed to the presence of very high phenolic contents in the extract which is already investigated qualitatively and through antioxidant activities as a preliminary part of current study and it has been reported.
Earlier in a study published in 2011 that cytotoxicity of the plant extract of
Polygonum avicularis is may be due to upregulation of p53 genes that play an important role in apoptosis of the cell and along with it due to high antioxidant activity of the plant of interest, extract can help prevent lipid peroxidation and
DNA damage which are common phenomenon in the cancer cells hence helping the cell maintain its homeostatic balance (Habibi et al., 2011; Batool et al., 2015;
Ahmad et al., 2013).
Moreover, we were not being able to identify ferulic acid, scopletin, beta galloyal glucose, epigallocatechin gallate, rutin hydrate, epicatechin, quercetin,
Reservatrol and kaempferol which are already reported from the same plant which could be since the optimal solvent was not used for extraction of the specific
59
compound or may be due to the climate dependent biochemistry of plant species used (Inass Leouifoudi et al., 2014).
4.3.1 UPLC Profiling of Rhizome Extract Ethanolic
Rhizome extract ethanolic was further subjected to UPLC profiling to identify the phenolic compounds that may attribute anticancer potential to plant.
Fourteen standard compounds were tested for qualitative analysis in REE and four compounds were found in the extract i-e gallic acid @0.8min, Caffeic [email protected], Chlorogenic acid @2.8min, Epicatechin@ 3.79mins and catechin@
2.5mins as shown in Table 5.
4.3.2 Determination of Stability of Extracts with Different Concentrations
Along with the identification of compounds from the extract, the stability of extract was also assessed to ensure unaffected drug loading into liposomes later.
Five different concentrations of the REE was made in the solvent A of the UPLC system viz; 0.5mg/mL, 2mg/mL, 3mg/mL, 4mg/mL and 5mg/mL shown in figure
11. The results showed that the extract compound profile was stable at all concentrations tested.
4.4 PREPARATION OF LIPOSOMES
4.4.1 Thin Film Method
Initially liposomes were prepared by thin film and fusion methods using egg phosphatidylcholine and cholesterol with 4mg/mL and 10mg/mL of REE. With
10mg/mL extract, nearly optimal range of liposomal size was obtained i-e
153.5±0.73nm but in the case of 4mg/mL extract size was 677.2±34.3nm with the
60
PDI of 0.28±0.007 and 0.6±0.033 shown in table 6. While preparation of liposomes big clumps was formed in the case of 10mg/mL loading of REE due to excessive extract so the film was hydrated with 15mL of PBS. But for 4mg/mL initially no clumps were seen and 5mL of hydration medium was used to make liposomes. Size and PDI indicates that there was aggregation in 4mg/mL liposomes and no uniform size distribution was seen but in 10mg/mL loading liposomes maintained the size and were uniformly distributed comparatively ( Asili et al.,
2012).
As it’s mentioned earlier that two different concentrations of REE were loaded in to liposomes using thin film method. Liposomes with the size less 200nm were considered good for drug formulations to achieve required therapeutic effects.
4.4.1.1 Scanning electron microscopy of prepared liposomes
Liposomes with loaded extract prepared by thin film method were analyzed in Scanning electron microscope. A mass aggregation was seen in 4mg/mL loaded liposomes which showed instability of prepared liposomes which could be due to electrosteric instability or may be due to lamellarity shown in figure 13 ( Asili et al., 2012).
4.4.2 Fusion Method
Fusion method was used to prepare liposomes using 10mg/mL and 4mg/mL extract loading. Size obtained was 3088±0.57nm with the PDI of 0.86±0.038 and
61
Table 3. Preliminary screening of Shoot, Rhizome and Leaf ethanolic and methanolic extracts with HCT-116
Concentration REE SEE LEE GAE REM LEM SEM GAM
(mg/mL)
0 100±0 100±0 100±0 100±0 100+/-0 100±0 100±0 100±0
0.25 14.4±0.3 91.2±3.2 79.5±3.5 16.5±1 89.7±17.2 100.7±15.2 0.5±0.4 0.7±0.4
0.5 14.3±1 97.2±1.6 65.8±2 15.8±1.2 109.2±22.2 102.4±31.1 0.0±0 1.2±1.2
1 12.7±0.2 38.0±2.8 15.4±1.5 19.4±3.1 99.16.4 46.6±20.2 0.4±0.07 0.0±0
2 14.6±0.5 12.1±0.3 14.8±0.8 20.0±0.8 55.5±44.8 21.1±9.8 15.2±0.5 4.6±0.2
IC50 0.8 0.9 0.6 0.8 3.21 1.48 6.03 1.08
Data represents triplicate values of percentage viability with SEM and IC50
62
2 0 0 R E M
L E M 1 5 0 S E M
) %
( G A M y
t 1 0 0
i l
i
b
a i V
5 0
0 0 .0 0 .5 1 1 .5 2
C o n c e n tr a tio n s (m g /m L )
Figure 7. Graphical representation of preliminary screening of Shoot, Rhizome and
Leaf methanolic extracts with HCT-116. Data represents triplicate
values of percentage viability with SEM and IC50
63
2 0 0
R .E .E
S .E .E 1 5 0 L .E .E
)
% G .A .E
(
y 1 0 0
t
i
l i
b
a i V 5 0
0 0 0 .2 5 0 .5 1 2
C o n c e n tra tio n (m g / m L )
Figure 8. Graphical representation of preliminary screening of Shoot, Rhizome and
Leaf ethanolic extracts with HCT-116. Data represents triplicate values
of percentage viability with SEM and IC50
64
Table 4. Percentage viability and IC50 of Rhizome extract ethanolic (REE) and
Gallic acid against HepG2 and MCF-7 cell lines
Concentrations HepG2 MCF-7
(mg/mL)
REE GA REE GA
0.5 21.7±2.3 9.7±1.9 12.5±1.8 5.7±2.9
0.25 14.7±2.4 5.5±1.6 5.3±1.7 9.7±1.5
0.125 10.3±1.2 2.7±1.7 11.3±0.7 11.7±2.8
0.0625 6.0±2.3 1.7±1.2 64.0±1.5 6.3±2.6
0.0312 23.7±0.3 49.3±22 100.7±3.5 95.3±2.6
0.0156 40.3±2.84 82.0±8.7 101.5±6.5 88.7±11.3
0 100±0 100±0 100±0 100±0
IC50 0.030 0.032 0.067 0.053
REE; Rhizome extract ethanolic, GA; Gallic Acid. Data represents mean of
triplicate readings with SEM
65
1 5 0 G A
R E E
) 1 0 0
%
(
y
t
i
l
i
b
a
i
V 5 0
0
.5 5 5 2 1 5 0 0 .2 2 6 3 1 0 .1 .0 .0 .0 0 0 0 0
C o n c e n tra tio n (m g / m L )
Figure 9. Percentage viability and IC50 of Rhizome extract ethanolic (REE) and
Gallic acid against HepG2. REE; Rhizome extract ethanolic, GA; Gallic
Acid. Data represents mean of triplicate readings with SEM
66
1 5 0 R E E
G A
) 1 0 0
%
(
y
t
i
l
i
b
a
i 5 0
V
0 0 .5 0 .2 5 0 .1 2 5 0 .0 6 2 0 .0 3 1 0 .0 1 5 0
C o n c e n tra tio n (m g / m L )
Figure 10. Percentage viability and IC50 of Rhizome extract ethanolic (REE) and
Gallic acid against MCF-7 cell lines. REE; Rhizome extract ethanolic,
GA; Gallic Acid. Data represents mean of triplicate readings with SEM
67
Figure 11. UPLC profile of ethanolic extract of rhizome of B. amplexicaulis; gallic
acid appears at 0.8 min, caffeic acid appears at 3.7 mins; Caffeic acid
appears at 2.5 mins chlorogenic acid appears at 2.8 mins. BEH C18
Column, 130Å, 3.5 µm, 2.1 mm X 100 mm (Waters). Mobile phase:
Solvent A was 94.9% water: 5%ACN: 0. 1 of Formic Acid, solvent B
was 98.9% ACN: 1% water: 0.1 % formic Acid. Flow rate:
0.45 mL/min
68
Table 5. Qualitative analysis of Rhizome extract ethanolic of B. amplexicaulis
through UPLC profiling
Sr Standards Identification
#
1 Gallic Acid +
2 Rutin -
3 Rutin Hydrate -
4 Caffeic acid +
5 Chlorogenic acid +
6 Epicatechin +
7 Chrysin -
8 Quercetin -
9 Catechin +
10 Reservatrol -
11 Kaempferol -
12 Scoploten -
13 Epi gallocatechin gallate -
14 Ferulic acid -
69
.
Figure 12. UPLC profile of ethanolic extract of rhizome of B. amplexicaulis; BEH
C18 Column, 130Å, 3.5 µm, 2.1 mm X 100 mm (Waters). Mobile
phase: Solvent A was 94.9% water: 5%ACN:0. 1 of Formic Acid,
solvent B was 98.9% ACN: 1% water:0.1 % formic Acid. Flow rate:
0.45 mL/min
70
4022±0.57nm with PDI of 1.1±0.088 shown in table 6. As compared to thin film method fusion method didn’t give required optimization criteria for the preparation of liposomes despite of sonication which may lead to the very low encapsulation of extract though, the optimal size may be achieved after longer hours of sonication.
Along with it formulation is always at a risk of degradation of lipids and the encapsulated material (Akbarzadeh et al., 2013; Asili et al., 2012).
4.4.3 Preparation of Second Generation; Stealth Liposomes
To improve the stability and to achieve better circulation inside the body liposomes have been engineered using long acyl chain containing lipids with changed level of unsaturation and head groups (Immordino et al., 2006).
Introducing extrusion as a vital step in the preparation with nitrogen flushing of the thin film further enhanced the encapsulation efficiency and rescued the extract from degradation (Gabizon et al., 1997; Gregoriadis, 1995).
4.5.1 Surface Morphology
To know the surface morphology of liposomes Scanning electron microscopy and Transmission electron microscopy was performed. The lipids were used in least toxic ratios.
4.5.1.1 Scanning electron microscopy (SEM)
As shown in the Figure 14 scanning electron microscopy of the freeze-dried samples were done to check the morphology of liposomes. Freeze drying of
71
Figure 13. Scanning electron microscope image of liposomes encapsulating
Rhizome Extract ethanolic (4mg/mL) with FEI Nova NanoSEM 450,
USA
72
Table 6. Screening the method of preparation of liposomes
Parameters REE(10) REE(4)
TLM SIZE(nm) 153.5±0.73 677.2±34.3
PDI 0.28±0.007 0.6±0.033
FM SIZE(nm) 3088±0.57 4022±0.57
PDI 0.86±0.038 1.1±0.088
TFM: thin film method, FM: fusion method; REE: rhizome extract ethanolic.
Results indicate triplicate readings with SEM.
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liposomes eliminate the water content up to 99.5% which converts the liposomal suspension in to a porous matrix which can easily be reconstituted upon addition of hydration medium (Lee et al., 2007). This is also shown in the Figure 14 Fine porous matrix like structures were seen.
4.5.1.2 Transmission electron microscopy (TEM)
Transmission electron microscopy reveals the uniformity of the size of the liposomes and it confirms the size and stability (Aisha et al., 2014). Transmission electron microscopy validates the PDI value and size distribution of loaded and empty vesicles as shown in the figure 14.
4.5.2 Zeta Potential
Zeta potential was determined using Zeta Sizer nano® by Malvern. It was found that 2mg/mL formulation of extract in liposomes have zeta potential of -
19.8±2.35, 4mg/mL formulation has zeta potential of -16.9±1.04 as compared to empty liposomes which were used as control with zeta potential -19.4 ± 0.23 respectively shown in table 7 and figures 15-18. This shows that prepared liposomes lie in incipient stability range.
There are various factors that caused the change in zeta potential of the liposomes after loading the extract which could be Adsorption of the contents on the exterior of liposome or the hydrolysis of the fatty acid chain due to the encapsulated contents. Less than -29mV are the ideal values for the stability and
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protection of integrity of liposomes form aggregation ideal values for the stability and protection of integrity of liposomes form aggregation (Chibowski and Szcze,
2016).
The results of zeta potential of the current study indicate that the liposomal formulation was stable with no mass aggregation was expected and encapsulation of extract didn’t react with the lipids to create any changes on the surface of liposomes.
4.5.3 Determination of Size using Dynamic Light Scattering (DLS)
Size of prepared formulations of liposomes encapsulating rhizome extracts were determined using dynamic light scattering (DLS) instrument. DLS is based upon the principle of photon light scattering correlation. The angled intensity of light is measured after being passed through the liquid sample which is directly affected by the Brownian motion of the particles present in the sample (Zhang et al., 2014).
The results obtained from tow formulations of 2mg/mL and 4mg/mL and the control depicts that size lie in the range of 140nm to 155nm. Smallest size was obtained with 4mg/mL formulation which was 143±4.58nm as compared to the
2mg/mL formulation which was 155±12.3nm which was statistically insignificant difference as shown in table 7. It could be due to extrusion using polycarbonate membrane filters with minimum size of 100nm. Whereas empty liposomes were of
152±3.52nm size. The size range of about 100nm and less are considered ideal for the drug delivery to the tumors other than hepatic tissues because of EPR effect and
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Figure 14: SEM images of freeze-dried 4 mg/mL liposomes, Top Center: SEM images
of freeze-dried 2 mg/mL Liposomes, Top Right: Freeze dried empty Liposomes;
Bottom left: TEM images of 4 mg/mL liposomes, Bottom Center: TEM images
of 2mg/mL Liposomes, Bottom Right: TEM images of Empty Liposomes
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Table 7. Characterization of stealth liposomal formulations.
Sr Liposomes Size (nm) PDI Zeta Potential (mV)
#
1 2mg/mL 155 ± 12.3 0.09±0.01 -19.8 ± 2.35
2 4mg/mL 143 ± 4.58 0.16±0.05 -16.9 ± 1.04
3 Empty Liposomes 152± 3.52 0.08±0.01 -19.4 ± 0.23
Data represent triplicate values with SEM.
77
Figure 15. Measurement of Zeta Potential of latex beads
78
Figure 16. Measurement of Zeta Potential of empty liposomes
79
Figure 17. Measurement of Zeta potential of 4mg/mL formulation of liposomes
80
Figure 18. Measurement of Zeta Potential of 2mg/mL formulation of
liposomes
81
as very low lymphatic drainage is there for tumor tissues, there are chances of accumulation of the liposomes in to the tumor tissues and enhance drug release in the required tumor site can be achieved. Doxil® has gone through all these challenges before commercialized (Barenholz, 2012).
4.5.4 Estimation of Encapsulation Efficiency
4.5.4.1 Using UPLC
Ultra-performance liquid chromatography is an advanced technique for chromatography to achieve better separation in minimal time and with nominal amount of mobile phase used (Herrero et al., 2010). Ethylene bridged hybrid column i-e BEH C18 Column, 130Å, 3.5 µm, 2.1 mm X 100 mm utilizing the principles of reversed phase. Better UPLC profile was obtained at 273nm and the highest peak was selected as the internal reference control. The calibration curve was obtained using internal reference control within the range of concentrations used in the liposomes i-e 2mg/mL and 4mg/mL by taking the area under the peak as one variable shown in the table 8, figure 19. From the current experiment by loading supernatant of the liposomes in to UPLC we get about 26±0.3% unentrapped content of extract in it. By estimating the relative amount of encapsulation into the liposomes by the calculations of unentrapped extract in supernatant 74% of encapsulation efficiency was obtained using thin film method
(Guilløn and Barroso, 2009; Kenny et al., 2013).
4.5.4.2 Using spectrophotometer
Spectrophotometer was used to calculate the un-entrapped extract in the supernatant obtained from ultracentrifugation of the liposomal formulation by
82
measuring the absorbance at 273nm and making standard calibration curve and finding the unknown concentration in the supernatant shown in figure 20 and table
9, 10 and 11. The results indicate the presence of about 19±0.06% of un-entrapped extract in the supernatant of 4mg/mL formulation. From the above-mentioned percentage of un-entrapped extract, relative entrapped extract was estimated, which is 81% in 4mg/mL formulation and 71% in 2mg/mL formulation (Da Silva, 2015).
Both methods show approximately similar findings with statistically insignificant difference which proves the validity of the experiment.
4.6 IN VITRO ANTICANCER ACTIVITY OF ENCAPSULATED
EXTRACT VS RAW EXTRACT ON HEPG2 CELLS
Anticancer activities of the plant of interest have already been reported against
MCF-7, SMMC-7721, HepG2, Huh-7, HCCLM3, MLF-7 and MDA-MB-435. In the present study, we aimed to encapsulate highly potent ethanolic fraction of highly promising rhizome part of plant into liposomes to check the increment in the anticancer activity of plant before and after encapsulation of extract. In the preliminary studies for screening ethanolic extract of Rhizome was found most promising against HepG2 cells with IC50 of 30µg/mL, after encapsulation the IC50 value was 8.78 and 8.3 for 2mg/mL and 4mg/mL formulations shown in table 12 and figure 21 and 22. Which shows that an increase of 31.6% in anticancer activity of the extract was seen in 72hrs after treatment. Since it has already reported that the molecular targets of the extract in HepG2 cells are STAT 3 proteins which are transcriptional factors through SHP-1 proteins which directs the cancer cells
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towards apoptosis. Along with it in MCF-7 cells it has been recently found that
Amplexicaule A, a flavonoid isolated from n-butanol extract inactivates Akt, downregulates MCL-1 along with BCL-2 expression and activates caspases3, 8 and
9 hence initiating apoptosis (Intisar et al., 2012a; Dong et al., 2014; Xiang et al.,
2015; Xiang et al., 2016).
4.7 EFFECTS OF ENCAPSULATED EXTRACT VS RAW EXTRACT
ON HUVEC ENDOTHELIAL CELLS IN VITRO
Raw and encapsulated extract was also tested on the normal human umbilical vein endothelial cells. It has been shown that the LD50 of the raw extract was 13µg/mL as compared to the LD50 of encapsulated extract in case of 4mg/mL it was 54µg/mL and with 2mg/mL it was 66µg/mL, which shows a protective behavior of the extract towards normal cells as compared to the cancerous cells once encapsulated in to liposomes shown in table 13and figure 23. This could be due to the difference between biochemical status of normal and cancerous cells
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Table 8. Standard calibration curve obtained from UPLC for supernatant
Concentrations of Area
Standard(mg/mL)
4 3652485
3 2705932
2 1803421
1.5 1291975
1 740156
0.5 343445
85
Figure 19. Calibration curve from the data obtained from UPLC for supernatant
86
Table 9. Data represents calculations for the encapsulation efficiency through UPLC
Liposomes Area(AU) conc by Eq (y= mg/mL Final % unentrapped
954070x – 151903) concentration
4mg/mL SupbatchIII 400929 0.58 1.16 28.9
SupbatchII 123350 0.29 0.58 14.4 25.8
SupbatchI 497637 0.68 1.36 34.0
2mg/mL SupbatchIII 26891 0.19 0.37 18.7
SupbatchII 233416 0.18 0.36 18.3 25.8
SupbtachI 22376 0.40 0.81 40.4
87
Figure 20. Calculations for encapsulation efficiency obtained from
Spectrophotometer at 273nm.
88
Table 10. Data represents absorbance readings against standard concentrations for
spectrophotometer
Sr # Sample Concentration(standard) Absorbance
1 0.25 2.81
2 0.125 1.43
3 0.063 0.65 Standard 4 0.031 0.34
5 0.016 0.15
6 0.008 0.0
89
Table 11. Data represents calculations for the encapsulation efficiency through Spectrophotometer
Sample Concentration Absorbanc Concentration Concentration % Un- Mean Percentage
(Spectrophoto e (Conc*DF) (Conc*2) entrapped Entrapped
meter)
4mg/mL supbatchI 0.1729 1.9434 0.5187 1.0374 25.935 19.6±0.1 81
supbatchII 0.1124 1.2529 0.3372 0.6744 16.86
supbatchIII 0.1076 1.1974 0.3228 0.6456 16.14
2mg/mL supbatchI 0.2034 2.2922 0.4 0.8 40 29.2±0.1 71
supbatchII 0.1518 1.7026 0.3036 0.6072 30.36
supBatchIII 0.086 0.9508 0.172 0.344 17.2
HBS 0.0035 0.0082 - - - - -
90
along with it the slow release of the extract to the normal cells and cancer cells which saved the normal cells from sudden burst of the extract which can damage the cells (Liu et al., 2010). It has already been reported that amplexicaule Aisolated from P. amplexicaule synonym B. amplexicaulis show no effects to the normal fibroblasts cells as compared to the breast cancer cells (Xiang et al., 2016). In raw form rhizome extract of the plant affects and damages the normal cells in a way similar to etoposide, doxorubicin and paclitaxel (Mailloux et al., 2001b). Another study depicts the similar results with Dexamethasone encapsulated Vs un- encapsulated against fibroblasts and macrophages showing less cytotoxicity upon liposomal encapsulation (Bartneck et al., 2014).
4.8 PREPARATION OF POLARITY EXTRACTS/FRACTIONS AND IN
VITRO CYTOTOXICITY ASSAY
Four different extracts/fractions in ethanol(REE), methanol(REM), 80% ethanol(80RE), 80% methanol(80RM) and acetone(RAC) of rhizome extract were prepared using the method of Batool et al., 2015 to investigate the highest promising fraction by in vitro cytotoxicity assays using HepG2 cell lines (Batool et al., 2015). The results showed no significant difference among all the tested extracts/fractions with following IC50 values; REE 0.031µg/mL, RAC 0.038µg/mL,
80RE 0.035µg/mL, 80RM 0.038µg/mL respectively shown in figure 24 and table
14. This may relevant to that fact that the solvents did not vary much in terms of polarity which is an important factor in extraction of phenolic, types of solvents with similar polarity extracts almost similar phenolic compounds which could be simple or complex polymers associated with carbohydrates and proteins (Dai and
Mumper, 2010).
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4.8.1 UPLC Profiling of Extracts/Fractions
To validate the results obtained from cytotoxicity profiling of extracts/fractions, the UPLC profiling was designed using the C18 columns with binary phase mobile system already mentioned. Almost similar compounds profile was obtained with all the fractions with slight peak shifts which indicates that the similar compounds were extracted using different solvents which were ethanol, methanol, acetone, 80% ethanol and 80% methanol respectively shown in figure 25
(Sasidharan et al., 2011).
4.9 DETERMINATION OF FLUORESCENCE FOR POLARITY
EXTRACTS/FRACTIONS OF REE
Fluorescence of prepared fractions of rhizome extract i-e REE, RAC, REM,
80RE and 80RM was determined using flourimetry. The highest intensity of fluorescence was detected in REE and the rest of all the fractions shows insignificant difference between the intensity of fluorescence since the class of compounds phenolics contains ringed structures of the compounds which shows absorption of specific wavelength of light upon excitation and emit the light later as emission spectra. The compounds present in the Rhizome extract shows excitation at 394 and emission at 421nm shown in figure 26-29 (Ferreira et al.,
2015;Talamond et al., 2015). This study warrants further research upon the fluorescence properties of the extract so that it may be used as autoflourescent in further cell based assays (Drabent et al., 1999; Dinoiu et al., 2011).
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Table 12. Cytotoxicity of encapsulated and un-encapsulated extract against HepG2 cell lines
Concentration 2mg/mL1 2mg/mL2 2mg/mL3 Extract 4mg/mL1 4mg/mL2 4mg/mL3 Extract
(µg/mL)
35 105.1±5.6 86.3±11.5 86.1±19.6 74.4±10.2 90.0±17.6 99.5±13.6 99.0±15.7 79.7±11.9
25 109.9±3.7 91.3±9.4 92.9±11.7 93.9±6.3 78±3.9 99.5±10.7 95.5±8.2 99.3±4.0
15 93.4±10.4 92.8±4.2 76.3±16.3 97.6±5.7 78.9±10.3 99.5±9.1 65.5±7.8 96.6±5.1
7 76.9±18.2 73.7±12.8 54.4±10.5 91.7±5.7 89.6±11.2 99.5±9.1 59.6±5.2 92.8±6.1
0 100±0 100±0 100±0 100±0 100±0 100±0 100±0 92.8±0
IC50 9.8 8.76 7.74 27.74 6.2 8.8 9.83 27.6
MeanIC50 8.78 27.74 8.3 27.6
Data is represented in mean ± SEM (n = 3)
93
150 4mg/ml1 4mg/ml2 100 4mg/ml3 Extract
) 50
%
(
y
t
i
l
i
b
a
i
v 0 5 5 5 7 0 3 2 1 Concentration (mg/mL)
Figure 21. Graphical representation of cytotoxicity of encapsulated and un-
encapsulated extract against HepG2 cell lines. Data is represented in
mean ± SEM (n = 3)
94
150 2mg/ml1 2mg/ml2 100 2mg/ml3 Extract
) 50
%
(
y
t
i
l
i
b
a
i
v 0 5 5 5 7 0 3 2 1 Concentration (mg/mL)
Figure 22. Graphical representation of cytotoxicity of encapsulated and un-
encapsulated extract against HepG2 cell lines. Data is represented in
mean ± SEM (n = 3)
95
Table 13. Cytotoxicity of encapsulated and un-encapsulated extract against HUVEC
cell lines.
Concentration 4mg/mL REE 2mg/mL REE
(µg/mL)
128 43.4±5.4 6.1±1.7 34.1±2.8 16.8±1.3
64 31.7±5.6 9.4±0.7 49.4±7.3 18.3±1.3
32 53.2±3.2 5.8±0.6 43.8±4.4 3.5±0.1
16 61.0±3.3 9.2±1.7 39.9±14.1 6.8±1.8
0 100±0 100±0 100±0 100±0
LD50 65.9 13.1 54.0 12.9
Data is represented in mean ± SEM (n = 3)
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HUVEC Liposomes
150 4mg/mL 2mg/mL 100 REE
) 50
%
(
y
t
i
l
i
b
a
i
v 0
28 64 32 16 0 1 Concentration (mg/ml)
Figure 23. Graphical representation of cytotoxicity of encapsulated and un-
encapsulated extract against HUVEC cell lines. Data is represented in
mean ± SEM (n = 3)
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Table 14. Percentage viability and IC50 of fractions of crude extract of B.
amplexicaulis.
Concentrations REE RAC 80RE 80RM
mg/mL
4 9±0.6 19.2±5.2 21±3.5 19.5±3.8
2 3.5±0.3 6.2±1.1 8.8±1.4 3±0.6
1 0.28±0.1 1.4±0.1 3.97±2.3 1.07±0.4
0.5 21.7±2.3 0.7±0.2 1.35±0 0.6±0.4
0.25 14.7±2.4 0.77±0.2 1±0 0.67±0.2
0.125 10.3±±1.2 0.47±0.1 0.8±0 0.37±0.2
0.0625 6±2.3 5.17±0.7 12.7±0.1 12±0
0.03125 23.7±0.3 56.5±1.4 67±0.6 68±0
0.0156 40.3±2.8 84±2.3 100±0 68±3.3
0 100±0 100±0 100±0 68±0
IC50 0.031 0.038 0.035 0.0
Data is represented in mean ± SEM (n = 3). Rhizome extract ethanolic (REE), RAC
(Rhizome extract in acetone, 80RE(80%vethanolic extract), 80RM(80% methanolic extract)
98
F r a c tio n s o f R h iz o m e E x tr a c t(H e p G 2 )
1 5 0 R E E
R A C
1 0 0 8 0 R E
) 8 0 R M
%
(
y t
i 5 0
l
i
b
a
i
v
0
0 6 5 5 5 5 .5 1 2 4 5 2 2 2 .2 0 1 1 6 .1 0 .0 3 .0 0 0 .0 0 0 C o n c e n tr a tio n ( m g /m L )
Figure 24. Graphical representation of percentage viability and IC50 of fractions of
crude extract of B. amplexicaulis. Data shows that there is no remarkable
difference among the IC50 obtained from each fraction. IC50 REE
0.031µg/mL, IC50 RAC 0.038µg/mL, IC50 80RE 0.035µg/mL, IC50 80RM
0.038µg/mL respectively. Data represents n=3 with SEM
99
Figure 25. UPLC profile of ethanolic extract of rhizome of B. amplexicaulis; BEH C18
Column, 130Å, 3.5 µm, 2.1 mm X 100 mm (Waters). Mobile phase:
Solvent A was 94.9% water: 5%ACN:0. 1 of Formic Acid, solvent B was
98.9% ACN: 1% water:0.1 % formic Acid. Flow rate: 0.45 mL/min. Sky
blue: 80RE, Green:80RM Pink: REE, Blue : RAC. Data shows that there
are only peak shifts but no remarkable difference was found among the
compounds profile.
100
Figure 26. Fluorescence intensity of Rhizome extract ethanolic fraction (REE). Blue
line shows emission and orange line shows excitation.
101
Figure 27. Fluorescence intensity of acetone fraction of rhizome (RAC). Blue line
shows emission and orange line shows excitation.
102
Figure 28. Fluorescence intensity of 80% methanol fraction of rhizome (80RM). Blue
line shows emission and orange line shows excitation.
103
Figure 29. Fluorescence intensity of 80% ethanol fraction of rhizome (80RE). Blue
line shows emission and orange line shows excitation.
104
SUMMARY
Current study was designed to investigate the liposomal encapsulation of ethanolic extract of B. amplexicaulis to improve its in vitro anticancer efficacy. Plant collection was done from Miran Jani Tract, Nathia Galli Abbottabad and Murree Hills,
Rawalpindi Pakistan which was shade dried later. Initially methanolic and ethanolic extracts were prepared from rhizome, leaf and shoot extract of plant namely; rhizome
Extract methanolic (REM), Shoot extract methanolic (SEM), Leaf extract methanolic
(LEM), Rhizome extract ethanolic (REE), Shoot extract ethanolic (SEE) and Leaf extract ethanolic (LEE). Preliminary screening for anticancer activities was performed with Human colon carcinoma cell lines HCT-116 and REE was found most potent.
The IC50 obtained REE against HCT-116 was 0.8mg/mL with percentage extraction yield of 20% compared to Gallic Acid (IC50 0.8mg/mL) which was used as standard.
Based on preliminary screening, the highly promising REE was further analyzed for anticancer activity against breast cancer cell line i-e MCF-7 and
Hepatocellular carcinoma cell line HepG2 cells and gallic acid was used as positive control. It was found that the REE shows highest activity against HepG2 cell lines with
IC50 of 30µg/mL with similar activity obtained using gallic acid whose IC50 was
32µg/mL. Whereas against MCF-7 cells, the REE showed IC50 67µg/mL which was found less potent than gallic acid with IC50 of 53µg/mL.
The REE was further subjected to UPLC profiling to identify the phenolic compounds that may attribute anticancer potential to plant. Fourteen standard
104
105
compounds were tested for qualitative analysis in REE and four compounds were found in the extract i-e gallic acid @0.8min, Caffeic acid @1.3mins, Chlorogenic acid
@2.8min, Epicatechin @3.79mins and catechin @2.5mins. Along with it concentration based stability of UPLC profile was also determined and REE was found stable at different concentrations ranging 0.5mg/mL to 5mg.mL.
The REE was encapsulated into liposomes using two different methods i-e fusion method and thin layer method using egg phosphatidylcholine and cholesterol with 4mg/mL and 10mg/mL loading. Liposomes prepared from thin layer method were initially characterized and found to be of optimal size with good polydispersity index.
The liposomal size with 10mg/mL extract loading was 153.5±0.73nm but in the case of 4mg/mL extract loading the size was 677.2±34.3nm with the PDI of 0.28±0.007 and
0.6±0.033 respectively.
Stealth liposomes were made by thin film method encapsulating 4mg/mL and
2mg/mL of extract with DPPC, PEG2000DSPE and cholesterol in the ratio of
1.85:0.15:1 with zeta potential of -19.8±2.35 for 2mg/mL formulation and for 4mg/mL formulation -16.9±1.04 as compared to empty liposomes with zeta potential -19.4 ±
0.23 which were used as control. Size was measured using DLS and smallest size was obtained with 4mg/mL formulation which was 143±4.58nm as compared to the
2mg/mL formulation which was 155±12.3nm and statistically insignificant difference was observed with the size of empty liposomes (152±3.52nm). Surface morphology of
106
liposomes was determined using SEM and TEM and uniform size and distribution was observed.
Relative Encapsulation efficiency was estimated using UPLC and spectrophotometer. With UPLC about 74% encapsulation efficiency was achieved with both 2mg/mL and 4mg/mL formulations. Whereas with spectrophotometer about 81% encapsulation efficiency was estimated for liposomes with 4mg/mL extract loading and 71% for 2mg/mL extract loading.
Encapsulated extract showed significantly good in vitro efficacy with the IC50 of about 8µg/mL with both formulations i-e 2mg/mL and 4mg/mL extract loading as compared to un-encapsulated extract with IC50 of about 30µg/mL. Whereas in case of normal endothelial cells (HUVEC), the liposomal encapsulation of extract was proven protective to normal cells (LD50 54-65.9 µg/mL) as compared to the raw extract which was highly cytotoxic with LD50 of about 13µg/mL. This may be attributed to the difference between biochemical status of normal and cancerous cells along with the slow release of the extract to the normal cells than the cancer
Furthermore, four different polarity extracts/fractions i-e REE for absolute ethanolic extract, 80%RE for 80% ethanolic extract, 80%RM for 80% methanolic extract and RAC for acetone extract were prepared and their cytotoxicity and UPLC profiling was accomplished. There was no significant difference found between the different polarity extracts/fractions of rhizome extract in terms of cytotoxicity against
107
HepG2 cells which was further validated through UPLC profiling where no significant difference between the chromatogram patterns was seen.
So, concluding the present research the Rhizome extract ethanolic (REE) from
B. amplexicaulis is very promising against HepG2 cell line with IC50 of 30µg/mL.
Liposomes of 140-155nm with PDI of 0.09 were successfully prepared by Thin film method. Liposome encapsulation enhanced the delivery of extract with 30 % increase in cytotoxicity against epG2 cell lines. Extract and polarity fractions have fluorescence properties as well with excitation wavelength of 349nm and emission wavelength of
421nm, out of which maximum intensity of fluorescence was shown by ethanolic fraction of rhizome extract. For HUVEC cells (primary cell), the liposomal encapsulation of extract was shown protective, which means that liposomal encapsulation reduces the toxicity of extract to normal cells. Hence, Nano-liposomal encapsulation could be used as a mean of targeting the B. amplexicaulis rhizome extract to the cancer cells to enhance its anticancer potential avoiding cytotoxicity to the normal cells. To our knowledge this is the first report of the liposomal encapsulation of an extract, enhanced uptake of extract and an improved activity against hepatocellular carcinoma (HepG2). We therefore suggest further investigations upon the cellular targets of the extract in HepG2 and MCF-7 cancer cell lines which could be through caspases or STAT signaling pathways and cellular toxicity to be investigated on a broader range of concentrations.
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