Thq) Coupled with Poly (Lactide-Co-Glycolide) Nanoparticles

ASSESSMENT OF ANTI-PROLIFERATIVE AND ANTI- INFLAMMATORY POTENTIAL OF THYMOQUINONE (TQ) AND THYMOHYDROQUINONE (THQ) COUPLED WITH POLY (LACTIDE-CO-GLYCOLIDE) NANOPARTICLES

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AYESHA SIDDIQUE BUTT

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DEPARTMENT OF ENVIORNMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2018 ASSESSMENT OF ANTI-PROLIFERATIVE AND ANTI- INFLAMMATORY POTENTIAL OF THYMOQUINONE (TQ) AND THYMOHYDROQUINONE (THQ) COUPLED WITH POLY (LACTIDE-CO-GLYCOLIDE) NANOPARTICLES

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A THESIS SUBMITTED TO LAHORE COLLEGE FOR WOMEN UNIVERSITY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENVIRONMENTAL SCIENCE

By AYESHA SIDDIQUE BUTT

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DEPARTMENT OF ENVIRONMENTAL SCIENCE LAHORE COLLEGE FOR WOMEN UNIVERSITY, LAHORE 2018

CERTIFICATE

This is to certify that the research work described in this thesis submitted by Ms. Ayesha Siddique Butt to Department of Environmental Science, Lahore College for Women University has been carried out under my direct supervision. I have personally gone through the raw data and certify the correctness and authenticity of all results reported herein. I further certify that thesis data have not been used in part or full, in a manuscript already submitted or in the process of submission in partial fulfillment of the award of any other degree from any other institution or home or abroad. I also certify that the enclosed manuscript has been prepared under my supervision and I endorse its evaluation for the award of Ph.D. degree through the official procedure of University.

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Dr. Numrah Nisar Prof. Dr. Tahira Aziz Mughal Dr. Nadia Ghani Supervisor Co-Supervisor Co-Supervisor

Date: Date: Date:

Verified By

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Prof. Dr. Tahira Aziz Mughal

Chairperson

Department of Environmental Science

Stamp

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Controller of Examination

Stamp

Date: ______AUTHOR’S DECLARATION

I Ayesha Siddique Butt hereby state that My Ph.D. Thesis titled “Assessment of Anti- Proliferative And Anti-Inflammatory Potential Of Thymoquinone (TQ) And Thymohydroquinone (THQ) Coupled with Poly (Lactide-Co-Glycolide) Nanoparticles” is my own work and has not been submitted previously by me for taking any degree from this university (Lahore College for Women University) or anywhere else in the country world. At any time if my statement is found to be incorrect even after my Graduate the university has the right to withdraw my Ph.D. degree.

Ayesha Siddique Butt

Date: ______

PLAGIARISM UNDERTAKING

I solemnly declare that research work presented in the thesis titled “Assessment of Anti-Proliferative and Anti-Inflammatory Potential of Thymoquinone (TQ) And Thymohydroquinone (THQ) Coupled with Poly (Lactide-Co-Glycolide) Nanoparticles” is solely my research work with no significant contribution from any other person. Small contribution/help wherever taken has been duly acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and University (Lahore College for Women University, Lahore) towards plagiarism. Therefore, I as an author of the above titles thesis declare that no portion of my thesis has been plagiarized and any material used as references is properly referred/ cited.

I undertake that if I am found guilty of any formal plagiarism in the above titled thesis even after award of Ph.D. Degree, the University reserves the rights to withdraw/revoke my Ph.D. degree and that HEC and the university has the right to publish my name on the HEC/University website on which names of students are placed who submitted plagiarized thesis.

Ayesha Siddique Butt

DEDICATION

I would like to dedicate this academic endeavor to

My Beloved Parents

Mr. & Mrs. Mohammad Siddique Butt

For their Prayers, Unconditional love and persistent support during my hard times

My Life Partner

Eng. Kamil Mahmood Butt

For his support, affection and patience to letting my dream come true

My Souls

Hareem, Iman, Muzammil and Ibrahim

For their deep love, Endless support, prayers and space to live my dream with all my motherly duties

ACKNOWLEDGMENTS

“He who taught (the use of pen), taught man which he knows not”

All praises and thank to Almighty Allah, only who owns the power to make thing easy and task successful for us. Without countless blessings of Allah I would not have been able to fulfill this difficult work. I pray that I and my loved ones always remain in the care and blessings of Allah. I would like to acknowledge Prof. Dr. Farkhanda Manzoor, Vice Chancellor Lahore College for Women University, Lahore (LCWU) for providing me opportunity and facilities for the accomplishment of my PhD degree. My sincere gratitude is to Prof. Dr. Shagufta Naz, Director Research, LCWU, for providing research facilities and her kind support throughout my research work. My highest acknowledge is towards Prof. Dr. Tahira Aziz Mughal, Controller Examination and Chairperson of Department of Environmental Science, LCWU, for her continuous support and efforts in solving academic as well as research related issues to complete this task. I sincerely express my thanks to Prof. Kausar J. Cheema and Prof. Dr. Arifa Tahir, Ex-Chairperson of Department of Environmental Science, LCWU, for their all intellectual support. My sincerest thanks are due to my supervisor Dr. Numrah Nisar, Assistant Professor, Department of Environmental Science, LCWU, for her magnanimous guidance, kind support and help in my research and completion of my PhD thesis. I am obliged to my co-supervisor Prof. Dr. Tahira Aziz Mughal, for her continuous selfless involvement, guidance and encouragement, despite of her busy schedule. I also express my thanks Dr. Nadia Ghani, Assistant Professor, Department of Environmental Science, LCWU, for her supportive co-supervision. I also thank all the staff members of Department of Environmental Science, LCWU, for their continuous encouragement to complete this task. I acknowledge the supports provided by Mr. Ali, Mr. Saeed, Mr. Adnan, Mr. Saleem and all other laboratory staff during my research work. I feel grateful to Dr. Atif Islam, Associate Professor, Department of Polymer Institute of Chemical Engineering, University of Punjab (PU), for providing research facilities and his kind guidance in my research work. I also acknowledge to Dr. Imran Altaf, Assistant Professor, Quality Operation Lab, University of Veterinary and Animal Sciences (UVAS) for providing laboratory facilities and support. I express my sincere thanks to my companions Mrs. Faiza Butt, Miss. Houda Javed, Mrs. Madiha Aftab and Miss. Misbah Ghulam Raool, for their supports and suggestions during all my research work and thesis write-up. I feel heartily indebted to my tremendously affectionate and adoring parents, Mr. and Mrs. M. Siddique Butt for giving me such confidence and care. They always comforted me in my tensions and encourage me to move forward. They were always there to looking after my kids. I would not able to complete this work without their prayers and loads of care for me and for my children. I have no words to convey my special thanks for my husband Eng. Kamil Mahmood Butt, for whatever he did and doing for me. My loads of love for my children, my soul Hareem, Iman, Muzammil and Ibrahim, may you all live long healthy life (ameen). I would love to express my sincere thanks to my In-laws especially mother- in-law Mrs. Nasir Butt for her prayers and my sister-in-law Gul-e-Rana for her continuous care and help whenever I needed. I really appreciate her selfless love and attention for my children. I deeply acknowledge all of them for being so enduring and understanding. I want to express my deep love and thanks to my sisters Faiza Butt, Dr. Saira Butt, Zainab Butt and Eng. Fatima Butt for their continuous support in all my worries. My express my affection and appreciation for my dear brother Mr. Abdul Rehman Butt, who is always been there with all his support, whenever I looked upon him.

As a final point, I acknowledge all of my well-wishers who have helped me directly or indirectly in the successful completion of my work. I also show gratitude to anyone who missed mistakenly in this acknowledgement.

Ayesha Siddique Butt CONTENTS

List of Tables…………………………………………………………………………………….i List of Figure……………………………………………………………………………………iii List of Abbreviation…………………………………………………………………………….vii Abstract…………………………………………………………………………………………xii Chapter 1: Introduction…………………………………………………………………………..1 1.1. Aims and Objectives……………………………………………………………………….11 Chapter 2: Review of Literature……………………………………………………………...... 12 Chapter 3: Materials and Methods……………………………………………………………..36 3.1. Collection, Preparation and Storage of Samples…………………………………………..36 3.2. Reagents and Chemicals…………………………………………………………………...37 3.3. Extraction, Quantification and Purification of TQ and THQ from N. sativa and T. vulgaris……………………………………………………………………………………....38 3.3.1. Extraction of Essential Oils of N. sativa and T. vulgaris………………………………..38 3.3.2. Identification of TQ and THQ…………………………………………………………...38 3.3.3. Quantification of Extracted TQ……………………………………………………….....39 3.3.4. Purification and Confirmation of TQ……………………………………………………39 3.4. Extraction and Characterization of Pectin from Citrus Waste Peels………………………40 3.4.1. Extraction Method Standardization of Pectin……………………………………………40 3.4.1.1. Conventional Heating Method…………………………………………………………40 3.4.1.2. Soxhlet Extraction Method………………………………………………………….....40 3.4.2. Characterization of Extracted Pectin………………………………………………….....41 3.4.2.1. Confirmatory Tests for Pectin………………………………………………………....41 3.4.2.2. Pectin Yield (%)……………………………………………………………………….41 3.4.2.3. pH……………………………………………………………………………………...41 3.4.2.4. Moisture Content (%)………………………………………………………………….41 3.4.2.5. Equivalent Weight Determination……………………………………………………..42 3.4.2.6. Methoxyl Content Determination……………………………………………………...42 3.4.2.7. Viscosity Average Molecular Weight………………………………...……………….42 3.4.2.8. FTIR Analysis………………………………………………………...……………….43 3.5. Synthesis of Pectin-PLGA Hydrogel and Loading of TQ/THQ PLGA Nanoparticles…………………………………………………………………………………...43 3.5.1. Synthesis of Pectin Hydrogels………………………………………………………….43 3.5.1.2. Series 1…………………………………………………………………………………43 3.5.1.3. Series 2…………………………………………………………………………………44 3.5.2. Preparation of novel cross-linked Pectin-PLGA-PEG (PPP)…………………………….45 3.5.3. Drug Loading in Pectin Hydrogel………………………………………………………..45 3.5.4. Preparation of TQ loaded PLGA-PEG Nanoparticles in Pectin Hydrogel (PPPT-NPs)……………………………………………………………………………………..45 3.6.Characterization of Synthesized Novel Pectin Hydrogels and Loaded Nanoparticles……………………………………………………………………………………45 3.6.1. Swelling Experiment of Synthesized Pectin Hydrogels………………………………….46 3.6.2. FTIR Analysis……………………………………………………………………………46 3.6.3. Thermal Gravimetric Analysis (TGA-DSC)……………………………………………..46 3.6.4. X-Ray Diffraction (XRD) Analysis……………………………………………………...47 3.6.5. Scanning Electron Microscopy (SEM)…………………………………………………..47 3.6.6. Drug Loading (TQ/THQ) Efficiency in Pectin hydrogel and Nanoparticles…………….47 3.6.7. In-Vitro Drug Release from PPP and PPP-NPs Loaded Hydrogels……………………...48 3.6.7.1. Preparation of simulated solutions……………………………………………………..48 3.6.7.2. Drug Release in SGF and SIF………………………………………………………….48 3.7. In-vitro Anti-inflammation and Anti-proliferation Activities of TQ/THQ Loaded PLGA Nanoparticles…………………………………………………………………...48 3.7.1. Anti-Inflammatory Activities…………………………………………………………….48 3.7.1.1. Ferric Reducing Antioxidant Power (FRAP) Assay…………………………………...48 3.7.1.2. DPPH Scavenging Activity…………………………………………………………….49 3.7.2. Anti-Proliferative Activities…………………………………………………………….49 3.7.2.1.Anti-proliferation against HeLa by MTT assay………………………………………...49 3.7.2.2. Anti-Proliferative Activities of Synthesized Hydrogel and Nanoparticles…………….51 3.7.2.3. Anti-proliferative activities against HCT116 by Sulforhodamine B (SRB) assay…….52 3.7.2.4. Western Blot Analysis PARP Apoptosis induction of TQ against HCT116………….54 3.8. Statistical Analysis………………………………………………………………………..54 Chapter 4: Results………………………………………………………………………………56 4.1. Extraction, Quantification and Purification of TQ………………………………………...52 4.1.1. Weight of Extracted Oil………………………………………………………………...52 4.1.2. Identification of TQ…………………………………………………………………….53 4.1.3. Quantification of TQ…………………………………………………………………...55 4.1.4. Purification & FTIR analysis…………………………………………………………..55 4.2. Extraction of Pectin, Confirmation and Characterization………………………………...57 4.3. Synthesis and Characterization of Pectin-PLGA Hydrogel and its Nanoparticles………..61 4.3.1. Optimization of Pectin Hydrogels………………………………………………………..61 4.3.1.1. Series 1…………………………………………………………………………………61 4.3.1.2. Series 2…………………………………………………………………………………62 4.3.2. Preparation of novel cross-linked Pectin-PLGA-PEG (PPP)……………………………63 4.4. Anti-inflammation Activities by FRAP and DPPH Assay………………………………...76 4.5. Anti-Proliferative Activities by MTT assay against HeLa cancer cell lines………...... 79 4.6. Anti-Proliferative Activities SRB Assay against HCT16 Cancer cell lines……………….88 4.5. Western Blot Analysis……………………………………………………………………...91 Chapter 5: Discussion………………………………………………………………………….105 References……………………………………………………………………………………..125 Annexures……………………………………………………………………………………...xiii Plagiarism Report……………………………………………………………………………....xl List of Publications and reprints……………………………………………………………….xli

LIST OF TABLES

1. Table 1.1: Taxonomic classifications of N. sativa and T. vulgaris……...... 4

2. Table 2.1: Summary of Different Extraction Techniques for TQ and THQ for Their Potent Activities………………………………………………………..14

3. Table 2.2: Summarizing Sources of Pectin Extracting from Different Methods in Literature…………………………………………………………………..29

4. Table 2.3: Types of Phytochemicals and Pharamceutical Drugs with Different Type Drug Carrier………………………...………………………………….32

5. Table 2.4: Studies of PLGA-NPs Loaded Different Drugs in Literature….....34

6. Table 3.1: Number of groups with different treatment of TQ against HeLa cancer cell lines………….…………..……………………………………….51

7. Table 3.2: Treatment groups of TQ loaded Hydrogel and Nanoparticles against HeLa Cancer Cell Lines……………………………………………………...52

8. Table 3.3: Treatment Groups of TQ against HCT116 cancer cell lines……...53

9. Table 4.1: Calculated mass of extracted oil from N. sativa and T. vulgaris with different solvent (H:M) mixtures. Data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 where p < 0.05….…………………………………………………….………………….57

10. Table 4.2: RT obtained from the HPLC chromatogram for each samples...... 60

11. Table 4.3: Amount of quantified TQ from N. sativa and T. vulgaris. Data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 where p < 0.05……………………………………………………..………..60

12. Table 4.4: Physical appearance and confirmatory tests for pectin extracted from citrus waste peel. Data was collected in triplicates...……………………………..…………………………………….65

13. Table 4.5: Comparative Summary of Pectin Extracted from Conventional Heating and Soxhlet Extraction Methods. All data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 where p < 0.05……………………………………………………………………….…..66

14. Table 4.6: Characterization of Commercial Pectin and Extracted Pectin from Citrus Waste Peels by Soxhlet Method……………….……………………...67

15. Table 4.7: IC50 of HeLa Cancer Cells Treated With Curative Group By MTT Assay After 24 And 48h Incubation. The Data was Collected in Triplicates and Determined by Non-Linear Regression Graph Pad Prism V.8…………………………………………………………………………..102 ii

16. Table 4.8: Cell proliferation (%) of HCT116 cancer cells treated with dilutions of Cisplatin (E15). The Data was Collected in duplicates and Represented by (Mean±SEM) Graph Pad Prism V.8………………………………………...106

17. Table 4.9: IC50 of HCT116 Cancer Cells Treated With Curative Group By SRB Assay After 72 h Incubation. The Data was Collected in Duplicates and Determined by Non-Linear Regression Graph Pad Prism V.8…………………………………………………………………………..107

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LIST OF FIGURES

1. Figure 1.1: Transition conversion of thymol to THQ and TQ………..………..3

2. Figure 1.2: Structure of pectin (Source: Chemdoodle)..……..…………...... 7

3. Figure 1.3: Structure of PLGA……………..……………………………….....8

4. Figure 1.4: TQ and THQ encapsulated in PLGA-PEG NPs…………..………9

5. Figure 1.5: Summary of current study………….…………………………….10

6. Figure 2.1: Therapeutic Activities of TQ (Source: Juthika et al., 2014; Effenberger et al., 2010; Effenberger et al., 2010; Kaseb et al., 2007; Khan and Sultana, 2005)……………………...…………………………………….22

7. Figure 2.2: Therapeutic Activities of THQ (Source: Majdalawieh et al., 2016; Ivankovic et al., 2006)………………………………………………………..23 8. Figure 4.1: HPLC chromatogram representing RT peak of (A) TQ std (B) N. sativa (C) T. vulgaris where x-axis Time (min) and y-axis absorbance (AU)…………………………………………………………………………..59 9. Figure 4.2: HPLC chromatogram representing RT (5.58 min) peak of purified TQ from N. sativa……….…………………….……………………………...61

10. Figure 4.3: IR spectrum of purified TQ from N. sativa. Data was represented by software Origin v.8……………………………………………..…………62 11. Figure 4.4: Extracted pectin from each citrus peel after alcoholic precipitation in hot acid samples from soxhlet extraction ………………..………………..64 12. Figure 4.5: FTIR Spectra of Commercial pectin and Extracted Orange Peel Pectin. Data was represented by software Origin v.8…………..……...……..68 13. Figure 4.6.: SEM (ZEISS LS 10, LCWU) Images of (A) Commercial Citrus Pectin Powder (B) Extracted Pectin Powder from Orange Waste Peels ………………………………………………………………………………..69 14. Figure 4.7: Swelling (g/g) of Pectin Hydrogel with Different MW of PEG (300, 600, 1500 And 6000) in Distilled Water in Time (min). Data represented by Software Origin v.8.…………………………………………...………….70 15. Figure 4.8: Swelling (g/g) in Distilled Water of Pectin Hydrogel with different Molar Concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin- PLGA-PEG (PPP) hydrogel in time (min). Data represented by Software Origin v.8…………………………………………………………………….71 16. Figure 4.9: Swelling (g/g) in Different pH medium (2, 4, 6, 7 and 10) of Pectin Hydrogel with Different Molar concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEG (PPP) hydrogel in time (min). Data represented by Software Origin v.8…………………………………………..72 iv

17. Figure 4.10: Swelling (g/g) in different molar concentrations of NaCl (0.2, 0.4, 0.6, 0.8 and 1) of pectin hydrogel with different molar dilutions of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEEG (PPP) hydrogel in time (min). Data represented by Software by Origin v.8……………………….....73 18. Figure 4.11: Swelling (g/g) in Different Molar Concentrations of CaCl (0.2, 0.4, 0.6, 0.8 and 1) of Pectin Hydrogel with Different Molar Concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEG (PPP) Hydrogel in time (min). Data represented by Software Origin v.8…………..73 19. Figure 4.12: IR Spectra of Synthesized Pectin Hydrogel with Pectin-TQ, Petin-0.75 M TEOS, Pectin-PEG (6000), Pectin-PLGA-PEG (6000)-TQ (PPPT), PPPT nanoparticles (PPPTNPs), TQ, PLGA and Citrus Orange Pectin. Data represented by Software Origin v.8………………………….....75 20. Figure 4.13: Control Release of Drug TQ Loaded Pectin-PLGA-PEG-TQ (PPPT) Hydrogel in SGF and SIF Medium. Data represented by Software Origin v.8……………………………………………………………………..76 21. Figure 4.14: Control Release of Drug TQ in Pectin-PLGA-PEG-TQ nanoparticles (PPPT-NPs) Hydrogel in SGF and SIF Medium. Data represented by Software Origin v.8…………………………………………..77 22. Figure 4.15: TGA of Different Concentrations of TEOS in Pectin Hydrogel and Synthesized Pectin-PLGA-PEG (PPP). Data represented by Software Origin v.8……………………………………………………………………..79 23. Figure 4.16: DSC of different concentrations of TEOS in pectin hydrogel and synthesized PPP and PPP-NPs. Data represented by Software Origin v.8…..79

24. Figure 4.17: XRD diffraction of Different Concentrations of TEOS in Pectin Hydrogel and Synthesized Pectin-PLGA-PEG (PPPT) and PPPT-NPs. Data represented by Software Origin v.8…………………………………………..80

25. Figure 4.18: SEM (ZEISS LS 10, LCWU) image of simple Pectin (2%) Hydrogel...……………………………………………………………………81 26. Figure 4.19: SEM Image of Pectin Hydrogel with (A) 0.25M TEOS and (B) 0.50 M TEOS at magnification of 200KX…………………………………...82 27. Figure 4.20: SEM Image of Pectin Hydrogel with 0.75 M TEOS (A) at magnification of 100KX (B) at magnification of 300KX……………………82 28. Figure 4.21: SEM (ZEISS LS 10, LCWU) Image Of Pectin Hydrogel with 1.0 M TEOS……………………………………………………………….....83 29. Figure 4.22: SEM Image of Pectin Hydrogel Loaded with PLGA-PEG 0.75 M TEOS (A) at magnification 500X (B) magnification of 100KX…………....84 30. Figure 4.23: SEM (JEOL JSM-6480, CASP) Image Of Pectin Hydrogel Loaded with PLGA-PEG-TQ (PPPT) (A) at Resolution of 50 µm (B) at Resolution showing 1 µm………………………………………………….....85 v

31. Figure 4.24: SEM (JEOL JSM-6480, CASP) Image of Nanoparticles of PLGA-PEG-TQ at Resolution of 50µm……………………………………...86 32. Figure 4.25. SEM (JEOL JSM-6480, CASP) Images showing the Size of Nanoparticles of PLGA-PEG-TQ at Resolution of 1 µm……………………87 33. Figure 4.26: SEM (JEOL JSM-6480, CASP) Image of PLGA-PEG-TQ of Resolution at 0.5µm showing size less than 200 nm of particles………….....88 34. Figure 4.27: SEM (JEOL JSM-6480, CASP) image showing PLGA-PEG-TQ NPs loaded in Pectin Hydrogel at Resolution of (a) 50 µm (b) 1µm………...89 35. Figure 4.28: Inhibition Activity (%) Of Standard TQ of Different Concentration (800, 600, 400, 200 and 100µg/mL) and Extracted TQ in Oil of N. sativa and T. vulgaris by FRAP assay. The Data was Represented by Graph pad prism V.8………………………………………………………………...90 36. Figure 4.29: Inhibition Activity (%) of Standard TQ and Extracted TQ N. sativa and T. vulgaris By DPPH Assay. Data was represented By Graph Pad Prism V. 8…………………………………………………………………….91 37. Figure 4.30: DPPH Assay Scavenging Activities (%) of Pectin Hydrogels blends with PLGA-PEG-TQ (PPPT) and PLGA-PEG-TQ (PPPT-NPs). Data Was Collected In Duplicate and Represented (Mean±SEM) By Software Origin V. 8……………………………………………………………………92 38. Figure 4.31: Average Cell Proliferation Ratio (%) of HeLa Cancer Cells Treated With Experimental/ Control Groups After 48h Incubation. Data Was Collected In Triplicates and Represented By (Mean±SEM) Software Graph Pad Prism V.8………………………………………………………………...93 39. Figure 4.32: Cell proliferation (%) of HeLa Cancer Cells Treated with all Groups after 48 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism v.8………………94 40. Figure 4.33: Cell Proliferation (%) of Each Group in Triple Fold Dilution against HeLa Cancer Cells after 48 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8……………………………………………………………………………95 41. Figure 4.34: Average Cell Proliferation Ratio (%) of HeLa Cancer Cells Treated with Experimental/ Control Groups after 24 h Incubation. Data was collected in Triplicates and Represented By (Mean±SEM) Software Graph Pad Prism V.8………………………………………………………………...96 42. Figure 4.35: Cell Proliferation (%) of HeLa Cancer Cells Treated with all Groups after 24 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8……………...97 43. Figure 4.36: Cell Proliferation (%) Of Each Group in Triple Fold Dilution against HeLa Cancer Cells after 24 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8..…………………………………………………………………………..98 vi

44. Figure 4.37: Average Cell Proliferation Ratio (%) of HeLa Cancer Cells Treated with Experimental/ Control Groups after 48 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8……………………………………………………………………..99 45. Figure 4.38: Cell Proliferation (%) of HeLa Cancer Cells Treated With All Groups after 48 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8…………….100 46. Figure 4.39: Cell proliferation (%) of each Group in Triple Fold Dilution against HeLa Cancer Cells after 48 h Incubation. Data was collected in triplicates and represented by (Mean±SEM) Software Graph Pad Prism V.8..…………………………………………………………………………101 47. Figure 4.40: Average Cell Proliferation Ratio (%) of HCT116 Cancer Cells by SRB Assay Treated with Experimental/ Control Groups after 72 h Incubation. Data was collected in Duplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8………………………………………………………..103 48. Figure 4.41: Cell Proliferation (%) of HCT116 Cancer Cells Treated with all Groups after 72 h Incubation by SRB Assay. Data was collected in Duplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8………..104 49. Figure 4.42: Cell proliferation (%) of each Groups (E10, E11, E12, E13 and E4) in Double Fold Dilution against HCT 116 cancer cells by SRB assay after 72 h Incubation. Data was Collected in Duplicates and Represented by Software Graph Pad Prism V.8……………………………………………..105 50. Figure 4.43: Nitrocellulose Membrane Showing Bands of PARP Cleavage in Western Blot Analysis after Treatment with TQ, TQ NPs and Cisplatin (5 and 10 µM)………………………………………………………………………108

51. Figure 5.1: Conversion of TQ to THQ and its oxidation to stabilized TQ dianion (Islam et al., 2016)……………………………………………...... 111

52. Figure 5.2: Assumed structures for the pectin-TEOS crosslinking by forming bridge between two pectin polymers (Vityazev et al., 2017)……………….116

53. Figure 5.3: A Suggested Structure of Hydrogel Pectin-PLGA-PEG-TQ Nanoparticles (PPPT-NPs) by Physical Crosslinking with TEOS………….118

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LIST OF ABBREVIATIONS

% Percentage & and °C Degree centigrade µg Microgram µg/L Microgram per liter µL Micro liter µL Micro liter µm Micro meter µM Micro Molar A. oryzae Aspergillus oryzae AA Acrylic acid AAm Acrylamide ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ACHN Renal cell adenocarcinoma cell line ACN Acetonitrile AFs Aflatoxin AOAC Association of Analytical Chemist APPH 2,2'-azobis(2-amidino-propane) dihydrochloride ATR Attenuated total reflection AUA Anhydrouronic acid B. japonicum Bradyrhizobium japonicum BaX BCL2 Associated X, Apoptosis Regulator Protein Coding gene Bcl B-cell lymphoma 2 C33A Human cervix carcinoma cell line

CaCl2 Calcium Chloride CASP Center for Advance Studies in Physics cav-1 Caveolin-1 CCD Central composite design CP Cold press Da Dalton DMEM Dulbecco’s modified eagle medium viii

DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic acid DOX 4-substituted-2,5-Dimethoxyamphetamines DPPH 2,2-Diphenyl-1-Picrylhydrazyl DSC Differential scanning calorimetry E Experimental EDTA Ethylenediaminetetraacetic acid EE Encapsulation efficeincy EGDMA Ethylene glycol dimethacrylate EOs Essential oils FCC Food complex code FDA Food and Drug Administration

FeCl3 Ferric chloride

FeCl3 Ferric chloride FRAP Ferric Reducing Antioxidant Power FTC Ferric Thiocyanate FTIR Fourier Transform Infrared Spectroscopy g gram g/g Gram per gram g/mol Gram per mole GCMS Gas Chromatography Mass Spectrometry GCU Government College University GIT Gastro intestinal tract GSH Intracellular glutathione h Hour HCl Hydrochloric acid HCT Human colon cancer cell line HD Hydro distillation HeLa Cervical cancer cell line Hep Hepatocyte related cell lines, HL- hemolysin HL- Human leukemia cell line HPLC High Performance Liquid Chromatography ix

IC50 Inhibition Concentration 50 ICE Institute of Chemical Engineering IL- Interleukin IUPAC International Union of Pure and Applied Chemistry

K3Fe(CN)6 Potassium ferricyanide kg Kilogram

KH2PO4 Potassium di hydrophosphate L Liter LC Loading capacity LCWU Lahore College for Women University LP Lipid peroxidation LUMS Lahore University of Management Sciences M Molar MAE Microwave assisted extraction MAPK Mitogen-activated protein kinases MCF breast cancer cell MCT Mercury cadmium telluride mg Milligram mg/L Milligram per liter mg/mL Milligram per milliliter MG63 Human osteosarcoma cell min Minute mL Milli Liter mM Milli Molar mm Millimeter MMP Matrix metallo proteinases mPEG Methoxy poly ethylene glycol MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide MUC4 Mucin 4 protein Mw Molecular weight N Normality N. sativa Nigella sativa NaCl Sodium choloride x

NaOH Sodium hydroxide NC Negative control NF-kB Nuclear Factor kappa-light-chain-enhancer of activated B cells nm Nanometer NPs Nanoparticles ODS Octadecylsilyl groups p53/ TP53 Tumor protein PA Peak area PARP Poly (ADP-ribose) polymerase PBS Phosphate buffer solution PC Positive control PCL Polycaprolactone PCR Polymerase chain reaction PDLA Poly D-lactic acid PEG Poly ethylene glycol PGA Poly glycolic acid PLA Poly lactic acid PLGA Poly lactic co-glycolic acid/ Poly lactic co-glycolide PLLA Poly L-lactic acid ppm Parts per million PPP Pectin-PLGA-PEG PPPT Pectin-PLGA-PEG-TQ PPPTNPs Pectin-PLGA-PEG-TQ nanoparticles PU Punjab University PVA Poly vinyl alcohol PVP Poly vinyl pyrrolidone QOL Quality Operation Laboratory ROS Reactive oxygen species rpm Revolutions per minute RSC Radicle scavenging activity RSC Radical scavenging activity RSM Response surface Methadology RT Retention time xi

S. typhi Salmonella typhi S/O/W Solid oil water SBASSE Syed Babar Ali School of Science and Engineering SE Soxhlet extraction SEM Scanning electron microscope SFE Supercritical extraction SGF Stimulate gastric fluid SHF Seeds hexane fraction SIF Stimulate intestinal fluid SPSS Statistical Package for the Social Sciences SRB Sulforhodamine B T. vulgaris Thymus vulgaris TEOS Tetraethyl ortho silicate TGA Thermogravimetric analysis TGF Transforming growth factor THQ Thymohydroquinone THY Thymol TLC Thin layer chromatography TNF- Tumor necrosis factor TPC Total phenolic content TQ Thymoquinone USA United State of America UV- Ultra visible absorption spectroscopy UVAS University of Veterinary and Animal Science v/v Volume per volume VEGF Vascular endothelial growth factor w/v Weight per volume w/w Weight per weight WHO World health organization XRD X-ray powder diffraction

xii

ABSTRACT

Current study was designed to extract phytochemical compounds, thymoquinone (TQ) and thymohydroquinone (THQ) from indigenous herbs i.e., Nigella sativa (seeds) and Thymus vulgaris (aerial parts). Extracted compounds were further subjected to evaluate their anti-proliferative and anti-inflammatory activities and loaded in novel biodegradable drug carrier system including pectin and poly lactide-co-glycolide (PLGA) to achieve a controlled drug release system. Drug carrier system was synthesized by extracting pectin from waste peels of citrus fruits (lemon, orange and grape fruit). To enhance the stability, FDA approved PLGA drug carrier was physically cross-linked with pectin hydrogel. This may potentially introduce an opportunity to utilize organic waste in pharmacology. Results showed that soxhlet extraction at ratio of n-hexane: methanol (1:4) has been the appropriate combination to extract maximum yield of TQ from N. sativa (56.13%) and T. vulgaris (22%). THQ extraction was not achievable due to its very less stability. HPLC quantification of essentials oil of N. sativa showed higher amount of TQ i.e., 368.55±0.12 mg/kg (Mean±SEM) and T. vulgaris had 197.58±0.7 mg/kg. The pectin was successfully extracted in maximum yield from waste peels of lemon as compared to other two samples i.e., 35.3±0.66 g (Mean±SEM). Characterization of pectin extracted from each samples showed orange pectin potentially more suitable for hydrogel formation. SEM analysis confirmed the nanoparticles of TQ efficaciously produced loaded in PLGA-PEG at average size of 280 nm. TQ has been proved to be potent anti-proliferative and anti-inflammatory agent by in-vivo antioxidant assays (DPPH and FRAP assay), MTT assay against HeLa cancer cell line and SRB assay against HCT116 colon cancer cell line. Inhibition concentration (IC50) of TQ against HeLa and HCT116 cancer cell lines showed less significant difference (p > 0.05) to the IC50 of control commercial anti-cancer drug (cisplatin). TQ and its nanoparticles loaded in pectin hydrogel showed delayed release of TQ thus enabling the controlled drug release from 24 to 72 hours. TQ thus obtained can be utilized against early treatments of variety of cancers as can be observed from IC50 obtained against HeLa and HCT116 cancer cell lines. Findings of the current study indicate the potential of local resources in effectiveness against cancer. Therefore, increased incidences of cancer can be reduced among impoverished areas.

CHAPTER NO. 1

INTRODUCTION 1

INTRODUCTION

Proliferation relates with cancer which is manifested by the change in expression or happenings of protein related cell cycles resulting in the alteration of tissue arrangement and the formation of nodules (Feitelson et al., 2015). Whereas, inflammation is commonly refers to the reaction against injury or infection which can lead to pre-cancerous lesion at several anatomic sites (Chen et al., 2018). Progression or initiation of cancer occurred consequential in cellular proliferation, chronic inflammation, increased genomic damage, amplified DNA synthesis, disruption of DNA repair pathways, apoptosis induction and the promotion of angiogenesis (Shay and Simon, 2012). Anti-inflammatory and anti-proliferative responses are normal body cell defense mechanisms against chemical, mechanical or pathogen injury or attack (Choi et al., 2014). These can potentially be treated by natural phytochemical compounds of herbs and plants are being considered for antiproliferative and anti- inflammatory activities (Iqbal et al., 2017). Nowadays, the interest in phytochemical compounds with potent anti-cancer activities is growing due to their relatively environmental friendly, economical and their bioavailability in an ingestive form. It has always been known that nature always carries cure to diseases, researchers are focusing on medicinal plants for their medicinal potentials, properties, safety evaluation, mechanism of action and toxicological studies (Thokar et al., 2017). World Health Organization (WHO) reported that in health care almost 75% world population has been consuming plant and traditional medicine as additional modality (Sharif et al., 2012). Around 75 % of antiproliferative and anti-inflammatory agents are derivatives of the natural products including plants, marine organisms and microorganisms (Newman and Cragg, 2016). Essential oil from medicinal plants is significantly considered to be a prospective source of drug discovery for the chemoprevention against cancer development including inflammation, mutagenicity, proliferation etc. Many chemotherapeutic agents are derived from the essential oil of herbal plants which could thus provide a hope for the discovery of anticancer molecules efficient towards apoptotic induction, as well as have anti-inflammatory, anti-proliferative or anti-cancer activities (Bayala et al., 2014; Newell, 2005; Liu, 2004). Most of the anti-cancer activities of phytochemicals involve inhibition of proliferation of cells, destruction of inflammatory process involving cyclo oxygenase-2 expression, angiogenesis, reserve 2 mitosis, and initiation of apoptosis at different stages of numerous types of cancers (Karikas, 2010). Other mechanism to suppress the growth of cancer is by inducing programmed cell death. This mechanism is indicated by the notable changes such as DNA damage (Wei et al., 2012), increase ROS generation (Das and Roychaudhury, 2014), release of cytochrome c (Guo et al., 2013), activation of caspases, cell cycle arrest (Kumar et al., 2014) and activation of nuclear factor NF-κB (Anand et al., 2010) along with visible morphological apoptotic changes (Kown et al., 2015). Varieties of phytochemicals components are present in a single plant and similar phytochemical can exist in various plant species (Tiwari et al., 2011). Thymoquinone (TQ) and Thymohydroquinone (THQ) are the effective quinones which exist in many plant species like Monarda didyma L, Monarda media Willd, Monarda menthifolia Graham, Satureja hortensis L., Satureja montana L., Thymus pulegioides L. , Thymus serpyllum L., Thymus vulgaris L. and Nigella sativa L. (Subramanian et al., 2015; Taborsky et al, 2012). Both phytochemical compounds can be extracted from the same plants but in different amounts varying with the extraction method, extracting solvents and temperatures (Subramanian et al., 2015; Taborsky et al, 2012). TQ (2-isopropyl-5-methyl-benzoquinone) is an active phytochemical component showing potent anti-proliferative and anti-inflammatory activities in vitro and as well as in vivo (Woo et al., 2012). The promising pharmacological and therapeutic effects of TQ against different disease models have grown research interest to evaluate its anticancer activity and mechanism of action against different types of cancer (Woo et al., 2012; Banerjee et al., 2010). THQ (2-Methyl-5-isopropylhydroquinone) is also the major plant derivative component along with the TQ. The present knowledge about the antitumor activity of THQ is very limited (Ivankovic et al., 2006; Sing et al., 2014). THQ is less stable in nature which can leads to the conversion of more TQ by oxidation process (Javed et al., 2013; Monira et al., 2012; Butt et al., 2018). Both quinones possess potent anti-inflammatory and anti-proliferative potentials in dose dependent manner where THQ has been observed to be lesser effective as compared to TQ (Agbaria et al., 2015). Agbaria et al., (2015) explained the transition between TQ and THQ by oxidation process leading to chemical changes and modification between compounds at specific temperature. The thermal processes within extracted oil of seeds/plants convert parent compound thymol to THQ and finally TQ. The consistent heating process leads to the increased oxidation of THQ into TQ resulting in increased amount of TQ (Figure 1.1). 3

Figure 1.1: Transition conversion of thymol to THQ and TQ

Nigella sativa L. (N. sativa) belongs to family Ranunculaceae (Table 1.1). It is commonly known as black seed (English), kalonji (India and Pakistan), black- caraway seeds (US), shonaiz (Persian), kalajira (Bangladesh), Al-habba Al-sawda (Arabic) and krishnajirika (Southeast Asia) with wide spectrum of pharmacological potential therapeutic activities (Abel-Salam et al., 2012). N. sativa is instinctive to Mediterranean region, Southern Europe, North Africa and Southwest Asia and it is cultivated in many countries in the world like Middle Eastern Mediterranean region, South Europe, India, Pakistan, Syria, Turkey, Saudi Arabia (Ahmed et al., 2013). It is an annual flowering plant of length 20-90 cm tall, with finely divided leaves, the leaf segments narrowly linear to threadlike. The flowers are with 5-10 petals usually white in color and also found in yellow, pink, pale blue or pale purple colors. The fruit with numerous black seeds in inflated capsule having 3-7 united follicles (Amin and Hosseinzadeh, 2016). Several phytochemical components of N. sativa seed oil have been identified, including TQ, THQ, thymol, dithymoquinone, nigellimine-N-oxide, nigellicine, nigellidine, and carvacrol (Amin and Hosseinzadeh, 2016; Morikawa et al., 2004). Significant therapeutic properties of this plant are mainly due to the presence of TQ comprising of 25-45% of the essential oil of seed (Ahmed et al., 2013; Woo et al., 2011; Salim et al., 2013). N. sativa has been widely studied for its potent potential against cancer proliferation, inflammation, hypertensive, diabetes, analgesic and microbial, hepatic, cardiovascular, respiratory, immune, endocrine systems, gastrointestinal disease (Abel-Salam et al., 2012; El-Tahir et al., 2006; Mbarek et al., 2007; Gilanie et al., 2004). Yet the knowledge about the exact molecular process involved in anti-inflammatory and anti-proliferation activities of these phyto- constituents is still partial. Forthcoming studies are required to elucidate the 4 comprehensive mechanisms of action that facilitate the anti-cancer properties of N. sativa phytochemicals (Majdalawieh and Fayyad, 2016). The genus Thymus of mint family Lamiaceae (Table 1.1) covers about 400 species of perennial aromatic, evergreen or semi-evergreen herbaceous plants with many varieties, sub varieties, sub-species and forms (de Martino et al., 2009). The most cultivated and medicinal important species of genus Thymus is Thymus vulgaris L. (T. vulgaris) which is a flowering plant and commonly termed as Thyme (Parasanth et al., 2014). It is native in Southern Europe, South East Asia and can be cultivated in many countries of Mediterranean region It can grow up to 15-30 cm with little leaves having hair covering and special scent (Hosseinzadeh et al., 2015).

Table 1.1: Taxonomic classifications of N. sativa and T. vulgaris Taxonomic Classification N. sativa T. vulgaris (Sultana et al., 2015) (Parasanth et al., 2014)

Kingdom Plantae Plantae

Class Magnoliopsida Magnoliopsida

Order Ranunculales Lamiales

Family Ranunculaceae Lamiaceae

Sub-family Ranunculoideae Nepetoideae

Genus Nigella L. Thymus L.

Species Nigella sativa L. Thymus vulgaris L.

For many centuries, thyme had been used as herbal medicine, flavouring agent and culinary herbs (Opara and Chohan, 2014). T. vulgaris is rich in phytochemical components which contribute in its medicinal importance as antibiotic, antifungal, antiseptic properties along with other beneficial efficacy to be anthelmintic, carminative, disinfectant, astringent and tonic. Other beneficial properties of thyme reported include appetite stimulant effect, liver function improvement, treatment of cartilaginous tube, bronchial infection, inflammation, urinary infections and treatment of laryngitis and lately anti-proliferative (Dauqan and Abdullah, 2017). Investigations on chemical and biological properties of T. vulgaris concluded that both yield amount and chemical components composition of essential oil are reliant on a number of factors such as the region of growth, environment, cultivation practices and extraction procedures (Venturini et al., 2012; Hudaib et al., 2007). Thymol is the predominate bioactive component of T. vulgaris comprising of total 56%, along with other high content of oxygenated monoterpenes, low contents of monoterpene hydrocarbon, sesquiterpene hydrocarbons and oxygenated sesquiterpenes (Venturini et al., 2012; Maqtari et al., 2011; Shukla and Gupta, 2010; Verma et al., 2009). With the advancement in the field of medicine up to nano level TQ and THQ, like other phytochemical compound, in nano drug delivery system showed potent target specificity and increased efficiency. For nano drug delivery, drug carriers carrying these nano drugs to the targeted destination without leaking or destroying them before they reach to the final destination (Bourzac, 2012). The nano sized drug carriers having controlled shape, size, chemistry and surface charges carry drugs to the specific sites and enhance their functions upto increase in 40 folds (von Maltzahn et al., 2011). United States Food and Drug Administration (FDA) approved materials may be selected as nano-scale drug carriers that have been proven to be non-inflammatory and non-toxic while enabling the delivery of highly localized concentrations of both hydrophilic and hydrophobic agents (Bertrand et al., 2014). In particular, polymer nanoparticle-based drug delivery systems have been evaluated as attractive options for efficacious delivery of agents such as drugs and genes with resulting treatment efficacy (Danhier et al., 2012). To avoid side effects of drugs there is crucial need for the development of safer drug delivery system to improve bioavailability and therapeutic response of drugs by targeted specific drug delivery system (Gujral and Khatri, 2013; Hoffman, 2008). For control drug release extracted 6

TQ and THQ from N. sativa and T. vulgaris were proposed to be loaded in fabricated nano drug delivery system. The category of biomaterials employed in drug delivery can be broadly classified as (1) naturally occurring polymers, such as complex sugars (pectin, hyaluronan, chitosan) and inorganics (hydroxyapatite) (2) synthetic biodegradable polymers, which includes relatively hydrophobic materials such as the α-hydroxy acids (A family that includes poly lactic-co-glycolic acid, PLGA), polyanhydrides and others (Makadia and Siegel, 2011). Natural polysaccharides like pectin, starch, alginate, chitosan have received popularity as drug carrier in the field of drug delivery systems (Liu et al., 2008). This is because of stimulating properties of natural polysaccharides like low environmental toxicity, varying chemical compositions; high auspicious property of natural polysaccharides also attributed to the fact that their hydrogels are already approved as pharmaceutical excipients (Jain et al., 2007). Recent research advancement explores the natural polysaccharide pectin as a promising drug carrier for pharmaceutical industry in specific drug delivery system (Efthimiadou et al., 2014). Pectin is a natural polysaccharide having galacturonic acid units in 300-1000 in each chain (Georgiev et al., 2012). It has 1,4 linked α-D-galactosyluronic acid residues, different neutral sugars such as rhamnose, galactose, arabinose and amounts of others non-sugar components acetic acid, methanol, phenolic acids and amide groups (Sharma et al., 2006). Pectin due to the presence of 100 to about 1,000 saccharide units in a chain-like configuration, its average molecular varies weight between 50,000 and 150,000 Da (Sriamornsak, 2003). Pectin being soluble as dietary fiber increase the passage time through gastrointestinal tract, short chain fatty acid production, fecal bulk and bile acid excretion (Wong et al, 2010). Nonetheless, one shall highlight that there is no clear picture on the exact complex structure of pectin (Figure 1.2) and the physicochemical characteristics of pectin varying with its source and manufacturing process (Fernandez and Fell, 1998; Glinsky and Raz, 2009).

Figure 1.2: Structure of pectin (Source: Chemdoodle) 7

Pectin is a natural, degradable polymer and mostly derived from waste of citrus fruit peels. These waste peels are usually generated from juice production, possesses several disposal and environmental issues. Citrus fruits belong to family Rutaceae, including various important fruits with high commercial prominence like limes, lemons, oranges and grape fruits. The favorable climates for citrus fruits growth accounts wild winters and temperate summers predominantly in Mediterranean agro- climate (Hussain et al., 2008). The current region, Pakistan being among the top ten citrus producers globally, yields citrus about 9.5 tons per hectare, as compared to largest citrus producer Brazil which produce 40-60 tons per hectare (Ashraf et al., 2012). There are estimates that for juices processing 34% of citrus fruits are processed, resulting in large volume of waste annually (Ghani et al., 2009). Since there is lack of adequate means for disposal management the waste of citrus peels is becoming gigantic problem (Abhay and Partap, 2013). The major problem for dumping citrus wastes on adjacent area to production premises results in large volume of methane gas and unpleasant smell due to fermentation. Percolating of these substances trigger severe environmental impacts including contamination of the under-ground water table (Lin, 2013). For the waste management of citrus fruits, many valuable products can be produced and utilized in food, pulp, paper, feed and many other areas including pharmaceutical aspects, biofuel production, anti-inflammatory and antioxidant compounds, dietary fibers and pectin production (Ali et al., 2015). Extensive citrus production locally has allowed deriving pectin based products. Citrus peels of fruits like orange; lemon contains 20-30% pectin which is more than pectin produce from apple pomace which contains 10-15% of pectin on dry matter basis (Srivastava and Malviya, 2011). Pectin has excellent ability to form gels by hydrolyzing it with acid like citric acid. This property of pectin increases the interest of many researchers to use the pectin gel in target specific drug delivery (Sadeghi, 2011). Stability of pectin gel can be enhanced by cross-linking with other polymer (synthetic/ natural) by employing chemical, physical blending or grafting techniques (Cheng et al., 2009). The novel cross-linked network of natural (pectin) and synthetic polymers (PLGA) results in biodegradable co-polymer. This had led to the formation of multifaceted hydrogels that enhance the stability and its efficacy in control drug release. Preferably degradable, nondegradable and biodegradable polymers are the choices for control release of polymer drug delivery system. PLGA with respect to its design and 8 performance is well-defined polymer available for drug delivery (Kamaly et al., 2016; Fu and Kao, 2010). Biodegradable polymer PLGA is the most successfully used nanoparticle due to its readily hydrolyzing properties to metabolite monomers i.e., lactic acid and glycolic acid (Kamaly et al., 2016; Rao and Geckeler, 2011) (Figure 1.3). PLGA can encapsulate molecules of all/any size and can be processed in almost any shape and size. It is solvable in wide range of common solvents including chlorinated solvents, tetrahydofuran (THF), acetone or ethyl acetate (Kamlay et al., 2016).

Figure 1.3: Structure of PLGA (Source: Chemdoodle)

The change in PLGA properties during polymer biodegradation influences the release and degradation rates of incorporated drug molecules. PLGA physical properties themselves have been shown to depend upon multiple factors, including the initial molecular weight, the ratio of lactide to glycolide, the size of the device, exposure to water (surface shape) and storage temperature (Houchin, 2009). The need for better delivery formulations that incorporate a variety in drugs and methods of administration has resulted in the development of various types of block copolymers of polyesters with poly ethylene glycol (PEG) (Kamlay et al., 2016). PLGA/PEG block copolymers have been processed as diblock (PLGA-PEG) (Cheng et al., 2007) or tri-block molecules with both ABA (PLGA-PEG-PLGA) and BAB (PEG-PLGA- PEG) types (Ghahremankhani et al., 2007). This layer of PEG acts as a barrier and reduces the interactions with foreign molecules by steric and hydrated repulsion, giving enhanced shelf life (Dhar et al., 2008). In order to avoid the inconvenient surgical insertion of large implants, injectable biodegradable and biocompatible PLGA particles (microspheres, microcapsules, nanocapsules, nanospheres) could be employed for controlled-release dosage forms (Kamaly et al., 2016). In addition to its biocompatibility, drug compatibility, suitable biodegradation kinetics and mechanical properties, PLGA can be easily processed and fabricated in various forms and sizes (Makadia and Siegel, 2011). This may lead to 9 the successful preparation of PLGA-PEG nanoparticles encapsulated with TQ and THQ to enhance the release efficiency to the targeted area (Figure 1.4).

Figure 1.4: TQ and THQ encapsulated in PLGA-PEG NPs

Green pharma routes have been gaining extensive attention following the fact that synthetic medicines are released to the environment as a result of waste (either urination or processing wastes). Switching towards the efficient natural resources can lead to the formation of drugs which are environmentally benign, least side effects associated and readily absorbed by the body. Further utilization of waste is very common nowadays, if research is shifted towards the utilization of organic waste in pharmaceutical industry, then more pharmaceutical components can be initiated in future. Current study was designed for evaluation of anti-proliferative and anti- inflammatory activities of extracted phytochemical compounds (TQ and THQ) loaded in novel biodegradable drug delivery system, based on extracting components of citrus fruits waste peelings, up to nano level (Figure 1.5). 10

Phase I Phase II

N. sativa seeds T. vulgaris aerial parts Waste dried citrus peels Extraction & Extraction Characterization & Purification

Pectin Thymoquinone (TQ) Thymohydroquinone (TQ) Hydrogel formation Phase III

Physical Cross- PLGA-PEG Linking with TEOS Pectin hydrogel Preparation of NPs of PLGA-PEG-TQ/THQ Pectin hydrogel with PLGA-PEG loaded with TQ/THQ TQ THQ and NPs

Phase IV

In-vitro Anti-inflammatory and Anti-proliferative activities

Figure 1.5: Summary of current study

1.1. AIMS AND OBEJECTIVES

Aim of the current study was to design an economical and environmental friendly drug from the available local green resources to combat growing cancer incidences. Further extraction of raw material from the waste citrus peelings will provide a new direction of waste utilization and explore green pharma routes in the pharmaceutical industry. Following objectives can be derived as  Extraction and Purification of phytochemical components TQ and THQ from selected medicinal herbs N. sativa and T. vulgaris.  Extraction of pectin from waste peels of citrus fruits (orange, lemon and grape fruits) and its characterization.  Synthesis of biodegradable pectin hydrogels cross-linked with PLGA to fabricate novel pectin-PLGA co-polymer as drug carrier.  Production of PLGA-PEG drug (TQ) loaded nanoparticle and their loading in synthesized hydrogels of pectin-PLGA.  Comparative efficacy of drug TQ and its nanoparticles loaded in pectin- PLGA hydrogel showing potent in-vitro anti-inflammatory and anti- proliferative activities against cancer cell lines.

CHAPTER NO. 2

LITERATURE REVIEW 12

REVIEW OF LITRATURE

Plants are used as herbal medicine since the start of civilization and considered as tradition medical system (Li et al., 2016). People are shifting to the natural products due to the many side effects of other ways of treatments and medicines (Shinwari and Qaiser, 2011). It is an estimation that by 2050 medicinal plant business will reach US $ 5 trillion (Shinwari, 2010). Regionally many research works are being carried out for the investigation of active ingredients and novel phytochemical compounds. The scientists are also focusing on investigation of new aspects of indigenous medicinal plants having anti-inflammatory, antiproliferative and antimicrobial activities along with their other proximate analysis (Shinwari and Qaiser, 2011; Ahmad et al., 2009; Hussain et al., 2009; Pong, 2003). Studies have shown that approx. 20% of plants have been used in pharmaceutical industry influencing health care system for the cure of many harmful diseases including cancer (Altemimi et al., 2017). Several clinical studies have reported the beneficial effects of phytochemical compounds from medicinal herbs, on the survival, immune adaptation and quality of life of cancer patients (Yin et al., 2013). The phyto-components thymoquinone (TQ) and thymohydroquinone (THQ) were considered from regional medicinal herbs (Nigella sativa and Thymus vulgaris) for anti-inflammatory and anti-proliferative activities against cancer. Extensive studies have been carried out for the extraction of TQ which occurs along with its reduced form THQ. TQ is distributed in many genera of Lamiaceae family such as Thymus, Monarda, Mosla, Satureja, Coridothymus, Origanum. Its presence has also been confirmed in the genera Nigella and Tetraclinis of the Ranunculaceae and Cupressaceae families, respectively (Toborsky et al., 2012; Woo et al., 2012). Studies have proven that N. sativa seed is a complex substance compromising of more than 100 compounds; certain of them have not yet been recognized or studied (Amin and Hosseinzadeh, 2016; Salem et al., 2005). The production yield of essential oil of N. sativa and chemical composition varies with the geographical distribution and extraction methods (Ahmad et al., 2013; Cheikh-Rouhou et al., 2007). Large numbers of researches have been carried out on the pharmacological benefits of EO of N. sativa which not only explains the pharmacological action of oil but also the active ingredient that interpret or explain the therapeutic activities (Gholamnezhad et al., 2015; Roy et al., 2006). 13

Review of literature revealed that T. vulgaris L. of family Lamiaceae is a well-known spice plant possessing excellent medicinal properties, due to presence of thymol, of antibacterial, antifungal, and antioxidant, antispasmodic, anti-rheumatic, antiseptic, antimicrobial, cardiac, carminative, expectorant and diuretic. Thyme oil compromising of thymol along with quinones TQ and THQ, extracted from T. vulgaris L. is beneficial in improving the immune system and helps to fight against colds, flu, many infectious diseases and chills (Al-Asmari et al., 2017; Kuete, 2017; Nikolic et al., 2014). Many studies also reported thymol as a major component which on employment of different extraction methods and oxidation processes results in the formation of TQ and THQ in varied amount in essential oil of T. vulgaris. Extracted amount of TQ and THQ in essential oil may vary but their small quantities enhance the anticancer and antioxidant activities of thymus oil (Butt et al., 2018; Javed et al., 2013; Monira et al., 2012). Extractions of essential oil from both species were extensively studied by utilizing different methods summarized in table 2.1. Khan et al., (2011) suggested that the N. sativa has been used as ancient medicine for centuries. TQ extracted from its seeds and oil are efficient against many diseases like cancer, cardiovascular complications, diabetes, asthma, kidney disease etc. It shows therapeutic effects against cancer in blood system, lung, kidney, liver, prostate, breast, cervix, skin with much safety. The molecular mechanisms behind its metastatic tumor role is still not evidently understood, however, some studies showed that TQ has antioxidant role and improves body’s defense system, induces apoptosis and controls Akt (Protein Kinase B) pathway. Relevant investigation reported TQ as major components of N. sativa essential oil which contributes a lot in therapeutic properties of this plant essential oil (Gharby et al., 2015; Ahmad et al., 2013; Rao et al., 2007). Anti-oxidant activity of N. sativa seeds measured by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric reducing antioxidant power (FRAP) showed that extraction techniques majorly affect the potential inhibition concentration (IC50). This research showed that a high level of natural antioxidants could be derived from N. sativa oil extracted by super critical fluid extraction (SFE) as compared to soxhlet (Mohammed et al., 2016).

Table 2.1: Summary of Different Extraction Techniques for TQ and THQ for Their Potent Activities

Plant Extraction method Potent Activities References species N. sativa Supercritical fluid extraction Anti-oxidant Mohammad et al., 2016

N. sativa Soxhlet extraction Quantification Kausar et al., 2017

N. sativa Microwave assisted Anti-oxidant Abedi et al., 2017 extraction

N. sativa Microwave assisted Inhibition of CYP1A Liu et al., 2013 extraction activity

N. sativa Supercritical CO2 Ant-oxidant and Anti- Venkatachallam et caner al. 2010

N. sativa Soxhlet extraction Anti-oxidant Sen et al., 2010

N. sativa Soxhlet extraction Anti-oxidant Dinagaran et al., 2016

N. sativa Soxhlet extraction Anti-oxidant Ashraf et al., 2011

N. sativa Soxhlet extraction Anti-cancer Tabasi et al., 2015

T. Ultrasonic extraction Anti-oxidant Chizzola et al., 2008 vulgaris

T. Hydrodistillation clevenger Anti-oxidant El-Nekeety, 2011 vulgaris apparatus

T. CO2 supercritical fluid Anti-inflammatory Ocana and Reglero, vulgaris extraction 2012

T. Soxhlet apparatus Anti-oxidant, Anti- Durgadevi and vulgaris mutagenic and Kalava, 2013 cytotoxic

T. Hydrodistillation clevenger Anti-oxidant, anti- Nikolic et al., 2014 vulgaris apparatus bacterial and anticancer

T. Soxhlet apparatus Anti-microbial Redfern et al., 2014 vulgaris

T. Reflux extraction Anti-cancer Al-menhali et al., vulgaris 2014

T. Hydrodistillation clevenger Anti-oxidant El-Nekeety, 2011 vulgaris apparatus

Burits and Bucar (2000) suggested that the essential oil of black cumin seeds, N. sativa was tested for a possible antioxidant activity. A rapid evaluation for antioxidants, using Thin Layer Chromatography (TLC) screening methods, showed that thymoquinone and the components carvacrol, t‐anethole and 4‐terpineol demonstrated respectable radical scavenging property. These four constituents and therefore the volatile oil possessed variable inhibitor activity when tested in the DPPH assay for non‐specific hydrogen atom or electron donating activity. Abedi et al., (2017) suggested that the essential oil of N. sativa seeds and its major active component, TQ, possess a broad variety of biological activities and therapeutic properties. The inhibitor capability of essential oils extracted by completely different strategies were evaluated using DPPH and FRAP assays, and compared with traditional antioxidants. The results showed that microwave extraction method was a viable alternative to hydro-distillation for the essential oil extraction from N. sativa seeds because of the superb extraction efficiency, higher TQ content, and stronger antioxidant activity. Sen et al., (2010) described that the antioxidant activity of the methanolic extracts of the black cumin seeds depends on the geographical areas of collection and extraction methods. Amongst six black cumin samples of various origins, the black cumin sample origins from Konya city showed the most potent radical scavenging activity in each assay. These sufficient results lead to employment of black cumin seeds as health‐promoting ingredients such as dietary supplements and nutraceuticals. Hassanien et al., (2015) suggested that the black cumin seeds and its essential oil have been widely used in functional foods, nutraceuticals and pharmaceutical products. Analysis of N. sativa essential oil using gas chromatography (GC) and gas chromatography- mass spectrometry (GC-MS) resulted within the detection of many bioactive compounds representing 85% of the total content. The main compounds included p-cymene, thymoquinone, α-thujene, longifolene, β-pinene, α-pinene and carvacrol. N. sativa essential oil exhibited different biological activities including antifungal, antibacterial and antioxidant potentials. N. sativa essential oil resulted in totally inhibition zones against different gram-negative and gram-positive bacteria including Penicillium citrinum, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa. The volatile oil showed stronger antioxidant capacity in comparison with synthetic antioxidants in a rapeseed oil model system. 16

Ashraf et al., (2011) suggested that the various beneficial properties have been attributed to N. sativa, including its antioxidant potential. N. sativa extracts proved to be efficient prevent protein carbonyl formation as well as depletion of intracellular glutathione (GSH) in fibroblasts exposed to toluene. It was also revealed by preparative silica gel and reverse phase chromatography that different fractions of SFE-extracted or soxhlet extracted N. sativa had different levels of protective effects with regards to GSH depletion in vivo as well as in cell culture. Though fractions rich in thymoquinone were found to be most efficient in terms of antioxidant capacity, the data indicates that the inhibition effects of N. sativa may not solely be due to TQ, but perhaps alternatives antioxidants. Bourgou et al., (2012) investigated N. sativa for antioxidant; anti‐inflammatory; anticancer and antibacterial activities of the shoots, roots and seeds methanol extracts from N. sativa were studied. The three organs displayed strong antioxidant activity using the oxygen radical absorbance capacity method and a cell‐based assay. Additionally, the seeds hexane fraction (SHF) of the methanol extract exhibited significant anti‐inflammatory activity. Tabasi et al., (2015) determined the anticancer activities of N. sativa and TQ against human renal cell carcinoma (ACHN) and fibroblast L929 cell lines. Cytotoxicity ratio was measured using 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. Cell death pattern by apoptosis induction was determined by annexin V and propidium iodine (PI)-staining methods. Expose to N. sativa extracts, TQ and cisplatin significantly repressed the growth of ACHN cells and showed significant increase of early apoptotic cells. Normal cells showed resistant effects to NSE and TQ-induced apoptosis. This study along with other previous study reported their potential to be used as a new therapeutic strategy for renal cancers. In spite of the fact that N. sativa anti-tumour activities have been revealed in several models but still its mechanism of action is unknown. An assumption has made the anti-tumor consequence of N. sativa and TQ may be facilitated by one or more of the following mechanisms i.e., anti-oxidant activity, cytotoxicity and immune-modulatory action (Gali-Muhtasib et al., 2006). Randhawa and Alghamdi, (2011) described that the N. sativa seed as an important nutritive flavoring agent and natural medicine for many ailments for centuries in ancient systems of medicine, e.g. Unani, Ayurveda, Chinese and Arabic Medicines. Many active components have been extracted from N. sativa, including thymol, TQ, 17

THQ, dithymoquinone,carvacrol, nigellimine-N-oxide, nigellicine, nigellidine and alpha-hederin. Moderately a few pharmacological effects of N. sativa seed and its oil active components have been recognized to study immune stimulation, anti- inflammation, hypoglycemic, antihypertensive, antiasthmatic, antimicrobial, antiparasitic, antioxidant and anticancer effects. It is evident from literature search that extensive studies have been lately carried out related to the anticancer activities of N. sativa and some of its active compounds, such as TQ (Gholamnezhad et al., 2016). Anti-inflammatory activities of extracted oil from T. vulgaris, T. zygis, and T. hyemalis significantly decreased production and gene expression of the pro inflammatory mediators IL-1B, TNF-α, and IL-6. Thyme extracted suggested as a potent anti-inflammatory agent and activities were dose and time dependent (Ocaña and Reglero 2012). Anti-oxidant activities of thyme oil of T. Vulgaris were determined with help of anti-oxidative assays Folin-Ciocalteu method, DPPH decoloration, and FRAP Fe3+ reduction (Chizzola et al., 2008). Antimicrobial activity and antioxidant capacity of thyme oil with combination with other oils of rosemary and laurel were investigated (Tural and Turhan, 2017). The antimicrobial activity was studied by the agar well diffusion method, while antioxidant capacity was measured using the FRAP and DPPH scavenging activity methods. All oils and their mixtures showed significant antimicrobial activity and antioxidant capacity. The results along with other investigations suggested that essential oils obtained from thyme, rosemary, laurel and their mixtures have potential to be used as natural antimicrobial and antioxidant agents in the food industry (Tural and Turhan, 2017; Bozin et al., 2006). Various other studies reported potent antioxidant properties of thyme oil with major components including TQ with antioxidant assay FRAP and DPPH ( Roby et al., 2013; Hussain et al., 2013; Rababah et al., 2010; Kulisic et al., 2005; Lee and Shibamoto, 2002; Zheng and Wang, 2001). El-Nekeety, (2011) evaluated the protective effects of this oil of T. vulgaris against aflatoxin-induced oxidative stress in rats. The oil constitutes of high amount of carvarcrol, thymol, β-Phellandrene, linalool, humuline, α-Phellandrene and myrcene. Therapy with AFs alone disturbs lipid profile in serum; decreases total antioxidant capacity, increase creatinine, uric acid and nitric oxide in serum and lipid peroxidation in liver and kidney accompanied with undo histological alteration in the liver tissues. The combined treatment showed significant improvements in all tested parameters 18 and histological pictures in the liver tissues. Moreover, this improvement was more pronounced in the group received the high dose of the oil. Thus, T. vulgaris concluded potential antioxidant and had a defensive effect against AFs toxicity and this protection was dose dependent. Bozin et al., (2006) described that the essential oil of Ocimum basilicum L., Origanum vulgare L., and T. vulgaris were analyzed by means of GCMS and assessed for their antioxidant and antimicrobial activities. The antioxidant activity was measured as a free radical scavenging capacity (RSC), together with effects on lipid peroxidation (LP). The essential oil of T. vulgaris L. exhibited the highest OH radical scavenging activity, although none of the examined essential oils reached 50% of neutralization (IC50). All of the tested essential oil strongly inhibited LP, induced 2+ 2+ either by Fe / ascorbate or by Fe /H2O2. The antimicrobial activity was significant when tested against 13 bacterial strains and 6 fungi. A significant rate of antifungal activity of all of the examined essential oils was also exhibited. Review of literature strongly reveals the anti-cancer activities of T. vulgaris against several cancers. Al-Menhali et al., (2014) reported that colorectal cancer (CRC) inhibits in time and dose dependent manner with T. vulgaris extract. Taken together, these data suggest that the extracts of T. vulgaris inhibits malignant phenotype of colon cancer cells. Therefore, T. vulgaris could have an anticancer effect and that some of its bioactive compounds may prove to be effective treatment modalities for human CRC. Durgadevi and Kalava, (2017) reported that to evaluate the in-vitro antioxidant, anti-mutagenic and cytotoxic property of hydro-alcoholic extract of T. vulgaris by subjecting it to free radical scavenging and MTT assay respectively. Powdered Thyme leaves were extracted in a Soxhlet apparatus using 70% ethanol to obtain a dry extract. The antioxidant potential of thyme was determined using DPPH, 2,2’-Azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS+), ferric thiocyanate (FTC) and metal chelating assays. The in-vitro cytotoxicity assay of thyme was examined following the MTT assay against Hep 2 and Hep G2 cell lines. The thyme extract effectively scavenged the DPPH, ABTS+ and metal ions in a concentration dependent manner. It also significantly inhibited the in-vitro lipid peroxidation in liver and kidney and also erythrocyte hemolysis. A decrease in the viability of cancer cells were observed with increasing concentrations of thyme extracts supporting its cytotoxic potential. This study reveals that the common thyme could serve as a 19 potential antioxidant and chemotherapeutic culinary herb which may be justified by further in-vivo studies (Durgadevi and Kalava, 2017). The assumed mechanism of TQ action involves manifold paths which play significant roles in proliferation of cancer. It was reported that TQ prompt intrinsic pathways of apoptosis through the activation of caspases cascade. The activation of caspase-8 highlights the effect of TQ on Bcl2 and the role of mitochondria in TQ-induced apoptosis in human squamous cell carcinoma, in human osteosarcoma p53-null MG63 cells and in p53-null HL-60 Myeloblastic leukaemia (Subhasis et al., 2012; El-Mahdy et al., 2005). The process of apoptosis in MCF7/DOX cells was also found to be mediated through a caspase dependent manner which triggered the intrinsic pathway through the activation of caspas-3, -7, -9 and the cleavage of PARP but not caspase-8. An increase of TP53 expression level in MCF7/DOX cells indicated the p53- dependent apoptosis after treatment with TQ resulting in reduction of Bcl2 protein and reduction in the Bcl2/Bax ratio (Arafa et al., 2011). Studies showed anti-proliferative and anti-inflammatory effect of TQ on different type of cancer cells through in-vitro and in vivo which indicate the involvement of TQ in different cell death signaling pathways including apoptosis, proliferation, angiogenesis and tumor induced immunosuppression (Marjaneh et al., 2015). Every type of cell can secrete transforming growth which is dependent on the cell response to the TGF-s receptors. Increase or decrease in their functions and its downstream pathway can lead to cancer. Abu-khader, (2013) had investigated new findings suggesting new mechanisms of anticancer activity of TQ against breast cancer in-vitro and in vivo models (Attoub et al., 2012). Number of studies has been carried out to indicate the pharmaceutics role of TQ in several diseases such as cancer, inflammation, diabetes and atherosclerosis (Woo et al., 2012). The study by Ivankovic et al., (2006) undertaken to demonstrate the antitumor effects of TQ and THQ in-vitro and in vivo and revealed that both exhibited good anti-proliferative activity against tumor cells in-vitro which is in agreement with other literature findings (Singh et al., 2014; Gali-Muhtasib et al., 2004; Shoaieb et al., 2003). Halawani, (2009) suggested that the two main components of black seed essential oil, TQ and THQ were investigated for their antibacterial activity against Escherichia coli (E.coli), Pseudomonas aeruginosa (P.aeruginosa), Shigella flexneri, Salmonella Typhimurium, and Staphylococcus aureus (S.aureus). Both TQ and THQ exerted antibacterial activity against gram- positive and gram-negative bacteria regardless to their susceptibility to antibiotics. S. 20 aureus, was highly susceptible to TQ, since, three and 6 µg/ml were enough to inhibit and kill the bacteria respectively. On the contrary the concentration of THQ required to inhibit and kill S. aureus was 400 and 800 µg/ml respectively which is 100 times more than that of TQ. This study demonstrated that both TQ and THQ have antibacterial activity and their activity could be potentiated by antibiotics especially in case of S. aureus. Khan et al., (2011) and Ravindarin et al., (2010) emphasized more investigation regarding anti-cancer and anti-inflammatory activities of TQ. More research works should be emphasized on TQ and THQ extraction from some important medicinal plants because it is a safe and promising anticancer component. Also, the exact molecular mechanisms of TQ and other components like THQ, which is considered in very few studies, on different cancers should be investigated with more emphasis because current understandings are mostly uncertain. Ravindarin et al., (2010) and Nallamuthu et al., (2013) in their research on TQ chemo-sensitization potential employed polymer-based nanoparticle approach to improve upon its bioavailability and effectiveness against cancer cells including colorectal cancer. They also concluded that encapsulation of TQ into nanoparticles enhances its anti-inflammatory, anti-proliferative, and chemo-sensitizing properties. THQ studies on such basis are not being reported yet. Marjaneh et al., (2015) in their study found an up-regulation of the TGF-s pathway after TQ treatment which would be due to the inhibition of p38-MAPK pathway. The down-regulation of cav-1 gene expression is reported in lung, breast, colon and ovarian carcinoma cell lines. So far, the anticancer mechanism of TQ is not fully understood; however, several modes of action have been described depending on the stimulus and the cellular context (Siveen et al., 2014). Gali-Muhtasib et al. (2004) in their study found TQ prompted apoptosis and arrest the phase G1 of cell cycle by increasing the inhibitory in a p53-dependent manner. Study by Kundu et al. (2014) revealed that TQ reduced the cell viability and convinced apoptosis in colon cancer HCT116 cells. El-Mahdy et al., (2005) reported that TQ exhibits anti-proliferative effect in human myeloblastic leukemia HL-60 cells. Products of TQ were tested in HL-60 cells and 518A2 melanoma by Effenberger et al., (2010) reported that TQ have anti-proliferative effects in HL-60 cells, 518A2 melanoma blood cancer, in MCF-7 breast carcinoma and in HCT116 colon cancer. They concluded that the derivatives of TQ induce apoptosis associated with DNA 21 laddering, a slight increase in reactive oxygen species (ROS) and cell death. Chehl et al., (2009) showed that TQ induced cell death i.e. apoptosis and inhibited proliferation in effected cells in pancreatic cancer. Banerjee et al. (2009) suggested TQ as a novel inhibitor of pro-inflammatory pathways providing a promising strategy that combines anti-inflammatory and pro-apoptotic modes of action in cancer. Torres et al., (2010) assessed the down-regulatory effect of TQ on MUC4 in pancreatic cancer cells. TQ reported as markedly apoptosis inducer by stimulating apoptosis effects on cervical cancer cell lines Shia and C3A. Quantitative PCR revealed that significant apoptosis induction in Shia cells was p53 dependent pathway in which increase level of p53 mediated apoptosis target genes. Moreover apoptosis induction in C33A cell line was resulted with activation of caspase 3 (Ichwan et al., 2014). Anti-proliferative, anti-inflammatory and anti-cancer effects of extracted TQ has been studied in recent years (Figure 2.1) in several cancers like renal cancer, colorectal, prostrate and cervical cancers (Kundu et al., 2014; Effenberger et al., 2010; Kaseb et al., 2007). Ravindarin et al., (2010) and Nallamuthu et al., (2013) in their research on TQ chemo-sensitization potential on employing polymer-based nanoparticle approach to improve upon its bioavailability and effectiveness against cancer cells including colorectal cancer. They also concluded that encapsulation of TQ into nanoparticles enhances its anti-inflammatory, anti-proliferative, and chemo-sensitizing properties. Studies have showed THQ as therapeutic agent (Figure 2.2) equivalent to TQ but still more researches are needed. 22

Figure 2.1: Therapeutic Activities of TQ (Source: Juthika et al., 2014; Effenberger et al., 2010; Effenberger et al., 2010; Kaseb et al., 2007)

Figure 2.2: Therapeutic Activities of THQ (Source: Majdalawieh et al., 2016; Ivankovic et al., 2006)

Khan et al., (2011) and Ravindarin et al., (2010) emphasized more investigation regarding anti-cancer and anti-inflammatory activities of TQ. More research works should be emphasized on TQ and THQ extraction from some important medicinal plants because it is a safe and promising anticancer component. Also, the exact molecular mechanisms of TQ and other components like THQ, which is considered in very few studies, on different cancers should be investigated with more emphasis because current understandings are mostly uncertain. Like other phytochemical compounds TQ and THQ as nano drug loaded in nano drug carrier shows potent target specificity and increase the efficiency (Qiu et al., 2013, Zhang et al., 2011; Ravindrin et al., 2010). Researches on drug delivery system have gaining attention towards the hydrogels because of their unusual characteristics that make them a perfect candidate for many pharmaceutical, biomedical and other related applications (Reddy et al., 2014; Ozay et al., 2010). For cancer drug delivery first step involves leaking of nano medicine out of the blood stream into the affected blood vessels (Jain and Srylianopouls, 2010). The usage of nanoparticles have solved the problem of estimation of right doses of the drug to the affected area by keeping the drug away from healthy tissues/cells and delivering to the targeted site (Hrkach, 2012). Diverse types of nano drug carriers are used to deliver drug in cancer therapy. This is because these nano carriers effortlessly target the cancer cells by fabricating them from normal cells (Ferrari, 2005). Delivery of drugs loaded in nano drug carriers at targeted areas depends on the small size and site-specificity. Riehemann, (2008) stated that all the biological procedures happening within the body, as well as the origin and prognosis of cancer, are supposed to occur in the nano-level. A great challenge in medical field and pharmaceutical industry is to design and develop a drug delivery system in terms of delivering drug to the targeted specific area. There are limitations in improvement of drug delivery or carrier as therapeutic agents that show potent consequences in in-vitro studies but weaken the effects in living body of animal/ human (Hoffman, 2008; Katdare and Chaubal, 2006). Limitations in drug delivery to targeted area, over doses of anti-cancer drug are administrated to the patients resulting in more strong side effects. Hence there is a necessity of development of effective drug delivery system with drug carrier that improve the delivery and the efficiency of drugs (Nagy et al., 2011). Hydrogel, with particular characteristics like biocompatibility, integrity, hydrophilic and flexible 25 nature, attained importance in carrier facilitated drug delivery in pharmaceutical, biomedical and other related applications (Reddy et al., 2014; Ozay et al., 2010). Pectin, in combination with a crosslinking agent or a polymer, may also be engaged itself as a delayed release coat to be applicable onto a drug core via film or compression coating technique (Wong et al., 2011). In precise, multiple coat layers can be applied onto the drug core and these coats can diverge in their chemical composition in order to modulate drug release at different sites of upper gastrointestinal tract. Pectin has also been utilized for preparation of pro drugs which will release the free drug upon arrival at targeted area. In drug delivery system with coat composed of pectin and hydrophobic polymer, the digestion of pectin results in the formation of pores for drug release (Ofori-Kwakye and Fell, 2003; Wei et al., 2007; Semde et al., 1999). Pectinolytic enzymes are involved, for pectin leaching and drug release in colon, in covering made of complex of water-soluble polymers (Maestrelli et al., 2008). The release of hydrophilic polymer counterpart of pectin from complex becomes freely solvated, swells, and leads to distortions in coat thereby further facilitating drug release. Enduring efforts by many researchers are made to design pectin-based delayed release dosage system in drug delivery system. Gelatin, alginate, and xyloglucan are some polymers explored in formulation of pectin-based drug delivery system (Pillay and Fassihi, 1999; Itoh et al., 2008). Pectin has a strong attention on its practical applications and implications in drug delivery. Nonetheless, one shall highlight that there is no clear picture on the exact complex structure of pectin and the physicochemical attributes of pectin vary with its source and manufacturing process (Fernandez and Fell, 1998; Glinsky and Raz, 2009). Further researchers are required in pectin drug delivery, to characterize the physicochemical qualities of pectin and evaluate its structure-activity relationship with orientation to delayed drug release (Wong et al., 2011). The option of pectin- based colon-specific dose form to achieve solely and precisely at a cancer site of cancerous colon is appealing challenge for pharmaceutics. Butte et al., (2014) explored combination of Pectin with other polymer Eudragit S100. This study the revealed that both these polymers have ability to protect the core in the upper gastro intestinal tract (GIT) and helps in attaining targeted release of curcumin in the colon the Combination of pectin with other hydrophobic polymers, like PLGA (Poly lactide- co-glycolide), require further investigation on their potential use as synchronized drug 26 carrier and chemotherapeutic agent through a multi-disciplinary approach. Danhya et al., 2012, successfully developed zein-Pectin nano drug carriers which were biodegradable, non-toxic nanoparticle, solely from natural polymers. Zein –pectin nanoparticle comprising of a hydrophobic zein core and a hydrophilic pectin core, loaded with model drug quercetin. Many researchers now days are interested in utilization of hydrogels as drug carrier for biomedical applications. Hydrogels are biodegradable and have pH sensitive characteristics, offer promising applications in target drug delivery. Ferrira et al., (2006) synthesized hydrogel, based on synthetic monomer methacrylate, as a drug carrier to enhance efficacy of drug delivery. Methacrylate based hydrogels were synthesized by crosslinking method following chemical induced polymerization. Consequently due to good swelling, bio-compatibility and mechanical characteristics methacrylate hydrogels show promising ability for utilization in drug delivery. Ranjha et al., (2011) conducted a study to develop biodegradable pH-sensitive polycaprolactone/acrylic acid (PCL/AA) hydrogels. They adapted free radical polymerization metheod to develop hydrogel in which they used benzoyl peroxide as initiator and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. Different parameters i.e. swelling measurements, sol–gel fraction analysis and percentage porosity of synthesized hydrogels were measured to assess their ability to be used in drug delivery. The results showed that swelling capacity was decreased by increasing the concentration of PCL and EGDMA on the other hand the porosity was increased on increasing the concentration of PCL. They also evaluated the shape of hydrogel beads by scanning electron microscope (SEM) which was found to be smooth and less porous and characterized the hydrogel functional groups by Fourier transform infrared spectroscopy (FTIR). Moreover drug release studies were carried out by using Tramadol hydrochloride as model drug. Hence it is confirmed that PCL/AA hydrogel can be used as effective pH-sensitive controlled release drug delivery vehicle. A comparable study was conducted by Mahmoud et al., (2014) where hydrogels were synthesized by using polyvinyl alcohol (PVA) and acrylamide (AAm) in different ratios as a raw material. The method used for preparation was radical polymerization following gamma radiations. They characterized the hydrogels by Fourier Transform Infrared Spectroscopy (FTIR), Thermal gravimetric Analysis (TGA), Differential scanning calorimeter (DSC) and swelling measurements. The results revealed that the thermal stability of hydrogel decreases with the increase in acrylamide content, on the 27 other hand swelling capacity of hydrogels were increased with the decrease in acrylamide content. Moreover, the drug release studies were carried out using ketoprofen as a model drug to inspect the drug release behavior and the results showed that hydrogels release drug at pH 7. This pH stimuli- responsive character of polyvinyl/ acrylamide based hydrogels recommends that such prepared hydrogels are of practical interest in the field of drug delivery system. However, most of the hydrogels used in target drug delivery are synthetically oriented such as polyesters, polyamides, polyacrylamides etc., which exhibited good physiochemical properties but poor biological performance. Accordance to this recently a trend of using natural polymers (starch, cellulose, pectin and chitin) in target drug delivery is growing attention because of their peculiar characteristics (Adikwa and Esimone, 2009). They are cheaper, less toxic, biodegradable, highly compatible nature, renewable and are comparable to synthetic drug carriers (Kulkarni et al., 2012). But there are some growing concerns of instability or poor mechanical properties of natural polymers consequently a new class of specifically designed bio- artificial polymeric material has been introduced (Adikwa and Esimone, 2009). These materials are combination of both synthetic and natural polymers that not only reduce the problem of instability and biocompatibility but also enhanced its efficacy, involved low cost, less environmental impact materials, low toxicity profile and are biodegradable (Tan and Marra, 2010). However, the main obstacle in using starch, chitosan, cellulose, collagen and alginate as drug carrier is that the resources of them are not readily available and they often require long processing protocol to convert them into useable form (Adikwu and Esimone, 2009). In consequence alternative natural polysaccharide pectin that is derived from citrus fruit peels have received considerable attention of many researchers (Sadhegi, 2011). In current region, citrus fruit production has reached up to 200 thousand metric tonnes per annum, which highly flourished the fruit processing industries (Aulja et al., 2007). The fruit processing industries produce the irrational amount of waste in the form of citrus peels and pulp that pose enormous environmental and disposal issues. However the dried citrus fruit peels is a rich source of pectin (Berry, 2001). Pakistan is an agricultural country and among the top ten citrus growing countries in the world (Haleem et al., 2005). The production of citrus fruit per year is is slightly less than 200 thousand metric tons (Iqbal et al., 2009). This manufacture pattern has 28 led to the development of ridiculous amount of fruit processing wastes from of many juice processing industries. These citrus wastes are highly consumable and account for 45-50% of the weight of original citrus fruit. This waste is a valuable resource of pectin which otherwise is a problem to the processing industries and may lead to enormous environmental and disposal issues. In order to prevent these problems waste must be properly processed and converted into value-added products like pectin which not only reduce the environmental issues but also improve the overall economics of processing units (Berry, 2001). Pectin is a miracle polymer and mostly find in cell wall of higher plants. It mainly consists of partially methoxylated polygalacturonic acid and is capable of forming gel under suitable conditions (Aina et al., 2012). Pectin gels have been studied long because of its safe history in food industry and now attracting attention of many pharmacist because of unique properties of pectin gel that have enabled it to be used as a matrix for the entrapment and or delivery of a variety of drugs, proteins and cells (Sadeghi, 2011). Pectin is mostly extracted from different sources like citrus fruit peels that contain 20-30% pectin on dry matter basis and apple pomaces that contain 10-15% pectin on dry matter basis (Salam et al., 2012). Alternative source to derive pectin is sugar beet waste obtained from sugar manufacturing industries. However citrus fruits like orange, lemon, pomegranate, plums contain much more pectin than other soft citrus fruits like grapes, cherries and strawberries (Srivastava and Malviya, 2011). Different sources of pectin and its extraction yield were summarized in table 2.2. Christy et al., (2013) in their study concluded fruit waste is a rich source of pectin and its production is effective to manage fruit waste. Aina et al., (2012) extracted pectin from waste peels suggested that it was comparable to commercial pectin and were suitable for industrial use. The study conducted by Khule et al., (2012) concluded citrus fruit peels are rich source of pectin and are readily available low cost and has effective application in pharmaceutical industry comparable to synthetic polymers. Hence review of literature shows that fruit waste is a rich source of pectin that can replace commercial pectin and has been studied long for its application in food industry and pharmaceutical industry (Sadeghi, 2011). Pectin has the ability to withstand under acidic conditions, at high temperature, resistant to protease and amylase enzymes which are active in upper gastrointestinal tract and is degraded only by microflora present in colon (Sinha and Kumria, 2001). Furthermore the peculiar 29

Table 2.2: Summarizing Sources of Pectin Extracting from Different Methods in Literature

Extraction Extraction Methods Maximum Yield (%) References sources Orange peel Soxhlet extraction 52.90% maximum yield Tiwari et al., 2017 Pumpkin Soxhlet extraction 7.72% yield Hameed, peel Elkhaider and Mustafa, 2017 Mango peel Soxhlet extraction 69.1% maximum yield Banerjee et al., 2016

Grape fruit Soxhlet extraction 25% maximum yield Mohamed, 2016 peel Citrus Conventional heating Significant yield Khan et al., orange peel 2015

Mexican lime Conventional heating, Pomace yielded maximum Sanchez-Aldana bagasse and Microwave assisted pectin from MAE et al., 2015 pomace

Passion fruit Conventional heating 14.60% yield Liew et al., peel 2014 Banana peel Soxhlet extraction Significant yield Bansal et al., 2014

Orange peel Optimized microwave with maximum pectin yield Parkash et al., assisted method (19.24%). 2013 Banana, Conventional heating maximum from mixture peel Christy et al., orange, 2013 lemon and papaya Lemon, Conventional heating Maximum form Lemon peel Aina et al., 2012 grapefruit and sweet orange Grape fruit Conventioanl heating, Highest from ultrasound Bagherian et al., peel Microwave assisted assisted method following 2011 and Ultrasound microwave assisted and lastly assisted by conventional heating Passion fruit Conventional heating Significant yield Pinheiro et al., peel 2008

Orange peel Soxhlet and Maximum from microwave Yeoh et al., Microwave assisted assisted method 2008 30 characteristics of pectin to form gels in presence of cations, reputed as non-toxic, degradable, low production cost and easily available make it an ideal candidate for drug delivery (Watts and Smith, 2009). Recently tremendous interest has been shown by researchers to develop pectin based formulations for controlled drug delivery (Liu et al., 2003). Mishra et al., (2008) synthesized hydrogels based on pectin blended with polyvinyl pyrrolidone (PVP). Different instrumental techniques were used to characterize the hydrogel like FTIR, X-ray diffraction (XRD), DSC, tensile strength test and scanning electron microscopy (SEM). The IR spectral peaks confirmed the strong crosslinking between pectin and polyvinyl pyrrolidone, the x-ray diffractogram of pectin showed sharp peak that depicted the crystalline structure of pectin whereas diffractogram of PVP showed two broad peaks which depicted that PVP had amorphous structure. As the content of pectin decreased in the blend, the crystallinity of compound was also decreased. The SEM micrographs displayed spherical shaped type topology. Moreover swelling studies in different pH buffer solutions and in-vitro drug release studies were also conducted. The swelling results revealed that as the pH of the medium increased the swelling was also increased from 175% at pH 1.4 to 295% at pH 7.4 similarly the drug release pattern was also based on pH as the pH increased (pH7.4) the drug release increased up to 65% within 15min. Furthermore the hydrogels formed were biocompatible as they did not produce any cytotoxic effects when incubated with B16 melanoma cells. Hence hydrogel showed pH responsive behavior and biocompatibility, these properties make pectin based hydrogels an effective drug delivery vehicle in target drug delivery system. Experimental studies to formulate novel hybrid based polymeric hydrogel of pectin and acrylic acid (AA) were conducted by Ranjha et al., (2011). The hydrogels were prepared in different proportions of pectin and acrylic acid by following catalytic polymerization reaction in the presence of benzoyl peroxide as initiator and N, Nmethylene bisacrylamide (MBAAm) as cross-linker. The synthesized hydrogel was then characterized by FTIR and for surface morphology by SEM. The IR spectral results confirmed the crosslinking of pectin with acrylic acid and scanning electron micrographs showed that gel had minute pores that facilitated the incorporation of drug. Furthermore swelling, sol gel fraction and porosity were calculated. 31

The results revealed that as concentration of pectin or acrylic acid increased the swelling, sol gel fraction and porosity was also increased, depicting direct relationship. The in-vitro drug release study was also carried out which showed that drug release was depend upon composition of hydrogel, as percentage of pectin increased the drug release was also increased. From the results it was concluded that pectin acrylic acid based formulations could be used as drug carrier in controlled drug delivery system. A parallel study was conducted by Sadeghi (2011) in which the hydrogel based on pectin and polyacrylonitrile was prepared in the presence of kaolin powder. The structure and surface morphology of hydrogels was characterized by FTIR and SEM respectively. According to FTIR results all the nitrile groups were converted into carboxylate and carboxyamide groups and the SEM micrographs showed that hydrogels exhibited porous structure which facilitated the permeation of water and incorporation of drug. Moreover TGA was also conducted which revealed that the thermal stability of pectin blend was more than the raw pectin. On the other hand, the hydrogel showed pH responsive behavior as pH increased the swelling was also increased. This pH responsive behavior of hydrogels clearly suggested that pectin based hydrogels could be used for colon target drug delivery. Pectin, in combination with a crosslinking agent or a polymer, may also be employed itself as a delayed release coat to be applied onto a drug core via film or compression coating technique (Wong et al., 2011). Pectin has also been used to prepare pro drugs which will release the free drug upon arrival at targeted area. In drug delivery system with coat composed of pectin and hydrophobic polymer, the digestion of pectin results in the formation of pores for drug release (Ofori-Kwakye and Fell, 2003; Wei et al., 2007; Semde et al., 1999). The hydrophilic polymer complement of pectin, once released from complex, similarly becomes freely solvated, swells, and leads to distortions in coat thereby further facilitating drug release. Ongoing efforts by many researchers are made to design pectin-based delayed release dosage system in drug delivery system. Gelatin, alginate, and xyloglucan are some polymers explored in formulation of pectin-based drug delivery system (Pillay and Fassihi, 1999; Itoh et al., 2008). Pectin receives an attention on its practical applications and implications in drug delivery. Different phytochemical and pharmaceutical drug loaded in different drug carrier were summarized in table 2.3. 32

Table 2.3: Types of Phytochemicals and Pharamceutical Drugs with Different Type Drug Carriers

Type of drugs Type of drug References carriers Phytochemicals Triptolide MePEG-PLA Zheng et al., 2011 copolymer micelle Honokiol MPEG-PCL star Dong et al., 2010 shaped micelle Luteolin MPEG-PCL micelle Qui et al., 2013 Curcumin Nano particles with Kumar et al., 2014 C18PMH-PEG Thymoquinone PLGA nano particles Mona and Mottaleb, 2016 Pharamceutical Paracetamol, Pectin-Chitosan Ghaffari et al., 2006 drugs Indomethacin coated tablet

Quercetin Zein-Pectin nano drug Danhya et al., 2012 carriers

5-Fluorouracil Eudragit S 100 citrus Subudhi et al., 2015 pectin nano particle

Ravindran et al., (2010) investigated anti-proliferative activities of TQ loaded in poly (lactide-co-glycolide) (TQ-PLGA) nanoparticles using human colon cancer HCT 116. The TQ encapsulated in PLGA nanoparticle had an encapsulation efficiency of 94% and size ranged between 150 and 200 nm in size. Apart from this the nanoparticles were active in inhibiting NFkB activation and in suppressing the expression of cyclin D1, matrix metalloproteinase (MMP-9), vascular endothelial growth factor (VEGF) when compared to the free TQ. Overall, the results demonstrate that encapsulation of TQ into nanoparticles enhances its anti-proliferative effects. Delivery of drugs loaded in nano drug carriers to targeted areas depends on the small size and site-specificity. The four kinds of focusing characteristics of nano drug carriers are active, passive, temperature and pH sensitivity (Torchilin, 2010; Bourzac, 2012). Subramanian et al., (2016) in their review concluded that for nano drug delivery preferred nano carrier is micelle with polymeric base. A micelle is a nano carrier having hydrophobic part and a hydrophilic part. The common polymeric bases employed for the micelle are poly ethylene glycol (PEG), poly lactic-acid (PLA), PLGA and methoxy poly ethylene glycol (mPEG), which are also known for their good biocompatible and biodegradable behavior. The nano-drug delivery of phytochemicals has been extensively investigated by a large number of cell studies and a few animal studies. Further investigations are needed to find outcome of these combination with nano-drug delivery for cancer. In addition to this, clinical studies on nano-drug delivery as well as the mode of administration should be carried out to promote them in the field of medicinal oncology. Many investigators have reported successful preparation of PLGA nanoparticles (Table 2.4). The parameters which are involved are small dispersed phase ratio, rate, time and speed of stirring. The emulsification-solvent evaporation technique is most common method used for the preparation of solid, polymeric nanoparticles. However, this method is mainly used in encapsulation of hydrophobic drugs. A modification on this procedure called the double or multiple emulsion technique has become the favored protocol for encapsulating hydrophilic compounds and proteins (Mundargi et al., 2008). Nano-precipitation is another successful technique for synthesis of nanoparticles. Polymer and drug are dissolved in acetone and added to an aqueous solution containing pluronic F68 or Poly vinyl alcohol (PVA). The acetone is evaporated at appropriate temperatures and reduced pressures leaving behind the polymer encapsulated nanoparticles with drug (Esfandyari-Manesh et al., 2015). 34

Table 2.4: Studies of PLGA-NPs Loaded Different Drugs in Literature Nanoparticles Drug Loaded/ Encapsulated References (NPs)

PLGA NPs Loaded N-Acetylcysteine Lancheros et al., 2018

PLGA NPs No drug McCall and Sirianni, 2013

PLGA NPs EtNBS (5-ethylamino-9-diethyl- Hung et al., 2016 aminobenzo[a]phenothiazinium chloride) drug encapsulated

PLGA/PLA For blood-brain barrier drug delivery Li and Sabliov, 2013 NPs

PEGylated Encapsulated docetaxrel for prostate Cao et al.,, 2016 PLGA NPs cancer

PLGA NPs Loaded with hydrophobic drug Yan et al., 2015

PLGA NPs Loaded TQ Nallamuthu et al., 2013

PLGA-PEG Encapsulated TQ Ravindarin et al., NPs 2010

PLGA NPs Loaded Paclitaxel Fonseca et al., 2002

Another method used in which a water-in-oil emulsion is first formed containing polymer, solvent (usually non chlorinated like acetone), salt (e.g., magnesium acetate tetrahydrate) and stabilizer is termed as salting out. Water is then added to the solution until the volume is sufficient to diffuse acetone into the water, resulting in nanoparticle formulations (Rafiei et al., 2016). Nallamuthu et al., (2013) conducted a study in which TQ was successfully encapsulated in PLGA nanoparticles using PVA as stabilizing agent. The particles were characterized for morphology, particle size, encapsulation efficiency, in-vitro release, anti-microbial activity and antioxidant activities. Solid-in-oil-in-water (S/O/W) solvent evaporation method was employed for the preparation of nanoparticles. Mean particle size determined was <200nm observed by Dynamic laser light scattering (DLS) and SEM studies. The successful encapsulation of TQ in PLGA nanoparticles was confirmed by FTIR, and the encapsulation efficiency (EE) of TQ was determined to be 62%. In-vitro release study of drug showed a maximum release of TQ at 75% and 54% respectively for artificial intestinal and gastric juices over the period of 7 days. DPPH radical scavenging activity at 1 mg/ml concentration of the nanoparticles was found to be 71%. It also exhibited antibacterial property against Salmonella typhi (S. typhi) strains, E. coli and S. aureus by using well diffusion method. This study showed that PLGA encapsulated TQ nanoparticle with constant release property has preserved antioxidant as well as anti-microbial activity, and therefore signifying its therapeutic applications in various food samples. The present work is aimed to extract TQ and THQ from the oil of native herbs of N. sativa and T. vulgaris and study anti-inflammatory and anti- proliferative activities of TQ/THQ and nano TQ/THQ in fabricated novel biodegradable polymer drug carrier system (Pectin-PLGA hydrogel). The underlying mechanism of action of TQ/THQ and its loading in biodegradable pectin (extracted from waste peels) physically cross- linked with PLGA as drug carrier are of particular interest. This study will encourage interested researchers to conduct further preclinical and clinical studies to evaluate the anticancer activities of T. vulagris and N. sativa, along with the novel Pectin-PLGA cross-linked hydrogel.

CHAPTER NO. 3

MATERIALS AND METHODS 36

MATERIALS AND METHODS Present study involved the extraction of phytochemical compounds thymoquinone (TQ) and thymohydroquinone (THQ) from plant source and their loading in synthesized poly lactide-co-glycolide (PLGA) nanoparticles. The phytochemical compounds considered to be cheap and environmental friendly therapeutic agents for the cure of many diseases including different cancers. For this purpose some native herbs were inspected that are widely used as medicinal herbs and food. The seeds of Nigella sativa L. and aerial parts of Thymus vulgaris L. are selected on the basis of their wide used as medicinal herbs from ancient and also as spice in regional cuisine. Both species have medicinal properties due to the presence of TQ. The impending phytochemical components TQ and THQ were considered due to their vast and potent anti-inflammatory and anti-proliferative activities. The extraction and purification of THQ was a challenge as it gets readily converted to TQ resulting in extensive quantity of TQ. The challenge was to deliver extracted drug to the targeted areas which can be achieved by loading drug and it’s nanoparticles in hydrogel. For environmental concern, waste management of citrus waste peels from the juice shops was considered for the production of biodegradable polymer pectin. To overcome the un stability and fragile nature of natural polymer, another synthetic polymer PLGA was considered for physical cross-linking to synthesized pectin- PLGA hydrogel. This ecofriendly natural drug and drug carrier system was evaluated by measuring inflammatory and proliferative activities against cancer cell lines up-to nano level. 3.1. Collection, Preparation and Storage of Samples The seeds of Nigella sativa L. (wild type) were purchased from the herbal medicinal shops Anarkali, Lahore, in the month of February, 2016. Plants of Thymus vulgaris L. were collected from Changa Manga in months of March - April, 2017. Both samples were identified by Prof. Dr. Tahira Aziz Mughal (Lahore College for Women University LCWU, Lahore). The samples were deposited in Prem Madan Herbarium, LCWU. The waste peels of lemon, orange and grape fruit were collected from different juice shops during the months of November – December, 2016. The peels of each fruit were chopped into small pieces (approx 5mm size) and washed twice with double distilled water to remove dirt and possible glycosides which are responsible for the bitter taste of the peels. After washing and air drying for about 72 h, the dried peels 37

were finely grounded in grinding laboratory mill (Food mixer National, Japan). These powdered samples were properly labeled (L.P for lemon peels, O.P for orange peels and G.P grape fruit peels) and were kept in polythene bags until further analysis. 3.2. Reagents and Chemicals Thymoquinone [TQ] (analytical standard 99%, purchased from Sigma Aldrich, Germany), Commercial Pectin (Galacturonic acid ≥ 74.0% purchased from Sigma Aldrich, Germany), Poly (lactide-co-glycolide) (50:50) [PLGA] purchased from Poly Science (23987-5), Polyethylene Glycol [PEG] different Molecular Weight (MW = 6000, 1000, 600,300) and Tetraethyl orthosilicate [TEOS 98.5%] MW = 208.329 g/mol (DAEJUNG chemical & metals Co. Ltd., Korea)], Polyvinyl alcohol [PVA], Acetonitrile [ACN] (analytical grade 95%, Sigma Aldrich), Citric acid (Sigma BDH, Pakistan), Methanol (99% pure HPLC grade Sigma Aldrich, Germany), Ethanol (99% pure HPLC grade Sigma Aldrich Germany), 2, 2-Diphenyl-1-picrylhydrazyl [DPPH], Ascorbic acid, 95% Methanol of HPLC grade (Sigma Aldrich, Germany), n-Hexane 98% Pure (Sigma Aldrich, Germany,) and isopropyl alcohol HPLC Grade (Sigma

Aldrich, Germany), Sodium chloride [NaCl], Calcium chloride [CaCl2], Hydrochloric acid [HCl], Potassium dihydrophosphate [KH2PO4], Sodium hydroxide [NaOH], Dimethyl sulfoxide (DMSO reagent ≥ 99.9% pure, MW = 78.13 g/mol, Sigma Aldrich, Germany), Dulbecco's Modified Eagle's Medium [DMEM] (prepared by mixing solid DMEM 3.4 g in 100 ml distilled water and sodium bicarbonate), MTT cell proliferation assay kit (Trevigen, USA), cisplatin (Chemotherapeutic drug), HeLa cancer cell lines (human cervical adenocarcinoma) HCT116 (human colorectal carcinoma cell line). All chemicals used were of analytical grade. PEG of different molecular weights (300, 600, 1500, and 6000) and TEOS were provided with courtesy by Department of Polymer, Institute of Chemical Engineering, University of Punjab, Lahore. HeLa cancer cell lines were obtained from Department of Microbiology, University of Veterinary and Animal Sciences (UVAS), Lahore. HCT116 colon cancer cell lines were provided with courtesy by SBSSE, Biology department, Lahore University of Management Science (LUMS), Lahore.

Phase I

3.3. Extraction, Quantification and Purification of TQ and THQ from N. Sativa and T. vulgaris. 3.3.1. Extraction of Essential Oils of N. Sativa and T. vulgaris. The collected samples of N. sativa seeds and T. vulgaris plants were washed, dried and finely ground in grinding laboratory mill (Food mixer National, Japan). The ground samples were subjected to the soxhlet extraction, by passing through sieve, following the procedure with modification (Taborsky et al., 2012; Ashraf et al., 2011). Three different ratio of solvent of n-hexane (HPLC grade) and methanol (HPLC grade) i.e., 4:1; 3:2; 1:4 (200: 50 mL, 150: 100 mL, 50: 200 mL) respectively, were used in the soxhlet extraction in separate 250 ml round bottom glass flasks. Powder of 25g N. sativa seeds and T. vulgaris aerial parts were placed in cotton cellulose extraction thimble of size (25 x 80 mm) in a Soxhlet with solvents combinations of n-hexane and methanol (as mentioned above) separately for 6 h. The extract of each solvent combination were collected and further re-extracted with 30 ml methanol (HPLC grade) three times in a separatory funnel. This re-extraction step would enhance the extraction of hydroquinone compounds (TQ and THQ) in oil samples. Extra solvent was removed by rotary evaporation at 40 °C under vacuum for 5 min. Each sample were than centrifuged at 4000 rpm for 30 min, the upper oily layer was separated and stored in a glass container with cap at 4 °C until analysis. Soxhlet extraction of both samples was carried out in triplicates. The yield (%) of extracted oils from different solvent combination of both samples was calculated by equation Eq. 1.

( ) ...... Eq.1

3.3.2. Identification of TQ and THQ Extracted oil was produced in a higher percentage from N. sativa and T. vulgaris were subjected to High Performance Liquid Chromatography (HPLC) for the identification of TQ and THQ. Dilution of analytical TQ standard (Sigma Aldrich, Germany) was prepared 100 ppm for mixing 100 µg of TQ in 1 mL methanol making it equivalent to 100mg/L. HPLC analysis on the samples, was carried out by Waters (600) HPLC (Environmental Analytical Lab, LCWU) coupled with Waters 600 controllers, Waters 39

600 pumps and 2996 PDA (photo diode array). The working was controlled by PC based empower software. Separation was achieved at ODS C-18 column with the particle size 5 µm (4.6 x 250 nm length). Elution was adjusted to isocratic mode using acetonitrile (solvent A) and methanol (solvent B) (30 A: 70 B) at the flow rate of 1.5 mL/min. Sample run time was 30 min and temperature of column was kept on 25 °C. The injection volume of sample was 20 µL with the detection obtained at 254 nm at the resolution of 1.2 nm. The column was then washed with acetonitrile (100%) and equilibrated for 25 min to remove any impurities before the injection of samples. 3.3.3. Quantification of Extracted TQ For quantification of extracted TQ from oil of both samples, standard run based calibration curve was used. Different dilutions of TQ standard were prepared to get the calibration curve (Annexure 3a). Dilutions were made by diluting the stock of 800 mg/L (TQ analytical standard in methanol) i.e., 600, 400, 200, 100 & 50 mg/L. Each dilution was injected separately and peak area was calculated. The signal to noise ratio (S/N) was measured by following optimized procedure of Chandran and Singh (2007). The signal to noise ratio was measured manually and by auto integrator of the instrument. The concentration of TQ with lower detectable peak was selected. The limit of detection (LOD) and limit of quantification (LOQ) was measured as 3:10 of signal to noise ratio (Shrivastava and Gupta, 2011). Purified TQ from both samples was quantified by the regression equation obtained from the calibration curve (Annexure 3a). The amount of quantified TQ was further confirmed by employing HPLC peak area calculations of respective samples (Annexure 3b) 3.3.4. Purification and Confirmation of TQ Structural confirmation of TQ obtained was assessed through IR Tracer - 100 Fourier Transform Infrared Spectrophotometer (FTIR) - Shimadzu with ATR accessory and MCT detector was used (Central Research Lab, LCWU). The resolution was set at 500-4000 cm-1 with a resolution 4 cm-1 at 100 scans. TQ purified from both N. Sativa and T. vulgaris along with standard TQ were recorded and wavelengths were compared to identify the functional groups and proposed structure of TQ.

Phase II

3.4. Extraction and Characterization of Pectin from Citrus Waste Peels. 3.4.1. Extraction Method Standardization of Pectin Two different methods were carried out for extraction of pectin i.e., conventional heating method and soxhlet extraction method. 3.4.1.1. Conventional Heating Method For this purpose, 25 g of each citrus peels powdered sample were blended separately with 500 mL distilled water which was acidified by using 40 % citric acid (pH=2). This acidified mixture was then heated at 80 ºC for around 2 h. After that, the sample was allowed to cool down at room temperature (37°C). The cooled mixture was then centrifuged at 26 °C for 25 min at speed of 4000 rpm. After centrifuging the slurry was discarded and supernatant (concentrated pectin liquid extract) was further used for the analysis. A mixture of ethanol (HPLC grade) and supernatant (2:1 v/v) was stirred well for 15 min. As a result gelatinous precipitate was formed which was left aside at room temperature for 2 h without stirring. The precipitates were washed twice with 10mL ethanol (HPLC grade) to remove any remaining impurity, consequently pectin gel was obtained. Finally, gel was subjected to drying in the air oven at 40 ºC for 12 h to generate dried pellets. These pellets were further crushed to obtain pectin in powdered form which was stored in desiccators till further use (Aina et al., 2012). This procedure was repeated in triplicates for all samples. 3.4.1.2. Soxhlet Extraction Method Grounded samples of citrus peel (25g) were taken in Whatman cellulose thimble with internal diameter of 33 mm and external length of 80 mm. It was subjected to acidic water soxhlet extraction at 80 °C for 6 h. The hot acid extracts from soxhlet extraction of each fruit peels were then subjected to alcohol-juice treatment using ethanol by 2:1 (v/v) with continuous stirring of 15 min. Each sample was left at room temperature (38°C) for 2 h to allow pectin to precipitates. Extracted pectin was then filtered with cheese cloth, washed with ethanol and further dried in the hot air oven at 40 ºC for 12 h to get hard pectin cake (Yapo and Koffi, 2013). All the samples were subjected in triplicates for soxhlet extraction. The obtained cakes after drying were then ground in marble mortar and pestle and subjected to further analysis.

3.4.2. Characterization of Extracted Pectin 3.4.2.1. Confirmatory Tests for Pectin The extracted pectin from different samples of L.P, O.P and G.P was confirmed by following tests i.e., (Sood and Mathur, 2014; Srivastava and Malviya, 2011). i. Color of Pectin of each sample was observed with naked eye. ii. Texture of the extracted pectin from each sample was also observed. iii. Solubility of extracted pectin was observed by mixing 0.1 g in NaOH (hot 60 °C and cold 25 °C) and water (hot 60 °C and cold 25 °C). iv. Precipitation test was performed by making pectin solution (0.1 g) in hot water (2 mL) and adding ethanol (95 %) drop wise (1 mL). v. Stiff gel test was performed by adding each extracted of 0.5 g in the hot water (5 mL) and allow cooling. vi. Iodine test was performed with each extracted samples of pectin by adding 0.2 mL of iodine solution. 3.4.2.2. Pectin Yield (%) Pectin extracted from all samples was then weighed and mean ± SEM of each sample was calculated. The mean yield (%) of extracted pectin was calculated according to the method of Seggiani et al., (2009) by formula in Eq. 2.

( ) ………………………Eq. 2 ( ) 3.4.2.3. pH pH of extracted pectin from each sample was measured by pH meter (WTW- Germany) using pectin solution (1 % w/v in water). pH meter was calibrated already. The meter probe in pectin solution determined the pH readings. 3.4.2.4. Moisture Content (%) Moisture content was measured according to the standard protocol by Association of Analytical Chemists (AOAC, 1980). Extracted pectin of each samples were weighed before drying in oven. The samples were dried in oven at the temperature of 40 °C for 10 min and again weighed. The percentage moisture content was calculated by employing following formula Eq. 3.

( ) …………………………………………Eq. 3

Where, M.C = Moisture Content (%). W1 = Weight of the empty crucibles (g) W2 = Weight of crucibles+ sample before oven drying (g) W3 = Weight of the crucibles+ sample after oven drying (g)

3.4.2.5. Equivalent Weight Determination For equivalent weight determination extracted pectin powder of each sample (0.5 g) was dissolved in 5 mL ethanol (HPLC grade). About 1 g of NaCl was added to each sample and dissolved in distilled water (100 mL). Finally 6 drops of phenol red was added and titrated against 0.1 N NaOH solution. End point was appearance of pink color indicated by Phenol red. The equivalent weight was determined by using the formula in Eq. 4 (Shukla et al., 2014). ( ) ………………….Eq. 4

3.4.2.6. Methoxyl Content Determination The colorless solution of each samples of pectin attained from the step of equivalent weight determination, was added with 25 mL of 0.25 N NaOH. After thoroughly solution, it was allowed to stand for 30 min. 25 ml of HCl was added before titrating against 0.1 N NaOH. Phenol Red was used as the indicator. The methoxyl content was calculated using the formula in Eq. 5 (Shukla et al., 2014).

………………..Eq. 5 ( )

3.4.2.7. Viscosity Average Molecular Weight For determination of average molecular weight of extracted pectin of each samples and commercial pectin (Sigma Aldrich, Germany), viscosity of each sample was measure with red wood viscometer by following the procedure of Rege and Block (1999). The procedure was repeated in triplets from each sample. Molecular weight of each sample was determined by the formula given in Eq. 6 (Zhou et al., 2010).

……………………………………………………….Eq. 6

Where, Ƞ = Viscosity of each sample = 0.665 and K (constant )= 2.14 x 10-3

3.4.2.8. FTIR Analysis FTIR analysis of all extracted pectin samples and commercial pectin was carried out by IR with ATR (Research Central Lab, LCWU). Wavelengths were measured out in the spectral range 550–4000 cm−1.

Phase III

3.5. Synthesis of Pectin-PLGA Hydrogel and Loading of TQ/THQ PLGA Nanoparticles 3.5.1. Synthesis of Pectin Hydrogels Extracted pectin from each sample of O.P, L.P and G.P and commercial pectin were subjected to form hydrogel by following method of Islam et al., (2014). About 2 g of each pectin sample was added to distilled water of 100 mL (making 2 % pectin solution) in 250 mL glass beaker at temperature 70 °C with continuous stirring on hot plate until it dissolves for about an hour. After an hour each pectin solution was poured in plastic petri dishes separately and allowed to cool. The hydrogels were formed after oven drying at temperature 40 °C for 12 h. The extracted pectin with high molecular weight resulted in suitable hydrogel. To enhance the hydrogel stability and properties, series of cross-linker Tetraethyl orthosilicate (TEOS) and stable agent polyethylene glycol (PEG) were blended in synthesized pectin hydrogel. Commercial pectin was used as control under similar conditions. 3.5.1.2. Series 1 Different molecular weights of PEG (300, 600, 1500 and 6000) were blended in pectin blend individually to enhance the gelling properties. Each PEG (0.5 g) was dissolved in distilled water (15 mL) under continuous stirring and poured in the pectin blending (9.5 g) in hot water (50 mL) on hot plate (Wisd laboratory Instruments, Model MSH 20A) for next 2 h at 50 °C. After this, the blends were poured on petri plates, dried, detached from the plates and stored in polythene bags. The strength of each PEG was noted in pectin hydrogel and appropriate PEG was selected for further work. 3.5.1.3. Series 2 In this series cross linker TEOS with molar different concentrations were used in pectin blend to evaluate its influence on hydrogel. The molar concentrations of TEOS 1.0, 0.75, 0.50 and 0.25 M were prepared in 50 % aqueous ethanol (HPLC grade) by 44

adapting method with modifications (Vityazev et al., 2016). About 50 % aqueous solution was prepared by adding 2 mL ethanol (HPLC grade) in 2 mL distilled water making it 4 mL of total volume in 10 mL glass beaker. The calculated molar volume of TEOS was added in aqueous ethanol with one drop of HCl. TEOS molar volume was calculated with help of its density and MW by Eq. 9.

………………………………………………………………Eq. 7

The calculated molar volume of TEOS per liter was used for the preparation of molar dilutions. About 1 g pectin was dissolved in 50 mL of water with continuous stirring on hot plate at temperature 70 °C in 250 mL glass beaker. Each concentration of TEOS (4 mL) was added drop wise to the pectin blends separately at continuous stirring and kept on stirring for next 3 h at about 60 - 65 °C. Each pectin hydrogel was named according to respective molar concentration of TEOS. Each cross-linked blends of pectin was then poured on plastic petri plates and oven dried at 40 °C. The dried films of cross-linked pectin with different concentration of TEOS were separated from petri plates and stored in polythene bags. The TEOS concentration resulting in increased stability of pectin hydrogel was selected for further analysis. 3.5.2. Preparation of novel cross-linked Pectin-PLGA-PEG (PPP) PEG with specific MW and TEOS on the basis of molar concentrations were selected from the series 1 and 2 respectively, were blended to prepare Pectin-PLGA-PEG blends. PLGA (0.25 g) was dissolved in acetonitrile (5mL) with continuous stirring and PEG (0.25 g in acetonitrile) was added to PLGA blend. The mixture was stirred for 2 h at room temperature. Precipitation was observed for when PEG (dissolved in water) was added to the PLGA blend, therefore the solvent for PEG (soln.) was kept to be acetonitrile. About 9.5 g of pectin was dissolved in 50 mL of water (70 °C) in a 100 mL glass beaker with continuous stirring on hot plate. The prepared PLGA-PEG blend was added drop wise to the pectin blend with continuous stirring for the next 2 h at 60 °C. At the end cross-linker TEOS (4 mL) with specified concentration (0.75 M) was added drop wise in the blend and continuously stirred for the next 3 h. Finally, the blend was poured in plastic petri plate, dried and separated as Pectin- PLGA-PEG (PPP) hydrogel for characterization.

3.5.3. Drug Loading in Pectin Hydrogel To load TQ as drug in PPP hydrogel, 50 mg of TQ was dissolved in 5 mL acetonitrile. Blend of PLGA-PEG was prepared by dissolving PLGA (0.25 g) in acetonitrile (10 mL) and PEG (0.25 g; in 10 mL acetonitrile) was added to the solution (PLGA blend) drop wise under continuous stirring at room temperature. After 2 h of stirring solution of TQ was added to PLGA-PEG blend drop wise and allowed to stir for the next 1 hr at room temperature. This drug loaded PLGA-PEG blend was poured drop wise in 1 % pectin blend in water at 70 °C and allowed to stir for next 2 h. TEOS (0.75 M) 4 mL was added drop wise to the blend and stirred for the next 3 h. The blend was then poured in plastic petri dish and allowed to oven dry. The dried hydrogel was then stored in the plastic bag until further use. 3.5.4. Preparation of TQ loaded PLGA-PEG Nanoparticles in Pectin Hydrogel (PPPT-NPs) For the preparation of drug loaded PLGA-PEG nanoparticles, emulsification-solvent evaporation (S/O/W) was adopted with modifications (Nallamuthu et al., 2013). For an oil phase 0.25 g of each PLGA-PEG blended in acetonitrile for 2 h in room temperature making total 0.50g on polymer weight. About 0.05g of TQ dissolved in acetonitrile was poured in the above organic solution of PLGA-PEG with continuous stirring for the next 2 h. The aqueous phase was obtained by making 1% PVA solution in the distilled water. PVA of 0.25 g was prepared in 25 mL of distilled water with continuous stirring at 50 °C. Organic phase, after sonication for 5 min resulting in S/O primary emulsion, was added drop wise to the aqueous solution under continuous stirring at room temperature. Resulting suspension was sonicated for another 5 min to formulate final S/O/W emulsion. Emulsion, thus produced, was kept on magnetic stirring for next 12 h at room temperature to remove organic solvent. The remaining solution after the evaporation of solvent was subjected to centrifugation at 10,000 rpm for 20 min at 4°C. Finally, the pellet of nanoparticles was settled at bottom. The supernatant was kept for evaluation of drug loading efficiency and pellet was filtered with the help of nano-filters. 3.6. Characterization of Synthesized Novel Pectin Hydrogels and Loaded Nanoparticles Synthesized hydrogels of pectin (extracted and commercial pectin, synthesized novel PPP, PP and PPP-NPs) were characterized by swelling behaviors (water, ionic 46

solution and pH medium), FTIR, X-Ray diffraction (XRD), Thermal gravimetric Analysis (TGA-DSC) and Scanning electron microscope (SEM). 3.6.1. Swelling Experiment of Synthesized Pectin Hydrogels For swelling behavior of synthesized pectin hydrogel in series 1 and 2 and novel physical cross-linked hydrogel PPP, were subjected to observed swelling in the distilled water, pH medium (2, 4, 7, 9, 10) and ionic solution of NaCl as well as CaCl2 (each with concentration of 0.1, 0.3, 0.5, 0.7, 0.9 and 1 mol/L) by following the method of Atta et al., (2015). Synthesized hydrogel were cut into small pieces of about 0.5 g, and immersed in 50 mL of respective swelling medium (water, ionic solution, and different pH media) in plastic petri dishes at room temperature. After every quantified time intervals (10 min), the respective solvent was removed from petri plate to remain the swollen hydrogel undisturbed. Excess of solvent was gently wiped with tissue paper and swollen gel was weighed. This procedure was repeated until the equilibrium condition was achieved by each synthesized hydrogel. All hydrogels were analyzed separately and data represented by mean of triplicate measurements. The results of swelling behavior were calculated by the following Eq. 8.

( ) …………………………..…………………………Eq. 8

Where, Ws= weight of swollen hydrogel at different time (t) interval Wd= weight of dry hydrogel

3.6.2. FTIR Analysis FTIR measurements of all synthesized hydrogel (extracted pectin, commercial pectin, series 1,2 and PPP, PPP-NPs) were carried out using a FTIR spectrometer IR Tracer-100 Fourier Transform Infrared Spectrophotometer- Shimadzu with ATR accessory and MCT detector in the spectral range 650–4000 cm−1. 3.6.3. Thermal Gravimetric Analysis (TGA-DSC) Thermal gravimetric analysis of synthesized hydrogels (series 2, PPP and PPP-NPs) was done with TGA-DSC analyzer (TA instruments SDTQ600) under nitrogen flow of 15 mL/min to eliminate unnecessary corrosive gas. Heating rate was maintained at 20°C /min from room temperature to 600°C. Each gel was cut into small size of about 0.50 g in weight.

3.6.4. X-Ray Diffraction (XRD) Analysis XRD of synthesized pectin hydrogels (series 2, PPP and PPP-NPs) were measured on D 8 Discover X-Ray Diffractor of 0.2 1/min from 10 to 60 (2θ). A piece of 1 cm was taken from all hydrogel for XRD analysis. 3.6.5. Scanning Electron Microscopy (SEM) Synthesized pectin hydrogels (extracted and commercial pectin, series 2, PPP and PPP-NPs) were observed by SEM (ZEISS LS 10, LCWU) and SEM (JEOL JSM- 6480, CASP, GCU). The morphology and texture of each pectin hydrogels were observed and images were recorded. Dry film of hydrogel was cut into 1cm piece to observe under SEM. For the TQ-PLGA, nanoparticle sample preparation for SEM analysis the nanoparticles of 0.1mg was vortexed in 1mL of acetonitrile for 1min and then sonicated at room temperature 30°C for 5 min. Drop of aggregated sonicated particles was placed on the silica plate piece (1 cm) by a dropper and solvent was allowed to evaporate. The dried silica plate was observed with SEM (JEOL JSM-6480, CASP) for confirmation of synthesis of Nanoparticles PLGA-PEG- TQ. 3.6.6. Drug Loading (TQ/THQ) Efficiency in Pectin hydrogel and Nanoparticles Drug (TQ) loading efficiency in PLGA-PEG nano particles was measured with Ultra Visible absorption spectroscopy (UV) at the wave length of 254 nm for drug TQ. Wavelength of standard TQ (100 ppm) in acetonitrile was also observed to calculated weight of loaded drug by its wavelength Eq. 9.

……..………………………………………………………………………………Eq.9 The supernatant obtained from the nano particle formation after centrifugation at 10,000 rpm was analyzed for free drug in it. The drug encapsulation efficiency and capacity was calculated with the equation 10 & 11 respectively (Sharani et al., 2017).

( ) …….Eq. 10

( ) …………Eq. 11

3.6.7. In-Vitro Drug Release from PPP and PPP-NPs Loaded Hydrogels 3.6.7.1. Preparation of simulated solutions Simulated gastric fluid (SGF) of pH 1.2 was prepared by mixing NaCl (1 g) in HCl (3.5 mL) and diluted the solution up to 500 mL with the help of distilled water.

Simulated intestinal fluid (SIF) having pH 6.8 was made by adding 0.2 M KH2PO4 solution (250 mL) to 0.2 M NaOH (118 mL). 3.6.7.2. Drug Release in SGF and SIF For the release of drug TQ, from both PPP and PPP-NPs, method of Islam et al., (2014) was followed. The drug loaded pectin hydrogel PPPT and nanoparticles loaded PPPT-NPs were placed in beaker at 37°C in SGF and SIF solutions (100 mL each). After every time interval of 10 min 5 mL was taken from the beaker and adding 5 mL from fresh solution (SGF or SIF) to make up the solution volume up to 100 mL. Amount of released TQ was determined at absorbance 257 nm by using UV spectrophotometer. Solutions of SIF and SGF were used as the reference standards. Release amount of TQ by pectin hydrogels were calculated with standard reference TQ amount of 100 ppm.

Phase IV

3.7. In-vitro Anti-inflammation and Anti-proliferation Activities of TQ/THQ Loaded PLGA Nanoparticles 3.7.1. Anti-Inflammatory Activities For the evaluation of anti-inflammatory activities of significant anti-oxidant tests Ferric Reducing Antioxidant Power (FRAP) Assay and DPPH radical Scavenging Activities Assay were performed. 3.7.1.1. Ferric Reducing Antioxidant Power (FRAP) Assay According to this method, the reduction of Fe3+ to Fe2+ is determined by the absorbance of Perl’s Persian blue complex. Different concentrations of TQ (600, 400, 200, 100, 50, 25 µg/mL) were prepared in dissolving solvent acetonitrile. The extracted HPLC quantified TQ oil of both samples i.e., N. sativa and T. vulgaris were evaluated for anti-oxidant activities. The modified method of Shanta et al., (2013) was adopted. Different concentrations of TQ were prepared with 0.2 M phosphate buffer (2.5mL, pH 6.6) and 1 % potassium ferric cyanide [K3Fe (CN)6] (2.5 mL). The prepared mixture was incubated in 50 °C for 20 min. An aliquot of 2.5mL of 10 % tri- 49

chloroacetic acid was added to incubated mixture, which was then centrifuged at 3000 rpm for 10min. Resultant supernatant was mixed with distilled water (2.5 mL) and 0.1

% ferric chloride [FeCl3] (0.5 mL). Reduction reaction occurred and the absorbance was measured at 700 nm in UV. Ascorbic acid was used as reference. FRAP inhibition activity was calculated by Eq. 12.

( ) ………………………………..Eq. 12

Where, A0= absorbance of control A1= absorbance of sample

3.7.1.2. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Scavenging Activity For the evaluation of DPPH scavenging activities, procedure of Chaudhry et al., (2015) was followed with modification. Reduction of DPPH purple color was observed in the samples of different concentrations (600, 400, 200, 100, 50, 25 µg/mL) of TQ and HPLC quantified TQ in extracted oil from samples of N. sativa and T. vulgaris. The pectin hydrogel blends with PLGA-PEG-TQ and their nanoparticles were also subjected to DPPH assay. The drug TQ loaded in pectin hydrogel blend was 4mg and compared the DPPH activities with the concentrations of TQ (4, 2, 1, 0.5, 0.25 mg/mL) All samples were added (1 mL) in 3 mL of 0.004% methanolic DPPH and blank (reference; ascorbic acid dissolve in water). All samples were placed in dark for 45 min at room temperature of 30°C. Absorbance was taken at 517 nm, lower absorbance of reaction mixture results in higher inhibition activity. The DPPH activity was calculated by following formula Eq. 13.

( ) ……………………………….Eq. 13

Where, A0= absorbance of control A1= absorbance of sample

3.7.2. Anti-Proliferative Activities For evaluation of anti-proliferative activities two available cancer cell lines HeLa and HCT116 were studied. 3.7.2.1. Anti-proliferation against HeLa by MTT assay HeLa line is cancerous cell line of cervical cancer. Respective cell line was grown in medium of DMEM for 24 h incubation period to make a monolayer adherent cell line culture. The extracted oil of N. sativa and T. vulgaris was evaluated for cell cytotoxicity against HeLa cancer cell line on the basis of quantified amount of TQ 50

from them and preparing dilution up to 200 µM (Annexure 3c). Purified TQ from N. sativa was considered as standard and curative agent in different dilution from 200 µM to 100, 50, 25, 12.5, 6.25 and 3.12 µM. The treatment samples termed as experimental groups and were added in each well by two fold dilution methods in triplicates. The negative control groups was designed on the basis of solvents used for dissolving TQ i.e., 0.1 % DMSO and for methanolic extracted oil i.e., methanol. The concentration of TQ (200 µM) was prepared by mixing 0.164 mg with 0.01 mL DMSO and diluting it with 10 mL DMEM media (making 0.1 % DMSO in media). Different dilutions of TQ from stock of 200 µM to 100, 50, 25, 12.5, 6.25 and 3.12 µM with media in cell 96 well plate. The groups were designed according to dilutions and treatment with different sample illustrated in Table 3.1. The plate was incubated for 48 h in incubator. After 48 h of incubation MTT staining procedure was done with the help of MTT cell proliferation kit (Trevigen) by following the protocol provided (Annexure 3d). About 20μl of 3-(4,5-dimethylthiazole-2-yl)– 2,5-Diphenyltetrazolium Bromide (MTT) dye was added in each treated group at 37ºC for 3 h and kept in dark. After the incubation period, 200μl of PBS (1X) was added and then aspirated carefully from all the samples. The yellow color of MTT reagent reduced to purple color in live mitochondria of cancer cell line. The cell viability was observed in each well plate with the help of 40x Nikon inverted Microscope (Eclipse Ts2, Nikon, Inc., USA). The optical density of cell culture plate was observed by ELISA plate reader Multi Skan Ex. (Thermo Electron Corporation, USA). Cell proliferation percentage was calculated by Eq. 12.

( ) ……Eq. 12

Table 3.1: Number of groups with different treatment of TQ against HeLa cancer cell lines

S. No. Treatment Experimental/Control

1 Untreated cancer cell culture Positive Control (P)

2 0.1% DMSO + DMEM Media + cancer Negative Control (C1) cell culture

3 Methanol + 0.1% DMSO + DMEM media Negative control (C2) + cancer cell culture

4 TQ pure (200 µM) + 0.1%DMSO DMEM Experimental (E1) Media + cancer cell culture

5 N. sativa oil + 0.1% DMSO DMEM media Experimental (E2) +cancer cell culture

6 T. vulgaris oil + 0.1% DMSO DMEM Experimental (E3) media +cancer cell culture

3.7.2. 2. Anti-Proliferative Activities of Synthesized Hydrogel and Nanoparticles Cell Proliferation (%) of HeLa cancer cell line was comprehensively calculated by treating it with different groups including TQ, TQ loaded hydrogel blends and nanoparticles after 24 and 48 h. Cisplatin a commercial cancer treating drug used for comparison of cytotoxic efficacy of TQ. Different groups designed were illustrated in Table 3.2. The pure TQ concentration was prepared 200 µM and accordingly concentrations of all other treated groups were prepared (Annexure 3e). The negative control group was treated with 0.1 % DMSO as all the concentrations were prepared in it. All the groups were treated in triplicates following double fold dilution with the well plate. The 96 well plate was marked with the group names and then incubated for 24 h and cell viability was observed in 40x Nikon inverted Microscope (Eclipse Ts2, Nikon, Inc., USA). After 24 h cell proliferation percentage was calculated with MTT cell proliferation kit (Trevigen). About of 10 µL of MTT dye was poured in each well of plate and left for 3 h. The optical density of cell culture plate was observed by ELISA plate reader Multi Skan Ex. (Thermo Electron Corporation, USA) at 570nm and cell proliferation (%) was calculated. 52

Table 3.2: Treatment groups of TQ loaded Hydrogel and Nanoparticles against HeLa Cancer Cell Lines

S. No. Treatment Experimental/Control

1 Untreated cancer cell culture Positive Control (P1)

2 0.1% DMSO DMEM Media + cancer cell Negative Control (C3) culture 3 TQ pure +0.1% DMSO DMEM Media + Experimental (E4) cancer cell culture

4 PPP (hydrogel blend 2%) 0.1% DMSO Experimental (E5) DMEM Media + cancer cell culture

5 Pectin-PLGA-PEG-TQ blend (PPPT) + 0.1% Experimental (E6) DMSO DMEM Media + cancer cell culture

6 PPPT-NPs blend + 0.1% DMSO DMEM Experimental (E7) Media + cancer cell culture

7 PLGA-TQ NPs + 0.1% DMSO DMEM Experimental (E8) Media + cancer cell culture 8 Cisplatin + 0.1% DMSO DMEM Media + Experimental (E9) cancer cell culture

3.7.2.3. Anti-proliferative activities against HCT116 by Sulforhodamine B (SRB) assay The HCT116 cancer cells were seeded 1500 per well and incubated for 24 h before treatment in cell culture plate. From MTT assay it was noted that high concentration of TQ (200 µM) was more toxic to the cancer cells. The treated groups were designed in table 3.3. The concentrations of treated groups were prepared accordingly (Annexure 3f). For this purpose the lower concentration of TQ (50µM) was considered and diluted in three dilutions fold 50, 16.66, 5.5, 1.8 and 0.62 µM in duplicate. The group cisplatin (E15) was prepared in concentration of 25 µM and treated in triple fold dilutions (25, 8.33, 2.77, 0.93 and 0.310) µM. The dilutions of other groups were prepared in accordance with concentration of TQ. The optimized protocol of Orellana and Kasinski (2016) was followed (Annexure 3g). SRB staining solution was added about 100 µl in each cell and left for 30 min. The extra dye was 53

removed by washing with PBS and then 10mM tris (100μl) was added to each well and shook plate gently for 20 min. After staining procedure viable cells appeared pink in color while dead cell emit no color. Cell viability of HCT116 against each treated group was observed by Elisa plate reader (Synergy HTX Multimode Reader-1353 OUH003-BIO-SH2) after 72h at 530 nm and cell proliferation (%) was calculated. Linea regression and IC50 of each group was originated from software Graph pad prism v.8.0.

Table 3.3: Treatment Groups of TQ against HCT116 cancer cell lines

S. No. Treatment Experimental/Control

1 Untreated cancer cell culture Positive Control (P2)

2 0.1% DMSO + DMEM Media + cancer cell Negative Control (C4) culture 3 TQ pure + 500µL DMSO + DMEM Media + Experimental (E10) cancer cell culture

4 PPP (hydrogel blend 2%) + 500µL DMSO + Experimental (E11) DMEM Media + cancer cell culture

5 Pectin-PLGA-PEG-TQ (PPPT) blend + Experimental (E12) 500µL DMSO + DMEM Media + cancer cell culture 6 PPPT-NPs blend+ 500µL DMSO + DMEM Experimental (E13) Media + cancer cell culture

7 PLGA-TQ NPs + 500µL DMSO + DMEM Experimental (E14) Media + cancer cell culture 8 Cisplatin (25 µM) + 0.1% DMSO DMEM Experimental (E15) Media + cancer cell culture

3.7.2.4. Western Blot Analysis PARP Apoptosis induction of TQ against HCT116 Western blot analysis for PARP apoptosis cleavage was carried out to study apoptosis phenomena involved against HCT116. The treatment groups of TQ, TQ NPs without hydrogel and cisplatin were considered. The dilutions of each group were selected on the basis of IC50 of TQ calculated from SRB assay i.e., 5 and 10 µM. DMSO treated group was considered negative control group as all samples dilution were prepared in it. The preparation of concentrations of treatment groups and the optimized protocol for western blot analysis (Bio Rad) was followed with modification. Colorectal cancer 54

cells HCT116 were seeded 0.70×106 in 6 well plates with 2 mL DMEM and incubated for 24 h at 37°C. The cells in each well was treated with all samples and incubated for 24 hours. From each 6 well 1 mL media was taken in eppendorf tubes and centrifuge in centrifuge machine (Minispin plus-Eppendorf) for 3min at 8000 rpm. After centrifugation, the pellet in each eppendorf was washed with 1mL of 1X PBS (Phosphate buffer saline) in eppendorf and again centrifuged. After centrifugation, all the supernatant was removed. The lysis buffer of 70 µl was added in each well for scrapping of cells in well. All the scraped cells from well was added in respective eppendorf and incubated on ice for 10min. After incubation, centrifugation at 14000 rpm was carried out to collect supernatant. BCA assay was performed by adding 200 µl bradford reagent (2 mL + 5 mL water) to quantify protein concentration. Reading was taken at 595 nm by Elisa plate reader to calculate protein estimation with Bio- Rad protein assay [Bio-Rad ChemiDoL XRS (with image lab software)]. A calculated amount of lysate (90 µl) with Laemmli buffer (4X) in each tube and heated at 95°C for 10 min. After heating tubes were vortex, then centrifuged at 14000 rpm for 2min and stored at -20°C. SDS/PAGE (Sodium Dodecyl Sulphate) gel (10/4 %) with 15 well comb was prepared. The protein was then transferred onto the gel on nitrocellulose membrane at 100 volts for 2h. Samples were incubated in the blocking buffer (5% non-fat milk) for 1h at room temperature. After blocking buffer the cells were incubated with 1:1000 antibody cleavage PARP and α-tubulin at 4°C overnight (24 h). The obtained membrane was washed with PBST (Phosphate buffer saline Tween) for 10min. After washing, the membrane was incubated with HRP secondary antibodies (1:1000) of rabbit for cleaved PARP and mouse for α-tubulin for 1h. The membrane after washing with PBST (1X), was examined for its chemiluminescence by ECL Western Blotting Detection Reagent kit (Amersham, EXL, GE Health care). The scanned blot was measure by Bio-Rad ChemiDoL XRS (with image lab software) considering α-tubulin as control. 3.8. Statistical Analysis All the data for the extraction of TQ (from N. sativa & T. vulgaris) and pectin (from citrus waste peels) were carried out in triplicates and tabulated in word excel sheet (Microsoft office 2010). All the data were statistically analyzed by SPSS V. 16 by calculating mean, percentages, standard deviation (SD) and standard error mean (SEM). The error bars were measured by considering Mean ± SEM and significant 55

value p < 0.05. All the characterization experiments for pectin hydrogel and nanoparticles were graphically presented by using software Origin V. 8.0. The evaluated data for anti-proliferative and ant-oxidant activities were statistically analyzed for mean, percentage, SD and SEM by SPSS v.16 and graphically presented by Graph pad V. 8.0.0 considering significant value p < 0.01.

CHAPTER NO. 4

RESULTS 56

RESULTS Current study elaborated the extraction of TQ and THQ and their anti-inflammatory and anti-proliferative effects up-to nano level loaded in PLGA cross-linked pectin hydrogel. Several studies have already considered for extraction of TQ from locally available herbs. Methanolic extraction for TQ and THQ from selected species, N. sativa and T. vulgaris was considered as there were limitations for the extraction of THQ due to its un-stability. The controlled drug delivery system was generated from synthesis of pectin hydrogel by utilizing waste peels of citrus fruits (lemon, orange and grape fruits). For the stability of pectin hydrogel, FDA approved PLGA drug carrier was physically cross-linked with hydrogel to achieved stable economical and ecofriendly drug carrier system loaded with herbal extracted drug.

Phase I

4.1. Extraction, Quantification and Purification of TQ 4.1.1. Weight of Extracted Oil Essential oils of both samples of N. sativa and T. vulgaris were extracted by different combination of solvents (soxhlet extraction). The ratio of n-hexane: methanol (H:M 1:4) resulted in significantly higher (p < 0.05) volume of oil from N. sativa and T. vulgaris i.e., (Mean± SEM) 15.8±0.18 mL and 9.7±0.02 mL respectively (Table 4.1). Total yield of extracted oil from both samples were calculated from the Eq. 1 and found that with high amount of methanol resulted in significantly higher (p < 0.05) weight of oil from N. sativa i.e., (Mean± SEM) 14.03±0.10 g (56.13% yield) and (Mean± SEM) 5.6 ± 0.08 g from T. vulgaris (22% yield). It was also observed that high amount of oil obtained from N. sativa from all solvent combinations as compared to T. vulgaris. As per yield, the oil obtained from (H:M 1:4) was further processed to attained TQ and THQ.

Table 4.1: Calculated mass of extracted oil from N. sativa and T. vulgaris with different solvent (H:M) mixtures. Data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 where p < 0.05

Solvent 4:1 3:2 1:4 (H:M)

*Wt **Vol ***Yield Wt Vol Yield Wt Vol Wt (g) (mL) (%) (g) (mL) (%) (g) (mL) (%)

N. 3.5± 3.6± 14 5.8± 6.8± 23.2 14.03 15.8± 56 sativa 0.10 0.12 0.14 0.14 ±0.10 0.18

T. 0.66± 1.34± 2.6 2.5± 4± 10 5.6± 9.7± 22 vulgaris 0.05 0.09 0.12 0.10 0.08 0.02

*Wt = weight **Vol =volume ***Yield (%) = calculated by Eq.1

4.1.2. Identification of TQ Acquisition of analytical TQ standard (run time =30 min) yielded the standard peak at retention time (RT) 5.5 min (Figure 4.1). TQ and THQ in the oil samples were evaluated afterwards. Due to un-stability of THQ and its potential hydrolysis to TQ, the peaks for THQ were below the limit of detection (LOD). Further unavailability of HPLC grade THQ standard also limited its detection within the samples. Therefore, THQ was not considered further for the current study. The calculated LOD of TQ was 0.3 mg/L and calculated LOQ was 1 mg/L. Identification of TQ in each sample was done by comparing with standard RT peak. N. sativa oil showed retention peak time at 5.49 min for TQ and 5.48 min for T. vulgaris (Figure 4.1). Thus, the both sample successfully extracted TQ (Table 4.2). 4.1.3. Quantification of TQ Quantification of TQ from both samples was done by HPLC standard curve method (Annexure 4a). Total extracted amount (w/w) of TQ from N. sativa and T. vulgaris was summarized in Table 4.3. TQ was extracted in significantly higher (p < 0.05) amount from N. sativa (Mean±SEM 614.25±0.12) as compared to T. vulgaris ((Mean±SEM 548.86±0.17).

B C

Figure 4.1: HPLC chromatogram representing RT peak of (A) TQ std (B) N. sativa(C) T. vulgaris where x-axis Time (min) and y-axis absorbance (AU).

Table 4.2: RT obtained from the HPLC chromatogram for each samples

S. No. Sample Name *RT (min)

1 TQ standard 5.5

2 N. sativa 5.6

3 T. vulgaris 5.6

*RT= retention time

Table 4.3: Amount of quantified TQ from N. sativa and T. vulgaris. Data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 (p < 0.05)

Samples Quantified amount of TQ by caliberation curve method

*mg/kg **mg/L N. sativa 368.55±0.12 614.25±0.12

T. vulgaris 197.58±0.17 548.86±0.17

*mg/kg = milligran/kilogran

**mg/L = milligram/liter

4.1.4. Purification & FTIR analysis TQ purification by HPLC fractionation was carried out from the oil of N. sativa, showed maximum quantification of TQ. The purified oily layer obtained was further subjected to HPLC and confirmed RT at 5.7 min (Figure 4.2). Amount of TQ was then quantified and observed to be 235 mg/kg (w/w) from N. sativa oil (15mL), obtained from raw mass (25g) of N. sativa seeds. This purified TQ fraction was evaporated under nitrogen gas until complete solvent evaporation. This purified compound was further confirmed by FTIR analysis through comparing its spectrum with that of TQ standard. The IR spectra showed prominent peaks at −1 −1 3498 cm which represented primary amines –NH2 groups, 3023 and 2897 cm −1 represented the presence of aliphatic C-H stretching (CH3), 1655 cm stood for Ester −1 C=O stretching, 1460 cm aliphatic C-H bending CH2, 1200 for Ester C-O stretching and 943 and 727 cm−1 for trans –CH=CH- (Figure 4.3).

Figure 4.2: HPLC chromatogram representing RT (5.58min) peak of purified TQ from N. sativa 62

100

Transmitance(%)

50 4000 3500 3000 2500 2000 1500 1000 500

-1 Wavelength (cm ) Figure 4.3: IR spectrum of purified TQ from N. sativa. Data was represented by software Origin v.8

Phase II

4.2. Extraction of Pectin, Confirmation and Characterization As a part of method standardization soxhlet method provided more yield of pectin from each citrus waste peel compared to the conventional heating method (Figure 4.4). Pectin extracted from different sources of waste peels were confirmed by performing confirmatory tests including color appearance, stiff gel formation test, iodine test, solubility (alkali and water) tests and precipitation with ethanol (Table 4.4). Both methods successfully resulted in extraction of pectin from each citrus peel. Two different extraction methods employing acid extraction for pectin from citrus waste peels were compared by yield (%), moisture content (%) and pH of pectin (Table 4.5). On the basis of highest yield (%) of pectin from all samples, only pectin obtained from soxhlet extraction was processed further. The characterization of extracted pectin were through determination of viscosity average molecular weight, equivalent weight, methoxyl content (%), degree of esterification (%), total anhydrouronic acid (AUA%) and pectin hydrogel formation in parallel with commercial pectin (Table 4.6). The properties of extracted pectin from each citrus peel were compared with the commercial citrus pectin. The physiochemical properties of pectin from orange waste peel was proved to be suitable as compared to other lemon and grape fruit waste peels due its high average molecular weight, methoxyl content (%) and AUA (%), which is required for the potent hydrogel forming properties (Table 4.6). FTIR of orange pectin was carried out showing similar pattern of peaks as were in IR spectra of commercial pectin (Figure 4.5). The morphology of orange pectin was observed in SEM showing similarity with commercial pectin (Figure 4.6). 64

Figure 4.4: Extracted pectin from each citrus peel after alcoholic precipitation in hot acid samples from soxhlet extraction

Table 4.4: Physical appearance and confirmatory tests for pectin extracted from citrus waste peel. Data was collected in triplicates.

Confirmatory Lemon Peel Orange Peel Grape Fruit Peel Tests

Physical Brown Color Light Brown Color Brown Color Appearance

Stiff Gel Test Gel Formed Gel Formed Gel Formed

Precipitation Precipitate Precipitate formed Precipitate formed Test with formed Ethanol

Iodine Test No blue color No blue color No blue color

Solubility test Insoluble but Insoluble but slightly Insoluble but in alkali slightly soluble in hot alkali slightly soluble in soluble in hot hot alkali alkali

Soluble test Insoluble Insoluble Insoluble in water (37°C)

Table 4.5: Comparative Summary of Pectin Extracted from Conventional Heating and Soxhlet Extraction Methods. All data was collected in triplicates. Statistical analysis (Mean±SEM) was carried out by SPSS v.16 where p < 0.05

Extraction Characterization of Lemon Orange Grape Methods Pectin Peel Peel Fruit Peel Conventional Weight of extracted 3.7±0.16 3.1±0.15 2.46±0.17 Heating Pectin (gram) Pectin yield (%) 14.8±0.46 12.4±0.50 9.8±0.7

Moisture Content (%) 5.2±0.17 4.2±0.21 4.8± 0.22

pH 2.3±0.14 3.5±0.21 4.13±0.20

Soxhlet Extraction Weight of extracted 8.8±0.11 7.63±0.18 6.96±0.27 Pectin (g)

Pectin yield (%) 35.3±0.66 30.5±0.93 27.8±1.09

Moisture Content (%) 3.24±0.15 2.05±0.20 2.65±0.27

pH 2.73±0.12 3.66±0.18 4.13±0.27

Table 4.6: Characterization of Commercial Pectin and Extracted Pectin from Citrus Waste Peels by Soxhlet Method

Characterization Commercial Lemon Orange Grape fruit Pectin Peel Peel Pectin peel Pectin Pectin

Viscosity Average 1,61,254.94 33,318.83 83,623.778 59,304.616 Molecular Weight

Hydrogel Yes No Yes No Formation in Water (70°C)

Equivalent Weight 983 384 784 582 (g/mol) Methoxyl Content 11.2% 7.8% 9.3% 9.7% (%)

Total 79.78% 63.9% 72.8% 68.9% Anhydrouronic Acid (AUA) %

Figure 4.5: FTIR Spectra of Commercial pectin and Extracted Orange Peel Pectin. Data was represented by software Origin v.8

A B

Figure 4.6.: SEM (ZEISS LS 10, LCWU) Images of (A) Commercial Citrus Pectin Powder (B) Extracted Pectin Powder from Orange Waste Peels

Phase III

4.3. Synthesis and Characterization of Pectin-PLGA Hydrogel and its Nanoparticles 4.3.1. Optimization of Pectin Hydrogels The hydrogel formation from extracted pectin samples of all waste peels showed that the pectin extracted from the orange peel was resulted in dry film with high elasticity in equivalence to the commercial pectin. 4.3.1.1. Series 1 In this series different molecular weight of PEG were blended in pectin hydrogel and their swelling (g/g) showed that PEG 6000 had high swelling efficiency (19.82 g/g) in water up to 40 min, as compared to other molecular weights of PEG (Annexure 4b). These results are illustrated in Figure 4.7.

PEG 300 PEG 600 PEG 1500 PEG 6000 20

Swelling (g/g) Swelling 10

0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

Figure 4.7: Swelling (g/g) of Pectin Hydrogel with Different MW of PEG (300, 600, 1500 And 6000) in Distilled Water in Time (min). Data represented by Software Origin v.8.

4.3.1.2. Series 2 The TEOS molar concentration was evaluated after the selection of PEG molecular weight. Increased addition of cross-linker to some extent reduced the stability and swelling as shown by 1M TEOS (Annexure 4c). The increased swelling was observed by 0.75 M TEOS pectin hydrogel i.e., 38.08 g/g in 60 min and lowest in 0.25 M TEOS pectin hydrogel i.e., 10 g/g in 30 min (Figure 4.8). The pectin hydrogel was prepared by blending PLGA-PEG6000 with 0.75M cross-linker TEOS.

45 0.25M 0.50 M 40 0.75 M 1.0 M PPP 35

Swelling (g/g) Swelling 20

5 10 20 30 40 50 60 70 Time (min)

Figure 4.8: Swelling (g/g) in Distilled Water of Pectin Hydrogel with different Molar Concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin- PLGA-PEG (PPP) hydrogel in time (min). Data represented by Software Origin v.8.

4.3.2. Preparation of novel cross-linked Pectin-PLGA-PEG (PPP) To enhance the stability and properties of pectin hydrogel, PLGA was added to the blend of pectin with cross-linker 0.75 M TEOS and PEG 6000. Swelling of hydrogel PPP increased in water up to 50.45 g/g in 75 min (Figure 4.8). PPP hydrogel showed highest swelling ratio among all hydrogel and considered for drug TQ loading. The pH swelling of pectin hydrogel with different concentration of TEOS showed increased swelling in basic medium of pH 10 (Annexure 4d). In acidic pH of 2 and 4 hydrogels showed very low swelling while swelling increased in neutral pH of 7 and further increased swelling ration in basic pH of 10 (Figure 4.9). Pectin hydrogel with different molar concentration of TEOS, were subjected in molar concentrations of salt

NaCl and CaCl2 (Annexure 4e & 4f). Swelling ratio decreased with increasing amount of TEOS and showed high swelling ratio with 0.75 M TEOS with increased time. PLGA physical linking with pectin in presence of 0.75 M enhanced swelling ratio of pectin hydrogel (Figure 4.10 & 4.11).

45 0.25M 40 0.50 M 0.75 M 1.0 M 35 PPP

Swelling (g/g) Swelling 20

pH2 pH4 pH6 pH7 pH10 pH

Figure 4.9: Swelling (g/g) in Different pH medium (2, 4, 6, 7 and 10) of Pectin Hydrogel with Different Molar concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEG (PPP) hydrogel in time (min). Data represented by Software Origin v.8. 73

20 1 M 0.75 M 0.50 M 0.25 M PPP

Swelling (g/g/) Swelling 10

5 0.2 0.4 0.6 0.8 1.0 NaCl Molar (M) Concentrations

Figure 4.10: Swelling (g/g) in different molar concentrations of NaCl (0.2, 0.4, 0.6, 0.8 and 1) of pectin hydrogel with different molar dilutions of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEEG (PPP) hydrogel in time (min). Data represented by Software by Origin v.8.

20 1 M 0.75 M 0.50 M 0.25 M PPP 15

Swelling (g/g) Swelling 10

5 0.2 0.4 0.6 0.8 1.0 CaCl Molar (M) Concentrations 2

Figure 4.11: Swelling (g/g) in Different Molar Concentrations of CaCl (0.2, 0.4, 0.6, 0.8 and 1) of Pectin Hydrogel with Different Molar Concentrations of TEOS (0.25 M, 0.50 M, 0.75 M and 1 M) and Pectin-PLGA-PEG (PPP) hydrogel in time (min). Data represented by Software Origin v.8. 74

The IR spectra of all hydrogels along with standards of PLGA, PEG 6000, TQ and pectin were compared (Figure 4.12). The IR of TQ represented sharp peaks at 600- 1600 and 2800-2900 cm-1. Pectin cross linked hydrogel loaded with TQ showed shifted peaks from 750-1000, 1000-1200 and 21—2200 cm-1. PLGA have the peaks at 1000-1200, 1600-1800 and 22-2300 cm-1. Hydrogel PPPT represented sharp peaks at 600-1200, short at 1500-1300 and 2200-2300 and broad peaks at 3100-3200 cm-1. Pectin hydrogel loaded with PLGA-PEG TQ nanoparticles (PPPT NPs) sharp short length peaks at 600-1200, sharp peak at 1600, 2000-2250 and 3100-3600 cm-1 broad short peaks. The finger print region 800cm-1 represented pyranose ring, 1200-1250 cm-1 represented C-O 1450 –CH3 or –CH2 symmetrical and 1550-1680 showed Amide I and II respectively. Other typical peaks of specific groups in pectin were also observed in IR spectrum. The intensive broad asymmetrical peak with a maximum at 3200–3600 cm-1 corresponded to valence oscillations of -OH groups in the pectin molecule. The area of approximately 2926 cm-1contained the peaks corresponding to oscillations of different groups containing C–H bonds. The area of 1500–2000 cm-1 corresponded to oscillations of C = O groups.

Figure 4.12: IR Spectra of Synthesized Pectin Hydrogel with Pectin-TQ, Petin- 0.75 M TEOS, Pectin-PEG (6000), Pectin-PLGA-PEG (6000)-TQ (PPPT), PPPT nanoparticles (PPPTNPs), TQ, PLGA and Citrus Orange Pectin. Data represented by Software Origin v.8.

Pectin hydrogel loaded with TQ was subjected to the control release of drug TQ in SGF and SIF (Figure 4.13). The release of drug TQ was 9.55% in SGF satisfying the maximum release of drug in stomach and it was 100% released in intestine in 2h. The hydrogel in SGF medium remained un-dissolved in 2 h but in SIF medium it completely dissolved in 2h time (Annexure 4g). NPs loaded pectin hydrogel when subjected to SGF and SIF medium they showed delayed release of TQ from encapsulated PLGA-PEG loaded (Figure 4.14). NPs release about 8.65% TQ drug in SGF medium and also delayed release in SIF medium in more than 2h (Annexure 4h).

Figure 4.13: Control Release of Drug TQ Loaded Pectin-PLGA-PEG-TQ (PPPT) Hydrogel in SGF and SIF Medium. Data represented by Software Origin v.8.

Figure 4.14: Control Release of Drug TQ in Pectin-PLGA-PEG-TQ nanoparticles (PPPT-NPs) Hydrogel in SGF and SIF Medium. Data represented by Software Origin v.8.

TGA analysis showed weight loss in pectin 0.75M TEOS at 280°C which gradually decreases with increase temperature from 400-600°C almost 25% loss from (100- 75%) and complete decomposition to 600° (Figure 4.15). Pectin loaded PLGA-PEG and TQ (PPPT) showed loss from 200-250°C with stability up to 300°C and then gradual decrease of weight from 300-600°C with complete degradation. TGAs of pectin curves revealed complete degradation at 900°C. DSC curves of pectin hydrogel cross-linked with 0.75 M TEOS endothermic curve up to 170°C and exothermic peak was from 200°C-300°C, the peaks lower down at temperature 350°C but again increase from 370-500 °C. The complete degradation of gel was at 550°C (Figure 4.16). DSC curves of PPPT showed that endothermic peak was up to 100°C and slight increase of heat at 200°C-250°C and again drop at 300°C. From 300°C-500°C endothermic pattern was achieved. These DSC curves are slightly different from pectin without PLGA-PEG cross-linked with TEOS 0.75M. The DSC peaks of polymer at 100°C indicate the water loss from the matrices, which is followed by decomposition at approximately 240°C. XRD diffraction of pectin hydrogel cross-linked with 0.75M TEOS showed intense peak at 22°θ and 29°θ. PPPT showed peak at 11 and near 30°θ short peaks and PPPNPs showing complete amorphous nature. The peaks at 11.55°θ and 21.36°θ, shows amorphous nature of hydrogel (Figure 4.17). It can be observed from the X-ray diffractogram of pure pectin that it shows sharp crystalline peaks at 2θ equals to 9°θ, 12.70°θ, 18.42°θ, 28.22°θ, and 40.14°θ, which clearly indicate the crystalline behavior of pectin.

Figure 4.15: TGA of Different Concentrations of TEOS in Pectin Hydrogel and Synthesized Pectin-PLGA-PEG (PPP). Data represented by Software Origin v.8.

Figure 4.16: DSC of different concentrations of TEOS in pectin hydrogel and synthesized PPP and PPP-NPs. Data represented by Software Origin v.8.

Figure 4.17: XRD diffraction of Different Concentrations of TEOS in Pectin Hydrogel and Synthesized Pectin-PLGA-PEG (PPPT) and PPPT-NPs. Data represented by Software Origin v.8.

The pectin hydrogel formed by simple blending of commercial pectin powder in water. This thin film was obsereved in SEM (ZEISS, LCWU) which shows clear surface of film with no crosslinking (Figure 4.18).

Figure 4.18: SEM (ZEISS LS 10, LCWU) image of simple Pectin (2%) Hydrogel.

Different pectin hydrogel with different molar concentration of TEOS were observed in SEM revelaing the cross linking in hydrogel. 0.25 M TEOS pectin hydrogel showed lower crossliking nature (Figure 4.19) when compared to 0.50 M TEOS (Figure 4.20). Maximum crosslinking was obsereved in SEM image of pectin hydrogel wiyh 0.75 M TEOS which results in increased stability and swelling ability of pectin hydrogel (Figure 4.20). 82

Figure 4.19: SEM Image of Pectin Hydrogel with (A) 0.25M TEOS and (B) 0.50 M TEOS at magnification of 200KX

Figure 4.20: SEM Image of Pectin Hydrogel with 0.75 M TEOS (A) at magnification of 100KX (B) at magnification of 300KX

SEM image of pectin hydrogel with 1.0 M TEOS showed crosslinking but its stability and swelling as compared to 0.75 M TEOS pectin hydrogel decreased. This showed that increasing amount of croslinking after some extent results in unstabilty of hydrogel (Figure 4.21).

Figure 4.21: SEM (ZEISS LS 10, LCWU) Image Of Pectin Hydrogel with 1.0 M TEOS

Pectin hydrogel loaed with PLGA-PEG (6000) crosslined with 0.75 M TEOS (PPP) in SEM showed crosslinking in Figure 4.22.

Figure 4.22: SEM Image of Pectin Hydrogel Loaded with PLGA-PEG 0.75 M TEOS (A) at magnification 500X (B) magnification of 100KX

The loading of drug TQ in hydrogel of PPP showed porous nature of hydrogel loaded with drug from SEM at high resolution (Figure 4.23).

Figure 4.23: SEM (JEOL JSM-6480, CASP) Image Of Pectin Hydrogel Loaded with PLGA-PEG-TQ (PPPT) (A) at Resolution of 50 µm (B) at Resolution showing 1 µm

4.3.3. Confirmation of Synthesis of NPs and Efficiency of Drug loading in PLGA- PEG NPs Synthesis of nanoparticles were confirmed by SEM images showing efficous encapsulation of TQ drug in PLGA-PEG nanoparticles (Figure 4.24).

Figure 4.24: SEM (JEOL JSM-6480, CASP) Image of Nanoparticles of PLGA- PEG-TQ at Resolution of 50µm.

Averages size of nanoparticles was calculated from observing 100 particles of different size varying from maximum nano-size (574 nm) to minimum (99.7 nm) of PLGA-PEG-TQ nanoparticles (Figure 4.25). The synthesis of nanoparticles was efficiently reported average size of 280 nm nanoparticles which were further loaded in pectin hydrogel with 0.75 M.

Figure 4.25. SEM (JEOL JSM-6480, CASP) Images showing the Size of Nanoparticles of PLGA-PEG-TQ at Resolution of 1 µm

The highest resolution of SEM showed successful synthesis of nanoparticles of PLGA-PEG-TQ of less than 200 nm (Figure 4.26).

Figure 4.26: SEM (JEOL JSM-6480, CASP) Image of PLGA-PEG-TQ of Resolution at 0.5µm showing size less than 200 nm of particles

The total weight of nanoparticles obtained was 80 mg in solid forms calculated from Eq. 9. The absorbance of supernatant of NPs from UV at 254 nm was 0.84 and absorbance of reference solution of TQ standard was 1.09 (Annexure 4k). Amount of free TQ drug was 0.076 mg in the supernatant. Drug loading efficiency by applying Eq. 10 showed the combination to be highly efficient of 99.84%. The drug loading capacity measured was 62.4% by Eq. 11; here loaded TQ in nanoparticles of PLGA- PEG was obtained to be 0.62 mg/ mg of PLGA-PEG (Annexure 4i). The synthesized PLGA-PEG-TQ nanoparticles were further loaded in pectin hydrogel and observed in SEM. The drug loaded was successfully indicated by porous hydrogel nature by SEM image (Figure 4.27) and size of nanoparticles loaded in pectin hydrogel ranges from 179 nm to 200 nm. 89

Figure 4.27: SEM (JEOL JSM-6480, CASP) image showing PLGA-PEG-TQ NPs loaded in Pectin Hydrogel at Resolution of (a) 50 µm (b) 1µm

Phase IV

4.4. Anti-inflammation activities Anti-inflammation activities were observed by scavenging activities assessed by antioxidant assay Ferric Reducing Antioxidant Power (FRAP) and 2, 2-Diphenyl-1- Picrylhydrazyl (DPPH) activities of extracted oil from N. sativa and T. vulgaris compared with activities of TQ with different concentrations (Annexure 4j). Reduction of Fe3+ to Fe2+ decreases with increase in concentration of TQ determined by applying Eq. 12. The inhibition activity was represented by Graph pad prism v. 8.0 (Figure 4.28). N. sativa and T. vulgaris have potent FRAP assay activities as compared to different concentration of standard TQ. The N. sativa showed 58% and T. vulgaris 62% reduction ability by FRAP assay (Figure 4.28).

110

100

)

% (

v i

t 80

o 70

i t

i T.vulgaris

b i

h 60 n I N. sativa 50 0 200 400 600 800 Concentration (µg/mL)

Figure 4.28: Inhibition Activity (%) of Standard TQ of Different Concentration (800, 600, 400, 200 and 100µg/mL) and Extracted TQ in Oil of N. sativa and T. vulgaris by FRAP assay. The Data was Represented by Graph pad prism V.8

DPPH assay for standard TQ showed scavenging activities dose response manner i.e. with increasing TQ concentration scavenging activity increased. N. sativa (86%) and T. vulgaris (89%) showed increased inhibition activity (%) as compared to TQ calculated by Eq. 13 (Figure 4.29). IC50 for TQ obtained was 146.8 µg/mL calculated by nonlinear regression curve with Graph pad prism v. 8.0.

100 T.vulgaris

80 N. sativa

)

(

y 60

c a

i b

i 20

n I 0 0 200 400 600 800 Concentration (µg/mL)

Figure 4.29: Inhibition Activity (%) of Standard TQ and Extracted TQ N. sativa and T. vulgaris By DPPH Assay. Data was represented By Graph Pad Prism V. 8.

The DPPH activities of hydrogel were also evaluated by comparing with TQ concentrations (Annexure 4k). Pectin hydrogel blend loaded with TQ (PPPT) and TQ NPs (PPPT NPs) also showed significant highest antioxidant activity as compared to the TQ concentrations (Figure 4.30).

Figure 4.30: DPPH Assay Scavenging Activities (%) of Pectin Hydrogels blends with PLGA-PEG-TQ (PPPT) and PLGA-PEG-TQ (PPPT-NPs). Data Was Collected In Duplicate and Represented (Mean±SEM) By Software Origin V. 8.

4.5. MTT assay against HeLa cancer cell lines Cytotoxicity assay of TQ and extracted TQ in oil from N. sativa and T. vulgaris grouped in Table 3.1 showing significant anti-proliferation of HeLa cancer cell line. The data was represented by graph pad prism v.8 (Annexure 4l). The average cell viability of cancer cell was represented in Figure 4.31, showing cell death was significantly highest in group treated with N. sativa i.e., 65 % and in T. vulgaris was 42 %.

Figure 4.31: Average Cell Proliferation Ratio (%) Of HeLa Cancer Cells Treated With Experimental/ Control Groups After 48h Incubation. Data Was Collected In Triplicates and Represented By (Mean±SEM) Software Graph Pad Prism V.8.

The trend of cell viability of HeLa cancer cell lines treated with different groups showed that maximum concentration of TQ (E1) showed lowest cancer cell viability which increased with decreasing concentration of TQ. Same pattern was observed in other treated groups.

Figure 4.32: Cell proliferation (%) of HeLa Cancer Cells Treated with all Groups after 48 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism v.8.

Inhibition concentration at 50% (IC50) was determined by nonlinear regression curve obtained from Graph pad prism v.8.0. IC50 of standard TQ was observed at 1.56 µM and N. sativa being highly toxic showed that its lower concentration showed more than 50% death 0.5 µM. IC50 of T. vulgaris determined was near to 15 µM (Figure 4.33). The figure 4.33 also showed that cell proliferation (%) increased with decreasing dilution of all group.

Figure 4.33: Cell Proliferation (%) of Each Group in Triple Fold Dilution against HeLa Cancer Cells after 48 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

The designed groups showed significant anti-proliferation activities against HeLa cancer cell lines after 24 h & 48 h incubation (Annexure 4m). Average cell proliferation (%) of HeLa cancer cells after 24 h was significantly higher in pectin hydrogel (PPP) showing this group E5 less toxic to cancer cells. The cell proliferation (%) was significant lower in cisplatin (E9) treated group and also showed average 48% cell proliferation in group treated with TQ (E4). The pectin blend loaded with PLGA-PEG-TQ nanoparticles (E7) showed delayed release of drug resulting in higher cell viability of cancer cells as compared to the E4 (Figure 4.34)

Figure 4.34: Average Cell Proliferation Ratio (%) of HeLa Cancer Cells Treated with Experimental/ Control Groups after 24 h Incubation. Data was Collected in Triplicates and Represented By (Mean±SEM) Software Graph Pad Prism V.8.

The trend line after 24 hour showed that loaded drug has low toxicity towards cancer cell line because of delayed release from hydrogel (Figure 4.35). Pectin hydrogel loaded with PLGA-PEG without drug was less toxic to cancer cell line among all groups after 24 hour. This trend showed TQ alone (E4) was highly toxic to cancer cell following the group treated with cisplatin (E9). Hydrogel loaded with drug and nanoparticles showed less cell death of cancer cell.

Figure 4.35: Cell Proliferation (%) of HeLa Cancer Cells Treated with all Groups after 24 h Incubation. Data was Collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

The HeLa cancer cells were significantly decreased with increasing concentrations of curative agents. The cisplatin (E9) in 24 h of incubation results in significant death of cancer cells in all dilutions. The pectin hydrogel blend with PLGA-PEG (E5) was less toxic to cancer cells among all groups after 24 h incubation (Figure 4.36). Groups (E6 and E7) with loaded hydrogel with drug and nanoparticles showed maximum cell proliferation (%) in all concentrations.

Figure 4.36: Cell Proliferation (%) of Each Group in Triple Fold Dilution against HeLa Cancer Cells after 24 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

The designed curative groups after 48 h incubation showed increased cell death of HeLa cancer cell line as compared to 24 h incubation. Significantly higher average cell death was observed after 48 h incubation, treated with all experimental groups showing less than 50% cell viability among all groups except the group treated with pectin hydrogel E5 (Figure 4.37).

Figure 4.37: Average Cell Proliferation Ratio (%) of HeLa Cancer Cells Treated with Experimental/ Control Groups after 48 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

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The trend line of cell proliferation (%) decreased in all groups after 48 h showing time dependent cell death. Cisplatin (E9) treated group was more toxic to cancer cell among all groups (Figure 4.38). The pectin blend without TQ drug (E5) showed less cell death of cancer cells of all but increased death as compared to 24 h incubation (Figure 4.38). The TQ (E4) showed significant cell death in three fold dilution from 200 µM to 0.78 µM. Drug loaded hydrogel (E6) and nanoparticle loaded hydrogel (E7) showed increased cell death after 48 h as compared to cell death ratio after 24 h.

Figure 4.38: Cell Proliferation (%) of HeLa Cancer Cells Treated With All Groups after 48 h Incubation. Data was collected in Triplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

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The 48 h incubation of cancer cell death showed decreased cell proliferation as compared to 24 h incubation treated with three frold dilution of all experimental groups (E4, E5, E6, E7, E8 and E9). The group E7 with PLGA-PEG-TQ loaded in hydrogel showed increased cell death as compared to alone nanoparticles (E8) showing the hydrogel enhances the anti-proliferative activity against cancer cells (Figure 4.39).

Figure 4.39: Cell proliferation (%) of each Group in Triple Fold Dilution against HeLa Cancer Cells after 48 h Incubation. Data was collected in triplicates and represented by (Mean±SEM) Software Graph Pad Prism V.8.

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Inhibition concentration (IC50) of each group after 24 and 48 h was determined with the help of software graph pad prism v.8.0 by applying non-liner regression (Annexure 4n &o). IC50 of cancer cells increased in each group after 48h incubation as compared to cell death after 24 h (Table 4.7).

Table 4.7: IC50 of HeLa Cancer Cells Treated With Curative Group By MTT Assay After 24 And 48h Incubation. The Data was Collected in Triplicates and Determined by Non-Linear Regression Graph Pad Prism V.8.

IC50 E4 TQ E5 PPP E6 PPPT E7 PPPT E8 TQ E9 (NPs) NPs Cisplatin After 24 h 9.9 257.9 6.7 20.29 20.73 5.87

After 48 h 7.32 59.01 3.53 3.76 6.59 4.37

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4.6. SRB Assay against HCT16 Cancer cell lines Cell viability of HCT116 was observed after 72 h incubation. The results of SRB assay was in agreement with the results of MTT assay showing significantly increased cell death as compared to 24 and 48 h incubation (Annexure 4p). The average cell death in each experimental group showed highest cell death in group E10 (TQ) and lowest in group E13 (hydrogel with NPs) due to delayed release of TQ (Figure 4.40).

Figure 4.40: Average Cell Proliferation Ratio (%) of HCT116 Cancer Cells by SRB Assay Treated with Experimental/ Control Groups after 72 h Incubation. Data was collected in Duplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

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Cell death increased with increased concentration of TQ i.e., 1.56 % at 50 µM. Decreased cell death was shown in TQ loaded in pectin hydrogel indicating the delayed release of TQ from drug carrier i.e., 50% death at drug conc. 50 µM. TQ NPs alone showed significant cell death i.e., 22% at higher concentration, as compared to loaded NPs in hydrogel i.e., 57.8%. Hydrogel without drug showed no potency against cancer cell line (Figure 4.41). Cell proliferation (%) decreases with increase in the concentration of drug from 0.0-50 µM (Figure 4.41).

Figure 4.41: Cell Proliferation (%) of HCT116 Cancer Cells Treated with all Groups after 72 h Incubation by SRB Assay. Data was collected in Duplicates and Represented by (Mean±SEM) Software Graph Pad Prism V.8.

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Minimum amount of drug released from the drug carrier pectin showing less cell death as compared to the drug alone. High cell proliferation (%) of HCT16 cancer cells treated with pectin blend (E14) in all dilutions was observed and low cell proliferation (%) was observed in pectin blend loaded with nanoparticles E13 (Figure 4.42).

Figure 4.42: Cell proliferation (%) of each Groups (E10, E11, E12, E13 and E4) in Double Fold Dilution against HCT 116 cancer cells by SRB assay after 72 h Incubation. Data was Collected in Duplicates and Represented by Software Graph Pad Prism V.8.

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The cisplatin (E15) was treated in concentration of 25 µM in triple fold dilutions (25, 8.33, 2.77, 0.93 and 0.310 M) against HCT116 cancer cells. The HCT116 cancer cells showed significantly higher cell death when treated with group E15 as compared to other groups (Table 4.8).

Table 4.8: Cell proliferation (%) of HCT116 cancer cells treated with dilutions of Cisplatin (E15). The Data was Collected in duplicates and Represented by (Mean±SEM) Graph Pad Prism V.8.

E15 Cisplatin Concentration (µM) Cell Proliferation 25 8.33 2.77 0.93 0.310 0.00 % (Mean±SEM) 4.3± 33.12± 56.25± 63.75± 65.6± 67.8± 0.001 0.006 0.000 0.002 0.001 0.001

107

The inhibition concentration (IC50) of HCT116 cancer cells treated with all designed groups after 72 h incubation of SRB assay was calculated by non-linear regression graph pad prism v.8 (Annexure 4q). Group E14 with TQ NPs showed IC50 at lowest concentration of 1.87 µM showing the complete release of TQ from PLGA-PEG nanoparticles in 72 h incubation period (Table 4.9).

Table 4.9: IC50 of HCT116 Cancer Cells Treated With Curative Group By SRB Assay After 72 h Incubation. The Data was Collected in Duplicates and Determined by Non-Linear Regression Graph Pad Prism V.8.

IC50 E410 TQ E11 PPP E12 E13 PPPT E14 TQ E15 PPPT (NPs) NPs Cisplatin (µM) (µM) (µM) (µM) (µM) (µM)

After 72 h 3.915 13.55 6.443 5.62 1.87 6.69

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4.7.Western Blot Analysis The band on SDS gel was transferred on Nitrocellulose and observed the PARP activity of each sample against DMSO and antibody α-tubulin. The clear PARP activity was observed in Cisplatin at 5 µM after 24 h incubation. TQ at concentration of 10 µM showed apoptosis as compared to lower concentration. TQ NPs also showed band for cleaved PARP at 10 µM but clear band resulted in Cisplatin 5 and 10 µM (Figure 4.43).

Figure 4.43: Nitrocellulose Membrane Showing Bands of PARP Cleavage in Western Blot Analysis after Treatment with TQ, TQ NPs and Cisplatin (5 and 10 µM)

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Thus, the TQ was successfully extracted and purified from the seeds of N. sativa and T. vulgaris for evaluation of its efficacy against cancer cell line up-to nano level. The synthesized pectin from waste peels resulted in effective control drug release carrier cross-linked with PLGA loaded with extracted drug TQ. Cancer cell proliferation (%) against HeLa and HCT116 proved TQ as potent anticancer agent and pectin-PLGA hydrogel as potent drug carrier for control drug release.

CHAPTER NO. 5

DISCUSSION 110

DISCUSSION

Current study was designed to extract phytochemical compounds Thymoquinone (TQ) and Thymohydroquinone (THQ) for utilizing as natural drug for anti- inflammatory and anti-proliferative activities. Herbal/ plant derived medicines are preferable over synthetic drugs due to their less reported side effects (Ahmad et al., 2013). Common local herbs Nigella sativa and Thymus vulgaris were considered for extraction of TQ and THQ. Seeds of N. sativa and aerial parts of T. vulgaris commonly used as ancient medicine and are selected for oil extraction from soxhlet. The present study was designed, to enhance the control drug release of TQ and THQ biodegradable drug carrier system was successfully developed. Hydrogel of natural polymer pectin was prepared loaded with synthetic PLGA loaded with TQ and its nanoparticles (NPs). Extracted drug proved to be a potent anti-inflammatory and anti- proliferative drug satisfying our hypothesis for the purification of anti-cancer component from selected plant species. TQ was successfully extracted and purified from seeds on N. sativa and T. vulgaris but THQ due to its un-stability converted to TQ. Optimized soxhlet extraction method was employed with n-hexane and methanol solvent at 70°C resulting in maximum amount of TQ. Solvent combination of n-hexane and methanol (H:M) ratio showed that amount of extracted oil increased with increased ratio of methanol. THQ with this continuous thermal process also converted to TQ which results in increased amount of TQ. Analytical standard of THQ was unavailable commercially and reduction of TQ to THQ requires a long and expensive procedure (Guin et al., 2011). TQ in presence of catalyst and specific pH resulted in conversion of THQ, however, for this conversion large amount of TQ is required and it’s a time-consuming process (Figure 5.1). As TQ has the ability to produce unstable and stable redox compounds in presence of internal and external factors such as catalyst, light and pH (Islam et al., 2016).

111

Figure 5.1: Conversion of TQ to THQ and its oxidation to stabilized TQ dianion (Islam et al., 2016).

Present finding that yield of THQ is in very low amount as compared to TQ from plants oil, is also reported by Toborsky et al., (2012). Halawani, (2009) and Ivanosky et al., (2006) showed that high amount of THQ is required to show its efficacy as compared to TQ. TQ was successfully extracted by HPLC analysis in the current study by comparing similar retention peak of analytical standard TQ and extracted TQ from both samples. The purification of TQ was successfully done by manual fraction collection, which was also earlier reported by Ashraf et al., (2011). From the chromatogram of both samples it was clear that the maximum amount of TQ has been recovered from the current experiment. Extraction of TQ from both samples proved to be as major bioactive component of oil supporting the similar findings about the chemical composition of N. sativa and T. vulgaris with TQ as the major bioactive components of their essential oils (Woo et al., 2011; Aziz et al., 2010). Yield of oil depend on the extraction method, solvents, time and temperature, which has great influence on the quantity of TQ (Khan et al., 2012; Mohammed et al., 2016). TQ was quantified in significantly higher amount from seed of N. sativa as compared to T. vulgaris aerial parts. This result was in agreement with the earlier findings in showing comparatively high amount of TQ from N. sativa as compared to T. vulgaris (Taborsky et al., 2012; Aziz et al., 2010). Maximum TQ quantified from N. sativa was 390 mg/kg and from T. vulgaris was 195mg/kg but maximum reported amount of TQ was 1881 mg/kg and 300 mg/kg respectively (Taborsaky et al., 2012). The amount of purified TQ was lower than quantified TQ in N. sativa samples might be due to loss of sample during HPLC run. The quantity of TQ as obtained from the current study, however, is lesser than the reported ones, which may be because of the geographical area and quality of seeds/plants. Seed fat also varies with the region where they are grown and this 112

affects the quantity of bioactive compounds in it (Singh et al., 2014). Two methods i.e., calibration curve method and HPLC peak concentration equation, were used and suggested both method equivalent as quantification of TQ from both methods was similar. Pectin was extracted from the selected citrus waste peels. Citrus fruit waste poses great environmental threat in terms of their discarding and dumping. To overcome such issues waste to wealth strategies are often employed to manage waste (Khalid et al., 2011). Waste peels of lemon, orange and grapefruit, due to their abundance usage in juice industries and discarding issues, were selected for the extraction of biodegradable pectin as drug carrier to introduce their potential application in the pharmaceutical industry. The amount of extracted pectin depends directly on extraction procedures, solvents, temperature, time and acid types. For this point of view all factors (solvents, temperature, time and acid types) were kept constant and two different extraction procedures i.e., conventional heating and soxhlet were compared. Although not all waste peels can be utilized for the extraction of pectin, however, this pathway has introduced a way to explore waste in pharmaceutics. The soxhlet extraction method produced significantly higher amount of pectin from all samples as compared to the conventional heating (Tiwari et al., 2017; Bagherian et al., 2011). Confirmatory tests for pectin showed that all samples yielded pectin from both extraction methods. This also followed the fact that amount of pectin extraction depends on raw sources, extraction method, time, temperature, acid type and time (Georgiev et al., 2012). Pectin extracted from waste peels of citrus fruits (orange, lemon and grape fruit) was yellow to light brown to brown color (IPPA, 2009). The color of pectin extracted from different sources depends on the contaminations on peels, fruit source types, extracting solvents, temperature and time of extraction and also on the amount of alcohol used for precipitation of pectin (Aina et al., 2014). Extracted pectin remained insoluble in cold alkali and cold water at room temperature as described earlier by Aina et al., (2014). Identification of polysaccharide was done with Iodine test resulting in no blue color changed in each sample of pectin (Sharma et al., 2013). Soxhlet extraction from current study resulted in large amount of pectin yield (%) from waste peel of lemon (Mean±SE) 35.3±0.66, orange 30.5±0.93 and grape fruit 27.8±1.09 as compared with heating method. These results are supporting preceding 113

studies on yield of pectin by soxhlet extraction from citrus peel which was found maximum of 18.2 % and 14.60 % reported by Seixas et al., (2014) and Liew et al., (2014) respectively. Similar result was reported by the maximum yield of citrus peel pectin (CPP) 21.95 % (Wang et al., 2014). The fabrication of pectin from each citrus waste peel was higher in this study when compared with the earlier studies (Khan et al., 2013; Pinheiro et al., 2008). In current study moisture content (%) of each pectin samples derived from conventional heating was more as compared to the moisture content of three pectin lemon, orange and grape fruit samples extracted from soxhlet. Orange pectin extracted from soxhlet had less moisture content. Extracted pectin from both methods proved that all were slightly acidic in pH secondary the earlier studies (Aina et al., 2014). The yield of extracted pectin when all other factors (time, temperature, solvent) kept constant depends strongly on pH (Koffi et al., 2013). Although maximum extraction of pectin was resulted from lemon peel but characterization showed pectin obtained from orange peel was appropriate among other samples when compared to commercial pectin. The viscosity average molecular weight of each extracted pectin was determined and found higher molecular weight of orange pectin i.e., 83, 623.77 g/mol with comparison with commercial pectin i.e., 1,61,254.94 g/mol and having lower molecular weight of lemon and grape fruit pectin (33,318.83 and 59,304.616 respectively). These results can be related with the findings that molecular weight of pectin ranges from 50,000-250,000 Da (Sriamornsak, 2003). Earlier studies also revealed that acid-extraction of pectin usually characterized by high viscosity average molecular weight with high methoxyl content (Yapo and Koffi, 2013). Higher molecular weight of pectin are more beneficial because of its ability to breakdown in lower molecular weight in human body for detoxification and importantly forming compatible hydrogel as drug carrier for targeting drug delivery (Sayah et al., 2016). The FTIR spectra of both commercial and orange pectin representing showed corresponding parallel peaks. The characteristics regions which reflect composition of both extracted and commercial pectin were identified by the peaks of carboxylate ions stretching bands at 1640-1620 cm-1, carbonyl ester at 1760-1645 cm-1. C-H stretching -1 at 3000-2800 cm indicating methyl ester -CH, -CH2 and -CH3 of galacturonic acid and O-H stretching at wide range 3600-500 cm-1 showing increased free hydroxyl 114

groups stretching bonds. Similar band pattern was also confirmed by earlier studies (Fracsso et al., 2017; Liu et al., 2010; Gnanasambandam and Proctor, 2000). Pectin IR spectrum signified the identity peaks associated to free carboxyl groups peaks at 1620 cm-1 and peaks with esterified carboxyl groups at 1700 cm-1 and, which is consistent with the literature (Sato et al., 2011). IR spectrum in the wavelength range of 800 and 1300 cm-1 are considered as the characteristic (finger print) region for carbohydrates which allows the identification of major chemical groups in polysaccharides (Urias-Orona et al., 2011). The characteristic peaks of ester and carboxylic C=O vibrations were observed at 1730–1760 cm-1 (COO–R) and 1600– 1630 cm-1 (COO–), respectively. It has been shown that the relative intensity of the last two peaks is related to the degree of methoxylation (Saberian et al., 2017). The spectral peak at 2931 cm−1 indicated –C–H stretching vibrations, whereas the peak at 1749 cm−1 suggested the presence of ˃C=O, those at 1628 cm−1 and 444cm−1 corresponding to C=C and –CH2 stretching vibrations. Importantly, the peak range of 1725–1700 cm−1 is assigned to carboxylic acid (1699 cm−1 indicates carboxylic acid) and the peak at 1633 cm−1 shows the C=H stretching vibrations. The peak range of 3570–3200 cm−1 is assigned to –OH stretching and peaks at 3457 and 3245 cm−1 indicated the hydrogen-bonded –OH cm−1 stretching vibrations. IR spectrum of PLGA (50:50) represented the characteristics peaks at 2950-2980 cm-1 were due to C–H, C–

H3 and C–H2 functional group stretching vibration of PLGA while the peaks at 1350- 1400, 1430-1450 cm-1 were attributed to bending vibration in relation to the spectrum connected with C=O groups (Singh et al., 2014). The morphology of commercial pectin powder observed by SEM revealed its compact structure and extracted orange pectin powder had compact structure with small pores on smooth surface. The morphology of pectin observed by SEM also relates with other earlier studies (Liew et al., 2014; Luo et al. 2014). The equivalent weight (784), methoxyl content (9.3%) and AUA (72.8%) of orange pectin showed higher range as compared to pectin extracted from lemon and grape fruit. Orange pectin have showed parallel characteristics with commercial pectin which make it enable to consider for further analysis among all three selected waste peels of citrus fruits. High methoxyl content, equivalent weight are the features that allow one to prepare good gels in the occurrence of high sugar as orange pectin hydrogel formed in hot water while grape 115

fruit and lemon pectin failed to result in gel formation showing low methoxyl content (Yapo and Koffi, 2013). To enhance the gel stability two series i.e. PEG of different molecular weight and TEOS of different concentrations were considered. Characterization of pectin hydrogel with different PEG and TEOS concentration showed that Pectin hydrogel with PEG 6000 and molar concentration of TEOS 0.75M was more stable and showed significant characterization. The cross-linking step, TEOS concentrations were prepared in ethanol with one drop of HCl which act as a catalyst (Viteyazev et al., 2013). The assumed mechanism involved in the preparation of pectin hydrogel by using TEOS, as silica source to enhance the stability, interrelates with residues of galacturonic acid resulting in a bridge of (-O-Si(OH)2-O-) by linking them to the second and third hydroxyl groups (Figure 5.2). The functional groups methyl ester and primary amide groups in pectin involved in the formation of hydrogel. These methyl ester groups affect the gelling properties by eliminating negative charges and amide groups play significant role in formation of strong intra and intermolecular hydrogen bonds (Bán et al., 2009). Different study concluded that increased concentration of TEOS increases the stability and thickness of gel (Viteyazev et al., 2013; Assifaoui et al., 2013). The TEOS with concentration 0.75M was also suggested good results in study conducted by Viteyazev et al., (2013). The stability and swelling activities of hydrogel increases with increase concentration of TEOS but at certain high concentration reverse occurs. In this study 0.75 M concentration of TEOS in hydrogel showed significant results but at higher 1M concentration of TEOS swelling ratio of hydrogel decreases. Different characterization analysis swelling ratio (water, molar salt concentration, different pH) FTIR, TGA-DSC, XRD, SEM showed 0.75 M TEOS enhanced the hydrogel properties as compared to other concentration of TEOS. PEG of different molecular weight showed that swelling ratio of hydrogel increased with increased molecular weight i.e., 6000 had the maximum swelling among the other molecular weights (300, 600 and 1500) of PEG. 116

Figure 5.2: Assumed structures for the pectin-TEOS crosslinking by forming bridge between two pectin polymers (Vityazev et al., 2017)

117

Previous study has shown that increased in PEG molecular weight resulted in an increased in equilibrium swelling ratio, probably due to an increase in the molecular weight between cross-links. This follows the thermodynamic first principles of hydrogel networks (Parlato et al., 2014) The current study designed the novel pectin hydrogel combination with the extracted phytochemical compound loaded in pectin hydrogel cross-linked with 0.75 M TEOS and PEG of Mw. 6000. IR spectrum showed the significant peaks showing the successful loading of TQ in hydrogel. The nanoparticles of PLGA-PEG encapsulated TQ were successfully formulated. The encapsulation efficiency was above 90% which was also reported earlier (Nallamuthu et al., 2014). Different studies on the formation of PLGA nanoparticles resulted in successful preparation of PLGA NPs with stabilizing agent PVA (Cao et al., 2016; Nallamuthu et al., 2014; Yan et al., 2015). TQ was encapsulated in PLGA-PEG, the molecular weight Of PEG used was 6000 which was earlier selected in hydrogel formation, loaded in pectin hydrogel with 0.75 M TEOS concentration. The analysis of Pectin-PLGA-PEG (PPP) hydrogel proved this combination as successful hydrogel showing maximum swelling ratio and controlled release of drug in SGF and SIF medium. Characterization of PPP by TGA-DSC, FTIR, XRD and SEM showed that this combination had the best results as compared to other hydrogels without PLGA. The IR spectrum of physical crosslinking of Pectin-PLGA- PEG in presence of TEOS showed shifter peaks of all compounds when compared to their respective spectrum individually. This novel drug carrier can be utilized in drug delivery system as it showed delayed released of drug TQ in SGF and SIF medium. The pectin hydrogel loaded with TQ loaded PLGA-PEG NPs in PVA stabilizing agents showed enhanced activities of delayed release of TQ as compared to the PPP hydrogel loading TQ. Othman et al., (2018) studied loading of TQ NPs in chitosan which showed high encapsulation efficiency of TQ and average size of particles from 7 to 35 nm. The formation of PLGA-PEG encapsulated TQ NPs were confirmed by FTIR, TGA-DSC, XRD and SEM. SEM observation clearly depict the encapsulation of TQ in PLGA-PEG and average size of NPs were > 200nm. For the first time PLGA was physically blended with pectin hydrogel in presence of PEG 6000 and TEOS (0.75 M). The proposed structure for pectin-PLGA-PEG-TQ (PPPT) illustrated in figure 5.3. 118

Figure 5.3: A Suggested Structure of Hydrogel Pectin-PLGA-PEG-TQ Nanoparticles (PPPT-NPs) by Physical Crosslinking with TEOS.

The release study of drug TQ in SGF and SIF medium showed delayed release of TQ from PLGA-PEG NPs. The ideal release percentage of TQ in stomach fluid is 10 % which was achieved by cross-linked pectin hydrogel loaded TQ (PPPT) in 2h. The release of TQ from the pectin hydrogel in intestinal fluid also followed the rule of 100% drug release in more than 2h (Ma and Coombes, 2014). The pectin hydrogel in 2h completely dissolved in intestinal fluid showing that physical linkage of pectin- PLGA with TEOS was less stable suggesting chemical linkage in future. The delayed release of TQ from NPs in hydrogel showed less than 10% release in SGF and similar delayed released in SIF medium. This delayed released support the pectin properties which showed complete degradation of pectin up to the colon with help of enzymes and microflora present there making it potential drug carrier to deliver drug to colon (Shukla et al., 2011). Successful formulation of physical cross-linked pectin hydrogel loaded with PLGA- PEG-TQ (PPPT) and pectin hydrogel with PLGA-PEG-TQ NPs were further subjected to assess the in-vitro anti-inflammatory and anti-proliferative activities parallel to the analytical TQ standard and extracted samples of N. sativa and T. vulgaris. The in-vitro anti-inflammatory potential of all samples were evaluated by anti-oxidant assays DPPH and FRAP. Both assays showed significant anti-oxidant activities of standard TQ and highest in T. vulgaris as compared to N. sativa. Antioxidants inhibit with the generation of reactive oxygen specie (ROS) and also play a crucial role in their inactivation. ROS are source impairment to cellular biomolecules such as proteins, nucleic acids, carbohydrates and lipids (Adjimani and Asare, 2015). 119

The anti-oxidant activities of TQ increases in dose dependent manner i.e., higher the TQ dose higher the activities. FRAP assay for evaluation of anti-oxidant activities showed influential anti-oxidant nature of TQ standard from both samples seconds the studies showing potent FRAP reducing activity of TQ from N. sativa and T. vulgaris (Mohammed et al., 2016; Grosso et al., 2010). The radical scavenging activities DPPH assay was determined on the basis of IC50 of TQ standard which was 146.8 µg/mL supporting the results of earlier study needed 2.26 mg/ mL of TQ for 50% inhibition activity DPPH by N. sativa (Solati et al., 2014). T. vulgaris proved to be more potent anti-oxidant as compared to N. sativa possibly due to the fact of presence of thymol as bio-active component (Chizzola et al., 2008). The ROS scavenging activities of pectin hydrogel (PPPT) and (PPPT NPs) showed enhanced activities as compared to TQ alone. This showed that hydrogel itself has anti-oxidant potential along with its released drug in solution. The gel loaded with NPs showed highest anti-oxidant potential among all the samples. This increase ROS activities might be due to the fact studied by that nano-materials have antioxidants activities. The study also suggested that nanoparticles, exhibit essential redox activity that is often linked with radical trapping and with catalase-like or superoxide dismutase-like activities. Extensive researches are being carried out for prevention and control of carcinogenic process by using naturally occurring phyto-component. Current study showed in- vitro cell proliferation of HeLa cancer cell line has significant cell death with dose dependent manner of TQ standard along with extracted TQ from N. sativa and T. vulgaris. Studies showed anticancer effect of TQ on different type of cancer cells through in vitro and in vivo which indicate the involvement of TQ in different cell death signaling pathways including apoptosis, proliferation, angiogenesis and tumor induced immunosuppression (Marjaneh et al., 2014). MTT assay is a standardized and recognized test to screen possible cytotoxic effects in adherent cancer cell lines (HeLa cancer cell line) though it does not give any comprehensive information other than membrane stability of the cell but help in determination of potent activity of drug dose and time dependent manner (Yazan et al., 2009). For anti-proliferative activities MTT assay was performed by evaluation of cell death against treatments with extracted TQ samples and different formulations of pectin hydrogel with TQ and its PLGA-PEG TQ nanoparticles in parallel with analytical TQ 120

standard. Experimental groups were designed according to the concentrations and samples types for the treatment against cancer cells. N. sativa showed significant cancer cell death in 48 h with potent IC50 at 0.5 µM as compared to T. vulgaris (IC50 15 µM) and TQ standard (IC50 1.56 µM), seconds the previous studies conducted for anti-proliferation activities of N. sativa against different cancer cell lines (Liu et al., 2013; Venkatachallam et al., 2010). Tabasi et al., (2015) also determined the significant anticancer activities of N. sativa and TQ against human renal cell carcinoma (ACHN) and fibroblast L929 cell lines by measuring cytotoxicity by MTT assay. Although complete inhibition action mechanism of TQ is still unclear (Salim et al., 2103) studies are suggested to revealed the TQ cytotoxicity and apoptotic induction for tumor inhibition in animal models. The cell viability of cancer cell lines when treated with pectin hydrogel alone showed no significant decrease after 48 h incubation pointing the fact that pectin hydrogel was not potent anti-proliferative. Although, there was reduction in cell viability in comparison with incubation time after 24 and 48h. Similar study on cytotoxicity of cancer against hydrogel formulation pectin-PVP hydrogel induced no significant cancer cell death (Mishra et al., 2008). The MTT assay also showed delayed release of TQ from pectin-PLGA formulations and decreased cell death in comparison with TQ standard and NPs alone. As pectin hydrogel is highly pH dependent and show significant release in basic pH but in-vitro cancer cell line need acidic pH for their growth. For this reason the gel formulation did not release significant amount of TQ in cancer cell well showing delayed response against cancer cell death. The incubation time 48 h showed significant cell death in comparison with 24 h incubation time. SRB assay was used for non-adherent HCT116 cancer cell for cell cytotoxicity against pectin gel formulation loading TQ and PLGA-PEG NPs in parallel with TQ standard. SRB and MTT assay both have equivalent importance and showed significant results for cell cytotoxicity (Sonia et al., 2014). SRB assay was evaluated after 72 h incubation keeping in mind that MTT assay had already assessed in 24 and 48 h. Similar, trend in activities of each sample were observed as were in MTT assay. Pectin hydrogel loaded TQ again showed delayed release and minimum cell death than the alone TQ. As the incubation time was 72 h so the every sample showed increased IC50 as compared to 24 and 48 h incubation by MTT assay. Hydrogel has 121

the ability to entrapped drug which enable it for control drug release on degradation of network within hydrogel allowing drug to diffuse out of hydrogel. The degradation of hydrogel can occur in backbone of polymer or at the crosslinks site (Li and Mooney, 2018). In the current study pectin hydrogel was cross-linked with PLGA-PEG loaded with TQ in presence of TEOS cross-linker. In anti-proliferative activities against HeLa and HCT116 cancer cell lines the hydrogel loaded with drug and its nanoparticles showed delayed release of drug which was time dependent and pH specific. Studies have shown that diffusion of drug on degradation of hydrogel network is mediated by enzyme activity or hydrolysis (Li and Mooney, 2016; O’Shea et al., 2015). Current drug can be very effective in controlling the initial stages of cancer as results of apoptosis predicted. Due to limitation in time and facility, this study is limited to just in vitro analyses, however, in vivo analysis is recommended for future extension of the current work. The western blot analysis was carried out to determine PARP cleavage by programmed cell death in HCT116 cancer cell against concentration of TQ analytical standard showing IC50 concentration in SRB assay. For this reason, 5 and 10 µM concentration of TQ standard was selected (IC50 was 3.95 µM) and also for Cisplatin (IC50 6µM). PLGA-PEG-NPs were also considered by taking amount making TQ amount 5 and 10 µM). The main purpose of western blot analysis is to identify the PARP cleaved protein from complex mixture of protein extracted in cells. Three main steps involved are (i) separation by size, (ii) transfer to a solid support, and (iii) marking target protein using primary and secondary antibodies to visualize (Mahmood and Yang, 2012). The band formed on nitrocelloluse showed that TQ at this lower amount did not significantly pose change in the protein band while cisplatin had clear PARP cleavage. This might be the fact that the previous studies conducted western blot analysis showing potent effects when treated TQ in higher amount 40 µM (Zhang et al., 2016; Woo et al., 2012).

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CONCLUSION Current study successfully conducted anti-inflammatory and anti-proliferative activities of TQ loaded in PLGA nanoparticles in-vitro. The oil extracted with soxhlet apparatus from seeds of N. sativa was potent source for TQ as compared to plant of T. vulgaris. Sudden conversion of THQ into TQ and lack of its standard availability left this experiment to be conducted for TQ alone. It was concluded that methanol as a solvent is favorable for the maximum extraction of TQ from plant sources. Notable antiproliferative activities against cancer cell line were shown by oil of N. sativa as compared to T. vulgaris. While oil obtained from T. vulgaris was potent anti-oxidant agent when DPPH and FRAP assay were performed for evaluation of anti- inflammatory activities. Pectin as biodegradable natural polymer was positively extracted from all selected waste peels and orange peel was considerable from all. Current study provided an opportunity to utilize waste in pharmaceutics and to make it more economically viable. This opportunity can be explored further. The innovative drug delivery system was introduced utilizing biodegradable pectin as drug carrier physically cross-linked by cross-linker TEOS with PLGA loaded TQ and their Nanoparticles. Nanoparticles of TQ efficaciously produced loaded in PLGA- PEG. TQ proved to potent antiproliferative agent by MTT assay, SRB assay and western blot analysis against HeLa and HCT116 cancer cell lines Pectin hydrogel with cross-linked PLGA-PEG proved to be effective ecofriendly drug release system, by control release of TQ loaded in hydrogel. TQ thus obtained can be utilized against variety of cancers and substituted with the expensive treatments in developing countries.

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RECOMMENDATION From the current study, following recommendations can be generated  Screening and utilization of other native herbs for their effectiveness and disease control.  Exploration of more pharmaceutical components from the waste to minimize waste.  Sustainable use of regional medicinal plants for cure of many serious diseases including cancers.  For large scale cultivation and conservation of important medicinal herbs policies should be planned.  Although many pharmaceuticals and health benefits can be attained by the extraction of essential oils of herbs and pectin from citrus waste peels which can be additionally accompanied in wide range of products such as jams, squashes and juices to increase the quality and market value.  In vivo studies must be considered in future to get further idea of apoptosis potential of the current drug.  Consideration should be made on the large scale production of eco-friendly and cost effective control drug delivery system to the targeted areas for the economical betterment of regional pharmaceutical industries.

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Annexure 3a

HPLC Calibration Curve Method

………………………………………………………………….Eq. Annex. 3a

Where r2 = 0.9976 y = absorbance of peak area x = concentration

Annexure Figure 3a: Dilution Curve of TQ std. obtanied from regression equation

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Annexure 3b HPLC Peak Areas Calculation

Annexure 3c: TQ standard 200 µM and dilution of N. staiva and T. vulagirs for MTT assay Molecular weight of TQ= 164.2 g/mol For preparation of 100µM TQ 1 Molar= 0.0001 µM To prepare 1M concentration of TQ= 164.2g/L of solvent For 200 µM concentration of TQ= 0.32 µg/10mL of DMEM media For preparation of 200µM of extracted TQ from T. vulgaris Concentration of TQ from T. vulgaris 9.7mL oil= 548µg/mL= 548 mg/L Concentration=mass/volume Mass= concentration × volume=548×0.0097=5.31mg=0.0053g Molarity= no. of moles (N)/ volume in liter (L) Where N= mass in gram/ mol. weight (g/mol) = 0.0053/164.2= 0.000032 Molarity= N/L= 0.0000207/0.0097= 0.003 M

M1V1=M2V2

M1= Molarity of T. vulgaris= 0.003

M2= Molarity to prepare 200µM or 0.0002 M

V2= volume to be prepared 10mL

V1= volume required

V1= 0.0002× 10/ 0.003= 0.66 mL= 0.66 mL oil in 10 mL medium For preparation of 200 µM of extracted TQ from N. sativa Concentration of TQ from N. sativa 15.8 mL oil= 625 µg/mL= 625 mg/L Concentration=mass/volume xv

Mass= concentration × volume (liter) =625×0.0158=0.0098g Molarity= no. of moles (N)/ volume in liter (L) Where N= mass in gram/ mol. weight (g/mol) = 0.0098/164.2= 0.00006 Molarity= N/L= 0.00006/0.0158= 0.0038 M

M1V1=M2V2

M1= Molarity of N. sativa= 0.0038 M

M2= Molarity to prepare 200µM or 0.0002M

V2= volume to be prepared 10mL

V1= volume required

V1= 0.0002 × 10/ 0.0038= 0.26 mL= 0.6 mL oil in 10 mL medium Annexure 3 d: Protocol of MTT assay Kit (Trevigin) Reagents 1. cell culture medium 2. microplate plate (flat bottomed) 3. sterile tubes (5 ml) 4. serological pipettes 5. sterile pipette tips Reagent Preparation 1. MTT Reagent The MTT Reagent (Cat# 4890-25-01) is supplied ready for use. The MTT Reagent is stable at 4 oC provided there is no contamination. Care should be taken not to contaminate the MTT reagent with cell culture medium during pipetting. It is recommended that the appropriate volume required for each experiment is aliquoted and placed into a separate clean tube under sterile conditions and the stock bottle is returned to 4 oC in the dark. If the MTT Reagent is blue-green in color do not use and refer to the Troubleshooting Guide on page 6. 2. Detergent Reagent The Detergent Solution (Cat# 4890-25-02) is supplied ready for use. If the Detergent Reagent has been stored at 4 oC, warm the bottle for 5 minutes at 37 oC then invert gently while mixing to avoid frothing. xvi

Assay Protocol -bottomed 96 well plate (tissue culture grade). The incubation period and the cell plating density should be determined well) and the plate is incubated for 2 to 12 hours to allow for intracellular reduction of the soluble yellow MTT to the insoluble purple formazan dye. Detergent reagent is added to each well to solubilize the formazan dye prior to measuring the absorbance of each sample in a microplate reader at 550 - 600 nm, depending upon the filters available. The complete protocol for optimizing the assay for your experimental system is given below.

Step Instructions Notes 1 Resuspend cells at 1 x 106 Harvest suspension cells per ml by cen-trifugation. Adherent cells should be released from their substrate by trypsinization or scraping. 2 Prepare dilutions of cells The number of cells per from 1 x 106 to 1 x 104 well re-quired for optimal cells per ml in order to results will vary plate cells at 103 – 105 depending upon cell type, per well. culture conditions, etc. 3 Distribute, in triplicate, Cells with medium alone provides the blank for the well. Include three con- absorbance read-ings. trols of medium alone. 4 Incubate the cells for 6 to The cells need time to 48 hours. recover and/ or adhere to the substrate. The time required will vary xvii

between cell types but 2 hours to overnight is sufficient for most cell lines. 5 reagent (Cat# 4890-25-01) medium was to each well. used per well increase the amount of MTT Reagent used accordingly e. g. for 250

of MTT reagent. To avoid con-tamination of the MTT reagent it is advisable to place a sample of MTT Reagent in a clean tube for aliquots and return the stock solution to 4 oC in the dark. 6 Return plate to cell culture Periodically view the cells incu-bator for 2 to 4 hours under an inverted until purple dye is visible. microscope for pre-sence of intracellular punctate purple precipitate. Longer periods of incubation of up to 24 hours may be required for some cell types.

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7 When the purple Samples can be read after 2 precipitate is clearly visible hours. If the readings are under the micro-scope, add low return the plate to the dark and incubate for a Reagent (Cat# 4890-25-02) longer period. The to all wells. Do not shake. solubilization time may be shortened by incubation at 37 oC but room temperature is usually adequate. 8 Leave plate with cover in Samples can be read after 2 the dark for 2 to 4 hours or hours. If the readings are overnight at room low return the plate to the temperature. dark and incubate for a longer period. The solubilization time may be shortened by incubation at 37 oC but room temperature is usu-ally adequate. 9 Remove plate cover and Absorbances can be read mea-sure the absorbance in with any filter in the each well, including the wavelength range of 550 - blanks, at 570 nm in a 600 nm. The reference microplate plate reader. wavelength should be higher than 650 nm. The blanks should give values close to zero (+/- 0.1). xix

10 Determine the average The cell number selected values from triplicate should lie readings and subtract the within the linear portion of average value for the the plot. blank. Plot absorbance against cell number/ml. Select a cell number that yields an absorbance of 0.75 - 1.25. 11 Analyze the experimental Perform appropriate system controls inclu-ding blanks to be tested using the cell (media only) and untreated number per well cells (refer to section VII). determined in step 10 above, in triplicate, and repeat MTT Cell Proliferation Assay steps 3 to 9.

Controls The minimum number of controls that should be included when running your assay are: 1. Blank wells containing medium only. 2. Untreated control cells. The absorbance range for the control cells (i.e. untreated) should typically be between 0.75 and 1.25. The cell number for plating should be determined using the procedure described in the Assay Protocol.

Annexure 3e: Calculation of Pectin hydrogel loaded TQ and NPs for MTT assay against HeLa cancer cell lines Pectin hydrogel loaded with PLGA-PEG6000-TQ (100 µM) 2% pectin hydrogel preparation= 1g of pectin in 50 mL water + 50 mg PLGA-PEG (25+25mg)+ 0.016mg TQ in 0.01 ml DMSO 5mL pectin loaded TQ hydrogel+ 5mL medium= making 1% pectin and 100µM Calculation for Nanoparticles PPT NPs was dissolved in 0.01 % DMSO and up to 10 mL media making its concentration nearly 200 µM of TQ, by calculating amount of TQ per mg of NPs. Concentration for Cisplatin 50mg/50mL Molecular weight= 300.01 g/mol 50mg/50mL Cisplatin = 1mg/mL Cisplatin = 3.333mM =3332µM Mass= Concentration × Volume × Molecular weight Diluting with 50 mL water= 66.64 µM To prepare 50 µM= 1mL of Cisplatin in 65mL water

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Annexure 3f: Calculations for SRB Assay against HCT116 TQ E4 TQ standard= 0.164mg+in 500microliter DMSO 50µM = 0.084+0.1mL DMSO+100mL DMEM 25, 12.5, 6.25, 3.12, 1.56

NPs PLGA-PEG-TQ NPs 0.62mg in 0.1mL DMSO+100 mL DMEM alone E8

PPP Pectin Hydrogel 2% (0.18g+10mL water) +PLGA-PEG (0.02mg in ACN) alone +0.1mL DMSO+100mL DMEM hydrogel E5

PPP Pectin hydrogel 2% (0.18g+10mL water) +PLGA-PEG (0.02mg in loaded Acetonitrile) + TQ (0.08mg+0.1mL DMSO)+50mL media TQ E6

PPP Pectin hydrogel (199mg+10 mL water)+ PLGA-PEG-TQ NPs dissolved in loaded 0.1mL DMSO+100mL DMEM NPs E7 Calculated TQ 0.62mg/mg of NPs..0.12mg NPs containing in 0.1mL DMSO

Cisplatin Cisplatin 50 µM= 2mL of Cisplatin in130 mL water (30mL+100mL E9 DMEM) 25 µM= 1mL of cisplatin in 130mL (30mL water+100mL DMEM)

NC Negative control 1 DMSO Treated

PC Positive Control Untreated

Calculations For TQ mol.wt 164.2g/L=1M 100µM=0.164mg/100ml 50 µM=0.08mg/100 ml Media+0.1mlDMSO to make 0.1% NPs calculations Weight of free drug in supernatant=

= xxii

76.67 = 0.076 mg free TQ

Drug loading Capacity=

= =62.4%

Drug Encapsulation efficiency=

=99.84%

Weight of NPs formed= 80mg Total loaded drug in NPs= = 49.9 mg of TQ calculated in 80mg of NPs Per mg of NPs load TQ= 49.9/80=0.62 mg TQ/ mg of NPs TQ NPs 0.62 mg /1mg of NPs Need 0.08mg TQ to make 50 µM 0.12 mg NPs have 0.08mg TQ in 0.1mL DMSO +100mL media Pectin hydrogel calculation PPP Pectin Hydrogel 2% (0.18g+10mL water) +PLGA-PEG (0.02mg in Acetonitrile) +0.1mL DMSO+100mL DMEM [to make 2% total amount of pectin+PLGA-PEG should be 0.2g] PPPT Required 1% pectin hydrogel (1g in 100mL water). As dilution are made so 2 % pectin hydrogel considered 0.18g pectin in 10mL water + 0.02g PLGA-PEG in acetonitrile+ TQ 0.08mg in 0.1mL DMSo+100 mL Media[t make pectin 1% and TQ 50 µM) PPPT NPs Pectin hydrogel (199.8 mg+10 mL water)+ 0.12 mg PLGA-PEG-TQ NPs dissolved in 0.1mL DMSO+100mL DMEM

Calculated TQ 0.62mg/mg of NPs.. Cisplatin Calculation Molecular weight= 300.01 g/mol 50mg/50mL Cisplatin = 1mg/mL Cisplatin = 3.333mM =3332µM xxiii

Mass= Concentration × Volume × Molecular weight Diluting with 50 mL water= 66.64 µM To prepare 50 µM= 1mL of Cisplatin in 65mL water Cisplatin 50 µM= 1mL of Cisplatin in 65mL water (15mL+50mL DMEM)

OR 2mL of cisplatin in 130mL (30mL water+100mL DMEM) So 25 µM= 1mL of cisplatin in 130mL (30mL water+100mL DMEM)

Annexure 3 g: Protocol for SRB Assay (Orellana and Kasinski, 2016)

Materials and Reagents

96-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3596)

384-well clear flat-bottom polystyrene tissue-culture plates (Corning, catalog number: 3701)

96-well PCR plates (Corning, Axygen®, catalog number: PCR-96-FS-C)

100 mm tissue-culture plates (Corning, catalog number: 430167)

1.5 ml Eppendorf tubes (VWR, catalog number: 89000-028)

15 ml Falcon tubes (Corning, Falcon®, catalog number: 352097)

Pipette tips (Mettler-Toledo International, catalog numbers: RT-L10FLR)

Pipette tips (Mettler-Toledo International, catalog numbers: RT-L200F)

Pipette tips (Mettler-Toledo International, catalog numbers: RT-L1000F)

Matrix™ pipette tips (1,250 μl) (Thermo Fisher Scientific, Thermo Scientific™, catalog numbers: 8245)

Matrix™ pipette tips (125 μl) (Thermo Fisher Scientific, Thermo Scientific™, catalog numbers: 7445)

Adherent cell line of interest

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Appropriate culture medium

Reagent reservoir sterile (Corning, Costar®, catalog number: 4870)

Reagent reservoir non sterile (VWR, catalog number: 89094-684)

Opti-MEM® (Thermo Fisher Scientific, Gibco™, catalog number: 31985070) or serum free medium (SFM, appropriate culture medium before adding fetal bovine serum)

Phosphate buffered saline (PBS) (GE Healthcare, catalog number: SH30256)

Trypsin solution (2.5%, wt/vol) (GE Healthcare, catalog number: SH30042.01)

Fatal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442)

Trypan blue (Sigma-Aldrich, catalog number: T9154)

Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: 91228)

Sulforhodamine B sodium salt (SRB) (Sigma-Aldrich, catalog number: S1402) in 1% (vol/vol) acetic acid

Acetic acid (Thermo Fisher Scientific, Fisher Scientific, catalog number: S25118)

10 mM unbuffered Tris base solution (Sigma-Aldrich)

DNAse/RNAse free water (Thermo Fisher Scientific, Ambion™, catalog number: AM9932)

Lipofectamine RNAimax (Thermo Fisher Scientific, Invitrogen™, catalog number: 13778150)

Mirvana miRNA mimics (Thermo Fisher Scientific, Ambion™) miRNA precursor molecules - negative control #2 (non-targeting scramble miRNA) (Thermo Fisher Scientific, Ambion™, catalog number: AM17111)

Software Statistical analysis software (GraphPad Prism, SPSS)

Procedure

A. Treatment solution preparation xxv

Volumes of treatment of choice should be enough for triplicates in 96-well plates (50 μl per replicate; final volume in well 100 μl) or six replicates in 384-well plates (10 μl per replicate; final volume in well 20 μl) and also account for pipetting variation. Treatment can be prepared in aqueous solution (Opti-MEM for transfections) or solvent of choice (e.g., DMSO).

B. Cell preparation Remove medium from cell monolayers and wash the cells once with sterilized PBS.

Remove PBS and add 1 ml (100 mm plates) 0.25% (wt/vol) trypsin to evenly cover the cell-growth surface.

Incubate at 37 °C for 5 min or until cells start to dissociate.

Next inactivate trypsin with 10 volumes of culture medium containing FBS, and mix up and down to obtain a homogeneous single cell suspension.

Transfer the cell suspension to a sterile Falcon tube. Determine the cell concentration by counting in a hematocytometer chamber under a microscope using a 1:1 mixture of cell suspension and 0.4% (wt/vol) trypan blue solution to determine cell viability prior cell seeding. Optional: before counting, spin down cells in order to wash trypsin and resuspend in growth medium.

Adjust the cell concentration with growth medium (10% FBS) to obtain an appropriate cell seeding density per well in a volume of 50 μl (96-well format) or 10 μl (384-well format). Note: Initial cell seeding density will depend on two main factors: 1) doubling time of the cell line used and 2) the day the plate will be read. Cells are typically at 70–80% confluency (2 × 104 for 96-well format and 8 × 103 for 384-well format) by the end of the experiment (day 5).

Transfer the cell suspension into a sterile reagent reservoir to make it easier to pipette with a multichannel pipette.

C. Treatment exposure

Mix the treatment solutions prepared in step A by pipetting. Dispense 50 μl (96-well format) or 10 μl (384-well format) of solution into each well. Mix cell suspension prepared in step B thoroughly and add 50 μl (96-well format) or 10 μl (384-well format) to each well already containing treatment solutions. Note: Ensure even cell distribution in the bottom of the plate and avoid shaking the plate to avoid ‘ring effects’. The best way to achieve this is to add the cell solution directly to the bottom of the well, avoiding touching the walls. We also recommend performing a short spin of the plate (20 sec, 10 × g) before placing it in the incubator.

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Set aside three wells in the plate containing only Opti-MEM or solvent of choice and cell suspension for an untreated or vehicle control. Also, leave three wells in the plate containing only medium for background subtraction.

Incubate the plate at 37 °C in a humidified incubator with 5% CO2 until plate is to be read. Note: The incubation time will depend on the type of compound being tested and has to be experimentally determined. For example, in our hands we observe the biggest difference in cell proliferation between negative control and a tumor suppressive miRNA 5 days following transfection.

D. Cell fixation and staining

Gently add 25 μl (96-well format) or 5 μl (384-well format) cold 50% (wt/vol) TCA to each well directly to medium supernatant, and incubate the plates at 4 °C for 1 h. Mixing is not required, as this could lead to some cells detaching from the bottom of the well. Wash the plates four times by submerging the plate in a tub with slow-running tap water and remove excess water by gently tapping the plate into a paper towel. After the last wash allow the plate to air-dry at room temperature. Note: Cell monolayer detachment can occur if water is forced into the wells. We recommend letting the plate dry completely before continuing to next step. If necessary, dried plates can be stored at room temperature indefinitely.

Add 50 μl (96-well format) or 20 μl (384-well format) of 0.04% (wt/vol) SRB solution to each well. Note: Ensure that the solution is in direct contact with the bottom of the well and that there are no bubbles in between. SRB solution is not light sensitive thus plate does not need to be covered during incubation.

Leave at room temperature for 1 h and then quickly rinse the plates four times with 1% (vol/vol) acetic acid (200 μl for 96-well format or 30 μl for 384-well format) to remove unbound dye. Allow the plate to air-dry at room temperature. Note: Non-homogeneous washes are a major source of error in this assay. Washes must be done quickly and homogeneously across the entire plate. This is particularly important for 384-well format. The small area of the wells leads to high superficial tension sometimes making it difficult to wash the plates uniformly. Visually check that all the wells in the plate have been injected with 1% (vol/vol) acetic acid and that there are no bubbles preventing the washes. We recommend washing with multi-channel pipet and injecting the solution indirectly into the wells using the walls of the wells.

E. Absorbance measurement Add 50 μl to 100 μl of 10 mM Tris base solution (pH 10.5) to each well and shake the plate on an orbital shaker for 10 min to solubilize the protein-bound dye (approximately 10 min).

xxvii

Measure the absorbance at 510 nm in a microplate reader.

Data analysis Subtract background absorbance from all wells. Calculate the percentage of cell-growth inhibition normalizing treatments to miRNA negative control using the following formula: SRB assay results can only be used if the data falls within the linearity limit of the assay. We recommend performing a cell number titration experiment to determine the linear dynamic range of the assay with the cell line used (Figure 2A). Generally, O.D. of > 2 are not within the linear range of the assay. Statistical tests for this type of assay can include t-tests for two group comparisons or ANOVA for multiple group comparisons.

Annexuere 4a: Comparison of quantified amount of extraced TQ from N. sativa and T. vulgaris from different methods (HPLC equation and Calibertaion curve method)

xxviii

Annexure 4b: Swelling (g/g) of pectin hydrogel with different molecular weight of PEG in pectin hydrogel

Time (min) PEG 300 PEG 600 PEG 1500 PEG 6000 10 8.82 9.5 5.9 16.9 20 9.89 10.96 6.98 17.69 30 6.87 11.78 5.6 18.50 40 8.89 19.82

Annexure 4c: Swelling (g/g) of pectin hydrogel with different concentrations of TEOS in water

Time (min) 0.25 M 0.50 M 0.75 M 1.0 M TEOS PPPT(Pectin- TEOS TEOS TEOS PLGA- PEG6000- TQ-0.75 TEOS) 15 10.02 12.5 22.66 20.9 25.3 30 13.89 15.96 28.98 25.9 32.09 45 12.87 14 37.64 26.2 39.89 60 10.88 12.89 38.08 24.2 46.12 75 7.06 8.83 22.78 19.65 50.45 80 - - - - 33.35

xxix

Annexure 4d: Swelling (g/g) of pectin hydrogel with varying TEOS concentrations in different pH pH 0.25 M 0.50 M 0.75 M 1.0 M TEOS PPPT(Pectin- TEOS TEOS TEOS PLGA- PEG6000- TQ-0.75 TEOS) pH2 9.65 10.33 15.98 11.94 17 pH4 9.89 11 14.78 12 17.98 pH6 12.78 20.33 23.98 18.94 18.98 pH7 14 21.78 33 19.9 28 pH10 25 28 38 29 43.6

Annexure 4e: Swelling (g/g) of pectin hydrogel with varying TEOS concentrations in NaCl molar dilutions

Molar Conc. 1.0 M TEOS 0.75 M 0.50 M 0.25 M PPP(Pectin- Of NaCl TEOS TEOS TEOS PLGA- PEG6000- 0.75 TEOS) 0.2 14.08 15.67 23.22 21.2 17.8 0.4 12.23 13.5 20.98 17.22 16.35 0.8 11.35 13.45 17.15 15 15.2 1 8.8 9.8 15.56 12.78 11.6

xxx

Annexure 4f: Swelling (g/g) of pectin hydrogel with varying TEOS concentrations in CaCl molar dilutions

Molar Conc. 1.0 M TEOS 0.75 M 0.50 M 0.25 M PPP(Pectin- Of CaCl TEOS TEOS TEOS PLGA- PEG6000- 0.75 TEOS) 0.2 13.08 14.87 15.22 15.62 16.2 0.4 11.23 13.9 14.8 13.22 15.35 0.8 10.35 13.09 13.15 12.09 14.2 1 9.8 11.0 12.56 10.78 12.6 Annexure 4g: Control release of TQ loaded in Pectin-PLGA-PEG(6000)- TEOS(0.75M) hydrogel

Time (min) SGF Release (%) SIF Release (%) 10 0.314 4.18 20 0.70 9.28 30 1.16 16.17 40 1.88 23.07 50 2.68 31.06 60 3.55 39.59 70 4.48 49.03 80 5.52 59.61 90 6.64 70.65 100 7.85 82.68 110 8.166 95.17 120 9.55 100 Reference (absorbance) 1.71 1.31

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Annexure 4h: Control release of TQ drug in PPPT-NPs (nanoparticles) hydrogel in SGF and SIF medium Time (min) SGF Release (%) SIF Release (%) 10 0.14 3.48 20 0.55 7.49 30 0.77 12.18 40 1.03 17.4 50 1.39 23.08 60 1.82 29.28 70 2.33 36.32 80 2.86 43.72 90 3.61 51.62 100 4.82 59.82 110 6.5 68.3 120 8.65 77.23 130 - 86.63 140 - 100 150 - Reference (absorbance) 1.72 1.32 Annexure 4i. calculation of TQ in nanoparticles, efficiency loading and weight.

Amount of TQ per mg of NPs= ……………………..Eq.b

= = 0.62 mg

Total loaded drug in NPs= = 49.9 mg of TQ

xxxii

Annexure 4j: FRAP Assay activities absorbance by UV-VIS

No. Of Concentration of Samples Absorbance Sample

Ascorbic Acid control 1.09

1 600µg/L 0.35

2 400µg/L 0.18

3 200µg/L 0.083

4 100µg/L 0.042

5 50µg/L 0.016

6 Nigella sativa 0.47

7 Thymus vulgaris 0.42

Annexure 4k: DPPH Assay activities absorbance by UV-VIS

No. Of Concentration of Samples Absorbance Inhibition (%) Sample

1 Ascorbic Acid control 1.09

2 600µg/mL 0.38 65.13

3 400µg/mL 0.45 58.71

4 200µg/mL 0.65 40.35

5 100µg/mL 0.83 29

6 50µg/mL 0.92 16.51

7 25µg/mL 1.02 6.4 xxxiii

7 Nigella sativa 0.15 86

8 Thymus vulgaris 0.20 89

Annexure 4l: Comparative Cell proliferation ratio in different concentrations of TQ standard and extracted TQ from N.S and T.V against HeLa cancer cell lines

Experimental Group after 48 h incubation Concentrations 200 100 50 25 12.5 6.25 3.12 1.56 0.78

E1 3.09 13.8 15.42 23.2 28± 30.1 38.6 50.7 95.55 Pure TQ Cell ±0.0 ±0.0 ±0.00 5±0. 0.02 ±0.0 ±0.2 3±0. ±0.00 Proliferation%+SE 07 06 4 179 4 2 4 05 6 M

E2 Extracted TQ in 0±0. 0±0. 0±0.0 3.57 7.5± 18.5 19.8 21.0 32±0. N.S Cell 006 01 5 ±0.0 0.00 7±0. ±0.0 7±0. 009 Proliferation% 5 7 005 07 008 (%+SEM) E3 21± 31.7 39.28 46.4 53.5 57.4 75± 82.1 96.42 Extracted TQ in 0.02 ±0.0 ±0.01 ±0.0 7±0. 2±0. 0.00 7±0. 0±0.0 T.V Cell 9 09 5 2 03 01 5 015 05 Proliferation% (%+SEM) Control groups

Negative control Group C1 0.08±0.003658 (Mean±SEM)

Negative control Group C2 0.10±0.006667 (Mean±SEM)

Positive control group P (Mean±SEM) 0.3±0.009545 xxxiv

Annexure 4m: Comparative Cell proliferation ratio (%) in different concentrations of TQ standard (200 µm), synthesized pectin hydrogel loaded with drug and NPs, TQ NPs and Cisplatin (50 µm) after 24 & 48 h incubation by MTT ASSAY

Experimental groups after 24 and 48 h incubation

Concentrations (µM) 200 100 50 25 12.5 6.25 3.12 1.56 0.78 E4 24 hour 3.5±0. 12± 25±0 35±0 58±0 58±0 66.02 75.5 95.5±0.0 Pure TQ 01 0.01 .005 .005 .005 .004 ±0.0 ±0.0 04 Cell 06 02 Proliferat 48 hour 0±0.00 0±0. 14±0 24.8 37.8 49.7 58.5 61.7 89.25±0. ion(%±S 4 012 .0026 ±0.0 ±0.0 ±0.0 ±0.0 5±0. 006 EM) 04 03 04 11 004 E5 PPP 24 hour 55±0.0 80± 82±0 84±0 86±0 95±0 100± 100 100±0.0 hydrogel 3 0.08 .02 .024 .04 .01 0.05 ±0.0 9 2% 4 (%±SEM 48 hour 35±0.0 550 82±0 84±0 86±0 95±0 100± 100 100±0.0 ) 03 ±0.0 .02 .0024 .003 .004 0.015 ±0.0 7 2 14

E6 24 hour 0±0.00 0±0. 0±0. 6.51 33±0 39±0 85±0 100 100±0.0 PPPT 12 014 030 ±0.0 .026 .016 .027 ±0.0 010 (200 11 01 µM)Cell 48 hour 0±0.00 0±0. 0±0. 3.65 18.38 19.4 47.97 91.6 100±0.0 Proliferat 2 024 030 ±0.0 ±0.0 ±0.0 ±0.0 6±0. 010 ion%(%± 16 26 16 07 001 SEM) E7 24 hour 0±0.02 0±0. 0±0. 35±0 87±0 95±0 100± 100 100±0.0 PPPT- 9 009 015 .02 .03 .01 0.005 ±0.0 04 NPs Cell .15 xxxv

Proliferat 48 hour 0±0.00 0±0. 0±0. 3.5± 18±0 19.7 47.97 100 100±0.0 ion%(%± 8 01 03 0.016 .03 ±0.0 ±0.0 ±0.0 04 SEM) 016 05 .15

E8 TQ 24 hour 0 ± 0±0. 0±0. 35±0 87.22 99±0 100± 100 100±0.0 loaded 0.0065 011 006 .025 ±0.0 .015 0.011 ±0.0 14 nanoparti 05 16 cles Cell 48 hour 0±0.00 0±0. 0±0. 0±0. 0±0. 60±0 99±0 100 100 ± Proliferat 4 005 006 005 001 .015 .006 ±0.0 0.001 ion% 06 (%±SEM ) E9 Conc. 50 25 12.5 6.25 3.12 1.56 0.78 0.39 0 Cisplatin (50 µM) 24 hour 0±0.00 0±0. 39.44 60.33 61.38 85.5 100± 100 99.77± Cell 7 02 ±0.0 ±0.0 ±0.0 ±0.0 0.025 ±0.0 0.047 Proliferat 24 10 07 25 47 ion% 48 hour 0±0.00 0±0. 0±0. 45±0 65.5 85.5 100± 100 100±0.0 (%±SEM 4 008 006 .028 ±0.0 ±0.0 0.025 ±0.0 07 ) 07 09 2

Control groups Negative control Group C3 (Mean ± SEM) 0.16 ± 0.07

Positive control group P1 (Mean ± SEM) 0.36 ± 0.008

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Annexure 4n: IC50 by MTT Assay After 24 H By Non-Linear Regression Graph Pad Prism 8.0

E4 E5 E6 E7 E8 E9 C3 P1 TQ PPP(H PPP PPPT( NP Cispl Negative Positive ydroge T NPs) s atin Control Control l) log(inhibitor) Perfect fit Perfect vs. normalized fit response Best-fit values LogIC50 0.998 2.412 0.829 1.307 1.3 0.765 1.933 17.90 7 5 17 3 IC50 9.970 257.9 6.753 20.29 20. 5.826 85.71 7.985e+0 73 17 Goodness of Fit Degrees of 8 8 8 8 8 8 0 0 Freedom R squared 0.946 0.8713 0.909 0.8607 0.8 0.933 1.000 1.000 9 5 54 2 2 Sum of 392.2 216.0 1376 2524 26 839.7 0.000 0.000 Squares 87 Sy.x 7.002 5.196 13.11 17.76 18. 10.25 33 Number of points # of X values 9 9 9 9 9 9 1 1 # Y values 9 9 9 9 9 9 1 1 analyzed xxxvii

Annexure 4o: IC50 by MTT Assay After 48 H By Non-Linear Regression Graph Pad Prism 8.0

E4 E5 E6 E7 E8 E9 C3 P1 Positive TQ PPP(Hydr PPP PPPT(N NPs Cisplat Negative Control ogel) T Ps) in Control log(inhibitor) vs. normalized response Best-fit values LogIC50 0.864 1.771 0.54 0.5761 0.81 0.6406 1.824 13.25 9 84 91 IC50 7.32 59.01 3.53 3.768 6.59 4.371 66.67 176099152 5 5 Goodness of Fit Degrees of Freedom 26 26 26 26 26 26 11 11 R squared 0.955 0.9000 0.90 0.8761 0.82 0.8955 0.000 -1.776e- 7 07 53 015 Sum of Squares 1233 2909 3745 5106 100 4436 0.6468 0.008448 01 Sy.x 6.888 10.58 12.0 14.01 19.6 13.06 0.2425 0.02771 0 1 Number of points # of X values 27 27 27 27 27 27 12 12 # Y values analyzed 27 27 27 27 27 27 12 12

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Annexure 4p: Comparative Cell proliferation ratio (%) in different concentrations of TQ standard (50 µm), synthesized pectin hydrogel loaded with drug and NPs, TQ NPs and Cisplatin (25 µm) after 72 h incubation by SRB ASSAY

Experimental groups for SRB assay after 72 h

Concentrations (µM) 50 16.66 5.556 1.852 0.617 0.00 E10 1.56±0. 3.125 26.25± 56.875± 62.5± 68.1 Pure TQ Cell 001 ±0.00 0.005 0.007 0.006 ±0.002 Proliferation(%±SEM) 4

E11 PPP hydrogel 2% 22±0.0 48.7± 58.1± 60.9± 63.12± 70±0.000 (%±SEM) 04 0.001 0.001 0.002 0.005

E12 50±0.0 56.8± 60±0.0 62.8± 65± 67.15± PPPT (200 µM)Cell 02 0.005 030 0.02 0.001 0.00 Proliferation%(%±SEM) E13 57.8±0. 62.5± 65.3±0 66.8±0.0 67.81± 70.6± PPPT-NPs Cell 004 0.002 .002 01 0.002 0.0001 Proliferation%(%±SEM)

E14 TQ loaded 54.3 ± 56.8± 59.06± 60.1±0.0 64.7± 67.5± nanoparticles Cell 0.004 0.002 0.002 03 0.005 0.001 Proliferation% (%±SEM) E15 Cisplatin (25 µM) Conc. 25 8.33 2.77 0.93 0.310 Cell Proliferation% 0.00 4.3±0. 33.12± 56.25±0. 63.7 65.6± 67.8±0. (%±SEM) 001 0.006 000 5±0. 0.001 001 002

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Control groups Negative control Group C4 (Mean ± SEM) 0.040 ± 0.002

Positive control group P2 (Mean ± SEM) 0.36 ± 0.008

Annexure 4q: IC50 by SRB Assay Non-Linear Regression Graph Pad Prism 8.0

E10 E11 TQ E12 E13 E14 PPP E15 Negative Std. TQ NPs PPP TQ PPP No TQ Cisplati Control C4 NPs n [Inhibitor] vs. normalized response Best-fit values IC50 3.915 13.55 6.443 5.622 1.876 3.679 27.54 logIC50 0.5928 1.132 0.8091 0.7499 0.2733 0.8247 1.440 95% CI (profile likelihood) Goodness of Fit Degrees of Freedom 11 11 11 11 11 11 11 R squared 0.9376 0.8811 0.9225 0.9127 0.9486 0.9179 -0.1672 Sum of Squares 1294 1538 1009 1090 753.3 1295 127601 Sy.x 10.85 11.83 9.578 9.952 8.276 10.85 107.7 Constraints IC50 IC50 > IC50 > IC50 > IC50 > IC50 > 0 IC50 > IC50 > 0 0 0 0 0 0 Number of points # of X values 12 12 12 12 12 18 12 # Y values analyzed 12 12 12 12 12 12 12

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LIST OF PUBLICATIONS

1. Butt, S. A., Nisar, N., Ghani, N., Altaf, I. and Mughal, A. T. 2019. Isolation of thymoquinone from Nigella sativa L. and Thymus vulgaris L., and its anti- proliferative effect on HeLa cancer cell lines. Tropical Journal of Pharmaceutical Research, 18 (1): 37-42.

2. Butt, S. A., Nisar, N. and Mughal, A. T. 2018. A review: Therapeutics potentials of phytochemical drugs and their loading in pH specific degradable Nano-drug carrier targeting colorectal cancer. Journal of Pakistan Medical Association, 68(4): 602-614.

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Tropical Journal of Pharmaceutical Research January 2019; 18 (1): 37-42 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.

Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v18i1.6 Original Research Article

Isolation of thymoquinone from Nigella sativa L. and Thymus vulgaris L., and its anti-proliferative effect on HeLa cancer cell lines

Ayesha Siddique Butt1*, Numrah Nisar1, Nadia Ghani1, Imran Altaf2, Tahira Aziz Mughal1 1Department of Environmental Science, Lahore College for Women University, Lahore, Pakistan, 2Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan

*For correspondence: Email: [email protected]; Tel: +92-321-9489301

Sent for review: 19 August 2018 Revised accepted: 15 December 2018

Abstract Purpose: To isolate thymoquinone (TQ) from Nigella sativa L. and Thymus vulgaris L., and investigate its anti-proliferative effect on HeLa cancer cells. Method: Pulverized dried samples of N. sativa seed (100 g) and aerial parts of T. vulgaris (1000 g) were subjected to Soxhlet extraction using methanol and n-hexane combined in different proportions. Thymoquinone (TQ) was then isolated from the extracts using high performance liquid chromatography (HPLC). The isolated TQ was further subjected to Fourier Transform Infrared (FTIR) spectroscopy to identify its functional groups. The anti-proliferative effect of TQ on HeLa cancer cells was evaluated using 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Results: Extract yield from N. sativa was significantly higher than from T. vulgaris, and also increased with increase in the proportion of methanol in the extraction solvent (p < 0.05). Methanol and n-hexane (4:1, v:v) yielded the highest amount of oil, with yields of 15.8 and 9.7 ml/25 g dry weight (d.wt.) from N. sativa and T. vulgaris, respectively. The results obtained from HPLC showed that the concentration of TQ isolated from N. sativa (388.61 µg/ml) was significantly higher than that from T. vulgaris (357.03 µg/ml, p < 0.05). The anti-proliferative effects of TQ standard and TQ isolated from N. sativa on HeLa cancer cells were dose-dependent, and was highest at the lowest concentration. The number of viable cells significantly decreased with increase in TQ concentration (p < 0.01). TQ from N. sativa significantly reduced the number of viable cells even at the lowest concentration when compared to TQ standard (p < 0.05). Cell death was significantly higher in TQ-treated groups than in untreated cancer cells. Conclusion: The results obtained in this study show that N. sativa is a potential source of TQ, with the yield enhanced by modifying the extraction procedure or solvent used. Furthermore, TQ isolated from N. sativa exerts a dose-dependent anti-proliferative effect on HeLa cancer cells.

Keywords: Thymoquinone, Nigella sativa, Thymus vulgaris, Anti-proliferative effect

This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts

INTRODUCTION Local herbs such as Nigella sativa and Thymus vulgaris have received lots of attention in recent Plants are sources of important phytochemical times due to the anti-tumor properties of their compounds that exhibit anticancer properties [1]. extracts. Nigella sativa which is native to

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Mediterranean regions such as South west Asia, (Japan). Nikon inverted microscope was Southern Europe and North Africa has gained purchased from Eclipse Ts2, Nikon, Inc. (USA), prominence due to the presence of bioactive while Multiskan Ex-microplate reader was a compounds that are effective against a number product of Thermo Electron Corporation (USA). of diseases [2]. Its oil possesses varied pharmacological activities such as anti-cancer, Plants anti-hypertensive, anti-diabetic, analgesic, anti- inflammatory, anti-microbial, hepato-protective, The seeds of N. sativa and T. vulgaris were gastrointestinal and antioxidant properties [3]. obtained from the seed stock of Botany The key components of the seed include fixed oil Department, Lahore College for Women (32 - 40 %), volatile oil, saponins, essential oils, University (LCWU), Lahore, Pakistan, and protein and alkaloids. The fixed oils contain nursed in the botanical garden under controlled eicosadienoic, linoleic, oleic, palmitic, myristic, conditions. Both plants were identified by Prof. and stearic acids [4]. Volatile oils from N. sativa Tahira A. Mughal. (0.40 – 0.45 %) contain carbonyl fractions, thymoquinone (TQ), dithymoquinone, carvacrol, Extraction p-cymene, t-anethole and thymohydroquinone (THQ) [5,6]. Pulverized dried samples of N. sativa seeds (100 g) and aerial parts of T. vulgaris (1000 g) were Thymoquinone (TQ, 2-isopropyl-5-methyl- subjected to Soxhlet extraction based on the benzoquinone) is also present in Monarda method described by Ashraf et al with some didyma L, Monarda media Wild, Monarda modifications, using methanol and n-hexane menthifolia, Satureja hortensis L., Satureja combined in different proportions [10]. About 25 g montana L., Thymus pulegioides L., Thymus of sample was placed in the cotton cellulose serpyllum L. and Thymus vulgaris L. Studies extraction thimble (25 x 80 mm) with the have shown that TQ is the major bioactive respective solvent combinations and extraction component in N. sativa and T. vulgaris oils, and lasted 6 h. Extracts from both samples were has been extensively studied [7]. The anti-tumor collected and re-extracted thrice with 30 ml activity of TQ is determined by targeting its mode methanol in a separating funnel to achieve of action. Some authors have speculated that its maximum extraction. The extracts were mode of action is via the induction of apoptosis, concentrated using a vacuum rotary evaporator because it promotes the expression of tumor at 40 °C for 5 min. In order to achieve maximum suppressor gene p53 in a time-dependent solvent decantation, the extracts were manner. Studies involving human cervical cancer centrifuged at 4000 rpm for 30 min, and the top cell lines have revealed dysfunctional p53 in C33A fatty layer was collected and stored at 4 °C. The and HT-3 cells, while in HPV-positive cells the procedure was repeated in duplicates for the expression of p53 is repressed by HPV-E6 other solvent combinations. The yield of oncoprotein [8]. It has been shown that the extracted oil was calculated as the ratio of the combination of selenomethionine and TQ quantity of oil obtained to the quantity of plant damaged Siha cells and reduced their material, expressed as a percentage. proliferation [9]. The aim of this study was to isolate TQ from N. sativa and Thymus vulgaris, HPLC analysis and investigate its anti-proliferative effect on HeLa cancer cells. Thymoquinone standard (100 µg/ml) was prepared in methanol and used to develop the EXPERIMENTAL calibration curve for the quantification and identification of TQ isolated from the extracts. Materials and reagents The analysis was carried out using Waters 600 HPLC coupled with controllers, pumps and 2996 Thymoquinone standard (98 %), methanol photo diode array (PDA), and the system was (HPLC grade), n-hexane, isopropyl alcohol controlled using Empower 3 Chromatography (HPLC grade), dimethyl sulfoxide (DMSO) were Data software. The column utilized for the of analytical grade and were products of Sigma detection and separation of TQ was a 4.6 x 250 Aldrich, Germany. Dulbecco's Modified Eagle's nm ODS C-18 with particle size of 5 µm. Elution medium (DMEM) and MTT assay kit were was adjusted to isocratic mode using acetonitrile products of Trevigen, (USA), while human (solvent A) and methanol (solvent B) (30 : 70 v:v) cervical adenocarcinoma (HeLa cell lines) were at a flow rate of 1.5 ml/min. The sample total run obtained from the Quality Operation Laboratory, time was 25 min, and the column temperature as Microbiology section, UVAS, Lahore. Grinding maintained at 25 °C. The injection volume was mill was a product of Food Mixer National 20 µl and detection was made at 254 nm at a

Trop J Pharm Res, January 2019; 18(1): 38

Butt et al resolution of 1.2 nm. The column was washed 24 h and the cells examined under a light with acetonitrile (100 %) and equilibrated for 25 microscope. The procedure was performed in min to remove impurities before the injection of triplicate, and the extent of cell proliferation was samples. The retention times of the extracted oils calculated. The wells were incubated in the dark were compared with those of TQ standard. for 2 h and absorbance of each well was measured within 20 min at 492 nm using Quantification of extracted TQ Multiskan Ex-microplate reader. Inhibition (H) of cell proliferation was determined as in Eq 1. A standard calibration curve of TQ was used to quantify the concentrations of TQ isolated from H (%) = {(AE - ANC)/(APC - ANC)}100 ……. (1) the plants, by extrapolation. This was further confirmed by comparing the peak areas of both where AE = absorbance of experimental well; ANC samples and standard. = absorbance of negative control well; and APC = absorbance of positive control well. The protocol Purification of TQ used for the MTT assay is shown in Table I.

Different eluate fractions from the HPLC were Statistical analysis identified by comparing their retention times with those of standard. The fractions were Data are expressed as mean ± SEM, and the concentrated using a vacuum rotary evaporator statistical analysis was performed using SPSS at 40 °C for 5 min. The remaining extracts from (version 16.0). Multiple comparison was both plants were re-purified using HPLC to performed using Tukey’s multiple comparison increase the purity of the isolated TQ. tests. Values of p < 0.01 were considered statistically significant. FTIR spectroscopy RESULTS FTIR spectra of TQ standard and HPLC fractions of N. sativa were obtained from IR Tracer - 100 Yield of extracted oil Fourier Transform Infrared Spectrophotometer equipped with ATR accessory and MCT detector. The yield of extracts from N. sativa was -1 The resolution was adjusted at 400 - 4000 cm significantly higher than that from T. vulgaris, and -1 with a resolution of 4 cm at 100 scans. The IR increased with increase in the proportion of spectrum of the isolated TQ was compared with methanol in the extraction solvent (p < 0.05; that of TQ standard. Table 2). Methanol and n-hexane (4 : 1) yielded the highest amount of oil. The oil yields were MTT assay 15.8 ml/25 g d.wt. and 9.7 ml/25 g d.wt. in N. sativa and T. vulgaris, respectively. This was performed to evaluate the anti- proliferative effect of TQ on HeLa cell lines. The Chromatograms of TQ cells were cultured in DMEM for 24 h to obtain a monolayer of adherent cells. Different dilutions of The results of HPLC analysis showed that TQ TQ standard (100, 50, 25, 12.5, 6.25, 3.12 & 0.78 standard (100 µg/ml) peaked at retention time of µM) were prepared in 96-well plates. Serial 5.5 min, and the concentration was 383.2 µg/ml. dilutions of TQ isolated from N. sativa were made The concentration of TQ isolated from N. sativa and also added to HeLa cells in DMEM for (388.61 µg/ml) was significantly higher than that comparison with those treated with TQ standard. of T. vulgaris (357.03 µg/ml). However, there Dimethyl sulfoxide (0.1 %) and methanol were was no significant difference in the concentration used to dissolve the TQ standard and isolated of TQ standard and PQ isolated from N. sativa (p TQ, respectively. Negative control groups were > 0.05). These results are shown in Figure 1. treated with the respective solvents, while the positive control group was left untreated. After treatment, the 96-well plates were incubated for

Table 1: Treatment protocol in the MTT assay

Group Treatment Experimental I (E1) TQ standard + 0.1 % DMSO + DMEM medium + cancer cell culture Negative control I (C1) 0.1 % DMSO + DMEM medium + cancer cell culture Experimental 2 (E2) TQ from N. sativa + 0.1 % DMSO + DMEM medium + cancer cell culture Negative control 2 (C2) Methanol + 0.1 % DMSO + DMEM medium + cancer cell culture Positive control (P) Untreated cancer cell culture

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Table 2: Yield of extracted oil (g)

Sample Solvent ratio (n-hexane : methanol) 4 : 1 3 : 2 1 : 4 N. sativa 3.23 ± 0.10 5.7 ± 0.14 12.23 ±0.10 T. vulgaris 0.3 ± 0.05* 2.9 ± 0.12* 5.82 ± 0.08* *P < 0.01, when compared to N. sativa

viable cells significantly decreased with increase in concentration of TQ (p < 0.01). The TQ from N. sativa significantly reduced the number of viable cells even at the lowest concentration, when compared to the TQ standard (Figure 3). Cell death was significantly higher in TQ-treated groups than in the untreated cancer cells (Figure 4).

DISCUSSION

The yields of seed oils depend on the extraction methods, solvents, time and temperature [12,13].

In the present study, the yield of oil from N. sativa was significantly higher than that from T. vulgaris, and increased with increase in the proportion of methanol in the extraction solvent. These results are in agreement with those previously reported [6,9].

Figure 1: HPLC chromatogram showing retention time peak of (a) TQ standard and (b) TQ isolated from N. sativa

FTIR spectra

To establish the identity of TQ isolated from the plant extracts, functional group analysis was carried out using FTIR and the results were compared with the IR spectrum of TQ standard and existing literature. Peaks at 3498 cm−1 Figure 2: IR spectrum of HPLC-purified TQ isolated from N. sativa represented primary amines –NH2, peaks at −1 3023 and 2897 cm showed aliphatic C-H −1 stretching (CH3), peaks at 1655 cm showed ester C=O stretching, and 1460 cm−1 peaks represented aliphatic C-H bending CH2. Peaks at 1200 showed ester C-O stretching, and peaks from 943 to 727 cm−1 showed trans–CH=CH-. The overall pattern depicted clearly that the purified sample was TQ (Figure 2).

Anti-proliferative effect of TQ

The anti-proliferative effects of TQ standard and TQ isolated from N. sativa on HeLa cancer cells Figure 3: Anti-proliferative effect of TQ standard (E1) were dose-dependent, and was highest at the and TQ isolated from N. sativa (E2) lowest concentration (Figure 3). The number of

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mechanisms involving cell death signaling pathways, proliferation, angiogenesis and tumor- induced immunosuppression [17,18]. It has been shown that MTT assay is a standardized and recognized test for screening possible cytotoxic effects of substances on cancer cells [20].

The yield and concentration of TQ depend on the extraction method and solvents used [19]. In a previous study, Tabasi et al evaluated the anticancer activities of N. sativa and its TQ isolate against human renal cell carcinoma (ACHN) and fibroblast L929 cell lines, and reported significant increases in cancer cell

death [20]. Although the complete anti- Figure 4: Microscopic images of HeLa cancer cells proliferation mechanism of TQ against cancer treated with: (a) TQ standard showing 15 % cell cells is still not fully elucidated, some authors proliferation; (b) TQ isolated from N. sativa showing 1 - have proposed TQ cytotoxicity and apoptotic 2 % viable cells; and (c) untreated cell culture induction as possible mechanisms of tumor inhibition in animal models [21,22]. These Studies have shown that TQ is the major mechanisms involve anti-oxidant activity, cell bioactive component in N. sativa and T. vulgaris cytotoxicity, immuno-modulatory action and [9]. In a previous study, the yield of TQ from N. apoptosis induction [23,24]. sativa (48.9 %) was shown to be significantly higher than that from T. vulgaris (23.2 %) [14]. CONCLUSION Some authors have reported that starting from 20 g of pulverized sample, the yields of TQ from N. The results obtained in this study show that N. sativa was between 856 and 1881 mg/g, using sativa is a potential source of TQ, and its yield is silica gel purification method [7, 10]. The yield of enhanced by modifying the extraction procedure TQ obtained in this study was lower than those or solvent used. Furthermore, TQ isolated from reported by Ashraf et al and Taborsky et al. This N. sativa exerts a dose-dependent anti- difference may be due to variation in geographic proliferative effect on HeLa cancer cells. location, and quality of seeds or plants. Seed fat usually varies with region where they are grown DECLARATIONS and this affects the concentrations of bioactive compounds in plants. In this study, the Acknowledgement concentration of TQ isolated from N. sativa was significantly higher than that from T. vulgaris. The authors wish to thank the staff of However, there was no significant difference in the concentration of TQ standard and TQ Microbiology Section, University of Veterinary isolated from N. sativa. and Animal Sciences (UVAS), Lahore, Pakistan, for providing facilities for this study. Special Several studies have shown that TQ isolated thanks to the Director, Botanical Garden, Heads from N. sativa is effective against a number of of Departments of Botany and Environmental cancers [15,16]. In this study, the anti- Sciences, LCWU, Lahore, for providing the plant proliferative effects of TQ standard and TQ growth facilities. isolated from N. sativa on HeLa cells were dose- dependent, and was highest at the lowest Conflict of Interest concentration. The number of viable cells was significantly decreased with increase in No conflict of interest associated with this work. concentration of TQ. The TQ from N. sativa significantly reduced the number of viable cells REFERENCES even at the lowest concentration when compared to TQ standard. Cell death was significantly 1. Bhadoriya SS, Mangal A, Madoriya N. Bioavailability and higher in TQ-treated groups than in the untreated bioactivity enhancement of herbal drugs by cancer cells. These results suggest that TQ “Nanotechnology”: a review. J. Curr. Pharm. Res. 2011; exerts dose-dependent anti-cancer effects 8(1): 1-7. against HeLa cancer cells. Studies have shown 2. Darakhshan S, Bidmeshki PA, Hosseinzadeh CA, that TQ exerts anticancer effect on different types of cancers through in vitro and in vivo Sisakhtnezhad S. Thymoquinone and its therapeutic potentials. Pharmacol. Res. 2015; 95: 138-158.

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Ashraf S, Rao M, Kaneez F, Qadri S, Al-Marzouqi AH, 21. Salim ZAL, Mohan S, Othman R. Thymoquinone induces Chandranath I, Adem A. Nigella sativa b Extract as a mitochondria-mediated apoptosis in acute lymphoblastic potent Antioxidant for Petrochemical-Induced oxidative leukaemia in vitro. Molecules. 2013; 18: 11219 - 11240. Stress. J. Chromatogr. Sci. 2011; 49: 221 - 228. 22. Ivankovic S, Stojkovic R, Jukic M, Milos M, Jurin M. The 11. Youa L, Liua, X, Fanga Z, Xub Q, Zhanga, Q. Synthesis antitumor activity of thymoquinone and of multifunctional Fe3O4PLGA-PEG nano-niosomes as thymohydroquinone in vitro and in vivo. Exp. Oncol. a targeting carrier for treatment of cervical cancer. Mater 2006; 28: 220 - 224. Sci. & Eng. C. 2018; 94 (2019): 291 - 302. 23. Rooney S, Ryan M. Modes of action of alpha-hederin and 12. Khan A, Chen H, Tania M, Zhang D. Anticancer activities thymoquinone, active constituents of Nigella sativa, of Nigella Sativa (Black Cumin). Afr. J. Trad. against HEp-2 cancer cells. Anticancer Res. 2005; 25: Complement Altern. Med. 2011; 8 (5): 226 - 232. 4255 - 4259. 13. Mohammed KN, Manap YAM, Tan PC, Muhialdin JB, 24. Kausar H, Abidin L, Mujeeb M. Comparative assessment Alheli MA, Hussain MSA. The effects of different of extraction methods and quantitative estimation of extraction methods on antioxidant properties, chemical thymoquinone in the seeds of Nigella sativa L by HPLC. composition, and thermal behavior of black seed International journal of Pharmacognosy and (Nigella sativa L.) oil. Evidence Based Complement phytochemical Research. 2017; 9 (12): 1425 - 1428. Alternat. Med. 2016; 25. Faheina-Martins GV, Silveira ALD, Cavalcanti BC, 14. Aziz S, Rehman H, Irshad M, Farina S, Asghar F, Ramos MV, Moraes MO, Pessoa C, Araújo DA. Hussain H. Phytotoxic and antifungal activities of Antiproliferative effects of lectins from Canavalia essential oils of Thymus serpyllum grown in the State of ensiformis and Canavalia brasiliensis in human Jammu and Kashmir. J. Essent. Oil Bear. Plants 2010; leukemia cell lines. Toxicol. Vitro. 2012; 26: 1161 - 13: 224. 1169.

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REVIEW ARTICLE A review: Therapeutics potentials of phytochemical drugs and their loading in pH specific degradable Nano-drug carrier targeting colorectal cancer Ayesha Siddique Butt, Numrah Nisar, Tahira Mughal

Abstract decapentaplegic homolog 4: A Cell signalling protein) and Increasing incidents of colorectal cancer have shifted K-ras genes. 4 Chemotherapeutic drugs are given as researchers' attention to the production and improvement combination of leucovorin, 5-fluorouracil and oxaliplatin of anti-cancer drugs by the scientific investigation of vast when colon cancer proceeds to lymph nodes. 2 pool of synthetic, biological and natural products. However, to improve the quality of cancer treatment, the Thymoquinone and thymohydroquinone are considered oral drug delivery of anti-cancer agents in comparison the ideal compounds for the cancer therapy as they are with injection was found to be more effective in terms of economically and environmental friendly and have less maximum absorption of drugs, patient life quality and toxicity level to the survival and diseased model up to cost-effectiveness. 2 Colon-targeted oral drug delivery, increased dosage level. For colorectal cancer, researches without being observed in upper gastro intestinal tract, are shifting towards the oral drug delivery instead of can increase drug bioavailability at target site, possibly injection, as administering drugs through oral route shows maximum absorption of drugs, improves patient life allowing minimum absorption in system resulting in the quality and is cost-effective. Naturally occurring reduction of the administered dose and decrease of 5 polysaccharides as oral drug carriers, such as pectin, have systemic side effects. Colon-targeted delivery by pro- the ability to break down completely in colon, making it drugs, potential of hydrogen (pH), azopolymers, time suitable for targeted drug delivery against cancer cells. and pressure-sensitive methods have respective pros Pectin with polymeric base is an efficient nano drug and cons. Each of them has limitations in site specificity, carrier. The current study reviews the delivery of ease of preparation, toxicity and reproducibility of thymoquinone/thymohydroquinone through pectin nano performance. 2 Numerous factors effect the colon drug carriers to treat colorectal cancer. delivery system, natural polysaccharides as a drug carrier can overcome the issues of toxicity, Keywords: Colorectal cancer, Thymoquinone, bioavailability and safety of drugs. 6 The polysaccharides Thymohydroquinone, Pectin, Nano drug carriers, as a drug carrier has the ability to hydrate and swell Phytochemical drugs. while passing through GIT and releases drug at the Colorectal Cancer targeted site in the colon in presence of colonic bacteria and enzymes. 7 Colon cancer, also referred to as colorectal cancer, is caused by the growth of cancerous cells in colon, rectum Phyto-Chemical Compounds or caecum. 1 In the United States, colorectal cancer is the The usages of plant-derived bioactive components are second largest reported cancer, and ranked fourth among increasing again in the medicinal field, especially against all cancers globally. 2 The threat of emerging sporadic cancer. 8 In cancer treatment, nanotechnologies are found colorectal cancer is significantly moderated by to improve bioavailability, solubility and specific targeting environmental factors and lifestyle attitudes including while reducing the doses, toxicity and achieving steady- obesity, physical inactivity, smoking, alcohol consumption state curative levels 9 (Figure-1). 10 Due to wide 3 and family history. The common pathways involved in the concentration of scientist in oncology, advancements are manifestation of colon cancer are the suppression of progressing towards nanotechnology, which combine tumour suppressor or instability in chromosomal pathway nano drugs and cancer. 11 Expected drawbacks of which is resulted by mutation of adenomatous polyposis traditional chemotherapeutic agents, scientists focused coli (APC), tumour suppressor protein p53, activated to develop molecular targeted treatments involving telomerase, deletion of SMAD 4 (Mothers against nanotechnologies which combines nano drugs and cancer. 11 This nano drug delivery system has potential for Lahore College for Women University, Lahore. enhancing drug concentration and efficiency in cancer Correspondence: Ayesha Siddique Butt. Email: [email protected] cells while avoiding any toxic effects to healthy cells. 9,11

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The role of chemotherapeutic drugs results in apoptosis Table: Types of phytochemicals and pharamceutical drugs with different type drug in cancer cells and the comprehensible drugs include carriers against Colorectal cancer. doxorubicin (DOX), 5-fluorouracil, paclitaxel, epirubicin, Type of drugs Type of drug carriers References oxaliplatin, cisplatin, etc. 10 Varieties of phytochemicals components are present in a Phyto Chemical drugs Triptolide MePEG-PLA copolymer micelle 14 single plant and the fact is that same phytochemical can Honokiol MPEG-PCL star shaped micelle 15 Luteolin MPEG-PCL micelle 16 exist in more than one plant. 12 Most of the anti-cancer Thymoquinone PLGA nano particles 17 activities of phytochemicals involve inhibition of Pharmaceutical drugs Quercetin Zein-Pectin nano drug carriers 64 proliferation of cells, angiogenesis, reserve mitosis, 5-Fluorouracil Eudragit S 100 citrus 65 destruction of inflammatory process involving cyclo pectin nano particle oxygenase-2 expression and initiation of apoptosis at MePEG-PLA: Methoxy poly(ethylene glycol)-poly lactic acid 13 different stages of different cancers. Zheng et al. noted MPEG-PCL: Monomethoxy poly(ethylene glycol)-poly( e-aprolactone) the cytotoxicity of triptolide and triptolide loaded PLGA: Poly-lactide-coglycolide. polymeric micelles against HT29 human adenocarcinoma cells. 14 Triptolide is extracted from the Chinese herb monomethoxy poly (ethylene glycol) (MPEG) and poly ( x- having anti-cancer activities Tripterygium wilfordii. For caprolactone) (PCL) by ultrasonication. The average synthesis of loaded polymeric micelles (TP-PM) methoxy particle size of obtained honokiol micelle was about poly (ethylene glycol)-poly lactic acid (MePEG-PLA) 40nm and was treated to CT26 murine colon carcinoma copolymer solvent evaporation method was used. cells. They showed anti-proliferative effect against the Both the free triptolide and the TP-PM had a dose- and CT26 cells in a dose dependent fashion. 15 time-dependent effect on the HT-29 cells, however, the Luteolin (Lu) is a flavonoid with anticancer activity loaded inhibitory effects of TP-PM on the tumour cell growth in monomethoxy poly (ethylene glycol)-poly ( e- were more significant for all incubation times and aprolactone) (MPEG-PCL) micelles in vivo to evaluate C-26 concentrations. Hence, the polymeric micelles served as colon carcinoma cells by self-assembly method. The 14 an excellent carrier of TP and reduced its toxicities. pharmacokinetics of free luteolin and Lu/MPEG-PCL Chemotherapeutic effects of phytochemical polyphenol micelles was studied in rats; it was found that the compound honokiol were studied by Dong et al. They bioavailable concentration of luteolin was more when the loaded drug into the bio degradable star-shaped micelles Lu/MPEG-PCL micelles were used. Moreover, the

Figure-1: Effective Mechanism of Nano drug system as compare to dietary intake of phytochemical compounds. 10

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Lu/MPEG-PCL micelles inhibited the growth of C-26 colon flowering plant, emerging as a marvel having wide carcinoma cells at IC50 of 12.62 ± 2.17 mg/mL. Hence, the spectrum of pharmacological potential. N. sativa is study recommends that the encapsulation of Lu into commonly known as black seed. N. sativa is native to MPEG-PCL micelles created an aqueous formulation of Lu Southern Europe, North Africa and Southwest Asia, and it with potential anticancer effect, increasing efficiency up is cultivated in many countries in the Middle Eastern to 95.6%. 16 Ravindran et al. studied anti-proliferative Mediterranean region, Southern Europe, and in India, activities of thymoquinone loaded in poly (lactideco- Pakistan, Syria, Turkey and Saudi Arabia. 24 glycolide) (TQ-PLGA) nanoparticles using human colon N. sativa has been widely studied for its natural and cancer HCT 116. The TQ-PLGA nanoparticle had therapeutic activities. It has potent potential against encapsulation efficiency around 94% and ranged cancer, hypertension, diabetes, analgesia and between 150 and 200 nm in size. Apart from this the inflammation, and has microbial, hepatic, gastrointestinal nanoparticles were active in inhibiting nuclear factor and many other antioxidant properties. 25 It is stated in kappa-light-chain-enhancer of activated B cells (NF-kB) many studies that the therapeutic properties of this plant and in suppressing the expression of cyclin D1, matrix are mainly due to the presence of most important phyto- metalloproteinase (MMP)-9, vascular endothelial growth compound, TQ, which is a main bioactive component of factor (VEGF) when compared to the free thymoquinone. the essential oil of seed. 26 On the whole, the results demonstrate that encapsulation of TQ into nanoparticles enhances its anti-proliferative Thymus Vulgaris 17 effects (Table). Thymus vulgaris (T. vulgaris) is a flowering plant of mint There has been a continued search for a new anticancer family Lamiaceae. It is native to Southern Europe and drug and a better method of administration. Natural South East Asia and can be cultivated in many countries of compounds are found to suppress the growth of cancer the Mediterranean region. 27 Thyme is major bioactive cells by inducing programmed cell death which is component of the essential oil of dried herb along with indicated by the notable changes such as other biochemical compounds including TQ and THQ deoxyribonucleic acid (DNA) damage, increase in reactive which shows a wide range of biological and medicinal oxygen species (ROS) generation, 18 release of cytochrome properties such as antiseptic, expectorant, antispasmodic, C, 19 activation of caspases, cell cycle arrest, 20 activation of anthelminthic, anti- inflammatory, antioxidants and lately as anti-cancer agents. 28 Thyme extraction from thymus NF- kB21 and down regulation of MMP, BaX (BCL2- Associated X Protein), cyclin D and VEGF, along with species as an essential oil, containing thymol as a main visible morphological apoptotic changes. 22 compound, can be easily converted to oil with TQ and THQ as the main component. With the appearance of However, there are enormous studies showing the even small quantities of these compounds, the thyme anticancer property of bioactive compounds of plants but essential oil becomes a more potent anticancer and this review particularly deals with the nanotechnology- antioxidant. 29 based drug delivery of TQ and thymohydroquinone (THQ) with the help of biodegradable citrus fruit-based TQ and THQ polysaccharide pectin drug carrier. Thymoquinone (2-isopropyl-5-methyl-benzoquinone) is a phytochemical component showing potent anti- TQ and THQ are the quinones existing in many plant cancer activities against different tumour models. 30 It species like Monarda didyma L, Monarda media Willd, has proved to be effective against several types of Monarda menthifolia Graham, Satureja hortensis L., cancer cell lines in which the classical hallmark of Satureja montana L., Thymus pulegioides L., Thymus apoptosis such as chromatin condensation, serpyllum L., Thymus vulgaris L. and Nigella sativa. 22 translocation of phosphatidyl serine across the plasma However, the occurrence ratio of both components may membrane, and DNA fragmentation have been vary with the species and also on the other environmental documented in TQ-treated cells. 31 There is an increasing factors like temperature, pH and laboratory extraction research interest in TQ to evaluate its anticancer activity methods. 10 For the particular review N. sativa and T. against different types of cancer. 32 Abu Khader vulgaris plants are considered due to their wide investigated new findings, suggesting new mechanisms distribution worldwide and extensive use in dietary as of anticancer activity of TQ against breast cancer in vitro spice in many cuisines. 23 and in vivo models. 33 Nigella Sativa Thymohydroquinone (2-methyl-5-isopropylhydroquinone) Nigella sativa (N. sativa) (family: Ranunculaceae) is annual is also the major component along with the TQ. The

Vol. 68, No. 4, April 2018 610 A. S. Butt, N. Nisar, T. Mughal present knowledge about antitumour activity of THQ is inducing apoptosis by conformational changes in the very limited and till today there is no data in vivo. 34 THQ, colorectal cancer targeting PAK1 (protein activated due to its less stable nature than TQ, may break earlier and kinase). 42 Many investigations are made on the anti- its oxidation results in the formation of more TQ. Both can proliferative, anti-inflammatory and anti-cancer effects of be extracted from the same plants but in different extracted TQ in recent years against cancers like breast, amounts. 22 renal cancer, colorectal, prostrate and cervical cancers by many investigators. 43 Anti-cancer mechanism of TQ and THQ The assumed mechanism of TQ action involves manifold Ravindarin et al. and Nallamuthu et al. performed paths which play significant roles in cancer development. research on TQ chemo-sensitisation potential on It was reported that TQ prompts intrinsic pathways of employing polymer-based nanoparticle approach to apoptosis through the activation of caspases cascade. The improve upon its bioavailability and effectiveness against activation of caspase-8 highlights the effect of TQ on Bcl2 cancer cells including colorectal cancer. They concluded and the role of mitochondria in thymoquinone-induced that encapsulation of TQ into nanoparticles enhances its apoptosis in human squamous cell carcinoma, in human anti-inflammatory, anti-proliferative, and chemo- osteosarcoma p53-null MG63 cells and in p53-null HL-60 sensitising properties. THQ studies on such basis are not myeloblastic leukaemia. 35 The process of apoptosis in being reported yet. 17,44 human breast cancer cell line (Michigan Cancer Khan et al. and Ravindarin et al. emphasised more Foundation-7) MCF7/DOX cells was also found to be investigation regarding anti-cancer and anti-inflammatory mediated through a caspase-dependent manner which activities of TQ. More research work is needed on TQ and triggered the intrinsic pathway through the activation of THQ extraction from some important medicinal plants caspas-3, -7, -9 and the cleavage of poly(ADP-ribose) because it is a safe and promising anticancer 36 polymerase (PARP) but not caspase-8. An increase of component. 45,17 Also, the exact molecular mechanisms of TQ TP53 expression level in MCF7/DOX cells indicated the and other components like THQ, which are considered in p53-dependent apoptosis after treatment with TQ very few studies, on different cancers should be investigated resulting in the reduction of Bcl2 protein and reduction in with more emphasis because current understandings are 36 the Bcl2/Bax ratio. Studies showed anticancer effect of mostly uncertain. Like other phytochemical compounds, TQ on different type of cancer cells through in vitro and in loading TQ and THQ in nano drug carrier shows potent vivo which indicate the involvement of TQ in different cell target specificity and better efficiency. 17 death signalling pathways including apoptosis, proliferation, angiogenesis and tumour-induced Nano Drug Delivery immunosuppression. 37 Every type of cell can secrete Scientists are focusing on the anti-cancer activities of transforming growth which is dependent on the cell phytochemicals leading to the delivery of these drugs to response to the transforming growth factor (TGF-s) specific sites via nano drug carriers, sized in nanometres. 46 receptors. Increase or decrease in their functions and The nano system having nano-materials may vary from its downstream pathway can lead to cancer. So far, the size of 1-100nm carrying drug to the specific site by anticancer mechanism of TQ is not fully understood; following the techniques of nanotechnology. 47 Nano however, several modes of action have been described drug carriers carry these drugs to the targeted destination depending on the stimulus and the cellular context. 38 without leaking or destroying them before they reach to the final destination. 48 The nano-sized drug carriers A study revealed that TQ reduced the cell viability and having controlled shape, size, chemistry and surface convinced apoptosis in human colorectal cancer cell line charges carry drugs to the specific sites and enhance their HCT116. 39 Products of TQ were tested in HL-60 cells and functions by up to 40 times. 49 518A2 melanoma by Effenberger et al., who reported that TQ has anti-proliferative effects in HL-60 cells, 518A2 For cancer drug delivery first step involves leaking of nano melanoma blood cancer, in MCF-7 breast carcinoma and medicine out of the blood stream into the affected blood in HCT116 colon cancer. They concluded that the vessels. 50 These nanoparticles have overcome the derivatives of TQ induce apoptosis associated with DNA problem of estimation of right doses of the drug to the laddering, a slight increase in reactive oxygen species and affected area by keeping the drug away from healthy cell death. 40 Woo et al. in their study showed that TQ tissues/cells and delivering to the targeted site. 51 induced cell death, i.e. apoptosis, and reduced Different types of nano drug carriers are used to deliver proliferation in xeno-grafted mouse model effected with drug in cancer therapy. This is because these nano carriers breast cancer. 41 El-baba et al. assessed the TQ ability of effortlessly target the cancer cells by fabricating them

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ROS: Reactive oxygen species VEGF: Vascular endothelial growth factor DNA: Deoxyribonucleic acid NF-kB: Nuclear factor kappa-light- chain-enhancer of activated B cells. Figure-2: Moleculare mechanism of nano drug loaded in nano drug carrier to the specific target (Cancer cell). 52 from normal cells (Figure-2). 52 methanol, phenolic acids and amide groups. 55 Delivery of drugs to the target sites, loaded in nano drug Due to the complete fermentation of pectin in colon, it is carrier, depends on the small size and site-specificity. The suitable for use as colon-specific drug delivery carrier in four kinds of focusing characteristics of nano drug carriers treatment of colon rectal cancer and other colon diseases. are active, passive, temperature and pH sensitivity. 53 Pectin in colon is digested in the form of short-chain fatty acid that regularise the micro-flora in gut by regulating Subranabiana et al. in their review concluded that for apoptosis in colonic crypts, enhancing the growth of nano drug delivery, micelle with polymeric base is ideal colocyte crypts, preventing pathogen overgrowth, nano carrier. For micelle the common polymeric bases lowering pH and affecting galectin network. 56 Pectin in considered are poly-lactide-coglycolide (PLGA), poly colon cancer can inhibit mutation and expressions of (ethylene glycol) (PEG), poly lactic-acid (PLA) and galectin-3 biological functions. Improved citrus pectin methoxy poly (ethylene glycol) (mPEG) due to their good with high degree of esterification and molecular weight 10 biocompatible and biodegradable behaviour. The increases the bioactivity and defensive roles against colon nano-drug delivery of phytochemicals has been cancer in mouse model by inhibiting galectin-3 in mouse extensively investigated, still further investigations are model. 57 needed to find outcome of the combination of nano-drug delivery and carrier system for cancer. Furthermore, In inventing colon-specific drug delivery systems, clinical studies on nano-drug delivery as well as the mode numerous preparation approaches have been taken in the of administration should be carried out to encourage past few years to prevent pectin-based matrix from them in the field of medicinal oncology. 48 undergoing early drug release in the upper GIT. Pectin, in combination with a cross-linking agent or a polymer, may Pectin Colon-Specific Drug Carrier also be employed itself as a delayed release coat to be Pectin is a natural polysaccharide predominantly found in applied onto a drug core via film or compression coating the cell wall of terrestrial plants. 54 It has 1,4 linked ?-D- technique. 2 Pectin has excessive ability to dissolve in water galactosyluronic acid residues, different neutral sugars creating problem colon targeted drug delivery which can such as rhamnose, galactose and arabinose, and amounts be overcome by preparing pro-drugs which will release the of other non-sugar components like acetic acid, free drug upon arrival at colon. Coating of hydrophobic

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TQ: Thymoquinone THQ: Thymohydroquinone PLGA: Poly-lactide-coglycolide

Figure-3: Proposed Model Nano drug delivery system of Pectin micelle matrix loaded with phytochemicals (TQ/THQ). polymer on pectin oral drug delivery system makes it with its manufacturing source, extraction temperature, unaffected in upper GIT which upon digestion of pectin pH and extraction acid type. 62 releases drug from developed pore. 58 Advancement in oral drug delivery as pectin drug carrier made by complex coat Future studies on pectin drug delivery may characterise made up of water-soluble polymers, the action of the physicochemical qualities of pectin and evaluate its pectinolytic enzymes on the coat complex results in structure-activity relationship with orientation to colon- leaching of pectin and drug release in colon. 59 specific drug delivery. 2 The option of pectin-based colon- specific dose form to achieve solely and precisely at a The hydrophilic polymer counterpart of pectin, once cancer site of cancerous colon is an appealing challenge released from complex, similarly becomes freely solvated, for pharmaceutics. Butte et al. explored combination of swells and leads to distortions in coat, thereby further Pectin with other polymer Eudragit S100. This study facilitating drug release. Enzymatic degradation of pectin revealed that both these polymers have the ability to components forms the primary mechanism of colon- protect the core in the upper GIT and helps in attaining specific drug release. In vivo assessments indicate that colon-specific pectin-based oral dosage forms can be targeted release of curcumin in the colon. The designed primarily through coating of drug matrix with combination of pectin with other hydrophobic polymers, pectin-ethylcellulose or Eudragit film having like PLGA, requires further investigation on their potential superdisintegrant Explotab® V17. 60 It is imagined that the use as synchronised drug carrier and chemotherapeutic reproducibility of matrix digestion and drug release at agent through a multi-disciplinary approach. 63 colon is an interaction effect of colonic pH and microflora Danhya et al. successfully developed zein-pectin nano environment with the physicochemical properties of drug carriers which were biodegradable, non-toxic drug, pectin and other excipients of the dosage form. 2 nanoparticles, made solely from natural polymers. Zein- The pectin- derived oral drug carriers are being extensively used in delayed release dosage system in pectin nanoparticle comprises a hydrophobic zein core colonic cancer. Gelatin, alginate and xyloglucan are some and a hydrophilic pectin core, loaded with model drug 64 polymers explored in formulation of pectin-based drug quercetin. Subudhi et al. developed effective Citrus delivery system resulting in different drug delivery pectin Nanoparticles coated on Eudragit S100 (E-CPNs) formulation like beads, pellets, film and upto micro and for the colon targeting drug 5-Fluorouracil (5-FU). This nano scale structures. 61 Pectin receives an overwhelming combination of drug carrier effectively guarded attention on its practical applications and Implications in nanoparticles upto the colonic region where these drug delivery (Table). However, the pectin has complex nanoparticles released drug for a prolonged period of structure and physiochemical properties which may vary time. 65

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A novel nano system having pectin with polymeric-based 14. Zheng S, Low K, Wagner S, Yang X, von Briesen H, Zou S. PLGA loaded with phytochemical drugs (TQ/THQ) to the Cytotoxicity of Triptolide and Triptolide loaded polymeric micelles in vitro. Toxicol In Vitro. 2011; 25: 1557-67. targeted site (colon) for studying their therapeutics 15. Dong PW, Wang XH, Gu YC, Wang YJ, Wang YJ, Gong CY, et al. Self- potentials and efficiency rate against colorectal cancer is assembled biodegradable micelles based on star-shaped PCL-b- proposed for future study (Figure-3). PEG copolymers for chemotherapeutic drug delivery. Colloid Surface A: Physiochemical and Engineering Aspects. 2010; 358: Conclusion 128-34. 16. Qiu JF, Gao X, Wang BL, Wei XW, Gou ML, Men K, et al. Phytochemicals TQ and THQ individually would be potent Preparation and characterization of monomethoxy poly (ethylene anti-cancer agents for different cancers, particularly for gly col)-poly ( a-caprolactone) micelles for the solubilization and colorectal cancer, when loaded in pH-specific drug in vivo delivery of luteolin. Int J Nanomedicine. 2013; 8: 3061-9. carriers. This review also concluded that pectin with 17. Mona M. A. and Mottaleb A. Biodegradable Thymoquinone Nanoparticles for Higher Therapeutic Efficiency in Murine polymeric base could be an efficient drug carrier up to the Colorectal Cancer. Ijppr. Human. 2016; 7: 436-50. nano level for oral delivery. 18. Paul A1, Das S, Das J, Samadder A, Khuda-Bukhsh AR. Cytotoxicity and apoptotic signalling cascade induced by chelidonine-loaded Disclaimer: None. PLGA nanoparticles in HepG2 cells in vitro and bioavailability of nano-chelidonine in mice in vivo. Toxicol Lett. 2013; 222: 10-22. Conflict of Interest: None. 19. 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