Optimization of panax notoginseng root extract hydrolysis by cordyceps militaris derived glycosidase and bioactivities of hydrolysis products

When major ginsenosides Rb1,Rb2 and Rc were administered to rats , some minor ginsenosides and compound k were recovered in rat blood [50] and later was shown that compound k derived fro

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

SIMURABIYE JEAN BAPTISTE

OPTIMIZATION OF Panax notoginseng ROOT EXTRACT HYDROLYSIS BY Cordyceps militaris DERIVED GLYCOSIDASE

AND BIOACTIVITIES OF HYDROLYSIS PRODUCTS

MASTER THESIS

KHANH HOA – 2020

MINISTRY OF EDUCATION AND TRAINING

NHA TRANG UNIVERSITY

SIMURABIYE JEAN BAPTISTE

OPTIMIZATION OF Panax notoginseng ROOT EXTRACT

HYDROLYSIS BY Cordyceps militaris DERIVED GLYCOSIDASE

AND BIOACTIVITIES OF HYDROLYSIS PRODUCTS

MASTER THESIS

Topic allocation Decision 192 /QD-DHNT

Decision on establishing the

Committee:

Defense date: 17/09/2020

Supervisors:

Dr Nguyen Duc Doan

Dr Le Thi Hoang Yen

Dr Trinh Dac Hoanh

Chairman:

Faculty of Graduate Studies:

KHANH HOA – 2020

UNDERTAKING

I undertake that the thesis entitled: “optimization of Panax notoginseng root extract hydrolysis by Cordyceps militaris derived glycosidase and bioactivities of hydrolysis products” is my own work The work has not been presented elsewhere for

assessment until the time this thesis is submitted

Khanh Hoa, 27/08/2020

SIMURABIYE Jean Baptiste

ACKNOWLEDGMENTS

I appreciate the conditions provided by Nha Trang University, especially

faculty of Food Technology for this master program to be completed

I especially thank my supervisors, Dr Nguyen Duc Doan; Lecturer at Vietnam

National University of Agriculture for selflessly contributing to the progress of this

thesis project until completion, and Dr Le Thi Hoang Yen; Member of the Institute of

Microbiology and Biotechnology of the Vietnam National University for theoretically

and practically guiding me and providing all the assistance that was required in every

step of this thesis

I would like to extend my gratitude to Dr Trinh Dac Hoanh from the Institute

of Chemistry and Material Science of Military Academy of Science and Technology

for his technical and financial contribution towards this project, and Dr Duy Nhan Vu

from the Institute of Chemistry and material science of Military Academy of Science

and Technology for financing my whole length of stay in Hanoi

In no particular order, I would also like to acknowledge the contribution of

Miss Nguyen Thi Huong Nhu, Miss Hoang Anh Dong, Miss Thanh Tran Huyen

and Miss Le Hong Anh and eventually express my appreciation

Lastly, I dedicate this work to each of my family members for the love and

support

This thesis was supported by “Đề án phát triển và ứng dụng công nghệ sinh học

trong lĩnh vực công nghiệp chế biến” (grant number DT.11.19/CNSHCB)

Khanh Hoa, 27/08/2020

SIMURABIYE Jean Baptiste

CONTENTS

UNDERTAKING iii

ACKNOWLEDGMENTS iv

CONTENTS v

LIST OF SYMBOLS viii

LIST OF ABBREVIATIONS xii

LIST OF TABLES xiii

LIST OF FIGURES xiv

ABSTRACT xvi

Chapter 1: INTRODUCTION 1

1.1 Overview 1

1.2 Objectives 4

1.2.1 Overall Objective 4

1.2.2 Specific Objective 4

Chapter 2: REVIEW OF THE LITERATURE 5

2.1 Ginseng and ginsenosides 5

2.1.1 History, evolution, botanical characteristics and use 5

2.1.2 Ginseng medicinal compounds, their structures, types and distribution in plant parts 7

2.1.1 Ginsenoside metabolism 11

2.2 Bioavailability of ginsenosides 13

2.2.1 Methods of biological conversion 13

2.2.2 Ginsenosides as antimicrobial agents 14

2.2.3 Anti-cancer and immune-modulatory effects of ginsenosides 15

2.2.3.1 Anti-cancer cell mediated immune system modulation by ginsenosides 16

2.2 Mushroom beta glucosidase 24

2.2.1 Overview 24

2.2.2 Properties of mushroom β-glucosidase 27

Chapter 3: MATERIAL AND METHODS 30

3.1 Material 30

3.1.1 Methods 32

3.1.2 Total saponin content determination 33

3.1.3 TLC analysis 33

3.1.4 HPLC analysis 33

3.2 Mushroom β-glucosidase 34

3.2.1 Mushroom cultivation and enzyme harvest 34

3.2.2 Mushroom enzyme purification 34

3.3 Mushroom enzyme activity assay 34

3.3.1 CMCase activity of mushroom enzyme 34

3.3.2 Enzyme activity test by DNS method 35

3.4 Ginsenoside fermentation 36

3.5 Bioactivities 38

3.5.1 DPPH Scavenging activity 38

3.5.2 Antibacterial activities 39

3.5.3 Cytotoxicity assay 39

3.5.4 Data analysis 40

Chapter 4: RESULTS AND DISCUSSION 41

4.1 Ginsenoside extraction 44

4.1.1 Extraction yield 44

4.1.2 Saponin content of P notoginseng fractions and crude extract 45

4.2 Ginsenoside fermentation 45

4.3 Bioactivities 58

4.3.1 DPPH scavenging activity 58

4.3.2 Antibacterial activity 60

4.3.3 Cytotoxic activity 62

REFERENCES 65 APPENDICES I

LIST OF SYMBOLS

(NH4)2SO4: Ammonium sulphate

∙OH: Hydroxyl radical

CaCl2: Calcium chloride

CaCO3: Calcium carbonate

cAMP: Cyclic adenosine monophosphate

CMCase: Carboxymethyl cellulase

CO2: Carbon dioxide

CT26: A murine colorectal carcinoma cell line

FeSO4: Iron sulphate

Glc: Glucose

GM-CSF: Granulocyte-macrophage colony-stimulating factor H2Ax: H2Ax gene

HOCl Hydrochloric radical

IBD: Inflammatory Bowel Disease

IFN- Interferon Gamma

iNOS: Indicible nitric oxide synthase

IP-10: Inducible protein-10 (also CCL10)

MDSC: Myeloid-derived suppressor cells

MgSO4.7H0: Magnesium sulphate heptahydrate

MHC-1: Major Histocompatibility class 1

MHC-2: Major Histocompatibility class 2

MiR-146a: MicroRNA-146a

MMP-2: Matrix metalloproteinase-2

MMP-7: Matrix metalloproteinase-7

MMP-9: Matrix metalloproteinase-9

Mn: Manganese

NADPH: Nicotinamide adenine dinucleotide phosphate

NaNO3: Sodium nitrate

NFB: Nuclear factor kappa light chain enhancer of activated B cells NK: Natural killer cells

OH: Hydroxyl radical

RNA: Ribonucleic acid

TNF- Tumor necrosis factor-

uPa : Urokinase receptor

VEGF: Vascular endothelial growth factor

xyl: Xylose

Zn: Zinc

 itric oxide

LIST OF ABBREVIATIONS

ANOVA: Analysis of variance

CCD: Central composite design

DNS 3,5-Dinitrosalicylic acid

DPPH 2, 2-diphenyl-1-picrylhydrazyl

GRAS: Generally regarded as safe.

HPLC: High pressure liquid chromatography.

IMBT/VNU: Institute of Microbiology and Biotechnology, Vietnam national UniversityPDB: Protein data bank.

PH: Potential of hydrogen

RGE: Red ginseng extract.

RSM Response Surface Methodology

RSM: Response surface methodology

TLC: Thin layer chromatography

USFDA: United States Food and Drug Authority

UV-HPLC: Ultra violet-high pressure liquid chromatography

WHO: World Health Organization

LIST OF TABLES

Table 2.1 The structures of PPD-type ginsenosides, PPT-type ginsenosides and

Ocotillol-type ginsenosides[46] 10

Table 3.1 Content of media for mushroom mycelial culture 31

Table 3.2 Mushroom strains subjected to DNS test 36

Table 3.3 Experimental factors and their respective levels 37

Table 4.1 Ratio of white circle to enzyme seat diameter in CMCase test 42

Table 4.2 Ginsenosides extraction yield 45

Table 4.3 Total saponin content of P notoginseng extract and fractions 45

Table 4.4 Central composite design of Ginsenoside hydrolysis 46

Table 4.5 ANOVA for the model of ginsenoside hydrolysis 47

Table 4.6 Cytotoxic activity of P notoginseng hydrolysis products by C militaris derived glycosidase 62

LIST OF FIGURES

Figure 2.1 Core structures of ginsenosides: A; PPD-type, B; PPT-type and C;

Ocotillol-type 9

Figure 2.2 Predicted damarane ginsenosides deglycosylation in gastrointestinal tract 12

Figure 2.3 A; Crystal structure of beta-glucosidase from mushroom Lentinula edodes in its tetramer form ( PDB code 6JBS (left)) and B; a superimposed structure of a substrate free form in gray and a binding form in green 27

Figure 4.1 Enzymatic activity of mushrooms by DNS method 43

Figure 4.2 Contour plot showing the effect of interacting factors on hydrolysis of major ginsenosides from P notoginseng extract by C militaris mushroom beta glucosidase with non-interacting factors hold at optimal values 51

Figure 4.3 A factorial plot giving an overview of the effect of changes in factors on the change in hydrolysis products 49

Figure 4.4 Overview of the optimal values of the individual factors 51

Figure 4.5 Standardized effects of the factors 52

Figure 4.6 Factorial plot showing optimal conditions for P notoginseng ginsenosides hydrolysis process by beta glucosidase from mushroom C militaris 52

Figure 4.7 TLC analysis of ginsenosides content in extract (left) and some chosen hydrolysates (right) with minor ginsenoside Rg3 as standard 55

Figure 4.8 HPLC chromatogram of the standard ginsenoside Rg3 56

Figure 4.9 HPLC chromatogram of hydrolysate (T30oC, pH6, t 30h, E/S 0.5) 56

Figure 4.10 HPLC chromatogram of hydrolysate (T60oC, pH8, t 30h, E/S 0.5) 56

Figure 4.11 HPLC chromatogram of hydrolysate (T30oC, pH8, t 60h, E/S 1) 57

Figure 4.12 HPLC chromatogram of hydrolysate (T60oC, pH8, t 80h, E/S 1) 57

Figure 4.13 HPLC chromatogram of hydrolysate (T15oC, pH7, t 54h, E/S 0.75) 57

Figure 4.14 HPLC chromatogram of hydrolysate (45oC, pH7, t 40h, E/S 0.75) 58

Figure 4.15 DPPH scavenging activity of hydrolysis products and crude extract 58

Figure 4.16 Growth inhibitory effect of ginsenosides hydrolysis products and extract

ABSTRACT

This thesis project examined the potential of Cordyceps militaris fungus for

ginsenoside transformation This mushroom was selected from seven mushrooms

namely, Cordyceps militaris, Coprinellus sp., Phillus sp., Ganoderma lucidum,

Agrocybe cylindi, Pleurotus sp., and Xerula sp The fruiting bodies of this fungus are

mostly used in making soups in Asia Enzyme extracted from this mushroom was used

to convert ginsenosides extracted from Panax notoginseng roots The enzyme activity

was evaluated using DNS method and it was found to be 1.55u/mL Ginsenosides were extracted by ultrasound assisted extraction with 1.284% yield The optimum

conditions for maximum hydrolysis of ginsenosides by enzyme derived from C

militaris was determined using response surface methodology The effects of factors

namely T (30-60oC), pH (6-7), time (30-80 h) and enzyme concentration (0.5-1%), on minor ginsenoside production Regression analysis was used to develop a second

degree polynomial model for minor ginsenosides production from P notoginseng root extract hydrolysis by C militaris derived enzyme Statistical significance of the model

was evidenced by coefficient of determination (R2=95.15%) and (P value=0.000) Minor ginsenosides production was affected by enzyme concentration, temperature and time respectively in order of magnitude PH effect was found insignificant Maximal minor ginsenosides were found at 0.86% enzyme concentration, 42.88oC, 62.63 h and pH 6.62

Bioactivities of produced minor ginsenosides were evaluated in comparison to the crude extract A significant difference between hydrolysates from different hydrolysis treatments was observed for DPPH scavenging activity The same

observation was also seen in antimicrobial activity with Staphylococcus aureus and

Escherichia coli and Pseudomonas aeruginosa The two former species were found

more susceptible to hydrolysates than the latter Hydrolysates have shown a strong cytotoxic activity against Human lung cancer cell line (SK-LU-1) and Michigan Cancer Foundation-7 (MCF-7); a breast cancer cell line The developed model for

Panax notoginseng hydrolysis by C militaris derived glycosidase can potentially be

used for producing minor ginsenosides with improved bioactivities for use in production of food and medicines

Keywords: P notoginseng, C militaris, hydrolysis, ginseng, ginsenosides, cancer

Chapter 1: INTRODUCTION 1.1 Overview

Health complications related to compromised immune system have been increasing over the years World health organization (WHO) predicts that the mortality related to those diseases will continue to increase for the next twenty years Sanitation and vaccination improvements have been a success in reducing the occurrence of infectious diseases but the recent viral strikes brought the world to realizing that it is not always ready to successfully control the spread and mortalities from heavily infectious species Studies have shown the role of plant extracts and phytochemicals in the fight against both infectious organisms and modulating the immune system to be more ready for the fight and prevention of infections to lead to more complications such as cancer [1]

Chemotherapy is always considered the first line of therapy in all health complications In cancer, surgery and radiotherapy are alternatives Depending on the target of chemotherapy, it has been shown to potentially exhibit lethal side effects for example killing normal vital fast growing cells such as the ones of bone marrow, mouth and intestines making it a significant limitation of their use in clinical settings [2] Recently, immunotherapy has gained public attention and accepted as a preference

in disease treatment especially in cancer treatment However, due to requirements in sophisticated equipment and intensive knowledge in other immunotherapy types, immune-modulation gained preference

In any case, before any extrinsic treatment our bodies use natural means of defense; the immune system to try to get rid of infectious agents and cancer cells Immune system of our bodies is divided into two major big components; namely innate and adaptive immunity Innate immunity acts as a first line of defense and consists of physical barriers like skin, cells of immune system and immune chemicals which mount mechanisms of inhibiting foreign materials from entering our bodies or destroying them once they managed to arrive in our bodies Similarly, cancer cells are recognized as foreign materials and destroyed by our immune system [3] Adaptive immunity is a more sophisticated system of defense which use innate cells and chemical component of immune system to specifically eliminate the pathogen or abnormal or damaged cells

The role of food nutrients and bioactive compounds on disease prevention has been elucidated [4].Understanding of mechanisms of these components has been a mind opening to food industry and made a liaison between food and pharmaceutical industries and a new food science area was born which is functional food or nutraceuticals The use of food technology innovations such as food fermentation, extraction, ingredient or specific nutrient addition, encapsulation and enzyme technology, ingredient stabilization, elimination or masking the undesirable flavors, manipulation of food components using advanced gene science and the use of nanotechnology are now acting as knowledge source for food technology industry to produce food with associated health promoting effects On economic point of view, the demand for these food products will continue to increase as the world population is getting more and more health conscious

Nutrients including essential fatty acids, essential amino acids, vitamins A, B6, B9, B12, C and E, mineral elements such as Cu, Fe, Zn, and Se and non-nutrient bioactive compounds from both plants and animals such as saponins, alkaloids, polyphenols, tannins and others are at the base of this revolution, however few food products available

at markets are accepted as immune modulating, though different compounds have shown their immune-modulating potential Different phytochemicals were investigated for health promoting properties, disease curing properties and their interactions with and modulation

of human immune functions with promising results [1]

One of the plants with a long history of use for therapeutic purposes is ginseng

Ginseng is a name of the plant of the genus Panax of the Araliaceae family

Ginsenosides; ginseng saponins are according to the literature, at the base of the plant bioactivities Like other saponins, ginsenosides are hydrophobic sapogenins to which hydrophilic saccharides or oligosaccharides are attached The presence of this structural antagonism in saponins is the root cause of the famous foam forming characteristics of these compounds Ginsenosides have been in use for health promoting purposes for millennia in Asia The increasing demand of ginseng plants due to these compounds has led to their exhaustion in their natural habitat As research technology improves it was shown that many orally ingested compounds can be recovered in their original forms in the faces and a small portion of them in urine but

in their transformed forms Examinations of the transformed forms showed that they are versions of original forms with reduced saccharide chains These original forms are mostly referred to in literature as major ginsenosides and transformed as minor or sometimes referred to as rare ginsenosides due to their scarcity in raw plants Knowing this with the pressure of increasingly disappearing source of these compounds due to overexploitation has led scientists to trying to maximize the bioactivities of these compounds, by converting the raw compounds into the transformed ones with added bioavailability As sugar molecules are removed solubility reduces due to increased hydrophobicity until a sugar free compound completely hydrophobic is obtained However this is not the most desired result, intermediary molecules are mostly preferable because they exhibit sufficient to maximum intestinal absorption.[5]

These forms can be obtained from the original forms of saponin by means of hydrolyzing sugar moieties, which can be physical, chemical or biological methods of transformation Biological methods are generally accepted in use for food or medicine related purposes because they are safe, fast, convenient, efficient and environmental friendly as contrary to chemical methods which can have undesirable side reactions Biotransformation gets use of enzymes and microorganisms to accomplish chemical hydrolysis [6] The use of microorganisms to produce minor ginsenosides for food or drug has been a hindrance because of their production of toxic substances such as biogenic amines, botulinum neurotoxins, aflatoxins, zearalenone, fumonisins, and ochratoxins which cause many physiologic defects [7] In an attempt to get this solved, several studies have been conducted on the use of food grade organisms, bacteria from fermented food [8], gut bacteria [9] and recently, different types of fungi [10,11]

Edible and medicinal fungi have had different applications and they are economically efficient and so can be used extensively on industrial scale as opposed to intestinal bacteria which are less productive and require expensive media Different types of fungi can produce mycotoxins even in the ones approved as generally

regarded as safe (GRAS) as in three species Aspergillus niger, Aspergillus oryzae,

and Trichoderma reesei known for their industrial use [12] This explains the necessity

of carefully finding species of fungi especially mushrooms that are safe for use in food

and medicine industry with the potential to convert major ginsenosides into minor ginsenosides; one of the least researched area

The major aim of this dissertation is to produce minor ginsenosides from P

notoginseng root extract using edible or medicinal mushroom derived glycosidase

Different species of mushrooms will be assayed for their enzymatic activity towards major ginsenosides so as to find out which mushroom species is suitable for ginsenoside transformation This will provide a safe and alternative means for producing more absorbable and hence more bioavailable minor ginsenosides with increased ability of delivery to their target tissues The investigation of bioactivities of the products of this hydrolysis will give an idea of how powerful is the candidacy of the mushroom specie(s) for major ginsenoside transformation Enzymes produced by some mushroom species were found to successfully produce minor ginsenosides [13], but with the large diversity of fungi kingdom, the scientific work available is minimal

and it is apparently an area which needs more attention

1.2 Objectives

1.2.1 Overall Objective

1 Conversion of panax notoginseng saponin extract to minor ginsenosides by

using edible and/ or medicinal fungi derived enzyme

2 Study the bioactivities of converted ginsenosides.

1.2.2 Specific Objective

1 Extraction of ginsenosides from P.notoginseng

2 Find an edible and/or medicinal mushroom with ginsenoside hydrolyzing ability

2 Subject the crude extract to hydrolysis by edible and/or medicinal mushroom enzyme

3 Analysis of hydrolysis products

4 Study anti-oxidant properties, antimicrobial and anticancer activities of hydrolysis products

Chapter 2: REVIEW OF THE LITERATURE

2.1 Ginseng and ginsenosides

2.1.1 History, evolution, botanical characteristics and use

Ginseng, a common name for all plants of the genus Panax, one of its kind was

discovered approximately 5000 years ago in the mountains of Manchuria, China It is believed that it was used primarily as food for farmers and labourers and approximately two millennia later, its health benefiting effects were recognised and started being used for medicinal purposes The belief in powers of ginseng by Chinese emperors made it so valuable that it was only reserved for them and often sold for more than its weight in gold This demand grew into ginseng production and export industry which dates back in the third century before Christ (BC) [14].The exploitation

of these plants led to their exhaustion in their natural habitat and started being

domesticated Domestication started by cultivating Panax ginseng by transplanting the

wild ginseng around 11 BC in Asia and propagating seeds of transplanted plants

There are fourteen known species of Panax namely, P notoginseng P bipinnatifidus,

P ginseng, P japonicus, P major, P quinquefolius, P sinensis, P wangianus, P zingiberensis P tricolours, P pseudoginseng, P stipuleanatus, P omeiensis and P vietnamensis all of which have been used as a source of medicine Phylogenetic

studies suggest that Asian species are only recently diverged as explained by the relatively low divergence of ITS sequence divergence and the centre of divergence is supposedly the Himalayas where eight of the fourteen species are found [15]

Biogeographically two species are present in Eastern North America whereas the rest twelve are distributed in Asia and Himalayas Phylogenetic studies by both cDNA and ITS sequences indicated that neither of the eastern Asian or the North American species form a monophyletic group Molecular sequence divergence studies suggest that this genus underwent two biogeographic disjunctions in different geologic times of which the older may have occurred in Oligocene and the younger estimated to

middle and late Miocene The diversification seen in Panax genus is believed to have

originated from the collision of indian plate with Asia in tertiary era [16]

Panax genus belongs to Araliaceae family informally named ginseng family Its species mostly grow in altitudes and warm freeze-free areas of Eastern Himalaya

through Southern China to North and Central highlands of Vietnam They are also

found in Russian far east, Canada and the United states of America [17] Ginseng

plants are naturally wild but were domesticated many years ago for their disease curative effects [18] Domestication and actions of humans towards the plant has caused the creation of high level of genetic diversity, alteration of some phenotypic traits and led to a plant with a seven years life cycle from a plant that can live for hundreds of years in its natural habitat [19] Despite many changes that took place, the general morphological characteristics and growth requirements seem not to have remarkably changed [20] As in natural habitat, cultivated ginseng requires shade, annual precipitation of 1000 to 1250 mm and for adequate seed dormancy, it requires cold temperature for several weeks They grow very slowly that they can develop a shoot of one leaf during the first year of development and may grow up to 1.2 m after several years approximated to 7 Ginseng is hermaphrodite and can grow both in light, medium and heavy soils but the root quality differs in different soil types They live well in moist well drained soils and do not respond much to changes in soil pH but prefer it slightly acidic (pH5.5) It is after three years that these plants start blooming and bear flat and oval shaped berries like fruits which turn red from green as they ripen Flowers have five petals with umble inflorescence The berries contain greyish-white seeds which are the port to the next plant generation The seeds do not germinate until twelve to eighteen months [21]

There are different types of ginseng based on the method of processing; fresh ginseng is eaten fresh and is harvested before reaching the fourth year, white ginseng

is harvested between the 4th and 6th year, peeled and air or oven dried, Red ginseng is a harvested when it is 6 years old and steamed When white ginseng is steamed at high temperature and pressure it changes the name to sun ginseng [22]

Even though ginseng has been a subject of American export to China since 1700s, Petkov was the first researcher to formally report the pharmacological potential of ginseng plants in the 1950s [21] and since then thousands of scientific reports about traditional use, chemical constituents and biological characteristics have been published Different plant parts have been used for disease curative effects including roots, flower buds, fruits, and leaves [23] Some of these applications include memory and physical function improvement [24,25], pain relieving effect [26], anti-cancer and

inhibition of tumor initiation[27,28], reduction of cancer chemotherapy tolerance and side effects [29], increase in immune function [30] anti-diabetic effects [31,32], liver function improvement [33], blood pressure adjustment[34], improvement of female climacteric disorder, anti-stress and fatigue [35], improving stress related sexual dysfunctions [36] inhibition of HIV propagation[37,38] and anti-aging and anti-oxidation abilities [35,36]

2.1.2 Ginseng medicinal compounds, their structures, types and distribution in plant parts

Ginseng contains different compounds namely polyphenols, flavonoids, polysaccharides, polyacetylene, peptides, individual amino acids and saponins [41], all believed to contribute to its attributed pharmacological properties Moreover, ginseng saponins called ginsenosides are the most studied and are believed to confer most of the benefits [42] Ginsenosides have a four or five ring structure and a steroidal body and sugar moieties These sugar moieties attach to hydroxyl groups of ginsenosides Each ginsenoside must have two or three hydroxyl groups at carbon 3, 6 and 20 to which monomeric, dimeric or trimeric sugars can bind Depending on the structure of their skeleton, they are divided into Damarane-type ginsenosides synthesized by hydroxylation of dammarenediol-II, a reaction which produces protopanaxadiol with

OH group at C-3 and C-20 at which sugars can bind, exemplified by ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, and Rh3, and protopanaxatriol with sugar moieties attached to

OH at C-3, C-6, and C-20 represented by ginsenosides Re, Rf, Rg1, Rg2, and Rh [25] Both proptopanaxadiol and protopanaxatriol have a 4 ring aglycone structure Oleanane-type ginsenosides are 5 ringed and are biosynthesized from β amyrin also derived from dammarenediol-II Oleanane type ginsenosides are rare but one mostly

detected in P.ginseng is ginsenoside Ro Ocotillol-type ginsenosides lack a glucosyl

moiety at 20 position [43], Apart from these compounds, ginseng has other nutritional elements such as Phosphorus (P), Potassium (K), Calcium (Ca), Thallium (Ti), Manganese (Mn), Iron (Fe), Copper (Cu), Zinc (Zn) and Strontium (Sr) with other minor components which include volatile oils, members of vitamin B complex, manganese, vanadium, copper, cobalt, fatty acids, amino acids, simple sugars and other carbohydrates [44]

Although the root is the most used ginseng part for medicinal purposes, ginsenosides are distributed in many other parts of the plant, including the root, leaf, and berry These different parts appear to exhibit distinct ginsenoside profiles which may exhibit different pharmacological activities Differences in profiles of ginsenosides were found to be influenced by the growth stage where accumulation of ginsenosides show opposite tendencies with age in leaves and roots, where higher accumulations in leaves are present in early growth stages whereas the roots achieve

their maximum accumulations in late growth stages [45]

Figure 2.1 Core structures of ginsenosides: A; PPD-type, B; PPT-type and C;

Ocotillol-type[46]

The types of ginsenosides are chemically inter-related and their structures are presented in (Figure 2.1) PPD and PPT type ginsenosides have the same skeleton and side chains they only differ from the presence or absence of hydroxyl group at position C-6 Dammarane and ocotillol types share the same parent structure, ocotillol ginsenosides are basically side chain double bond oxidative cyclization product of PPT-type ginsenosides Generally, the difference in ginsenoside structures is brought

about by pattern structure of their R group substituents

Table 2.1: The structures of PPD-type ginsenosides, PPT-type ginsenosides and

Note: “glc” represents glucose, “arap”; arabopyranose, “araf”; arabinofuranose,

“rham”; rhamnose and “xyl”; xylose C-24 of ocotillol-type ginsenosides is R

2.1.1 Ginsenoside metabolism

Ginsenosides are not human nutrients and so human digestive system lacks a metabolic function for their digestion However they are consumed orally as food supplements and get metabolised by colonic bacteria, predominantly species of Bacteroides, Fusobacterium and Eubacterium [47] These microorganisms use their enzymes to transform major ginsenosides into minor ginsenosides sometimes through intermediates For example ginsenoside Rb1 is hydrolysed to 20(S)-protopanaxadiol (PPd) via intermediates gypenoside XVII, ginsenoside Rd, ginsenoside Rg3, ginsenoside Rh, ginsenoside F2, and ginsenoside compound K by stepwise cleavage of sugar moieties at position C-3 and C-20 [48] There, deglucosylated ginsenosides are taken by passive diffusion into the bloodstream Deglycosylation studies in mice have shown that PPD type ginsenosides are hydrolysed at C-20 until one sugar moiety remains, creating ginsenoside Rd and then deglycosylation takes place at C-3 followed

by complete removal of the remained sugar at C-20 making an aglycone PPD, and for PPT-type ginsenosides, deglycosylation appears to occur preferentially at C-6, with further deglycosylation taking place at either C-6 or C-20 to form the final metabolite PPT Studies have shown however that most individuals depending on their intestinal microbiota may have these compounds not delivered to their targets Ginsenosides, let alone primary ginsenosides with higher molecular weights, have natural traits which hinder their intestinal membrane permeability which include high total number of hydrogen bonds, high Topological polar surface area (TPSA) and high flexibility As the number of sugar moieties are reduced, these hindrances are removed but also significantly reduces solubility The final products of ginsenoside hydrolysis, sugarless PPD and PPT tend to be more lipophilic than their mother ginsenosides, making them insoluble in intestinal fluids and hence limiting their absorption [49] The same report also observed that the poor membrane permeability of ginsenosides hinders them to reach target tissue cells, they persisted in organ capillaries and interstitial fluids rather than organ cells except the liver which showed higher ginsenoside concentrations than plasma though the significant part is also eliminated by biliary secretion leaving little for the actual target exposure However the biliary excretion of PPD ginsenosides was seen to be slower

Figure 2.2: Predicted damarane ginsenosides deglycosylation in gastrointestinal

tract[46]

2.2 Bioavailability of ginsenosides

It was reported that ginsenosides Rg1, Re, Rb1, Rc, Rb2 and Rd count for more

than 85%of total saponins in the notoginseng roots The bioavailability of these major ginsenosides is low as it is for all glycosides However it can be increased by hydrolysis of the sugar moiety Many researchers reported the poor intestinal absorption of ginsenosides both in vivo and in simulated models [5] The lower intestinal absorption may be due to their physicochemical properties, characteristic of their big size, poor solubility and permeability across the intestinal cell membrane [50] When major ginsenosides Rb1,Rb2 and Rc were administered to rats , some minor ginsenosides and compound k were recovered in rat blood [50] and later was shown that compound k derived from stepwise hydrolysis of ginsenoside Rb1 through intermediates Rd, and F2 [51] Compound K is a minor ginsenoside; a deglucosylated form found in trace amounts and sometimes absent in raw ginseng [50] Nonetheless,

it is with other minor ginsenosides like Rg3, Rh2, F2, Rg2, Rh1, and F1; all deglucosylated forms of major ginsenosides, more pharmacologically active and bioavailable than their parent ginsenosides [52,53] There are methods of transforming major to minor ginsenosides; physical methods that are based on treatment of ginsenosides with elevated temperatures and include steaming, sulphur fumigation and microwave Chemical methods are acidic hydrolysis with which ginsenosides are subjected to acidic conditions or alkaline hydrolysis with which major ginsenosides are treated in alkaline conditions with high temperature and pressure Biotransformation gets use of enzymes and microorganisms to accomplish chemical hydrolysis [6]

Due to drawbacks associated with physical and chemical transformation such as lack of selectivity, unwanted side reactions and environmental pollution; biological methods have gained general acceptance because they are fast, convenient, efficient and environmental friendly

2.2.1 Methods of biological conversion

Biological methods of ginsenoside transformation make use of different enzymes capable of hydrolysing the sugar moiety of ginsenosides It was revealed that one or all of the four molecules of glycosides may be present in ginsenoside sugar

moieties, and those are glucose, L-arabinopyranoside, L-arabinofuranoside, D-xylose, and/or L-rhamnose [54], and to ensure enzyme-substrate specificity, this dictate the use of either of , β-glucosidase (EC 3.2.1.21), β-xylosidase (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), and α-L-rhamnosidase (EC 3.2.1.40) for ginsenosides hydrolysis Various microorganisms were found to transform major ginsenosides into minor ginsenosides and they include bacteria, unicellular fungi and mushrooms [47,55] however the use microorganisms to produce minor ginsenosides for food or drug use has been a hindrance as most microorganisms which perform this activity do not have generally regarded as safe (GRAS) status for their production of potentially toxic substances such as biogenic amines, botulinum neurotoxins, aflatoxins, zearalenone, fumonisins, and ochratoxins which cause many physiologic defects [7] In an attempt to get this solved, several researches have been conducted on the use of food grade organisms, bacteria from fermented food [8], gut bacteria [9] and recently, different types of fungi [10,11] Edible and medicinal fungi have had different applications and they are economically efficient and so can be used extensively on industrial scale as opposed to intestinal bacteria which are less

productive and require expensive media Three species of fungi Aspergillus niger,

Aspergillus oryzae, and Trichoderma reesei extensively used in industries were found

to produce mycotoxins [12] This explains the necessity of carefully finding species of fungi especially mushrooms that are safe for use in food and medicine industry with the potential to convert major ginsenosides into minor ginsenosides; one of the least researched area

2.2.2 Ginsenosides as antimicrobial agents

Ginsenosides were found to inhibit growth and exhibit toxicity to many human pathogenic microorganisms including both yeasts and bacteria Antimicrobial activity

of ginsenosides seems to depend on their polarity as for example polar ginsenosides from white ginseng did not show any antimicrobial activity whereas the heat treated less polar ginsenosides was shown to exhibit activity against bacterial species such as

Fusobacterium nucleatum, Clostridium perfringens, and Porphyromonas gingivalis,

and the less polar ginsenosides from P notoginseng in Rk3, Rh4, Rh5 obtained

through heating polar ginsenosides ;notoginsenoside-R1, ginsenoside-Rg1, -Re, -Rb2,

-Rd exhibited inhibitory activities against fungal species including as Epidermophyton

floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes [56] Apart from

antibacterial and antifungal activities, extracts from ginseng roots have also shown activity against RNA viruses both in viral cultures and in animal models Antimicrobial activities of ginsenosides are dependent on both bacteriostatic and bactericidal abilities of these compounds which include disruption of biofilms,

inhibition of quorum sensing and virulence factors and motility alteration [57]

2.2.3 Anti-cancer and immune-modulatory effects of ginsenosides

Anti-carcinogenic effects of ginsenosides were found both in vitro and in vivo

and are exerted through various mechanisms, including direct cytotoxicity, growth suppression, induction of differentiation and inhibition of metastasis These activities are evidenced for example by ginsenoside Rh2 which was shown to inhibit human breast cancer cell growth by causing cell cycle arrest at G1 phase [58] and several

other types of cancers including Leukemia, prostate cancer and colon cancer [59] In

vivo studies in human and mice have shown that ginsenoside Rh2 administered orally

or subcutaneously injected were able to inhibit human ovarian cancer cell growth and improved overall survival time also administration of ginsenoside Rg3 successfully inhibited tumor progression in lung cancer and colorectal cancer by suppressing the activity of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) activity [60] Ginsenoside Rb1 the known angiogenesis inhibitor both in vivo and in vitro, its mechanism was associated with regulation of pigment epithelium-derived factor via the oestrogen beta receptor [61] Angiogenesis and metastasis inhibition was also found with ginsenosides Rg3 and Rb2 both in vivo and in vitro and they are important anti-cancer properties [62] Rb1 and its intestinal microbial metabolite compound K were shown to be cytotoxic to cancer stem cells or tumour initiating cells and improved sensitization of some otherwise resistant cancerous cells to chemotherapy especially ovarian cancer stem cells and inhibited cancer recurrence with no toxicity to normal cells [63]

Immune-modulation effects of ginsenosides are at the base of curing many immune system function related diseases including autoimmune disorders especially due to their immune-suppression abilities and most of the time anti- cancer properties

of ginsenosides are not separated with their immune-modulatory activities and it is of a great relevance since immune-therapy is taken as the best and preferred method of cancer therapy because of the generally conceived idea that natural is always better Our bodies have the ability to fight against infections and other health threatening hazards From the hematopoietic stem cells, a number of different cells is created with

a specific role of protecting the body These cells come from lymphoid and myeloid progenitors and play their role as the first mode of action of the immune system called

“Cell mediated immunity” as opposed to “humoral immunity” which make use of chemicals especially immunoglobulins Cell mediated immunity is made up of macrophages, natural killer cells, neutrophils, T cells, dendritic cells, Eosinophiles, basophiles and mast cells together with the B cells which also pray an important role

in Humoral immunity [64] These cells work collectively using different mechanisms

of action which can be broadly categorized as phagocytosis, antigen presentation, release of chemicals and antigen memorization One of the potential and easiest type

of immunotherapy is based on immunomodulation Immune-modulators are chemical compounds having ability to either increase or suppress the immune response Ginsenosides were reported by many researchers to have this property which proved to

be of great significance in cancer and autoimmune diseases

2.2.3.1 Anti-cancer cell mediated immune system modulation by ginsenosides

Neutrophils modulation

In the case of cancer, as it is in the theory of tumor immune-surveillance, neutrophils are always found in tissues and blood When they come in contact with a mutated cell or a cell with a tumor antigen expressed on their surface, they either phagocytize it or destroy it by releasing reactive oxygen species (ROS) Recent investigations however have found that the role of neutrophils in cancer can be both pro-tumorigenic and anti-tumorigenic depending on the type of cancer and the studied model system When ginsenoside Re was investigated for its role in gastric mucosal lesion induced by compound 48/80; a chemical agent of mast cell degranulation which causes releases of histamine, an inflammatory mediator which also act as a signaling molecule to recruit other cells of the immune system including neutrophils, it was found to regulate neutrophils infiltration in inflammation site [65], which would otherwise aggravate inflammatory responses leading to tumorigenesis

Macrophages modulation

Other important immune cells in tumor micro-environment are macrophages These versatile cells come from monocytes and exhibit a range of different fates Following development, macrophages function is determined by activation stimuli it encounters Macrophage activation is was described by a dichotomous model where classical macrophage (M1) activation is induced by cytokines of type 1 such as IFN-γ, TNFα, molecules associated with pathogens such as lipoproteins, lipoteicoic acid etc and endogenous signals like extracellular matrix components and heat shock proteins

To these types of activating agents, macrophages respond by releasing 12 and

IL-23 in high amounts and IL-10 in low levels The presence of these substances promotes responses of Th1 cells which exert their cytotoxic activities by releasing ROS such as NO, hydrogen peroxide and superoxide along with cytokines such as TNF, IL-1, and IL6 which exert pro-inflammatory functions which if persist promote tumorigenesis [66]

M2 the alternative activation method is achieved by Th2 cytokines IL-4, IL-10,

TGF-, hormones, such as the glucocorticoids and vitamin D3, and apoptotic cells and results into macrophages capable of exerting pathological functions to tumor cells and other eukaryotic parasites [67] Evaluation of ginsenoside Rp1 effect in J774A One macrophage cell line stimulated by a lipopolysaccharide endotoxin, showed the interference of this ginsenoside in NO production NO induces DNA damage by suppressing the expression of genes H2AX, CHK1, and CHK2 whose expression products are pathophysiology-related

In addition, this ginsenoside prevented macrophage infiltration in CT26 colon cancer cells [68] Another investigation of the same type but with treatment of RAW264.7 macrophage cells with crude ginsenoside extract from Korean red ginseng landed to the same effect of

NO and IL-1 inhibition and suppression of different DNA damaging factors at the level of gene expression [69] However, treatment of the same cells (RAW264.7 macrophage) with

Panax ginseng root stimulated production of NO in a dose dependent manner [70]

Intra-tumoral macrophage derived inflammation is inhibited by orally administered ginsenoside

Re which inhibits the binding of lipopolysaccharide (LPS) on macrophage surface toll –like receptors which additionally prevent the NF-B activation [70] LPS stimulated macrophages produce different cytokines with inflammatory effects of potential threat to cells genetic material (TNF- , IL-6, and IL-1) Treatment of RAW264.7 macrophage with

notoginseng has resulted into cancer preventing events; inhibition of these cytokine production and attenuation of COX-2 and IL-1 mRNA expression [71] Rb1 and compound K also down-regulated the production of NO in the same cell line [72] Still in these cells, the flower extract of notoginseng has inhibited the LPS stimulated production of

NO, PGE2, TNF- and IL-1 and stopped expression of iNOS, COX-2, TNF- and IL-1genes [73] Ginsenoside Rb1 also regulate the phagocytic abilities and cytokine production

by macrophages and reduces production of NO This ginsenoside was also found to regulate the phagocytosis functions of non-stimulated macrophages for a better innate body protection against pathogens and tumorigenesis [74] Microglial cells; nervous system residing macrophages when activated can produce signaling molecules up-streaming the brain inflammation Ginseng extract, total saponin from ginseng and individual ginsenosides Rh2, Rh3 and compound K were tested on their influence on microglial activation Both crude extract and total saponins repressed the expression of iNOS, MMP-9 stimulated by LPS, production of pro-inflammatory cytokines and suppression of NF-B and MAP kinase activities and individual ginsenosides both significantly reduced the expression of LPS-induced iNOS and cytokines [75] In another study which evaluated the effect of ginsenoside Rg5 in microglia cells, it suppressed LPS induced NO production, secretion of TNF- and exhibited inhibitory activities on expression of iNOS, TNF-, IL-

up-1, COX-2 and MMP-9 induced by LPS and binding abilities of NF-B and AP-1 on DNA [76] Ginsenoside Re inhibits activation of NF- - and IL-1in LPS stimulated murine macrophages [77]

Modulation of dendritic cells and T cells

The tendency of double antagonistic actions was also observed in dendritic cells At the positive end, these cells are potentially equipped with the ability to

initiate the anti-tumor response With MHC complex molecules, they present both

endogenous and exogenous antigens to T cells priming both nạve and memory T cells during Tumor development [78] Interaction of ginsenoside Rg1 and dendritic cells was observed to result in the dose dependent production of IFN -interleukins; IL-6, IL-1,TNF- and chemokines such as IL-8 and IP-10 in murine model lymphoma [79] Rg3 induced an immunogenic apoptosis of tumor cells and production of IFN-an important signaling molecule for anti-tumor

activities of T cells, and suppressed the pro-angiogenic activities of TNF-and immunosuppressive activities of TGF-by blocking its secretion [80] Rg3 when enhanced, it influenced Th1 differentiation by inhibiting DCs production of IL -12 and at the same time suppressing the secretion of IFN- by T cells [81] Ginsenoside Rd induces differentiation of regulatory T cells by up-regulating Foxp3 expression and it increases the generation of TGF-1, IL-10 and IL-35 [82] Ginsenosides Rg1 and Rh1 contribute their anti-tumor activity by stimulating dendritic cells to promote T cell proliferation [83] , In cancer bearing mice ginsenosides were seen to activate DCs and promote anti -cancer immune

response [84]

Natural killer cells (NK) modulation

Natural killer cells are effector lymphocytes of the innate immune system and are

of great significance in inhibiting tumor growth and metastasis Its efficiency is said to come from its ability to directly kill tumor cells without prior education as it is in T cells, and recognition of tumors with human leukocyte antigen (HLA) on the surface

of tumors which manage to evade T cell killing It produces a number of stimulatory cytokines for adaptive immunity sensitization including IFN- which eliminate cancer cells by preventing cell proliferation and angiogenesis leading to apoptosis [85] NK cells can also kill tumor cells collaborating with humoral immunity

immune-by antibody dependent cellular cytotoxicity; a mechanism which involve binding of antibodies by FC fragments to Fc receptors, FcRIII (CD16) on NK cells inducing a series of events which include the release of cytokines such as IFN- and cytotoxic granzyme-containing granules, the mechanism supported by Cartron and the colleagues who related the silencing of FcRIII encoding gene with reduced response

in patients with follicular non-Hodgkin‟s lymphoma [86] Cytotoxicity of NK cells was reported to be increased by the effect of Protopanaxadiol ginsenosides A different study reported an increase by 44-150% killing activity of NK cells induced by aqueous

extract of Panax ginseng C.A Meyer [87] Astonishingly, Panax ginseng was able to

restore the activity of NK cells against YAC-1tumor cells in immune- compromised mice [88] In invivo tests, NK cells have shown the increased activity in individuals treated with ginseng extracts [89]

Modulation of Mast cells

Mast cells(MCs) stand between innate and adaptive immunity and are associated with anti-tumor effects and positive influence on other cells both of innate and adaptive immune system all for the same target; fighting cancer The mediators produced by these cells namely TNF-, IL-1, IL-6 and IFN- have the ability to suppress tumor metastasis and chemokines such as CCL5 attract effector T cells contributing against the tumor growth However a lot of evidences relate these cells with tumorigenesis and tumor development [90] Mast cells were seen to interact with different immunosuppressive cells especially myeloid-derived suppressor cells (MDSC) in tumor micro-environment resulting in changes in development and switching from anti to pro-inflammatory roles [70,71] Mast cells treated with Re show reduced production of histamines and cytokines IL-1, IL-8, IL-10 [93] Ginsenoside Rg3 regulates the mitogen-activated protein kinases/nuclear factor-kappa B (MAPK/NF- and receptor-interacting protein kinase 2 (RIP2)/caspase-1 signaling pathway in mast cells and thus suppress the production of pro-inflammatory cytokines

in tumor micro-environment, it also modulate levels of cAMP and calcium influx which result in decreased release of histamine [94]

Modulatory effects of ginsenosides on inflammations

Inflammation is the most powerful defense weapon of the bodies of vertebrates

It is designed to help the body get rid of parasites, deformed or injured cells and chemical irritants However it came to scientific knowledge that inflammation has its dark side which contributes to the development of the tumor, helping in its growth and metastasis Cells of the innate immunity respond to infections and damaged or mutated cells by mounting inflammatory responses These responses, if not stopped are thought

to contribute to genetic errors which lead to formation of abnormal cancer-ancestor cells, kick-starting the tumor genesis If the tumor genesis is through another process other than this, the growing tumor still has another mechanism of creating inflammatory responses in favor of its growth and metastasis [95] In response to inflammatory mediators, neutrophils are directed to the site of inflammation and add more power to inflammation, in an attempt to clear off the pathogens by producing reactive oxygen species and phagocytically engulfing them and sending signals to

other immune cells; with the inflammatory progress, as more cytokines, chemokines and growth factors are highly released, other effector cells such as macrophages and lymphocytes also come into play [96] When this inflammation persists, it has been shown to cause the genesis of different types of tumors including colorectal carcinoma which follows chronic inflammatory bowel disease (IBD), esophageal cancer which comes after reflux esophagitis [97], gastric cancer as a result of a series of

inflammatory responses to H pylori and hepato-carcinoma associated with hepatitis C

virus infection and colon cancer and bladder cancer associated to chronic and

persistent Schistosoma and Bacteroides infections respectively [98], and cancers

related to environmental factors such as tobacco smoking and asbestos, and obesity are all initiated by chronic infection [99] During the time of inflammation, cells on the site, generate a number of oxygen radicals namely •O− 2, NO, hydrogen peroxide, hydroxyl radical (∙OH), peroxynitrite (ONOO−), and hydrochlorous acid (HOCl∙) These radicals cause protein damage and interact with DNA causing a number of mutations such as point mutation, rearrangement or deletions which produce cells regarded as precursors of cancer and causing even more inflammation and stimulating oncogenes [100] In cancers intrinsically initiated, oncogenic activation initiate expression of different inflammatory events leading to the buildup of the inflammatory environment [101] The tumor interact constantly with the surrounding environment and exert its influence by releasing extracellular signals which promote angiogenesis and induce tumor to avoid an attack of immune responses The signals released by a growing tumor which include cytokines, chemokines and hydrogen peroxide, recruit neutrophils in the micro-environment Once in the tumor micro-environment, neutrophils start empting their inflammatory products which include reactive oxygen species and granular content which rather play a role in cancer cell proliferation, immune suppression, angiogenesis and recruitment of more immune cells [102]

Inflammation is a major player in cancer and play in all the stages of cancer development In both intrinsic and extrinsic ways of collaborating between inflammation and cancer, the result is activation of transcription factors as can be exemplified by NF-B, STAT-3, and HIF-1 and accumulation of tumorigenic factors

in the tumor micro-environment [103] The key inflammatory mediators involved in modulating the expression of these factors are IL-1, IL-6, TNF, and PGHS-2, the role which makes them important in cancer development When stimulated, NF- B, STAT-3 interact with each other at many different levels of cancer development scaling up tumor associated inflammation which help in suppressing immune response against the tumor They involve in activation and recruitment of other effector cell populations such as macrophages, mast cells, eosinophils, and neutrophils

Accumulation of inflammation causing antigens activate transcription of important malignancy factors as for example pro-angiogenic factors (IL-8, VEGF), growth factors (IL6, GM-CSF), anti-apoptotic factors (Bcl-XL, c-FLIP), invasion promoting factors (MMP-2, MMP-7, MMP-9, uPA), inflammatory enzymes (PGHS-2, LOX), prostaglandins, iNOS, chemokines (CCL2,CCL20, IL-8), and pro-inflammatory cytokines (IL-1, IL-6, IL-23, TNF, TGF-, EGF) under regulation of NF-B iNOS in its role of stimulating macrophages to produce NO acts also as a link of inflammation

to tumorigenesis as it was shown that NO is a potential DNA damaging agent and through its transactivation with HIF-1 cause the down-regulation of tumor suppressor protein p53 and induction of pro-angiogenic VEGF [104]

Many researchers have reported that ginsenosides have a role to play into all this cascade of events and exert a good effect on growth of cancer cells In a series of cancer preventing effects of ginseng studies by both experimental models and epidemiological trials, the results suggested the inhibitory effects in some aspects of carcinogenesis, for instance, cell proliferation and apoptosis, immune-surveillance, and angiogenesis, and in different types of cancer which include stomach, liver, pancreas, colon and pharynx cancers [105] Red ginseng extract was shown to be associated with regression of transcriptional expression of NF-B resulting in reduced expression of IL-8 gene thus protecting cell damage that would result in tumorigenesis [106] The

toxicity caused by NO in H pylori infected gastric epithelial walls was prevented by

inhibition of red ginseng extract by suppressing the activation of NADPH oxidase and Jak2/Stat3 which inhibited the expression of MCP-1 and iNOS Red ginseng extract suppressed the activity of NADPH oxidase by blocking the translocation of p67phox and p47phox from cytosol to the cell membrane, hence reducing levels of ROS [106]

Gastric H pylori inflammation related oncogenicity is mainly a result of infiltration of

phagocytes; neutrophils and macrophages which produce large amounts of ROS These radicals activate the transcription factor NF-B which in return activates the expression of cell cycle regulators, inflammatory mediator genes and oncogenes Supplementing food containing red ginseng extract to Mongolian gerbils for 6 weeks inhibited phagocytes infiltration significantly and expression of inflammatory mediators namely iNOS, KC, and IL-1 induced by H Pylori infection through

moderating the expression of NF-B [106] Endothelial progenitor cells promote angiogenesis by producing angiogenesis promoting cytokines in early tumors and therefore contributing to tumor growth These activities were shown to be inhibited by ginsenoside Rg3 as discussed in murine model experiments [107] The rare ginsenoside R6 obtainable only in black ginseng was found to play huge anti-inflammatory roles including among others, the inhibition of neutrophils infiltration, inducing infiltrated macrophages expression of MiR-146a, anti-inflammatory micro-RNA thereby reducing chances of DNA damage, reduction of NF-B activation which would otherwise induce production of pro- inflammatory cytokines such as TNF-, interleukin-6 IL-6, and IL-1 [108] A mountain grown ginseng increased the level of macrophage cytotoxicity and induced TNF- independent apoptosis [109] In inflammation-to-cancer sequence, many other mediators are important in promoting different activities, ginseng was shown to inhibit some of the pathways leading to the production of these mediators As an example when Matrix metalloproteinase-9(MMP-9), a major contributor in invasion and angiogenesis in brain tumor because of its ability to degrade extracellular matrix, when activated by phorbol esther in human astriglioma cells, ginseng metabolite compound K was found to inhibit its expression

by inhibiting activator protein-1(AP-1) and MAPK signaling Pathways [110] Further investigation with Rh2 also was found to repress the MMP-9 expression blocking the transcription of genes mmp-1, 3, 9 and 14 It also repressed DNA binding and transcription of NF-B and AP-1 and phosphorylation of MAP kinases in human

glioma cells [111] Compound k of P.ginseng meyer induces ROS dependent cell death in neuroblastoma cells both in vitro and in vivo [112] and in colon cancer cells

[113] The same effect was reported for Rh4 in colorectal cancer cells [114] Ginsenoside Rh1 suppresses the production of ROS, NO and TNF- in microglia

stimulated with IFN-[115] Ginseng radix rubra extract has a transforming role in

TGF- a growth factor with a variety of functions including the extracellular matrix synthesis, cell growth inhibition, cell migration and differentiation and immune-suppression and inhibit or promote inflammation-to-cancer sequence of events [116] 20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol (M1) an intestine metabolite of ginseng stops tumor metastasis through its down-regulating influence on NF-B signaling in colon 26 cancer cells [117] Ginsenosides Rb1 and its metabolite are through their anti-inflammatory activities tumor initiation preventing agents They inhibited NF-B activation and expression of IL1b, TNF-, and IL-6 and promoted that of IL-10, inhibited protein expression of COX-2 and iNOS in TNBS-induced colitis mice [118] The same effects were found with oral administration of Rg1 and its metabolites Rh1, F1 and 20(S)-protopanaxatriol [119] Rg3 inhibited the proliferation

in 3 human colorectal cancer cell lines in vitro and in a xenograft of the same cells in a

mouse through inhibition of transactivation of C/EBP and NF-B, and nuclear interaction between f C/EBP with p65-NFB [60] In bladder cancer, tumor cells are killed by ROS obtained from p38 MAPK Pathway [120] Oral administration of ginsenoside Rp1 reduces metastasis significantly in lung cancer

2.2 Mushroom beta glucosidase

2.2.1 Overview

glucosidases are basically enzymes which catalyze the hydrolysis of glucosidic linkages in aryl-, amino-, or alkyl-β-D-glycosides, cyanogenic glycosides, and oligo- or disaccharides subsequently releasing β-D-glucose [121] They are ubiquitous enzymes as they are found in bacteria, plants animals and fungi They are a part of cellulose enzyme complex and work synergistically with endoglucanases and exoglucanases to completely hydrolyze cellulose to glucose where endo-𝛽-1,4-glucanases cleave the amorphous regions of the cellulose chain; exoglucanases or cellobiohydrolases the non-reducing or reducing chain ends to leave cellobiose for β glucosidase hydrolysis [122] Their specificity vary with different substrates and has shown dependency on the enzyme source [123] β glucosidases are of practical importance in different industries including feed where they break down fiber-rich cell walls to release reducing sugar that can be digested and absorbed by livestock and