Chapter – 1 

INTRODUCTION 

Introduction

1

1.1 Tobacco - Origin and History

Among the crops classified as stimulants, Tobacco is by far the most

important commercial crop grown in many countries of the world. Apart from the

major food-producing plants, cotton and tobacco are the largest cultivated plants

all over the world. Tobacco has been used by the Red Indians for medicinal and

ceremonial purposes. The English word “Tobacco” takes its origin from the Red

Indian word “tobaco” (Spanish) which was the name of the pipe used by the Red

Indians for smoking. The people of the whole world came to know about tobacco

and its products only after Columbus and his party landed on the shores of the

islands of Tobag, now known as Cuba, in the course of American voyage in

1492. It was he, who after observing the narcotic values of tobacco, introduced it

into Europe. Tobacco is purely, an American plant, dates back to as early as 400

A.D. (Massie, 1981). The genus Nicotiana, named in the memory of the French

diplomat Jean Nicot who introduced the usage of tobacco in France, is one of five

genera of five large genera of solanaceae and it is represented by sixty six

presently recognized species (Durbin, 1979). The extension of tobacco culture to

practically all parts of the world began with the introduction in Europe, especially

France in 1556, Portugal in 1558, Spain in 1559 and England in 1565. However,

cultivation of tobacco was introduced in India by British in Botanical Gardens of

Calcutta for export purpose. Virginia tobacco was first introduced in India by

Indian Leaf Tobacco Company in 1920.

In spite of its adversaries, the use of tobacco and its products is still

associated with peace, relaxation, contentment and employment to millions of

people through cultivation, trade and industry as well as ancillary industries and

transport. Tobacco is consumed in one form or the other all over the world and as

such, it is a rich man’s solace a poor man’s comfort and a lonely man’s

companion (Gopalachari, 1984).

Introduction

2

1.1.1 Importance

Tobacco is an annual, herbaceous plant belonging to the family

Solanaceae and genus, Nicotiana which was established by Linnaeus in 1753. It

is the native of tropical North America. Tobacco was introduced in India by the

Portuguese in the beginning of 17th

century A.D. during Akbar’s regime. Today,

the crop is grown in USA, China, Brazil, Zimbabwe, India, Malawi, Turkey, and

Japan and in several other countries providing livelihood to millions of people

and influencing the economy of several nations.

Tobacco growth is restricted, by environmental factors, to about the

latitudes of 60 degrees north to 45 degrees south, with the majority of the tobacco

entering the world trade produced in the latitudes between 45 degrees north and

30 degrees south. Limits to its growth are figured by the number of frost free

days. Almost all continents are capable of growing tobacco but the United States,

China, India and Brazil are the leading countries growing tobacco. Although,

there are 66 species in genus Nicotiana, only two species i.e. N.tabacum and

N.rustica are widely cultivated throughout the world, among these two species,

N.rustica requires cooler climate and occupies a small area while N.tabacum

occupies larger area. The progenitor species of N.tabacum were shown as

N.sylvestris and N.tomentosiformis (Gray et al., 1974). Description is available

for all the 60 species which are grouped in 3 sub-groups.

1. Sub-genus - Rustica

2. Sub-genus - Tabacum

3. Sub-genus - Petuniodes

Introduction

3

1.1.1: Photograph of typical tobacco plants

1.1.2 Tobacco Situation in India

India is the second largest producer of tobacco in the world with China

and Brazil occupying the first and second places, respectively. The total area

under all types of tobaccos is around 4 lakh hectares which accounts for ~ 0.23%

of total arable land and the total annual production is around 750 million kg.

Flue-cured Virginia (FCV) tobacco, the exportable type is cultivated in about 1.5

lakh hectares with production of around 300 million kg.

About 50% of the produce is exported and India stands at 2nd position in

tobacco exports. Tobacco earns about Rs.4000 crores as foreign exchange and

Rs.14000 crores in the form of central excise to the national exchequer. Tobacco

is an important commercial crop in view of revenue generation, export earnings

and employment potential. Its cultivation is mainly concentrated in Andhra

Pradesh, Karnataka, Gujarat, Tamil Nadu, Orissa, West Bengal, Bihar and Uttar

Pradesh. Different types of tobacco, flue-cured, Bidi, Burley, Chewing, Hookah,

HDBRG, Cigar-wrapper, Cigar-filler, Lanka, Cheroot and Natu used in the

Introduction

4

manufacture of different tobacco products, are being produced in varied agro-

climatic conditions in India. The production pattern in India is oriented towards

non-cigarette types of tobacco, constituting nearly 65%. Tobacco offers

significant employment opportunities both at on-farm and off-farm situations

apart from providing raw material to the industry. Conventionally tobacco is used

for the manufacture of cigarettes, bidis, cigars, cheroots, hookah paste, snuff and

chewing products (Gutka, Zarda and Quiwam). Up till now, the main thrust of

tobacco scientists has been to identify the factors conducive to the production of

best quality tobacco with concomitant yield improvement. However, a threat is

looming large over the tobacco scene in recent years due to health risk factors

associated with tobacco consumption in different forms. There is a growing

awareness and growing concern about the likely adverse effect of the health risk

factors on the future of this commercial crop of great economic importance.

Tobacco is a polymorphic crop and at least ten types of tobacco are

cultivated all over India. At present, India produces 2 types of tobacco in which

all tobaccos can be broadly classified into two major types (Gopalachari, 1984):1.

Flue-Cured Virginia (FCV) and 2. Non-Flue Cured Virginia (Non-FCV).

FCV tobacco is to most important type of tobacco produced in India.

Currently it is produced both in light and black soils in the country. FCV tobacco

grown in the light sols of the country is preferred not only in domestic market but

also for exports. It is used in the manufacture of cigarettes. The area under FCV

tobacco production in light soils is divided into three major zones: 1. Northern

Light Soils (NLS) of Andhra Pradesh in East and West Godavari and Khammam

districts; 2. Southern Light Soils (SLS) of Andhra Pradesh in Prakasam, and

Nellore districts and 3. Karnataka Light Soils (KLS) of Karnataka in Shimoga,

Hassan, Hunsur districts.

Introduction

5

Fig. 1.1.2: Tobacco Production in India

The tobaccos other than FCV are called Non-FCV tobacco (Table 1.1.2).

They are mainly grown in Andhra Pradesh, Karnataka, Gujarat, West Bengal,

Bihar, Uttar Pradesh, Tamil Nadu, Orissa and Maharastra. Non-FCV tobaccos are

used for Bidi, Cigar, Cheroot and Hookah paste and for manufacturing Chewing

tobacco products. However, Burley, HDBRG and Natu tobacco are used in

blending with FCV tobacco in the manufacture of cigarettes.

Table 1.1.2: Types of Non-FCV Tobacco

Types of non-FCV Tobacco States

Natu Andhra Pradesh and Orissa

Bidi Gujarat, Andhra Pradesh, Karnataka

Wrapper West Bengal Bihar and Uttar Pradesh

Hookah and Chewing Tamil Nadu, Gujarat, Bihar, Uttar

Pradesh and West Bengal

Cigar Tamil Nadu and Uttar Pradesh

Burley Andhra Pradesh and Karnataka

Cheroot Orissa, Tamil Nadu and Andhra Pradesh

HDBRG Andhra Pradesh

Oriental Andhra Pradesh

Introduction

6

1.2 Alternative Uses of Tobacco

Investigations on alternative uses of tobacco are attaining prominence all

over the world in view of the alleged health risk associated with tobacco

consumption. Estimates are that the total number of chemical constituents in leaf

exceeds 4000 and there are over 6000 in tobacco smoke (Leffingwell, 2001). A

wide range of chemicals like alkaloids, carbohydrates, nitrogenous compounds,

polyphenols, inorganic constituents, structural constituents, non-volatile acids,

fatty acids, sterols and terpenes etc., are present in tobacco.

Fortunately, tobacco contains several phytochemicals of pharmaceutical

and industrial importance. Hence, in order to sustain tobacco cultivation, the

concept of growing tobacco for alternative uses is being seriously contemplated.

According to G.Robert Di Macro “The tobacco plant, often called the white rat of

the in exhaustive plant world because its genetics has been studied for so long,

offers untold opportunities to produce valuable products, the world is looking

for”.

Tobacco is a complex plant material from a chemical composition

standpoint. No other plant material has been studied more extensively in the

history of man. Yet even today, the quality of tobacco is judged largely by

empirical experience and subjective sensorial evaluation. We are still unable to

replicate the smoking properties of the tobacco leaf synthetically. Yet, through

chemical knowledge and genetic advances, we now have a much better

understanding of tobacco growth and important constituents that allows us to

make improvements in quality using the best laboratory in the world - through

optimization of the biology of tobacco itself in the tobacco growing field.

Although tobacco consumption is controversial and considered to be a

major health concern, it has been a fruitful field for natural product chemists

because of the insight it has given into the formation of flavor ingredients found

Introduction

7

in many common foodstuffs and the fragrance materials found in flowers and

other botanicals. Research work at various institutions indicated that among the

major chemical compounds present in tobacco, solanesol, a tri-sesqui-terpenoid

alcohol; nicotine - an alkaloid; organic acids – malic & citric and crude protein

containing fraction–I protein have the potential for chemurgical development.

There is also a possibility of extracting oil from seed and utilizing the stalk for

card-board making and furfural production. Generally, the presence of active

compounds in very small quantities takes comparatively longer time for their

development as therapeutic agents. However, tobacco contains solanesol and

nicotine in appreciable quantities and is, therefore, an attractive starting material

for developing value-added products which will be of Intellectual Property Rights

(IPR) value. Apart from the utilization of tobacco waste, the possibility of

growing tobacco for recovery of phytochemicals has excellent prospects. Thus,

extraction of solanesol and nicotine from tobacco is a very promising proposition

in the area of alternative uses of tobacco.

1.2.1 Value - Added Products from Tobacco

Narasimha Rao and Krishnamurthy (2007) gave a comprehensive account

of different value-added products from tobacco. Nicotine, solanesol and organic

acids (malic and citric) have been identified as potential chemicals which can be

converted to value-added products. Nicotine is the source for the botanical

pesticide, nicotine sulphate. Recent scientific evidence suggests that nicotine and

nicotine-like compounds may slow the progression of, or ameliorate the

symptoms of, certain diseases like, Tourette’s syndrome, Alzheimer’s,

Parkinson’s disease, Ulcerative Colitis and Attention Deficit Disorder (ADD).

Solanesol is the starting material for many high-value biochemicals like vitamin-

K analogues, vitamin-E and coenzyme Q9 (Colowick et al., 1975). There are

several reports in the literature indicating the utilization of solanesol and other

polyprenyl alcohols in the preparation of anti-ulcer compounds. Preparation of

(22)-solanesol as anti-hypertensive, anti-hyperlipidemic and anti-tumor agent has

Introduction

8

also been reported. Japanese scientists have used a solanesol derivative, N-

solanesyl - N, N1-bis (3, 4- di methoxy benzyl) ethylene diamine for potentiation

of anti-tumor drugs against multi-drug resistant and sensitive cells. In pure form,

malic acid and citric acid find use in food and pharmaceutical industries and in

crude form they can be used for solubilisation of rock phosphate to release the

phosphorus for plants. Apart from these chemicals, there are other possibilities

such as edible protein recovery from green leaf, oil from seed, rutin from the

cured leaf and furfural from the stalk. Thus, almost all parts of the tobacco plant

- seed to stalk – can be harnessed to extract value-added chemicals. Fraction I

protein is the most abundant protein in tobacco and accounts for 50% soluble

protein containing essential amino acids. About 35% of oil is present in tobacco

seed and this can find use in paint industry. Refined tobacco seed oil, having

similar physical and chemical properties to edible oils, is being used for edible

purpose in Turkey and Tunisia. The residual tobacco can be used for making

sheet to be recycled into cigarette manufacture or in combination with certain

non-edible de-oiled cakes can be utilized for making organic manure.

At present, the country is exporting 40% nicotine sulphate and crude

solanesol (10 - 15%) to countries like Japan, UK etc. As the export market is not

steady, emphasis is also laid on the indigenous utilization of these chemicals.

With the advent biotechnology, new vistas are opened for utilization of

tobacco crop for production of bioengineered products like functional food

proteins and highly desirable enzymes, flavors and pharmaceuticals. Thus,

prospects of growing tobacco for alternative uses seem to be encouraging.

However, to exploit the immense economic potential, evolving proper marketing

systems for all the phytochemicals or their value-added products is imperative.

There is also every need to take up research on finding new uses for these

chemicals and undertake pilot-plant studies so that the bench-scale technologies

can be perfected for effective transfer of technology. The product isolation

scheme is given in Figure 1.2.1.

Introduction

9

All over the world, there is growing concern about the likely adverse

effects of tobacco consumption on health and hence, the future of this

commercial crop of great economic importance is causing anxiety. Keeping this

in view, working out a strategy for the tobacco crop for alternative uses attained

importance. In addition to the internal market, the global market for the

phytochemicals in tobacco like nicotine and solanesol must be explored. In view

of the significant potential of these compounds, identification of tobacco rich in

solanesol and development of a novel process for recovery of solanesol has

significantly enhanced the scope for alternative uses of tobacco. The proper

exploitation of solanesol is only possible when basic requirement of natural

product exploitations are met. Hence, the apparent need to discover optimum and

economic process for obtaining solanesol, its purification, chemical assay and

knowledge of chemical reactivity, and to find out how solanesol is influenced by

genetics, agricultural practices, soil type and nutrients, weather conditions, plant

diseases, stalk position, harvesting and curing procedures are prerequisites.

Fig. 1.2.1: Product isolation scheme from tobacco

Introduction

10

1.2.2 Nicotine

The chemical structures of nicotine and some other alkaloids found in

tobacco are shown in Fig.1.2.2. India is currently exporting nicotine derivatives,

nicotine salts and nicotine sulphate mainly to Japan, Europe, Russia, Canada and

China.

Fig. 1.2.2: Important chemicals present in tobacco plant

1.2.3 Solanesol

Solanesol mainly exists in plants of solanaceae family, especially in

tobacco leaves. Solanesol, the long- chain terpenoid alcohol is the starting

material for many high-value bio-chemicals, including Coenzyme Q9 (CoQ9),

Coenzyme Q10 (CoQ10) and vitamin K analogues (Colowick and Kaplan, 1975).

Solanesol itself can be used as a cardiac stimulant, lipid anti-oxidant and

antibiotic. Clinical trials are also going on for the usage of solanesol as anti-

Introduction

11

cancer drug. Studies indicate that by introducing solanesol radical into the

structure of some medicines, the effects increase noticeably. Solanesol

derivatives can be further developed as wound healing agents (Srivastava et al.,

2009). With solanesol as its primary material, CoQ10 is used in cosmetics and in

the treatment of heart diseases, cancer and ulcers.

CoQ10 is a physiologically active substance with high pharmaceutical

activity against cardiac insufficiency, muscular dystrophy and anaemia. Thus,

solanesol has immense potential in the pharmaceutical industry, as evidenced by

the voluminous patent literature.

1.2.3.1 Isolation & Characterization of solanesol from Nicotiana tabacum L.

The faith and popularity in the use of herbal medicine is growing

continuously worldwide. High activity profile drugs are developed by extraction

and characterization of active phyto-chemicals from medicinal plants. For

example vincristine, vinblastine and taxol are some of the drugs extracted from

herbs (Huie, 2002). Tobacco is also one such plant which contains many

alkoloids, fatty acids, sugars and the best known of which is nicotine (NIH

Publication, 1993). The medicinal uses of tobacco as anti - diarrhoeal, narcotic,

pain reliever, healing wounds and burns were well reported (Dickson et al.,

1954). Nicotine analogs are beneficial on Alzheimer’s disease, Parkinson’s

disease, chronic pain, obesity, and depression. Recently it was found that nicotine

hydrogen tartrate reduces Parkinson’s disease. The patients of post-encephalitic

parkinsonism were treated with subcutaneous injections of nicotine for immediate

improvement in muscular movement (Moll, 1926). In the treatment of scabies

nicotine salicylate was used (Silvette et al., 1958). The tobacco leaves and juices

were much used for skin disorders including basal cell cancer. The pyridine

content of the tobacco smoke destroys the comma bacillus of cholera or germs

responsible for diphtheria and typhus (Charlton, 2004). The products developed

of tobacco include tobacco leaf proteins, tobacco seed oil, phytochemicals and

pharmaceuticals.

Introduction

12

1.3 Coenzyme Q10 (Ubiquinone)

Coenzyme Q10 (also known as ubiquinone, ubidecarenone, coenzyme Q

and abbreviated at times to CoQ10 – pronounced like "ko-cue-ten" , CoQ, Q10, or

simply Q is a 1,4-benzoquinone (Ernster et al., 1995), where Q refers to the

quinone chemical group and 10 refers to the isoprenyl chemical subunits. This

oil-soluble vitamin-like substance is present in most eukaryotic cells, primarily in

the mitochondria (Dutton et al., 2000). It is a component of the electron transport

chain and participates in aerobic cellular respiration, generating energy in the

form of ATP. Ninety-five percent of the human body’s energy is generated this

way. Therefore, those organs with the highest energy requirements such as the

heart and the liver have the highest CoQ10 concentrations (Okamoto et al., 1989;

Aberg et al., 1992).

Fig. 1.3: Chemical structure of CoQ10: 2,3-dimethoxy-5 methyl-6-decaprenyl benzoquinone.

Coenzyme Q10 (CoQ10) is an essential vitamin-like nutrient for cell

respiration and electron transfer to control the production of energy in the cells of

heart (Palamakula et al., 2004; Kang et al., 2004). It is present in most human

cells except red blood and eye lens cells. It is responsible for the production of

the body’s own energy. In each human cell, food is converted into energy in the

mitochondria with the aid of CoQ10. Ninety-five percent of all the human body’s

energy requirements (Adenosine triphosphate (ATP)) is converted with the aid of

CoQ10 (Ernster et al., 1995; Dutton et al., 2000). Therefore, those organs with

the highest energy requirements such as the heart and the liver have the highest

CoQ10 concentrations (Okamoto, 1989; Aberg et al., 1992; Shindo et al., 1994).

CoQ10 acts as a powerful antioxidant and membrane stabilizer in

preventing cellular damage resulting from normal metabolic processes. It is

Introduction

13

naturally synthesized and occurs in all cells in the human body, but its rate of

production falls with age. It is found in food, especially meat, but in very small

amounts as thermal processing destroys it (Turunen et al., 2004). The use of

CoQ10 as a dietary, nutraceutical supplement has increased dramatically in the

last decade (Tarnopolsky et al., 2001; Hermann, 2002). It has potential preventive

and therapeutic effects in many diseases like cancer (Gaby, 1996; Roffe et al.,

2004), cardiovascular (Jones et al., 2004; Bhagavan et al., 2005) and

neurodegenerative disorders (Beal, 2002), acquired immunodeficiency syndrome

(AIDS) (Gaby, 1996) and Parkinson’s disease (Sharma et al., 2004; Muller et al.,

2003; Lieberman et al., 2005). It is also known to be an energy booster and

immune system enhancer (Folkers et al., 1985). Recently, the commercial

formulations containing CoQ10 have gained increasing popularity in health

management (Buettner et al., 2007).

1.3.1 Synthesis of Coenzyme (CoQ10)

The production of CoQ10 follows one of the three routes: extraction from

(i) animal tissues (Ouyang et al., 1994), (ii) fermentation of microorganisms

(Yuan et al., 2004; Sasaki et al., 2005), (iii) chemical synthesis (West et al.,

2004). Recently High-speed counter-current chromatography (HSCCC) in the

purification of CoQ10 from a fermentation broth extract was reported (Cao et al.,

2006). Non-aqueous two-phase solvent system composed of heptane–

acetonitrile–dichloromethane (12:7:3.5, v/v/v) was used for purification of CoQ10

from 500 mg of crude extract and the results were compared with those obtained

by a column chromatography and subsequent recrystallization. The method

yielded 130 mg of CoQ10 with HPLC purity of > 99%. The isolation involved

CH2Cl2 as one of the solvents of mobile phase, which is toxic and expensive. The

fermentation followed by purification makes the process lengthy and costly. The

chemical synthesis was widely used from cheapest source of solanesol as a

starting material.

Introduction

14

The synthesis of CoQ10 (V) involves 4 key stages. In first stage, solanesol

(II) was isolated from tobacco dust (Narasimha Rao et al., 1979). In the second

stage solanesol (II) was reacted with phosphorus tribromide in the presence of

pyridine to give solanesol bromide. Further, it was treated with ethyl acetoacetate

to give solanesyl acetone (III). It was treated with a Grignard reagent, vinyl

magnesium bromide in presence of NH4Cl and THF to give isodecaprenol (IV).

The equimolar portion of (IV) and 2,3 dimethoxy 5-methyl hydroquinone (I)

were stirred at 430C for 10 minutes in hexane. A 2.5% sodium sulfate solution

was added and the hexane layer was separated, dried over magnesium sulfate,

concentrated and chromatographed on silica gel (hexane/ether 10:1) to give

ubiquinone (V) as a yellow solid.

The chemical structures of CoQ10 and its related substances are shown in

Fig.1.3.1.

H

MeMe

OH8

H

Me Me Me

O8

H

Me Me Me

8OH

H3CO

H3CO

O

O

H9

(II)

(III)

(V)

(IV)

H3CO

H3CO

O

O

(I) HexaneNa2SO4

NH4Cl/THF

N

O

Br

Mg

O O

Fig. 1.3.1: Chemical structures of CoQ (V) and its related substances

(I) 2,3-Dimethoxy-5-methyl-p-benzoquinone (II) Solanesol

(III) Solanesyl acetone and (IV) Isodecaprenol.

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15

1.4 Determination of Ubiquinone and its Process Related Impurities

by High Performance Liquid Chromatography

Quinones and hydroquinones such as ubiquinones, plastoquinones,

phylloquinones (vitamin K1), and menaquinones (vitamin K2), are widely

distributed in plant and animal tissues. In addition to the vital role of promoting

electron transfer in respiratory chains and photosynthesis, these compounds

exhibit various pharmacological activities (Mamura et al., 1977a; 1980b; 1981c;

1983d). Recently, Ubiquinone 10 (Coenzyme Q10), which is used clinically as a

cardiovascular agent has attracted the attention of organic chemists due to a major

synthetic challenge (Lipshutz et al., 2005).

Table 1.4: Chemical structures, activity and adverse effects of some of

the common quinones.