Formulation and Evaluation of Chitosan Nanoparticles of Ethanolic Extract from Lentinula Edodes (Shiitake Mushroom) Ankur Raj Sh

FORMULATION AND EVALUATION OF CHITOSAN NANOPARTICLES OF ETHANOLIC EXTRACT FROM LENTINULA EDODES (SHIITAKE MUSHROOM)

ANKUR RAJ SHARMA

Under the supervision of Dr. Maneesh Jaiswal Assistant Professor (Grade-I)

In partial fulfillment of the Degree of

MASTER OF PHARMACY In PHARMACEUTICS

Department of Pharmacy Jaypee University of Information Technology Waknaghat, Distt. Solan, H.P

MAY -2014 TABLE OF CONTENTS

Chapter No. Topics Page No. Certificate from the Supervisor III Acknowledgements IV Abstract V Lists of Figures VI Lists of Tables VII List of Abbreviations VIII Chapter-1 INTRODUCTION 1-3 Chapter-2 OBJECTIVE 4 Chapter-3 LITERATURE REVIEW 6-46 3.1. Background 7-17 3.2. Fungi 17-18 3.3. Shiitake Mushrooms, Lentinus Edodes 18 3.3.1. Taxonomical Classification 18 3.3.2. Extracts from Shiitake Mushrooms, Lentinus 19-20 Edodes 3.3.3. Lentinan - β-D-Glucan 20-21 3.3.4. The Medicinal and Therapeutic Value of L. 22-29 Edodes 3.3.5. Clinical Studies of L. Edodes in Humans 29-30 3.3.6. Shiitake Products in Market 30-31 3.3.7. Toxicity and Side Effects of L. Edodes 31 3.4. Phytochemical Screening and Extraction 34 3.5. Chitosan Nanoparticles: A Promising System for Drug 34-36 Delivery 3.5.1. Chitosan 36 3.5.2. Preparation Method 37-45 3.5.3. Applications of Chitosan Nanoparticles 45-46 3.6. Instrument Used in Evaluating the Chitosan 46 Nanoparticles

Chapter-4 MATERIALS AND METHODS 47-55 4.1 Extraction of Extract from Lentinus Edodes 49 4.2 Scanning for Maximum Wavelength of Extract 50 4.3 Calibration Curve of Extract 50-51 4.4 Phytochemical Screening of Extract 51-53 4.5 Preparation of Chitosan Nanoparticles 53 4.5.1 Optimization of Formulation 53-54 4.6 Evaluation of Extract-loaded Chitosan Nanoparticles 54 4.6.1 Particle Size and Zeta Potential Analysis 54 4.6.2 Drug Entrapment Efficiency 54 4.6.3 In Vitro Drug Release Studies 54-55 4.6.4 Morphological Analysis 55 Chapter-5 RESULTS AND DISCUSSION 56-61 Chapter-6 CONCLUSION 62-63 REFERENCES 64-76 PUBLICATIONS 77 BIO-DATA 78-79

III

LISTS OF FIGURES

Figure 1: Normal vs. Cancer cell division 8 Figure 2: Some medicinal mushrooms with anti-cancer potential 13 Figure 3: Structure of anti-cancer compounds isolated form mushrooms 14 Figure 4: Anti-cancer mechanism of mushroom bioactive compounds 17 Figure 5: Mechanisms of antitumor activity of lentinan 21 Figure 6: Medicinal importance of Lentinus edodes 22 Figure 7: Early phase of the mechanism of action of lentinan and possible pathway 25 for inflammatory and immune reactions Figure 8: Examples of products marketed with claimed immune stimulatory and 33 anti-cancer properties containing mushrooms or their extracts Figure 9: Various types of drug loaded nanoparticles 35 Figure 10: Schematic representation of the method of ionic gelation 38 Figure 11: Schematic representation of the method of reverse micellisation 39 Figure 12: Schematic representation of the method of emulsion solvent diffusion 40 Figure 13: Schematic representation of the method of emulsification and cross- 41 linking Figure 14: Schematic representation of the method of emulsion droplet coalescence 42 Figure 15: Schematic representation of the method of modified ionic gelation 43 with radical polymerisation Figure 16: Schematic representation of the method of desolvation with sodium 44 sulphate Figure 17: Extraction of shiitake mushroom 49 Figure 18: Preparation of chitosan nanoparticles 53 Figure 19: Wavelength scanning of extract 58 Figure 20: Calibration curve of extract 59 Figure 21: Particle size of blank and extract-loaded CS NPs 60 Figure 22: The release profile of extract-loaded CS NPs 60 Figure 2 3 : Scanning electron micrograph of extract-loaded CS NPs 61

LISTS OF TABLES

Table 1 Reason to study cancer 8 Table 2 Cancer treatment types 9 Table 3 Chemotherapy drugs and side effects 11 Table 4 Bioactive compounds from mushrooms that exert anti-cancer effects 14 Table 5 Structure and physiochemical properties of Lentinan 20 Table 6 Therapeutic effects and bioactive bompounds of L. edodes reported 23 in the literature Table 7 Examples of marketed products of mushroom extracts with claimed 31 immunostimulatory activity Table 8 Criteria for ideal polymeric carriers for nanoparticles & nanoparticle 36 delivery systems Table 9 Methods used for the production of chitosan-based nanoparticles and 45 composition of the carriers’ matrix Table 10 Lists of materials used 48 Table 11 Lists of instruments used 48 Table 12 Preparation of sample solution 50 Table 13 Optimization of formulation 53 Table 14 Phytochemical screening of extract 57 Table 15 Particle size, zeta potential, PDI, and EE of extract-loaded 60 chitosan nanoparticles

VII

LIST OF ABBREVIATIONS

λmax. Absorption maxima

Conc. Concentration

0C Degree centigrade cm Centimetre

CS Chitosan

NPs Nanoparticles

PDI Polydispersity index

EE Entrapment efficiency

DSC Differential scanning calorimetry g Gram

Mol.wt Molecular weight

µg Micro gram mg Milligram min Minute mL Millilitre

μm micrometer

% Percent

SEM Scanning electron microscopy

UV-VIS Ultraviolet- visible

VIII

CHAPTER-1 INTRODUCTION

Written records of the use of plants as medicinal agents dated back thousands of years. The oldest records come from Mesopotamia, 2600 BC. The scientists have been making use of medicinal plants and practicing “Herbalism” for many years to cure illness, cancer and other intractable diseases. Plant or herbal remedies are in fact “a sensitive subject” to mainstream medicine and for most part remained discredited. Nature possesses vast and unknown sea of “miracles”. Modern drugs revealed significant number of newly approved drugs constituted from natural products rather than the synthetic sources.

Chronic diseases have become a growing burden throughout the world. Chronic diseases are conditions that persist for a year or more and result in lifelong disability (limit the activities associated with daily living), thus causing a decreased quality of life. They require ongoing medical attention. Several chronic diseases such as heart disease, stroke, various cancers, chronic respiratory diseases and diabetes, are by far the leading cause of mortality in the world. As a chronic disease may cause death or have long lasting effects throughout the lifetime of an individual, finding a cure for cancer is a major challenge faced by the whole world in this century.

In this context, some prized herbs with validated anti-cancer properties and their active compounds are of immense interest. From time immemorial, mushrooms have been valued by humankind as a culinary wonder and folk medicine in Oriental practice. The last decade has witnessed the overwhelming interest of western research fraternity in pharmaceutical potential of mushrooms. The chief medicinal uses of mushrooms discovered so far are as anti-oxidant, anti-diabetic, hypocholesterolemic, anti-tumor, anti-cancer, immunomodulatory, anti-allergic, nephroprotective, and anti-microbial agents. The mushrooms credited with success against cancer belong to the genus Phellinus, Pleurotus, Agaricus, Ganoderma, Clitocybe, Antrodia, Trametes, Cordyceps, Xerocomus, Calvatia, Schizophyllum, Flammulina, Suillus, Inonotus, Inocybe, Funlia, Lactarius, Albatrellus, Russula, and Fomes. The anti-cancer compounds play crucial role as reactive oxygen species inducer, mitotic kinase inhibitor, anti-mitotic, angiogenesis inhibitor, topoisomerase inhibitor, leading to apoptosis, and eventually checking cancer proliferation.

Lentinus edodes is the first medicinal macrofungus to enter the realm of modern biotechnology. It is the second most popular edible mushroom in the global market which is attributed not only to its nutritional value but also to possible potential for therapeutic applications. Lentinus edodes is used medicinally for diseases involving depressed immune function (including AIDS), cancer, environmental allergies, fungal infection, frequent flu and

2 colds, bronchial inflammation, heart disease, hyperlipidemia (including high blood cholesterol), hypertension, infectious disease, diabetes, hepatitis and regulating urinary inconsistancies. It is the source of several well-studied preparations with proven pharmacological properties, especially the polysaccharide lentinan, eritadenine, shiitake mushroom mycelium, and culture media extracts (LEM, LAP and KS-2). Antibiotic, anticarcinogenic and antiviral compounds have been isolated intracellularly (fruiting body and mycelia) and extracellularly (culture media). Some of these substances were lentinan, lectins and eritadenine.

The efficacy of many drugs is often limited by their potential to reach the site of therapeutic action. In most cases (conventional dosage forms), only a small amount of administered dose reaches the target site, while the majority of the drug distributes throughout the rest of the body in accordance with its physicochemical and biochemical properties. Therefore, developing a drug delivery system that optimizes the pharmaceutical action of a drug while reducing its toxic side effects in vivo is a challenging task. Chitosan nanoparticles are a drug carrier with wide development potential and have the advantage of slow/controlled drug release, which improves drug solubility and stability, enhances efficacy, and reduces toxicity. Because of their small size, they are capable of passing through biological barriers in vivo (such as the blood–brain barrier) and delivering drugs to the lesion site to enhance efficacy. As a natural product, chitosan is a renewable pharmaceutic adjuvant with good biocompatibility. Chitosan and its derivatives have strong potential for application as drug carriers.

CHAPTER-2 OBJECTIVE

The objective of this project work is to formulate and evaluate chitosan nanoparticles encapsulating ethanolic extract of shiitake mushroom and to overcome the limitation of the present marketed formulations.

To achieve these objectives, the project works were divided into three phases:  Extracting ethanolic extract from fresh shiitake mushrooms, lentinus edodes,  Phytochemical screening of the extract for the presence of active constituents,  Preparation and characterization of chitosan nanoparticles

2.1 RATIONALE OF THE STUDY  The estimates reported by WHO indicate that 84 million people will die of cancer between 2005 and 2015 if the disease is untreated. As of 2012, cancer is the number one cause of death, contributing to 29.6% of all deaths in the country.  Lentinus edodes is an edible mushroom, but some individuals may experience side effects or allergic reactions.  High doses of shiitake extract were used for treating certain diseases.  Shiitake extract is a high molecular weight polysaccharide, and is usually administered by the intraperitoneal / intravenous route because of poor oral bioavailability and rapid metabolism.  High costs of extract and marketed products.

2.2 RATIONALE BEHIND CHITOSAN NANOPARTICLES  Better stability, low toxicity, simple and mild preparation method, and providing versatile routes of administration.  Polymeric nanoparticles possess a better reproducibility and stability profiles.  Most of nanoparticles prepared from water-insoluble polymers are involved heat, organic solvent or high shear force that can be harmful to the drug stability.  Water-soluble polymers offer mild and simple preparation methods without the use of organic solvent and high shear force.  Biocompatible, biodegradable, nontoxic, and inexpensive. Furthermore, it possesses positively charge and exhibits absorption enhancing effect.

CHAPTER -3 LITERATURE REVIEW

3.1 BACKGROUND Written records of the use of plants as medicinal agents dated back thousands of years. The oldest records come from Mesopotamia, 2600 BC. The scientists have been making use of medicinal plants and practicing “Herbalism” for many years to cure illness, cancer and other intractable diseases. Plant or herbal remedies are in fact “a sensitive subject” to mainstream medicine and for most part remained discredited [1]. Nature possesses vast and unknown sea of “miracles”. It was not until the early 1800’s that the active principles from plants were isolated. From this point onwards, the effectiveness of medicinal natural products began gain scientific recognition. Nowadays, modern medicine in the U.S.A. placed an emphasis on the isolation and purification of active compounds [2].Nutraceuticals (a comprehensive term including foods, dietary supplements and medical foods that have a health-medical benefit including the prevention and/or treatment of disease) and functional foods now occupy the world’s newest natural products market. Modern drugs revealed significant number of newly approved drugs constituted from natural products rather than the synthetic sources. Chronic diseases have become a growing burden throughout the world. Chronic diseases are conditions that persist for a year or more and result in lifelong disability (limit the activities associated with daily living), thus causing a decreased quality of life. They require ongoing medical attention. Several chronic diseases such as heart disease, stroke, various cancers, chronic respiratory diseases and diabetes, are by far the leading cause of mortality in the world [3-5]. Cancer is a broad term that encompasses a complex group of more than 100 different types of cancerous diseases that can develop in the body. Most of these can be consider as chronic diseases, which represent one of the main health problems of mankind in the 21st century and have become the leading cause of death around the world [3, 4]. The estimates reported by WHO indicate that 84 million people will die of cancer between 2005 and 2015 if the disease is untreated [4, 5]. In the United States, cancer is the second most common cause of death among children between the years of 1 and 14 [6]. Leukemia (particularly acute lymphocytic leukemia) is the most common cancer causing death in these children, followed by cancer of the brain and other parts of the nervous system [6]. As a chronic disease may cause death or have long lasting effects throughout the lifetime of an individual, finding a cure for cancer is a major challenge faced by the whole world in this century.

Why is important to study Cancer? Table 1: Reason to study cancer Rank Cause of death No. of deaths % of all deaths 1. Heart Diseases 685,089 28.0 2. Cancer 556,902 22.7 3. Cerebro-vascular diseases 157,689 6.4 4. Chronic lower respiratory diseases 126,382 5.2 5. Accidents 109,277 4.5 6. Diabetes mellitus 74,219 3.0 7. Influenza and pneumonia 65,163 2.7 8. Alzheimer disease 63,457 2.6 9. Nephritis 42,453 1.7 10 Septicemia 34,069 1.4 Source: ICMR bulletin, 2010 Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade other tissues. Cancer cells can spread to other parts of the body through the blood and lymph systems [7].

Figure 1: Normal vs. Cancer cell division [7]

Cancer types can be grouped into broader categories. The main categories of cancer include [7]:  Carcinoma - cancer that begins in the skin or in tissues that line or cover internal organs. There are a number of subtypes of carcinoma, including adenocarcinom, basal cel carcinoma,squamous cell carcinoma, and transitional cell carcinoma.  Sarcoma - cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.  Leukemia - cancer that starts in blood-forming tissue such as the bone marrow and causes large numbers of abnormal blood cells to be produced and enter the blood.  Lymphoma and myeloma - cancers that begin in the cells of the immune system.  Central nervous system cancers - cancers that begin in the tissues of the brain and spinal cord. Cancer is often perceived as a disease that strikes for no apparent reason. While scientists don't yet know all the reasons, many of the causes of cancer have already been identified. Besides intrinsic factors such as heredity, diet, and hormones, scientific studies point to key extrinsic factors that contribute to the cancer's development: chemicals (e.g., smoking), radiation, and viruses or bacteria [7] Today, conventional cancer therapies mainly consist of: surgery, chemotherapy and radiation therapy, depending on the type of cancer and the stage of tumor development inside the body [8]. Table 2: Cancer treatment types Surgery Surgery can be used to diagnose, treat, or even help prevent cancer in some cases. Most people with cancer will have some type of surgery. It often offers the greatest chance for cure, especially if the cancer has not spread to other parts of the body. Chemotherapy Chemotherapy (chemo) is the use of medicines or drugs to treat cancer. Radiation Radiation therapy uses high-energy particles or waves to destroy or Therapy damage cancer cells. It is one of the most common treatments for cancer, either by itself or along with other forms of treatment. Targeted Therapy Targeted therapy is a newer type of cancer treatment that uses drugs or other substances to more precisely identify and attack cancer cells, usually while doing little damage to normal cells. Targeted therapy is a growing part of many cancer treatment regimens.

Immunotherapy Immunotherapy is treatment that uses your body's own immune system to help fight cancer. Hyperthermia The idea of using heat to treat cancer has been around for some time, but early attempts had mixed results. Today, newer tools allow more precise delivery of heat, and hyperthermia is being studied for use against many types of cancer. Stem Cell Bone marrow transplants and other types of stem cell transplants that Transplant are used to treat cancer. Photodynamic Photodynamic therapy or PDT is a treatment that uses special drugs, Therapy called photosensitizing agents, along with light to kill cancer cells. Lasers in Cancer Lasers, which are very powerful, precise beams of light, can be used Treatment instead of blades (scalpels) for very careful surgical work, including treating some cancers. Blood Product Transfusions of blood and blood products temporarily replace parts of Donation and the blood when a person's body can't make its own or has lost them Transfusion from bleeding. Herbs, Vitamins, Compounds derived from plants that are used for prevention or and Minerals treatment, as well as everyday vitamins and minerals. Examples include mistletoe, black cohosh, vitamin D, selenium, mushroom etc.

The major problem arising from these treatments, especially radiotherapy and chemotherapy are [9]:  Result in damage or weakening of the patient’s natural immunological defenses (which may already have been damaged by the cancer itself)  Radiation therapy can cause side effects by damaging normal, healthy cells near the cancer  Common side effects of chemotherapy are fatigue, nausea & vomiting, hair loss, anemia, infection, blood clotting problems, mouth, gum and throat problems, diarrhea and constipation, nerve and muscle effects, effects on skin and nails, flu-like symptoms, effects on sexual organs and sexuality  Emotional side effects are coping with cancer in everyday life, distress in people with cancer, anxiety, fear, and depression, coping with the loss of a loved one

Table 3: Chemotherapy drugs and side effects Chemotherapy drug Possible side effects Cisplatin (Platinol, Platinol-AQ) › decrease in blood cell counts › allergic reaction, including a rash and/or › usually given intravenously (IV) labored breathing › used for cancers of the bladder, ovary, › nausea and vomiting that usually occurs for and testicles 24 hours or longer › ringing in ears and hearing loss › fluctuations in blood electrolytes › kidney damage Cyclophosphamide (Cytoxan, Neosar) › decrease in blood cell counts › nausea, vomiting, abdominal pain › can be given intravenously (IV) or orally › decreased appetite › used for lymphoma, breast cancer, and › hair loss (reversible) ovarian carcinoma › bladder damage › fertility impairment › lung or heart damage (with high doses) › secondary malignancies (rare) Doxorubicin (Adriamycin) › decrease in blood cell counts › mouth ulcers › given intravenously (IV) › hair loss (reversible) › used for breast cancer, lymphoma, and › nausea and vomiting multiple myeloma › heart damage Fluorouracil (5-FU) › decrease in blood cell counts › diarrhea › given intravenously (IV) › mouth ulcers › used for cancers of the colon, breast, › photosensitivity stomach, and head and neck › dry skin Gemcitabine (Gemzar) › decrease in blood cell counts › nausea and vomiting › given intravenously (IV) › fever and flu-like symptoms › used for cancers of the pancreas, breast, › rash ovary, and lung

Methotrexate › decrease in blood cell counts (Folex, Mexate, Amethopterin) › nausea and vomiting › mouth ulcers › may be given intravenously (IV), › skin rashes and photosensitivity intrathecally (into the spinal column), or › dizziness, headache, or drowsiness orally › kidney damage (with a high-dose therapy) › used for cancers of the breast, lung, › liver damage blood, bone, and lymph system › hair loss (reversible) › seizures Paclitaxel (Taxol) › decrease in blood cell counts › allergic reaction › given intravenously (IV) › nausea and vomiting › used with cancers of the breast, ovary, › loss of appetite and lung › change in taste › thin or brittle hair › joint pain (short term) › numbness or tingling in the fingers or toes Vincristine › numbness or tingling in the fingers or toes (Oncovin, Vincasar PFS) › weakness › loss of reflexes › usually given intravenously (IV) › jaw pain › used for leukemia and lymphoma › hair loss (reversible) › constipation or abdominal cramping Vinblastine (Velban) › decrease in blood cell counts › hair loss (reversible) › given intravenously (IV) › constipation or abdominal cramping › used for lymphoma and cancers of the › jaw pain testis and head and neck › numbness or tingling in the fingers or toes

In this context, some prized herbs with validated anti-cancer properties and their active compounds are of immense interest. Herbs that can fight cancer are:  Mushrooms  Caraway  Aloe  Cinnamon  Arnica  Clove  Astragalus  Garlic  Aveloz  Ginger  Black Cohosh  Rosemary  Black Walnut  Saffron  Bromelain  Curcumin  Basil Many mushroom-derived extracts are therefore recognized as immunomodulators or as biological response modifiers (BRMs) [10, 11]. In particular, medicinal mushrooms not only act as strong immunostimulators but also as a source of good anti-cancer agents, capable of interfering with particular cellular signal transduction pathways linked to cancer development and progression [12].Currently, more than 30 species of scientifically identified medicinal mushrooms have demonstrated anti-tumor activity in experimental studies [13].

Figure 2: Some medicinal mushrooms with anti-cancer potential [13]

The active components in mushrooms responsible for conferring anti-cancer potential are, krestin, hispolon, lectin, calcaelin, illudin S, psilocybin,Hericium polysaccharide A and B (HPA and HPB), ganoderic acid, schizophyllan, laccase, etc. (Fig. 3) Some of them are listed in table 4. Anti-cancer mechanisms of mushroom bioactive compounds are summarized in Fig. 4.

Figure 3: Structure of anti-cancer compounds isolated form mushrooms [13] Table 4: Bioactive compounds from mushrooms that exert anti-cancer effects Mushroom Bioactive compounds Anti-cancer effects References species Albatrellus Grifolin Inhibition of tumor cell growth by inducing [14, 15] confluens apoptosis Induction of cell-cycle arrest in G1 phase via the ERK1/2 pathway Agaricus Caffeic acid phenethyl Inhibition of NF-κB binding to DNA [15] bisporus ester (CAPE) Suppression of aromatase activity Ganoderma Triterpenoid (ganoderic Inhibition of tumor metastasis by the [16] lucidum acid T) suppression of NF-κB activation likely

abrogates the expression of matrix metalloproteinase MMP-2 and MMP-9 Lentinula Lentinan Inhibition of NF-κB binding to DNA [15] edodes Suppression of aromatase activity Panus Panepoxydone Panepoxydone inhibits the TNF-α- or TPA- [17] conchatus induced phosphorylation and degradation of IκB Marasmius Sesquiterpenes Anti-tumor effects through the blockage of [18] oreades NF-κB activation at the IκB kinase (IKK) activation pathway Antrodia 4- Acetylantroquinonol B Inhibition of proliferation and growth of [19] cinnamomea hepatocellular carcinoma cells (HCC) Armillaria Arnamial and related Induction of apoptosis in different cancer [20, 21] mellea (Lepiota sesquiterpene aryl esters cell lines mellea) Clitocybe Clitocine [6-amino-5- Growth inhibitory activity against lung, [22] alexandri nitro-4-(β-D colon and gastric human cancer cells ribofuranosylamino) pyrimidine] Cordyceps 5α,8α-Epidioxy-22E- Cytotoxic effects on promyelocytic [23-25] sinensis ergosta-6,22-dien-3β-ol leukemia HL-60 cells Ergosterol Induction of apoptosis through activation of caspases-3/7 Cordyceps Cordycepin (3′- Induction of apoptosis of human leukemia [26] militaris deoxyadenosine cells through a Reactive oxygen species (ROS)-mediated caspase pathway Trametes Methanol extract In-vivo anti-melanoma activity through [27] versicolor (terpenoids & anti-proliferative, cytotoxic effects on polyphenols) tumor cells and promotion of macrophage activity Ganoderma Lucidenic acids Inhibition of HepG2 cancer cell invasion [28] colossum Colossolactones A-G by acting as inhibitor on the phorbol-12- myristate-13-acetate (PMA)-induced

matrix metalloproteinase (MMP-9) expression Ganoderma Lucidenic acid B, Induction of the apoptosis through a [29-31] lucidum sensu triterpenoid compounds, signaling cascade of death receptor- lato ganoderic acids, mediated and mitochondria-mediated, lucidimol, caspase pathways associated with ganodermanondiol, inactivation of the Akt signal pathway ganoderiol and ganodermanontriol Grifola Triacylglycerols (1- Inhibition of Cyclooxygenase activity [32] frondosa oleoyl-2-linoleoyl-3- palmitoylglycerol) Hericium Ethanol extracts Apoptosis, suppression of the cell [33, 34] erinaceus containing terpenoids, proliferation via activation of sterols and phenols mitochondria-mediated caspase-3 and -9 Lentinus Panepoxydone Interferes with the NF-κB mediated signal [35] crinitus by inhibiting phosphorylation of IkB Lepista inversa Clitocine [6-amino-5- Anti-tumor effects through the induction of [36, 37] nitro-4-(β-D- apoptosis ribofuranosylamino) pyrimidine] Leucopaxillus Clitocine [6-amino-5- Induction of apoptosis by the activation of [38] giganteus nitro-4-(β-D caspase-8, 9, and 3, release of cytochrome ribofuranosylamino) C from mitochondria, decrease of the Bcl-2 pyrimidine] level, and increase of the Bax level Omphalotus Irofulven (6 Inhibition of DNA synthesis, cell cycle [39] illudens hydroxymethyl arrest in S phase and induction of caspase- acylfulvene) mediated apoptosis Phellinus Phelligridins (pyrano[4,3- Cytotoxic activity against several human [40, 41] igniarius c][2]benzopyran-1,6 cancer cell lines dione) derivatives Phellinus Hispolon Induction of apoptosis by ROS mediated [42] linteus caspase pathway leading to cytochrome C

release & mitochondria dysfunction Polyporus Cytotoxic steroids Anti-cancer activity against HepG2 cells [43] umbellatus ergones (22E, 24R)- ergosta-7, 22-dien-3β-ol Poria cocos Lanostane-type triterpene Inhibitory effect on skin tumor promotion [44] acids Taiwanofungus Terpenoids(zhankuic acid Induction of apoptosis via suppression of [45-47] camphorates A,C) maleic and succinic the expression of apoptosis associated acid derivatives proteins Thelephora Thelephantin O, vialinin Anti-cancer activity against HepG2 and [48] aurantiotincta A, (p-terphenyl human colonic carcinoma cells derivatives)

Figure 4: Anti-cancer mechanism of mushroom bioactive compounds [13]

3.2 FUNGI For many years, the filamentous fungi mushrooms have been valued both as a food and medicine. In many cultures, especially in the East, the application of mushrooms to maintain health was formally recorded as early as 100 A.D. in China. They are healthy food due to the many beneficial components. Some mushrooms belonging to the family, Basidiomycetes were used in Japan as folk remedies for the treatment of malignant tumours. Among the

17 natural sources, fungi are of great importance, as a source of important pharmaceuticals for more than fifty years. Fungi are eukaryotic organisms which are heterotrophic; either saprophytic or parasitic. Many fungi break down organic matters such as dead wood, plants and animals, helping in the recycling process and contributing significantly to the ecosystem [49]. The study of the medicinal and nutritional values of mushrooms has become a matter of great interest. Among the approximately 10,000 species from 550 genera and 80 families, only 700 are edible and 200 are thought to have medicinal values. They are unique, stationary like a plant, yet built from chitin, the same material contained in the shell of a lobster. Mushrooms have been incorporated into health tonics, tinctures, teas, soups, and healthy food dishes, as well as herbal formuli. Within the framework of traditional medicine, mushrooms have been applied to lubricate the lungs (Tremella fuciformis), tonify the kidneys (Cordyceps sinesis), reduce excessive dampness (Grifola umbellate), and invigorate the spleen (Poria cocos) [50]. Mushrooms were shown to prevent/treat cancer, viral diseases (e.g. influenza, polio), hypercholesterolemia, blood platelet aggregation, and hypertension [51]. As a result, several thousand species of mushrooms (Basidiomycetes and Ascomycetes) are now being studied for their nutritional and flavor properties with approximately two hundred species of mushrooms that have been found to markedly inhibit the growth of different kinds of tumours [52]. It could therefore, be interesting to unravel the potentials of mushrooms for the benefits to mankind.

3.3 SHIITAKE MUSHROOMS, LENTINUS EDODES The curative powers of shiitake are legendary. It was stated in “Ri Yong Ben Cao”, Volume 3 [(1620) (written by Wu-Rui of the Ming dynasty, China, 1368 – 1644), that “Shiitake accelerates vital energy, wards off hunger, cures colds, and defeats body fluid energy”. Thus, shiitake is treated as an elixir of life, but without scientific verification [51]. 3.3.1 Taxonomical classification Kingdom: Fungi Division: Basidiomycota Class: Agaricomycetes Order: Agaricales Family: Marasmiaceae Genus: Lentinula Species: L. edodes

Shiitake mushrooms, Lentinus edodes, are currently the second most common and commercially produced edible mushrooms. Its medicinal and pharmacological properties are well known and extensively studied by a wide group of people [51].

Shiitake mushrooms have excellent nutritional values. Their raw fruit bodies include 88-92% water, proteins, lipids, carbohydrates as well as vitamins and minerals [53].

History: - The Japanese syllable ‘Shii’ refers to the type of host tree, perhaps oak or a tree in the same family as birch in America. ‘Take’ means the fruit of the mushroom. In a natural forest, shiitake spores are released from fruiting mushrooms in spring or autumn. For centuries the Chinese picked shiitake wild and dried them. The Japanese learned to cultivate them. They placed fresh mushrooms on dead log and let itself inoculate [53]. One ancient physician called it “The Elixir Of Life.” During the Ming Dynasty (1368-1644) the shiitake was believed to keep people vigorous and young. Because of the special power of the shiitake, it was decreed in ancient oriental history that only the emperor and his family could eat them. It was widely known as the “Emperor’s Food.” During times of war, only the most trusted of the guards were assigned to guard the shiitake supply. Shiitakes are deeply rooted in Asian history with the first mention of them dating back to 199 AD, when natives of Kyushu presented the emperor with the gift of the prized woodland gathering [53].

3.3.2 Extracts from Shiitake Mushrooms, Lentinus Edodes In 1969, Chihara and co-workers isolated a water-soluble antitumour polysaccharide from fruiting bodies of Lentinus edodes, which was named “Lentinan” after the generic name of this mushrooms and found complete regression of the solid type tumours in synergic host- tumour system [54]. The most important component extracted from many mushrooms is a β-D-glucan, a polysaccharide yielding D-glucose by acid hydrolysis. β- D-glucan could exist with heterosaccharide chains of xylose, mannose, galactose, uronic acid. Some of them showed remarkable carcinostatic effects. β-D-glucan, gluconoglucan, xycoglucan, mannoglucan, xylomannoglucan, and other active heteroglucans and their protein complexes were extracted and purified using salts and alkali [55, 56]. Another active component α-mannan peptide (KS- 2) extracted from cultured mycelium of Lentinus edodes was shown to be effective on sarcoma 180 and Ehrlich’s carcinoma. Antitumour activity of KS-2 in mice has demonstrated tumouricidal effect of macrophage, though the actual mechanism of KS-2 is not clear [57].

Two other fractions obtained from L. edodes, LEM [a glycoprotein containing glucose, galactose, xylose, arabinose, mannose, fructose and various nucleic acid derivatives, vitamin B compounds, especially B1 (thiamine), B2 (riboflavin) and ergosterol [58] and LAP [a glycoprotein containing glucose, galactose, xylose, arabinose, mannose and fructose [59]. Both displayed strong antitumour activity when administered to animals and humans. The mechanism was postulated through activation of the host’s immune system. The LEM fraction was prepared from an extract of the powdered mycelia of L. edodes through partial enzymatic hydrolysis and water extraction. The LAP fraction resulted from ethanol precipitation of the water solution of LEM. Other immunoactive substances are EP3, a lignin complex was obtained by fractionation of LEM. Water extract of shiitake spores produced an RNA fraction that is effective against mouse influenza A/SW 15 infection. In addition, a basic protein (named “Lentemine”) containing 5% carbohydrate isolated from shiitake mycelia appeared to inhibit TMV infection. It has also been claimed that a glycoprotein, KS-2 extracted from shiitake mycelia has a higher therapeutically effect against AIDS virus than azidothymidine [(AZT) [51]. Another substance isolated from the 80% ethanol extract of shiitake fruiting bodies known as “Lentinacin” (also called “Lentysine: or “Eritadenine”) that possessed decholesterol activity.

3.3.3 Lentinan-β-D-Glucan Lentinan is a high molecular weight (5x105 Da); [α] D +20–22° (NaOH) polysaccharide [(C6H10O5)n] extracted from cell wall of fruiting body in a triple helix structure containing only glucose molecules with mostly β-(1-3)-glucose linkages in the regularly branched backbone, and β-(1-6)- glucose side chains. The configuration of the glucose molecule in a helix structure is considered to be important for the biological and pharmacological activity. Lentinan is completely free of any nitrogen (and thus protein), phosphorus, sulphur and any other atoms except carbon, oxygen and hydrogen. It is water soluble, heat stable and alkali labile. The properties of lentinan are listed in Table 5 [60]. Table 5: Structure and physiochemical properties of Lentinan Properties Descriptions Primary structure

Higher structure Right-handed triple helical structure (by X-ray analysis).

Molecular formula (C6H10O5)n (by elementary analysis) Sugar component Glucose only (by gas chromatography) Molecular weight Distribution in a range between 4 X 105 – 8 X 105 daltons (by gel permeation chromatography and Laser Raman light scattering) Physical constants Specific rotation: [α]20 D, 13.5 – 14.5o (in 2% NaOH), 19.5 – 21.5o (in 10% NaOH) IR spectra: 890 cm-1 (β-glucose) Ultracentrifugation: One peak High voltage electrophoresis: One spot Solubility Soluble in hot water and dimethyl sulfoxide; and soluble in aqueous alkali and formic acid.

Mechanisms of antitumor activity of lentinan as a β-D-glucan The carcinostatic effect of lentinan results from the activation of the host’s immune system. ß-D-glucan binds to lymphocyte surfaces or serum-specific proteins, which activate macrophage, T-helper cells, natural killer (NK) cells, and other effector cells. All these increase the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN- γ) released upon activation of effector cells.

Figure 5: Mechanisms of antitumor activity of lentinan [13]

3.3.4 The Medicinal and Therapeutic Value of L. Edodes [61] L. edodes is one of the well-known macrofungus used in several therapeutic applications. It is the source of several well-studied preparations with proven pharmacological properties. The medicinal properties of L. edodes (Fig. 6) have been studied since Ming Dynasty (1369- 1644). The elders from Japanese Empire considered shiitake as the “elixir of the life” increasing vigor and energy. Antibiotic, anti-carcinogenic, anticancer, antifungal, antibacterial and antiviral, antidiabetic, hypolipidemic compounds have been isolated intracellularly (fruiting body and mycelia) and extracellularly (culture media) from L. edodes. Some of these substances were lentinan, lectin and eritadenine. The shiitake mushroom is used medicinally for diseases involving depressed immune function (including AIDS), cancer, diabetes, environmental allergies, fungal infection, frequent flu and colds, bronchial inflammation, and regulating urinary incontinence. A summary of the therapeutic effects and bioactive compounds of L. edodes which is reported in the literature till 2013 has been represented in Table 6. Major therapeutic effects of this wonderful mushroom are being elaborated below.

Figure 6: Medicinal importance of Lentinus edodes [61]

Table 6: Therapeutic Effects and Bioactive Compounds of L. edodes Reported in the Literature Therapeutic effects Bioactive compounds References Antitumor Lentinan (β -D-glucans), KS-2-α-mannan- [62-66] peptide, LEM,LAP (heteroglucan-protein), EP3 Immunomodulation Mannoglucan, polysaccharide [63, 67-71] protein complex, glucan, Lentinan, polysaccharide L-II, (1-3)-β-D-glucan Antimicrobial Lentinamicin [72] Antiviral Lentinan, LEM, JLS-18, EP3, EPS4 [62, 73-75] Antibacterial LEM, Lenthionine , chloroform and ethylacetate [74-76] extracts Antifungal Lentin [77] Cardiovascular and Eritadenine, lentinacin, lentysine [78-83] Hypolipidemic Hepatoprotective Lentinan, LEM, hot-water extraction and ethanol [84, 85] extraction Hemagglutinating Lectin [86-88] Antioxidant Methanol and water extracts, [89-91] polyphenolic compounds

3.3.4.1 Antitumor and Anticarcinogenic Activity The antitumor polysaccharide ‘Lentinan’ was first isolated and studied by Chihara et al. [68] who demonstrated that its anti-tumor effects were greater than other mushroom polysaccharides and was active for some types of tumors [92]. The antitumor effect of lentinan was confirmed by using Sarcoma-180 transplanted in CD-1/ICD mice [63]. Later, it showed prominent antitumor activity not only against allogenic tumors, such as Sarcoma-180, but also against various synergic and autochthonous tumors, and it prevented chemical and viral oncogenesis [93]. The tumor inhibitory effect of lentinan was highly striking. In 1-5 mg/kg x 10 doses, the inhibition ratio was 95 to 97.5%, and in dosages of 0.2 mg/kg x 10, the tumors underwent complete regression in 6 out of 10 mice [68]. Combination treatment of L. Edodes mycelium extracts with 5-Fluorouracil represent a novel chemotherapeutic strategy in colon cancers and that p53, p21/Cip1 and p27/Kip1 may play some important roles for the

23 involvement in antitumor activity [94]. Four antitumor (1/3)-β-D-glucans coded as L-I1, L-I2, L-I3 and L-I4 with high molecular weight (1.47x106–1.67x106) were isolated from four kinds of fruiting bodies of Lentinus edodes [95]. Exo-biopolymer from rice bran cultured with L. edodes [rice bran exo biopolymer (RBEP)] induced the activation of NK cells in a dose- dependent manner when administrated orally [96]. The carcinostatic effect of lentinan results from the activation of the host’s immune system. ß-D-glucan binds to lymphocyte surfaces or serum-specific proteins, which activate macrophage, T-helper cells, natural killer (NK) cells, and other effector cells. All these increase the production of antibodies as well as interleukins (IL-1, IL-2) and interferon (IFN- γ) released upon activation of effector cells [97]. The antitumor studies conducted with L. edodes thus far are very interesting and do show a potential for providing therapeutic control of cancer. It is, however, difficult to say whether L. edodes could have preventive effects against cancer when consumed as part of the diet.

3.3.4.2 Immunomodulating Effects Lentinus edodes has attracted a lot of attention owing to its immunomodulatory effects. Lentinan is well known as a type of biological response modifier (BRM). Augmentations of NK (Natural Killer), CTL (Cytotoxic T Lymphocyte), LAK (Lymphokine Activated Killer) activities and DTH (Delayed Type Hypersensitivity) responses against tumor antigen were observed after administration of Lentinan [98]. These activities are responsible for the antitumor effects of lentinan. Antitumor polysaccharide L-II was isolated and purified from the fruiting body of L. edodes [69]. The antitumor activity of the polysaccharide L-II on micetransplanted sarcoma 180 was mediated by immunomodulation by inducing T-cells and macrophage-dependent immune system responses. Kupfahl et al. [99] evaluated the effect of lentinan in the well established model system of the murine Listeria monocytogenes infection. The results showed that the lentinan enhances the protective CD8 T-cell response against L. monocytogenes probably by a mechanism that involves the IL-12-mediated augmentation of the specific antilisterial CD8 T-cell response. Fruit body and mycelial extracts of L. edodes are able to enhance the proliferation of rat thymocytes directly and act as co-stimulators in the presence of the T-mitogen PHA [100]. Many interesting mechanisms of action of Lentinan and possible pathways for inflammatory and immune reactions have been represented in Fig. 7 [101].

Figure 7: Early phase of the mechanism of action of Lentinan and possible pathway for inflammatory and immune reactions [61]. IL-1, IL-2, IL-3, and IL-6: interleukin-1, -2, -3, and –6. APPIF: acute phase protein-inducing factor VDHIF: vascular dilation and hemorrhage-inducing factor; CSF: colony-stimulating factor; MIF: migration inhibition factor;’ TNF: tumor necrosis factor; VPF: vascular permeability factor; NKF: natural killer cell-activating factor; MAF: macrophage-activating factor; CTL: cytotoxic T lymphocyte; LAK: lymphokineactivated killer cell G.

3.3.4.3 Antimicrobial Activity Antimicrobial activity has been found in liquid cultures, chloroform, ethyl acetate, water and dried fruit body extracts of L. edodes [72]. These extracts are active against gram positive and gram-negative bacteria, yeasts and mycelia fungi, including dermatophytes and phytopathogens [102]. Mycelial-free culture of L. edodes exhibited greater antimicrobial effect against grampositive than gram-negative bacteria with Bacillus subtilis and Staphylococcus aureus among the most highly inhibited [103]. Antimicrobial compounds isolated from L. edodes liquid cultures include lentinamicin.

Antiviral Activity L. edodes had been believed to cure the common cold for hundreds of years. More recently, some scientific evidences have been obtained to support this belief. L. edodes showed an activity (expressed as the percentage decrease in lung lesion score compared with the control) of 46%, which was of the same magnitude as for amantadine hydrochloride, a common drug against influenza (40%). A watery extract from L. edodes was also reported to prevent the multiplication of polio virus [104]. Lentinan enhanced the host resistance against infections with bacteria, fungi, parasites, and viruses, including the agents of AIDS [105]. Lentinan reduced the toxicity of azidothymidine AZT (a drug commonly used for treating HIV carriers and AIDS patients). Prevention of the onset of AIDS symptoms through potentiation of host defense is now being actively investigated both experimentally and clinically [106]. In addition to lentinan, other substances from L. edodes have also been shown to have antiviral activity. The mechanism of their effect is in most cases via induction of interferon [78]. Lentinan has also shown: (a) antiviral activity in mice against VSV (vesicular stomatis virus), encephalitis virus, Abelson virus, an adenovirus type 12; (b) stimulated nonspecific resistance against respiratory viral infection in mice; (c) conferred complete protection against an LD75 challenge dose of virulent mouse influenza A/SW15; (d) increased resistance to the protozoal parasites Schistosoma japanicum, Sch. mansoni; (e) exhibited activity against Mycobacterium tuberculosis bacilli resistant to antituberculosis drugs, Bacillus subtilis, Staphylococcus aureus, Micrococcus lenteus, Candida albicans and Saccharomyces cerevisiae; (f) increased host resistance to infections with potentially lethal Listeria monocytogenes [62]. LEM and a new lignan-rich compound ‘JLS-18’ derived from LEM, blocked the release of infectious Herpes simplex virus in animals [73] and it has been suggested because of its high activity that JLS-18 could be of value in the treatment of hepatitis B and AIDS patients [74]. Water-soluble lignins from EP3 and EPS4 from shiitake mushroom mycelium have shown antiviral effects [75].

Antibacterial Activity Anti-bacterial activity is an exciting result, with increasing bacterial resistance to antibiotics, improving host immunity may be the way forward in fighting bacterial infection. The antibacterial activity of L. edodes against Bacillus subtilis was evaluated in cell-free filtrates obtained after growth in 14 different culture media [107]. Lenthionine, a sulphur containing peptide from shiitake has antibacterial and antifungal activity and bis [(methylsulfonyl) methyl] disulphide [76], a derivative of lenthionine, has strong inhibitory effects against

Staphylococcus aureus, Bacillus subtilis and Escherichia coli [108]. The chloroform and ethylacetate extracts of the dried shiitake mushroom have bactericidal activity against both growing and resting Streptococcus mutans and Prevotella intermedia [109].

Antifungal Activity A novel protein designated lentin with potent antifungal activity was isolated by Ngai and Ng[77] from the fruiting bodies of the L. edodes. Lentin, which had a molecular mass of 27.5 kDa, inhibited mycelial growth in a variety of fungal species including Physalospora piricola, Botrytis cinerea and Mycosphaerella arachidicola. Lentin also exerted an inhibitory activity on HIV-1 reverse transcriptase and proliferation of leukemia cells.

3.3.4.4 Cardiovascular and Hypolipidemic Activity Cardiovascular diseases are among the main causes of death in our society and there is a strong correlation between enhanced blood cholesterol levels and the development of such diseases. The popular macrofungus L. edodes, has been shown to produce blood cholesterol lowering compound designated eritadenine [2(R), 3(R)-dihydroxy-4-(9-adenyl)- butyric acid] [79]. The hypocholesterolemic action of this compound has been quite extensively examined in rats. Eritadenine is suggested to accelerate the removal of blood cholesterol either by stimulating tissue uptake or by inhibiting tissue release [82]. It was demonstrated that when rats were fed a diet supplemented with 5% (dry weight) of L. Edodes fruiting bodies for 10 weeks the plasma cholesterol levels of the animals decreased significantly. Eritadenine works by lowering levels of all lipoprotein types, i.e., high-density as well as low-density lipoproteins [78]. In addition to animal tests, the effectiveness of L. Edodes in lowering blood serum cholesterol was also tested in human subjects. A daily intake of 90 g of fresh shiitake, 9 g of dried shiitake, and 9 g of UV-irradiated dried shiitake for 7 days lowered the mean serum cholesterol levels in young women by 12, 7 and 6%, respectively [110]. All three diets decreased serum cholesterol levels of older persons (60 years of age) by 9% over 7 days. Eritadenine reduces blood serum cholesterol in mice, not by the inhibition of cholesterol biosynthesis, but by the acceleration of the excretion of ingested cholesterol and its metabolic decomposition. It has been shown to lower blood levels of cholesterol and lipids in animals. When added to the diet of rats, eritadenine (0.005%) caused a 25% decrease in total cholesterol in as little as one week. The cholesterol-lowering activity of this substance is more pronounced in rats fed a high fat diet than in those on a low-fat diet. Although feeding studies with humans have indicated a similar effect [111, 67]. The amount of cholesterol

27 reducing agent (eritadenine) in L. edodes, in search of a potential natural medicine against blood cholesterol was quantified [82, 83].

3.3.4.5 Antidiabetic Activity The hypoglycemic effect of an exo-polymer produced from a submerged mycelium culture of L. edodes was investigated in streptozotocin- induced diabetic rats [111], the administration of the exo-polymer (200 mg/kg BW) reduced the plasma glucose level by as much as 21.5%, and increased plasma insulin by 22.1% as compared to the control group. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively. The hypoglycaemic effect of L. edodes has been also demonstrated and proved its potential in lowering the blood glucose and triglyceride (TG) levels in the serum of rats [112]. It also lowered the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively. Exopolysaccharide (EPS) produced from submerged mycelia culture of Lentinus species was evaluated for hypoglycaemic activity in streptozotocin (STZ)-induced diabetic rats [113]. In dose-dependent study, orally administered L. Strigosus EPS, at the dose of 150 mg/kg, exhibited a considerable hypoglycemic effect in STZ-induced diabetic rats. Plasma insulin levels of STZ-induced diabetic rats decreased as compared to control group rats. Although insulin levels slightly increased in the EPS-treated groups. The hypoglycaemic potential of the EPS was further supported by histological observations of pancreatic islets of Langerhans.

3.3.4.6 Hepatoprotective Activity A polysaccharide fraction from L. edodes showed liver protective action in animals together with improved liver function and an enhanced production of antibodies to hepatitis B [65]. Lentinan and LEM have given favourable results in treating chronic persistent hepatitis and viral hepatitis B patients [84]. Lentinan improved serum glutamic pyruvic transaminase (SGPT) and completely restored GPT levels in the livers of mice with toxic hepatitis. Crude extracts of shiitake mushroom cultures have demonstrated liver-protecting actions [62, 67, 66]. The hot-water extraction and ethanol extraction of the mycelia of L. edodes were examined for their hepatoprotective effect on dimethylnitrosamine-injured mice [85]. Both fractions decreased the blood aspartate aminotransferase and alanine aminotransferase levels, partially inhibited the over accumulation of collagen fibrils, and suppressed the over expression of genes for alpha-smooth muscle actin and/or heat-shock protein 47 in the mice. Both fractions also inhibited the morphologic change and proliferation of isolated rat hepatic stellate cells (HSCs), which play a central role in liver fibrosis, in a dose-dependent manner and without

28 cytotoxicity. The direct interaction between the extracts and HSCs appears to be important for the hepatoprotective activity. Polyphenols contained in both fractions are considered to be potential candidates for expressing the hepatoprotective effects.

3.3.4.7 Hemagglutinating Activity The agglutinating activity of different morphogenetic structures of L. edodes F-249, including mycelium, brown mycelial mat, primordia, and fruiting bodies was studied. Data showed that mycelial mat was found to possess the maximum hemagglutinating activity, which can be explained by the possible involvement of agglutinins in the formation of mycelial mat, which is composed of glued hyphae. The changes of the hemagglutinating activity of intracellular lectins of the basidiomycete L. edodes at various stages of its morphogenetic development depending on erythrocyte type, growth medium, and lectin purification degree was studied [86- 88].

3.3.5 Clinical Studies of L. Edodes in Humans [61] Lentinan was demonstrated to have antitumor activity and increased the survival time of patients with inoperable gastric cancer and women with recurrent breast cancer following surgical therapy. When the polysaccharide of L. edodes was administered once or twice a week with chemotherapy to a patient with progressive cancer but with no serious liver, kidney, or bone marrow dysfunction, it produced a statistically significant improvement in immune and anticancer activity when compared to chemotherapy alone. Two hundred seventy-five patients with advanced or recurrent gastric cancer were given one of two kinds of chemotherapy (mitomycin C with 5- fluorouracil or tegafur) either alone or with lentinan injections. The best results were obtained when lentinan was administered prior to chemotherapy and in patients with a basis of prolongation of life, regression of tumors or lesions, and the improvement of immune responses. Lentinan was injected into malignant peritoneal and/or pleural effusions of a group of 16 patients with advanced cancer. Eighty percent of the lesions showed probable clinical responses, with an improvement in performance demonstrated in seven subjects. The survival time for those who responded immunologically to the treatment was 129 days and 45 days for those who did not respond. Mycelium extract of L. edodes was used to boost the immune response in AIDS patients. When it was used to treat HIV-positive patients with AIDS symptoms, the Tcell count rose from a baseline of 1250/mm3 after 30 days up to 2550/mm3 after 60 days. Forty patients with chronic viral hepatitis B and seropositive for Hbe antigenemia were given 6 g of LEM daily (orally) for 4 months. The study focused on the number of patients seroconverting from Hbe

29 antigen positive to antiHbe positive, which was 25% after LEM therapy, and was higher in patients with chronic active hepatitis (36.8%). In addition, 17 patients (43%) became seronegative for Hbe antigen. Liver function tests improved even for patients who remained seropositive, and they had raised plasma albumin, and adjusted protein metabolism. Dried shiitake mushroom (9 g/day) decreased 7–10% serum cholesterol in patients suffering with hypercholesterolemia. For many patients 60 years of age or older with hyperlipidemia, consuming fresh shiitake mushroom (90 g/day in 7 days) led to a decrease in total cholesterol blood level by 9–12% and triglyceride level by 6–7%.

3.3.6 Shiitake Products in Market 1) Shiitake Extract [114]: Shiitake extract have a beneficial effect on: blood pressure; blood sugar; cholesterol; kidney tonic; liver tonic; stress; breast cancer; liver cancer; prostate cancer. It is an excellent immune system booster and enhances cellular defences. 2) Shiitake Polysaccharide Freezing – Drying Powder Capsule [115]: Strengthens the immune system, improves its ability to fight infection and disease such as influenza and other viral diseases. It even improves the immune status of individuals infected with HIV, the virus that can cause AIDS, has also been shown to have anti-cancer activity. 3) Shiitake Mushroom Extract Capsule [116] It contains compounds known as alpha and beta-glucans which appears to have some positive effects in enhancing immune function, healthy cholesterol profiles and healthy blood pressure levels. 4) Shiitake Mushroom Tablet [117]: It increases host’s resistance to infections by viruses, bacteria and parasites. It is approved for the treatment of gastric cancer. 5) Shiitake Polysaccharide Capsule [118]: Given for the treatment of: immune dysfunction, cholesterol, gallstone, rickets, hepatitis B, liver cirrhosis, cancer patients. 6) Shiitake Polysaccharide Electuary [119]: Enhances immunity, protects liver. It is an anti-cancer, anit-viral preparation, improves blood circulation, increases supply capacity of blood and oxygen to heart and lungs, increases physiological functioning of cell and organs.

8) Shiitake Mushroom P.E Plant Extract [120]: It is found to be particularly valuable for treating all forms of hepatitis. It acts as a powerfully antiviral agent. It can also lower blood levels of cholesterol and lipids. 9) Natural Shiitake Mushroom Extract [121]: It is a hemostyptic. 10) Shiitake Tea Bag, Supplement [122]: Strengthens immunity.

3.3.7 Toxicity and Side Effects of L. Edodes [61] Lentinus edodes is an edible mushroom, but some individuals may experience minor side effects or allergic reactions. Allergic reactions to the spores of L. edodes have been reported in workers picking mushrooms indoors. Symptoms include fever, headache, congestion, coughing, sneezing, nausea, and general malaise. A water extract of the fruiting body was found to decrease the effectiveness of blood platelets in initiating coagulation. L. edodes mycelium has shown no evidence of being acutely toxic, even in massive doses of over 50 mg/day for 1 week, though mild side effects such as diarrhea and skin rash may occur. Lentinan has no known serious side effects. However, in clinical trials of patients with advanced cancer, minor side reactions occurred such as a slight increase in glutamateoxaloacetate transminase (GOT) and GPT liver enzymes and a feeling of mild pressure on the chest. But these changes disappeared after lentinan administration was stopped.

Table 7: Examples of marketed products of mushroom extracts with claimed immunostimulatory activity [123] Product name Product function claim Fungus/extract present Dr Myko San – Health from Reducing the risk of the Different mushroom species Mushrooms occurrence of malignant diseases. Anti-tumor activity, without any toxic side effects. Ganoderma Lucidum Spore Anti-tumor and immuno- Ganoderma lucidum spore potentiating properties extract (enhancing the functioning of the immune system)

Grifron Maitake Mushrooms Overall health and immune Grifola frondosa support I’m-Yunity® Maintaining white blood cell COV-1® strain of Coriolus levels and versicolor increasing immune proteins MC-S (Metabolic Cell Significantly enhances white Ganoderma Lucidum, Support) blood cell (immune cell) Lentinus edodes (mycelia) proliferation while Coriolus versicolor simultaneously suppressing cancer cell growth. MycoPhyto® Complex Potential therapeutic value in Blend of mushroom mycelia the treatment of human from Agaricus subrufescens, breast cancer Cordyceps sinensis, Coriolus versicolor, Ganoderma lucidum, Grifola frondosa and Polyporus umbellatus Red reishi Overall immunity and Ganoderma lucidum fruit suppression of tumors body and spores Super Royal Agaricus Immune and kidney support Agaricus subrufescens- Mushroom fruting body extract Grifola frondosa-Maitake TD fraction

Figure 8: Examples of products marketed [123] with claimed immune stimulatory and anti- cancer properties containing mushrooms or their extracts. 1. Dr Myko San—Health from Mushrooms 2. Ganoderma Lucidum Spore 3.Grifron Maitake Mushrooms 4. I’m-Yunity® 5. MC-S (Metabolic Cell Support) 6. MycoPhyto® Complex 7. Red reishi 8. Super Royal Agaricus Mushroom.

3.4 PHYTOCHEMICAL SCREENING AND EXTRACTION Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs [124]. Extraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the solvent) in dry powder form, and are intended for oral or external use. Extraction methods used pharmaceutically involves the separation of medicinally active portions of plant tissues from the inactive/inert components by using selective solvents. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity [125]. The purpose of standardized extraction procedures for crude drugs (medicinal plant parts) is to attain the therapeutically desired portions and to eliminate unwanted material by treatment with a selective solvent known as menstrum. The extract thus obtained, after standardization, may be used as medicinal agent as such in the form of tinctures or fluid extracts or further processed to be incorporated in any dosage form such as tablets and capsules. This product contains complex mixture of many medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, flavonoids and lignans [126].

3.5 CHITOSAN NANOPARTICLES: A PROMISING SYSTEM FOR DRUG DELIVERY

The efficacy of many drugs is often limited by their potential to reach the site of therapeutic action. In most cases (conventional dosage forms), only a small amount of administered dose reaches the target site, while the majority of the drug distributes throughout the rest of the body in accordance with its physicochemical and biochemical properties. Therefore, developing a drug delivery system that optimizes the pharmaceutical action of a drug while reducing its toxic side effects in vivo is a challenging task [127]. One approach is the use of colloidal drug carriers that can provide site specific or targeted drug delivery combined with optimal drug release profiles. The idea of using submicron drug delivery systems for drug targeting was conceived and developed after Paul Ehrlich originally proposed the idea of tiny drug-loaded magic bullets over a hundred year ago [128].

Nanoparticles are solid colloidal particles with diameters ranging from 1-1000 nm. They consist of macromolecular materials and can be used therapeutically as adjuvant in vaccines or drug carriers in which the active ingredient is dissolved, entrapped, encapsulated, adsorbed or chemically attached. Polymers used to form nanoparticles can be both synthetic and natural polymers. There are two types of nanoparticles depending on the preparation process: nanospheres and nanocapsules [129]. Nanospheres have a monolithic-type structure (matrix) in which drugs are dispersed or adsorbed onto their surfaces (Fig. 9). Nanocapsules exhibit a membrane-wall structure and drugs are entrapped in the core or adsorbed onto their exterior (Fig. 9).The term “nanoparticles” is adopted because it is often very difficult to unambiguously establish whether these particles are of a matrix or a membrane type.

Figure 9: Various types of drug loaded nanoparticles [127]

Nanoparticles not only have potential as drug delivery carriers as they offer non-invasive routes of administration such as oral, nasal and ocular routes, but also show to be good adjuvant for vaccines. Most of nanoparticles prepared from water-insoluble polymers are involved heat, organic solvent or high shear force that can be harmful to the drug stability. Moreover, some preparation methods such as emulsion polymerization and solvent evaporation are complex and require a number of preparation steps that are more time and energy consuming. In contrast, water-soluble polymers offer mild and simple preparation methods without the use of organic solvent and high shear force. Among water-soluble polymers available, chitosan is one of the most extensively studied. This is because chitosan possesses some ideal properties of polymeric carriers for nanoparticles (Table 8) such as biocompatible, biodegradable, nontoxic, and inexpensive. Furthermore, it possesses positively charge and exhibits absorption enhancing effect. These properties render chitosan a very attractive material as a drug delivery carrier. In the last two decades, chitosan nanoparticles (chitosan NP) have been extensively developed and explored for pharmaceutical applications.

Table 8: Criteria for ideal polymeric carriers for nanoparticles & nanoparticles delivery systems Polymeric carriers Easy to synthesize and characterize Inexpensive Biocompatible Biodegradable Non-immunogenic Non-toxic Water soluble Nanoparticles delivery systems Simple and inexpensive to manufacture and scale-up No heat, high shear forces or organic solvents involved in their preparation process Reproducible and stable Applicable to a broad category of drugs; small molecules, proteins and polynucleotides Ability to lyophilize Stable after administration Non-toxic

3.5.1 Chitosan Chitosan is a modified natural carbohydrate polymer prepared by the partial N-deacetylation of chitin, a natural biopolymer derived from crustacean shells such as crabs, shrimps and lobsters. Chitosan is also found in some microorganisms, yeast and fungi [130]. The primary unit in the chitin polymer is 2-deoxy-2-(acetylamino) glucose. These units combined by β- (1,4) glycosidic linkages, forming a long chain linear polymer. Although chitin is insoluble in most solvents, chitosan is soluble in most organic acidic solutions at pH less than 6.5 including formic, acetic, tartaric, and citric acid [152]. It is insoluble in phosphoric and sulfuric acid. Chitosan is available in a wide range of molecular weight and degree of deacetylation.

3.5.2 Preparation method Over the past 30 years, chitosan NP preparation technique has been developed based on chitosan microparticles technology. Methods for the preparation of chitosan nanoparticles are: A. Ionotropic gelation, B. Microemulsion method / Reverse micellisation C. Emulsification solvent diffusion D. Polyelectrolyte complex. E. Emulsification and cross-linking, F. Emulsion droplet coalescence, G. Modified ionic gelation with radical polymerisation H. Desolvation The most widely developed methods are ionotropic gelation and self assemble polyelectrolytes. These methods offer many advantages such as simple and mild preparation method without the use of organic solvent or high shear force. A. Ionotropic gelation [131] Chitosan NP prepared by ionotropic gelation technique was first reported by Calvo et al., [131] and has been widely examined and developed. The mechanism of chitosan NP formation is based on electrostatic interaction between amine group of chitosan and negatively charge group of polyanion such as tripolyphosphate. This technique offers a simple and mild preparation method in the aqueous environment. First, chitosan can be dissolved in acetic acid in the absence or presence of stabilizing agent, such as poloxamer, which can be added in the chitosan solution before or after the addition of polyanion. Polyanion or anionic polymers was then added and nanoparticles were spontaneously formed under mechanical stirring at room temperature. The size and surface charge of particles can be modified by varying the ratio of chitosan and stabilizer.

Figure 10: Schematic representation of the method of ionic gelation [131]

B. Microemulsion method / Reverse micellisation [132] Chitosan NP prepared by microemulsion technique was first developed by Mitra et al [132]. This technique is based on formation of chitosan NP in the aqueous core of reverse micellar droplets and subsequently cross-linked through glutaraldehyde. In this method, a surfactant was dissolved in N-hexane. Then, chitosan in acetic solution and glutaraldehyde were added to surfactant/hexane mixture under continuous stirring at room temperature. Nanoparticles were formed in the presence of surfactant. The system was stirred overnight to complete the cross-linking process, which the free amine group of chitosan conjugate with glutaraldehyde. The organic solvent is then removed by evaporation under low pressure. The yields obtained were the cross-linked chitosan NP and excess surfactant. The excess surfactant was then removed by precipitate with CaCl2 and then the precipitant was removed by centrifugation. The final nanoparticles suspension was dialyzed before lyophilyzation. This technique offers a narrow size distribution of less than 100 nm and the particle size can be controlled by varying the amount of glutaraldehyde that alters the degree of cross-linking. Nevertheless, some disadvantages exist such as the use of organic solvent, time-consuming preparation process, and complexity in the washing step.

Figure 11: Schematic representation of the method of reverse micellisation [132]

C. Emulsification solvent diffusion method [133, 134] El-Shabouri [133] reported chitosan NP prepared by emulsion solvent diffusion method, which originally developed by Niwa et al. employing PLGA [134]. This method is based on the partial miscibility of an organic solvent with water. An o/w emulsion is obtained upon injection an organic phase into chitosan solution containing a stabilizing agent (i.e. poloxamer) under mechanical stirring, follow by high pressure homogenization. The emulsion is then diluted with a large amount of water to overcome organic solvent miscibility in water. Polymer precipitation occurs as a result of the diffusion of organic solvent into water, leading to the formation of nanoparticles. This method is suitable for hydrophobic drug and showed high percentage of drug entrapment. The major drawbacks of this method include harsh processing conditions (e.g., the use of organic solvents) and the high shear forces used during nanoparticle preparation.

Figure 12: Schematic representation of the method of emulsion solvent diffusion [133-134]

D. Polyelectrolyte complex (PEC) [135] Polyelectrolyte complex or self assemble polyelectrolyte is a term to describe complexes formed by self-assembly of the cationic charged polymer and plasmid DNA. Mechanism of PEC formation involves charge neutralization between cationic polymer and DNA leading to a fall in hydrophilicity as the polyelectrolyte component self assembly. Several cationic polymers (i.e. gelatin, polyethylenimine) also possess this property. Generally, this technique offers simple and mild preparation method without harsh conditions involved. The nanoparticles spontaneously formed after addition of DNA solution into chitosan dissolved in acetic acid solution, under mechanical stirring at or under room temperature. The complexes size can be varied from 50 nm to 700 nm.

E. Emulsification and cross-linking As mentioned above, this method was the first to be used to form chitosan nanoparticles and involves the preparation of a W/O emulsion (this first report described the emulsification of chitosan aqueous solution in toluene, using Span 80® as emulsifier), with subsequent addition of a cross-linking agent that has the function of hardening the formed droplets. The reactive amino groups of chitosan undergo a covalent cross-linking with the aldehyde groups of glutaraldehyde (Fig. 13), which is added after the emulsion formation and, consequently, after nanoparticles production [136]. Those authors pioneered the production of chitosan nanoparticles, which were used to deliver 5-fluorouracil. Other authors used the method for the same purpose of drug delivery, but modified the oil phase composition to liquid paraffin

40 and petroleum ether [137]. The final particle size was demonstrated to be highly dependent on stirring speed, as well as on the extent of cross-linking. Several drawbacks have been progressively pointed out for this method, including the need of tedious procedures and the application of harsh cross-linking agents [138]. In fact, cross-linkers such as glutaraldehyde were found to cause overt toxicity and to compromise drug integrity, contributing to a progressive shift of interest towards less aggressive procedures. Consequently, the application of this method to obtain chitosan nanoparticles was restricted to a few works.

Figure 13: Schematic representation of the method of emulsification and cross-linking [136]

F. Emulsion droplet coalescence This method is a derivation of the emulsification and cross-linking method described above and was first reported for microparticles preparation [139]. The same authors later adapted the method to prepare chitosan nanoparticles loaded with gadolinium, as a strategy for neutron- capture therapy of cancer [140]. Chitosan is dissolved in the aqueous solution of gadolinium and a small aliquot (1 mL) of this is added to 10 mL of liquid paraffin containing sorbitan sesquiolate (Span® 83). The mixture is stirred with a high-speed homogeniser, thus forming an W/O emulsion (Fig. 14). In parallel, another W/O emulsion is prepared by adding 1.5 mL NaOH to 10 mL of a similar outer phase. Both emulsions are then mixed using a high-speed homogeniser, leading to droplet coalescence. This results in the solidification of chitosan particles by action of NaOH, which acts as precipitating agent. Afterwards, a further set of washing and centrifugation steps is applied using toluene, ethanol and water [140]. A similar procedure was used in a different study to encapsulate 5-fluorouracil [141]. This method

41 exploits the fact that, when two emulsions with equal outer phase are mixed together, droplets of each collide randomly and coalesce, resulting in final droplets with uniform content. The nanoparticles are formed within the emulsion-droplets. Decreasing chitosan deacetylation degree was shown to increase particle size and to reduce nanoparticle capacity for drug association, as a consequence of the diminished capacity of ion-pair formation and de- swelling [140].

[140] Figure 14: Schematic representation of the method of emulsion droplet coalescence

G. Modified ionic gelation with radical polymerisation This method is derived from ionic gelation, but introduces a modification, because chitosan gelation occurs concomitantly with the polymerisation of acrylic acid monomers. The first step occurs at room temperature and consists in stirring an aqueous monomer solution of acrylic or methacrylic acid with an aqueous solution of oppositely charged chitosan. In some cases, polyethylene glycol (PEG) or polyether (polethylene glycol-polypropylene glycol- polyethylene glycol) are also added to the reaction medium, either separately into the monomer solution or following mixing with chitosan. As depicted in Fig. 15, the opposite charges of chitosan and acrylic or methacrylic acid lead to an ionic interaction, while radical polymerisation of the latter is initiated by the addition of potassium persulfate. This reaction takes place under a nitrogen stream and the temperature is usually raised to 60 - 70 ºC. The

42 polymerisation reaction lasts for approximately 6 h, after which the formed suspension of nanoparticles is allowed to settle overnight. Finally, unreacted monomers are removed by dialysis or subsequent washes of the formed particles with distilled water. Nanoparticles obtained by this method have been used to administer insulin, bovine serum albumin and silk peptide through the oral route [142-144].Increasing chitosan molecular weight was shown to induce an increase in the molecular weight of polyacrylic acid present in the nanoparticles, indicating the occurrence of a template polymerization of acrylic acid in the chitosan solution. Parameters such as chitosan/acrylic monomers ratio and polymers concentration have been found to strongly affect the physicochemical characteristics of the nanoparticles properties. More specifically, it was described that the zeta potential increases with the increase in chitosan/acrylic acid ratio, and nanoparticles size was smaller for the lowest polymer ratio (1/1) [142].

Figure 15: Schematic representation of the method of modified ionic gelation with [142] radical polymerisation

H. Desolvation The method of desolvation is also frequently referred to as simple coacervation or phase separation and involves a macromolecular aggregation brought about by partial desolvation of fully solvated molecules [145]. The use of desolvating agents to produce chitosan particles was reported for the first time for the preparation of micron-sized carriers [146] but, nowadays, this procedure is frequently applied to the production of chitosan nanoparticles. Substances such as sodium sulphate [147] and non-solvents miscible with water, like acetone [148], have been proposed as precipitating agents, although the former has been used more frequently.

The preparation of chitosan nanoparticles by this method is very simple and mild as it involves the dropwise addition of the solvent competing agent of greater hydrophilicity (e.g. sodium sulfate) into a previously formed chitosan solution (Fig. 16). As the salt enters in contact with the aqueous environment of chitosan solution, a progressive elimination of salvation water surrounding chitosan occurs as a consequence of the higher affinity of water for the salt. Eventually, this process leads to the polymer insolubilisation and its consequent precipitation [149]. This effect is observed because water-salt interactions are more favourable than those occurring between the water and the polymer, inducing the partial desolvation of chitosan. This, in turn, leads to increased interactions between chitosan molecules, forming the nanocarriers [145]. It is very frequent to include a stabiliser such as polysorbate 80 in the preparation medium, to stabilise the nanoparticle suspension. A subsequent process of cross- linking, for instance with glutaraldehyde, has been described, in order to harden the nanoparticles [149]. Factors such as chitosan molecular weight, chitosan concentration, amount of desolvating agent and stirring rate have been found to strongly affect the final characteristics of nanoparticles. Therefore, it is necessary to undergo an optimisation of these parameters. In addition, a correlation was identified between the amount of sulphate ions needed and chitosan properties, like the molecular weight and the deacetylation degree [150].

Figure 16: Schematic representation of the method of desolvation with sodium sulphate[160]

Table 9 provides summarised information of the methods applied to produce chitosan-based nanopartcles, as well as on their matrix composition, and displays a list of secondary materials that can be combined with chitosan to form the nanoparticulate systems.

Table 9: Methods used for the production of chitosan-based nanoparticles and composition of the carriers’ matrix

Production method Matrix composition

Emulsification and crosslinking Chitosan, glutaraldehyde

Emulsion droplet coalescence Chitosan

Emulsion solvent diffusion Chitosan

Reverse micellisation Chitosan, glutaraldehyde

Ionic gelation Chitosan, tripolyphosphate Polyelectrolyte complexation Chitosan, alginate, Arabic gum, carboxymethyl cellulose, carrageenan, chondroitin sulfate, cyclodextrins, dextran sulfate, polyacrylic acid, poly-γ-glutamic acid, insulin, DNA Modified ionic gelation with radical Chitosan, acrylic acid, methacrylic acid, polymerisation polyethylene glycol, polyether Desolvation Chitosan

3.5.3 Applications of chitosan nanoparticles [151] The applications of chitosan nanoparticles are:  As antibacterial agents, gene delivery vectors and carriers for protein release and drugs  Used as a potential adjuvant for vaccines such as influenza, hepatitis B and piglet paratyphoid vaccine  Used as a novel nasal delivery system for vaccines. These nanoparticles improve antigen uptake by mucosal lymphoid tissues and induce strong immune responses against antigens.  Chitosan has also been proved to prevent infection in wounds and quicken the wound- healing process by enhancing the growth of skin cells.  Chitosan nanoparticles can be used for preservative purposes while packaging foods and in dentistry to eliminate caries.  It can also be used as an additive in antimicrobial textiles for producing clothes for healthcare and other professionals.  Chitosan nanoparticles show effective antimicrobial activity against Staphylococcus saprophyticus and Escherichia coli.

 These materials can also be used as a wound-healing material for the prevention of opportunistic infection and for enabling wound healing.  The nanoparticles have also been proven to show skin regenerative properties when materials were tested on skin cell fibroblasts and keratinocytes in the laboratory, paving the way to anti-aging skin care products.

3.6 INSTRUMENT USED IN EVALUATING THE CHITOSAN NANOPATICLES A. Dynamic Light Scattering: Dynamic light scattering (also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS)) is one of the most popular technique used to determine the size and size distribution of molecules or particles in the submicron region. B. Scanning Electron Microscope (SEM): Scanning electron microscope is a type of electron microscope that produces images of a sample by scanning it with a focused beam of electrons. The electrons interact with atoms in the sample, producing various signals that can be detected and that contain information about the sample's surface topography and composition. The electron beam is generally scanned in a raster scan pattern, and the beam's position is combined with the detected signal to produce an image. SEM can achieve resolution better than 1 nanometer. Specimens can be observed in high vacuum, in low vacuum, and (in environmental SEM) in wet conditions. C. UV/Visible Spectroscopy: Ultraviolet/Visible molecular absorption spectroscopy is based upon electromagnetic radiation in the wavelength region of 160 to 780 nm (UV region: approximately 200-400 nm, visible region: approximately 400-800 nm). Absorption measurements based upon UV/Vis radiation find widespread application in quantitative and qualitative analysis of a substance, structure elucidation of organic compounds and detection of impurities.

CHAPTER-4 MATERIALS AND METHODS

MATERIALS Table 10: Lists of materials used

S.No. Materials Source 1 Shiitake mushroom Vikas mushroom farm, Solan 2 Chitosan Fisher Scientific , Bangalore 3 Acetic acid Fisher Scientific 4 Sodium tripolyphosphate Fisher Scientific 5 Disodium hydrogen phosphate, anhydrous Fisher Scientific 6 Potasium dihydrogen phosphate Fisher Scientific 7 Sodium Chloride (NaCl) Fisher Scientific

INSTRUMENTS Table 11: Lists of instruments used S.No. Equipments Model/Manufacturer 1 Digital electronic balance Citizen 2 Centrifuge Remi, Mumbai 3 Dynamic light scattring Malvern 4 Ultrabath sonicator Sonics 5 Water purification system Milipore 6 Hot air oven Tempo, Mumbai 7 Magnetic stirrer Remi motors,Mumbai 8 Scanning electron microscope Joel- LV-5600, USA 9 UV-Visible spectrophotometer Systronic

COLLECTION: The shiitake mushroom was collected from the Vikas Mushroom Farm, Solan.

4.1 EXTRACTION OF ETHANOLIC EXTRACT FROM LENTINUS EDODES [152] One hundred grams of fresh mushrooms with stalks removed were washed with deionised water and dried with paper towel. They were then homogenized with 300 mL of deionised water, using a warring blender. The resulting mixture was boiled for 3 hours, cooled and placed in a container with liquid nitrogen, freezing it. The frozen mixture was lyophilized (freeze-drying) for three days to remove excess water. The sample was collected and extracted in boiling water (100oC) for approximately 8 hours, with the total amount of water kept constant at 300 mL. The suspension was centrifuged at a low speed (5,000 rpm X 20 minutes) to remove all insoluble particles. The supernatant was retained and an equal volume of ethanol was added to precipitate out the polysaccharides. Precipitation was carried out overnight in cold (~4oC). The precipitated extract was collected via centrifugation and dried by lyophilization. The obtained precipitate was light brown in colour.

Lentinus edodes (fresh fruit bodies) 100 g

Homogenisation with hot water (100oC)

Freezing with liquid nitrogen

Lyophilisation

Extraction with boiling water (100oC)

Centrifugation to remove insoluble matters

Clear liquid

Precipitation with equal volume of 95% ethanol in cold overnight (4oC)

Repeatedly centrifugation

Extract (225 mg) Figure 17: Extraction of shiitake mushroom [152]

4.2 SCANNING FOR MAXIMUM WAVELENGTH For preparing standard curve, first we need to find out the maximum wavelength (λmax) and then standard curve of extract were plotted between absorbance & concentration so that the unknown conc. of extract can be determined in the sample solution. Procedure 1. 2mg of extract is dissolved in 20 mL of distilled water, so that the concentration of the solution will be 100μg/mL. This is the stock solution. 2. From the stock solution .5mL, 1mL, 1.5mL, 2mL, 2.5 mL is taken in different 10 mL volumetric flask & make up the volume by distilled water, so the concentration of the solutions will be 5, 10, 15, 20, 25 μg/mL. These are the sample solution. 3. The scanning for wavelength is done by UV-Spectrophotometry for stock solution and each sample solution. 4. A graph has been plotted between absorbance & concentration and the absorbance which is highest for stock solution and each sample solution corresponding to wavelength is considered as maximum wavelength for extract.

4.3 CALIBRATION CURVE OF EXTRACT The principle is to plot a calibration curve between absorbance & concentration, so that the unknown concentration of extract can be determined in the sample solution. Procedure 1. 2mg of extract is dissolved in 20 mL of distilled water, so that the concentration of the solution will be 100μg/mL. This is the stock solution. 2. From the stock solution .5mL, 1mL, 1.5mL, 2mL, 2.5 mL is taken in different 10 mL volumetric flask & make up the volume by distilled water, so the concentration of the solutions will be 5, 10, 15, 20, 25 μg/mL. These are the sample solution.

3. The absorbance is observed by UV-Spectrophotometry for each samle solution at ƛmax 340nm. 4. A graph has been plotted between absorbance & concentration. The line intersecting the maximum no. of points is drawn. It will give us the calibration curve. Table 12: Preparation of sample solution Concentration Stock solution Water 5 μg/mL 0.5 mL 9.5 mL 10 μg/ mL 1 mL 9 mL

15 μg/ mL 1.5 mL 8.5 mL 20 μg/ mL 2 mL 8 mL 25 μg/ mL 2.5 mL 7.5 mL

4.4 PHYTOCHEMICAL SCREENING [125] Phytochemical examinations were carried out for all the extracts as per the standard methods. 1. Detection of alkaloids: Extracts were dissolved individually in dilute Hydrochloric acid and filtered. A. Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.

B. Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.

C. Dragendroff’s Test: Filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.

D. Hager’s Test: Filtrates were treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids confirmed by the formation of yellow coloured precipitate. 2. Detection of carbohydrates: Extracts were dissolved individually in 5 mL distilled water and filtered. The filtrates were used to test for the presence of carbohydrates. A. Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates. B. Benedict’s test: Filtrates were treated with Benedict’s reagent and heated gently. Orange red precipitate indicates the presence of reducing sugars. C. Fehling’s Test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars. 3. Detection of glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides. A. Modified Borntrager’s Test: Extracts were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with

ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides. B. Legal’s Test: Extracts were treated with sodium nitropruside in pyridine and sodium hydroxide. Formation of pink to blood red colour indicates the presence of cardiac glycosides. 4. Detection of saponins A. Froth Test: Extracts were diluted with distilled water to 20mL and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

B. Foam Test: 0.5 gm of extract was shaken with 2 mL of water. If foam produced persists for ten minutes it indicates the presence of saponins. 5. Detection of phenols Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols. 6. Detection of tannins Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins. 7. Detection of flavonoids A. Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

B. Lead acetate Test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids. 8. Detection of proteins and aminoacids A. Xanthoproteic Test: The extracts were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of proteins. B. Ninhydrin Test: To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid. 9. Detection of phytosterols A. Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes.

B. Libermann Burchard’s test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.

4.5 PREPARATION OF CHITOSAN NANOPARTICLES (CS NPs) [153] Original chitosan was dissolved in acetic acid 1 (w/v) %. The pH of the solutions was raised to 4.6–4.8 by addition of appropriate amount of NaOH. Aqueous sodium tripolyphosphate solutions were prepared by dissolving in distilled water. The tripolyphosphate (TPP) solution was then added dropwise to a chitosan solution until opalescent suspension was formed while stirred with magnet stirrer at room temperature to form chitosan nanoparticles spontaneously. The formation of the particles was a result of the interaction between the negative groups of the TPP and the positively charged amino groups of chitosan (ionic gelation).

Figure 18: Preparation of chitosan nanoparticles [153] 4.5.1 Optimization of formulation

The effects of chitosan concentration (CCS) and tripolyphosphate concentration (CTPP) on the particle size distribution were investigated. A 2-factor design was used to optimize formulation using Design expert (version 8.0.0, Stat-Ease Inc., Minneapolis, Minnesota) Table 13: Optimization of formulation Formulation Affected parameters Extract (mg/mL)

CCS (mg/mL) CTPP (mg/mL) F1 1.0 1.0 3 F2 1.0 2.0 3 F3 1.0 3.0 3 F4 2.0 1.0 3

F5 2.0 2.0 3 F6 2.0 3.0 3 F7 3.0 1.0 3 F8 3.0 2.0 3 F9 3.0 3.0 3

4.6 EVALUATION OF EXTRACT-LOADED CHITOSAN NANOPARTICLES

4.6.1 Particle Size and Zeta Potential of CS NPs Mean particle size, polydispersity index (PDI), and zeta potential of CS NPs were measured by ZS-90 Zetasizer (Malvern Instruments) which was based on the Photon Correlation Spectroscopy (PCS). Samples were dispersed in distilled water prior to measurement. All measurements were performed in triplicate at 25◦C with a detection angle of 90◦, and results were reported.

4.6.2 Drug Entrapment Efficiency Extract was dissolved in chitosan solution. Tripolyphosphate was slowly added into the solution under magnetic stirring. Then, the chitosan nanoparticles were separated by centrifugation at speed of 20,000 g and temperature of 4oC for 30 min. Finally, the extract concentration in supernatant was measured by UV spectrophotometry. The entrapement efficiency (EE) was then calculated from Eq.

EE= (A-B)/A×100

Where A is total amount of extract (mg/ml); B is free amount of extract (mg/ml).

4.6.3 In vitro Drug Release In vitro drug release studies from the extract loaded nanoparticles were performed using a cellulose ester dialysis membrane, which was 20 mm in diameter and previously wetted for 24 hours with the release medium consisting of phosphate-buffered saline (PBS, pH 7.4). 5 mL of pure extract in water and extract loaded nanoparticles were placed into a membrane dialysis bag separately. The bag was tied and put into 100 mL of release medium consisting of phosphate-buffered saline (PBS, pH 7.4). The entire system was incubated at (37 ± 0.5)oC with stirring at 100 rpm for 24 h. At scheduled time intervals (0, 2, 4, 6, 8, 10, 12, 16, 20 and 24 hours), 1 mL samples of the release medium were removed, and the same volume of blank

54 medium, with the same temperature as that of the tested medium, was immediately added. Drug release patterns were obtained by analyzing the dissolution medium samples. The amount of extract in the release medium was analyzed by a UV-Vis spectrophotometer at a wavelength of 340 nm. All measurements were performed in triplicate.

Preparation of Release Medium (Phosphate-Buffered Saline, PBS, pH 7.4): Dissolve 2.38 g of disodium hydrogen phosphate R, 0.19 g of potassium dihydrogen phosphate R and 8.0 g of sodium chloride R in water. Dilute to 1000.0 mL with the same solvent.

4.6.4 Morphological analysis The morphology and surface appearance of extract-loaded chitosan nanoaparticles were examined by scanning electron microscopy (SEM, XL-30E, Philips). The nanoparticle samples were coated with gold and observed at an accelerating voltage of 15 kV with SEM.

CHAPTER–5 RESULTS AND DISCUSSION

5.1 EXTRACTION: BIOCHEMICAL METHOD (Yap and Ng, 2001) The method was performed accordingly to maximize yield and purity. From 100g of fresh shiitake mushrooms, 225mg of extract were produced. The extracts were cottony powder in light brown colour. The whole process of extraction took about five days to perform. The method was comparably easier to carry out and involved much lower cost. The only precaution would be to handle the liquid nitrogen, with care.

5.2 PHYTOCHEMICAL SCREENING OF EXTRACT Carbohydrates, alkaloids, phytosterols, flavonids, proteins and amino acids were present in the extract. Table 14: Phytochemical screening of extract

Tests of Positive Negative Tests of phytosterols Positive Negative carbohydrates

Molisch test + Salkowski’s test +

Phenol- + Libermann test + sulphuric acid test

Tests of Test of phenols - alkaloids

Mayer’s test + Tests of flavonids

Tannic test + Alkaline regeant test +

Tests of Lead acetate test + saponins

Froth test - Tests of proteins and amino acids

Foam test - Xanthoproteric tests +

Tests of - Ninhydrin test + tannis

5.3 SCANNING FOR MAXIMUM WAVELENGTH OF EXTRACT A graph has been plotted between absorbance & concentration and the absorbance which is highest for stock solution and each sample solution corresponding to wavelength is considered as maximum wavelength for extract. The maximum wavelength for the extract was found to be 340 nm from the graph.

λ Absorbance Stock 25μg/mL 20μg/mL 15μg/mL 10μg/mL 320 0.1825 0.057 0.0359 0.0191 0.0062 325 0.1986 0.0683 0.0455 0.0277 0.0113 330 0.2102 0.0721 0.0467 0.0296 0.0129 335 0.2164 0.0721 0.0461 0.0273 0.0151 340 0.2205 0.0727 0.0483 0.0283 0.0151 345 0.2094 0.0642 0.0418 0.0256 0.012 350 0.2091 0.0612 0.0392 0.0223 0.0124 355 0.2101 0.0612 0.0395 0.0228 0.0122 360 0.2088 0.0573 0.0347 0.021 0.0091 365 0.1945 0.0507 0.0305 0.0169 0.007 370 0.2006 0.05 0.0305 0.0198 0.0063 375 0.2076 0.0496 0.0307 0.019 0.0045 380 0.0734 0.0044 0.0007 -0.0042 -0.0058

0.25

0.2 25mcg 20mcg 0.15 15μg/ml

10μg/ml

0.1 stock (100mcg)

Linear (25mcg) Absorbance Linear (20mcg) 0.05 Linear (15μg/ml) Linear (10μg/ml) 0 315 320 325 330 335 340 345 350 355 360 365 370 375 380 385

-0.05 Wavelength

Figure 19: Wavelength scanning of extract

5.4 CALIBRATION CURVE OF EXTRACT A. The calibration curve helps us in quantitative determination of a sample. B. It is a useful method in pharmacokinetic study which helps us to know about the concentration of the drug in urine, saliva or in blood. C. It is useful to know the half life, plasma binding ability of the drug & many more pharmacokinetic data.

0.14

0.12 λmax: 340nm y = 0.0044x - 0.0082 0.1 R² = 0.9991 0.08

0.06

Absorbanc e 0.04

0.02

0 0 10 20 30 40 Concentration Figure 20: Calibration curve of extract

5.5 PARTICLE SIZE, ZETA POTENTIAL, PDI, AND EE OF EXTRACT-LOADED CHITOSAN NANOPARTICLES

Chitosan nanoparticles loaded with extract were successfully prepared by the ionic cross- linking of chitosan with TPP. As shown in Figure 23, the extract-loaded nanoparticles maintained a round shape with almost homogeneous structure. The optimum concentrations obtained for chitosan were 2 mg/mL, extract 3mg/mL, TPP 1 mg/mL and chitosan/TPP ratio 2:1. The optimized extract-loaded chitosan nanoparticles showed 203.3 nm size with PDI 0.332 and +36.9 mV zeta potential, entrapment efficiency of nanoparticles was 74%. The particles size of nanoparticles increased along with increasing concentration of polymer matrix density and this may be due to the increased viscosity of the inner phase and which leads to increased cross-linking. There was a steady decrease in the entrapment efficiency on increasing the polymer concentration in the formulation. The high entrapment efficiency is likely due to electrostatic interactions between the drug and the polymer.

Table 15: Particle size, zeta potential, PDI, and EE of Extract-loaded CS NPs

Formulation Particle size, nm PDI Zeta potential Entrapment Blank CS Extract- CS Efficiency NPs NPs (%) F5 127.9 203.3 0.332 + 36.9 74 F8 156.9 231.1 0.314 + 41.6 71 F9 159.8 262.1 0.255 + 45.8 68

Figure 21: Size distributions of CS NPs 5.6 In-Vitro Drug Release of Pure Extract and Extract-Loaded CS NPs Fig. 22 illustrates that the release of extract could be divided into two stages based on the release rate. In the first stage, the extract was rapidly released from the CS NPs and showed a burst release in the first 2h. This resulted in a 28% cumulative release. In the second stage, extract was slowly released from 2 h up to 18 h, resulting in a cumulative release of more than 68%. While pure extract in water rapidly released and showed a brust release in the first 4h, resulting in a cumulative release of 29%.

80 Extract-loaded CS NPs 70 60 50 40 %CR 30 %CR 20 Pure Extract 10 0 Cumulative release (%) Cumulativerelease 0 5 10 15 20 25 30

Time (h)

Figure 22: The release profile of extract-loaded CS NPs

5.7 MORPHOLOGICAL CHARACTERISATION OF EXTRACT-LOADED CS NPs

The morphological examination of nanoparticles was performed by scanning electron microscopy (SEM). As shown in Figure 23, the extract-loaded nanoparticles maintained a round shape with almost homogeneous structure.

Figure 23: Scanning electron micrograph of extract-loaded CS NPs

CHAPTER-6 CONCLUSION

In the present study, chitosan nanoparticles loaded with shiitake mushroom extract were prepared based on recently optimized ionotropic gelation method, which employed TPP as the crosslinker and to overcome the limitation of the present marketed formuations. The optimum concentrations obtained for chitosan were 2 mg/mL, extract 3mg/mL, TPP 1 mg/mL and chitosan/TPP ratio 2:1. The optimized extract-loaded chitosan nanoparticles showed 203.3nm size with PDI 0.332 and +36.9 mV zeta potential, entrapment efficiency of nanoparticles was 74%. The particles were found to be spherical in shape by SEM analysis. The in vitro release study revealed that the release of extract from chitosan nanoparticles could be better sustained. Extract was slowly released from 2 h up to 18 h, resulting in a cumulative release of more than 68%. These results indicate that there is possibility to formulate chitosan nanoparticles of extract from shiitake mushroom and it is readily bioavailable via nanoformulations.

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PUBLICATION

 Ankur Raj Sharma* and Afroz Khan “Gastroretentive Drug Delivery System: An Approach to Enhance Gastric Retention for Prolonged Drug Release” Int J Pharm Sci Res 2014; 5(4): 1095-06.

WORKSHOP, SEMINARS & PRESENTATIONS

 Participated in “IP Connect”: A workshop on Intellectual Property Rights organized by JP Cell, JUIT, Waknaghat, (August 2013)

 Presented poster entitled “Hypertension” in 49th National Pharmacy Week, organized by JUIT, Waknaghat (Nov 2010)

 Presented Poster entitled “Delivery of Peptide Based Therapeutics through Liposomes” in AICTE sponsored National Seminar on “Recent Trends in Development of Novel Drug Delivery Systems”, Jaipur College of Pharmacy (March, 2013)

BIO-DATA

ANKUR RAJ SHARMA Phone +91-8988248571 / 9816320355 M.PHARM (Pharmaceutics) Email [email protected]

Permanent Address II/G-4, Nehru Nagar, Hapur Road Near B.D Arya Nursing Home Ghaziabad (U.P.)- 201001

Career Objective

To accept all challenges and assignments assigned to me in the industry, with great responsibility and accomplish them with utmost sincerity by well coordinated teamwork.

Academic Profile M. Pharm, 2014 Jaypee University of Information 82% (7.9* CGPA) Pharmaceutics (expected) Technology, Waknaghat till date (3rd semester) 2012 Jaypee University of Information B. Pharm 82% (7.9* CGPA) Technology, Waknaghat 2008 Ch.Chhabil Dass Public School, Class XII,CBSE 69% Ghaziabad Class X,CBSE 2006 Dewan Public School, Hapur 72.2%

Publication

Ankur Raj Sharma* and Afroz Khan “Gastroretentive Drug Delivery System: An Approach to Enhance Gastric Retention for Prolonged Drug Release” Int J Pharm Sci Res 2014; 5(4): 1095-06.

Workshop, Seminars & Presentations

 Participated in “IP Connect”: A workshop on Intellectual PropertyRights organized by JP Cell, JUIT, Waknaghat, (August 2013)  Presented poster entitled “Hypertension” in 49th National Pharmacy Week, organized by JUIT, Waknaghat (Nov 2010)  Presented Poster entitled “Delivery of Peptide Based Therapeutics through Liposomes” in AICTE sponsored National Seminar on “Recent Trends In Development of Novel Drug Delivery Systems”, Jaipur College of Pharmacy (March, 2013)

Achievements and Awards

 Participated in National Children’s Science Congress in class XII  Actively participated in U.S.O., India quizes  Actively participated in Green Olympiad  Selected and performed as school captain in class X

Project

 M. Pharma “Formulation and Evaluation of Chitosan Nanoparticles of Ethanolic Extract from Lentinula Edodes (Shiitake Mushroom)” under the supervision of Dr. Maneesh Jaiswal  The objective of this project work is to formulate and evaluate chitosan nanoparticles encapsulating ethanolic extract of shiitake mushroom and to overcome the limitation of the present marketed formulations  B. Pharma “Design and Synthesis of New Antimicrobial Agent” under the supervision of Dr. Kuldeep Singh  The objective of this project is to synthesis of new antimicrobial agents.

Interest and Activities

Painting, Travelling, Playing badminton, Football

Personal Profile

Date of Birth Jan 01, 1991 Father’s Name Mr. Rajkumar Sharma Mother’s Name Mrs. Madhu Bala Sharma Languages Known English, Hindi Nationality Indian

Declaration

I hereby declare that all the information mentioned above is true to the best of my knowledge.

Shimla (ANKUR RAJ SHARMA)