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INTRODUCTION - HEC

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									Chap.1: INTRODUCTION

                            INTRODUCTION

1.1. CHEMISTRY OF NATURAL PRODUCT: SCIENCE OF ALL
TIMES
       Natural products, as the term implies, are those chemical compounds derived
from living organisms and the study of natural products is the investigation of their
structure, formation, use and purpose in the organism. Drugs derived from natural
products are usually secondary metabolites and their derivatives. Today those must
be pure and highly characterized compounds. Since prehistoric times, the humans
have relied on natural products as a primary source of medicine. Plants and animals
were used to bring back the health of sick and frail. Plant were found to be beneficial
as food, fodder, medicine etc. but also harmful as being poisonous and toxic (Fuller
and Hemrick, 1985). The application of herbs for external and internal use has
always been a major factor in practice of medicine (Steiner, 1986). The experience
and knowledge gained in using the traditional medicines in different regions over the
millennia resulted in the complex science of modern medication.


The various approaches to drug discovery from nature are:
            Ethnobotanical: Ethnic and traditional medicine
            Random screening: Bioassay guided routes
            Chemotaxonomic: Screening of relatives




1.1.1. HISTORIC BOOKS ON HERBAL MEDICINES
       The use of drugs can be divided broadly into five periods. The early periods
covers the Indians, Chinese, Sumerian, Egyptian and Assyrian civilization followed
by the Greco-Roman, Arabian, Medieval and modern periods. Rig Veda, the claimed
oldest religious book, had mentioned the medicinal use of plants. Records from as
early as 2700 B.C. from China, traced to the Emperor Shennung, indicate the
usefulness of plants for treating disease and the Ebers papyrus, written in about 1550
B.C., includes many of the plants used in Egyptian medicine. First Materia Medica
of the world was developed by the Greeks. Ibn al-Baitar (1197-1248) listed over
1400 drugs and medicinal plants in his Corpus of Simples. Al-Qanun fi al-Tibb (The

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

Canon of Medicine) is a 14-volume Arabic medical encyclopedia written by a
Persian scientist and physician Ibn Sina (Avicenna) and completed in 1025 (Stanley,
1994). Famous books Firdous al Hikmat, Deen-e-Doulat and Hifz al-Shehhat of
Ali ibn Rabban are kept at European libraries for their importance (Jacquart, 2008).
In Europe, after the tenth century, much of the medicinal lore was based in the
church, particularly the monastic orders, but by the 1500‘s, after the invention of the
printing press, herbals available to the general public were popular, particularly in
England. By the late 1700‘s, studies like William Withering‘s An Account of the
Foxglove and its Medicinal Uses (1785) began to appear. These studies included
case histories as well as specific doses and administration instructions for herbal
remedies. In the United States, before the advent of specific pharmaceuticals, herbal
medicine was relied upon to treat many illnesses. Development of drugs based on
natural products has had a long history in the United States, and in 1991, almost half
of the best selling drugs were natural products or derivatives of natural products.
There has recently been a resurgence of interest in herbal remedies and the U.S.
market for natural supplements is increasing by as much as 10% per year (courtesy
of TCM Physicians Clinic website visited on 20-05-2009).


1.1.2. FAMOUS HISTORIC MEDICINAL SYSTEM
       The traditional Chinese medicine system and Ayurveda (traditional medicine
system of India) were fully as sophisticated and as documented system as western
medicine systems. The Sushruta Samhita and the Charaka Samhita were influential
works on traditional medicine during this era (Sahu and Mishra, 2003). The
extensive records of Chinese medicine about response to Artemisia preparations for
malaria also provided the clue to the novel antimalarial drug artemisinin. The
therapeutic properties of the opium poppy (active principle morphine) were known in
Ancient Egypt and those of the Solanaceae plants in ancient Greece (active
principles atropine and hyoscine). The snakeroot plant was well regarded in India
(active principle reserpine), and herbalists in medieval England used extracts from
the willow tree(salicin) and foxglove (active principle digitalis - a mixture of
compounds such as digitoxin, digitonin, digitalin) for cure of malaria. The Aztec and
Mayan cultures of Mesoamerica used extracts from a variety of bushes and trees
including the ipecacuanha root (active principle emetine), coca bush (active principle
cocaine), and cinchona bark (active principle quinine) (Bensky et al., 2003).

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.1.3. HERBAL MEDICINE AND ISLAM
        ALLAH Almighty has repeatedly directed and encouraged people to ponder
on and investigate thoughtfully the happenings in this universe through Holy Quran.
For example:
       Verily! It is Allah Who causes the seed-grain and the fruit-stone (like date-
 stone) to split and sprout. He brings forth the living from the dead, and it is He
 Who brings forth the dead from the living. Such is Allah, then how are you deluded
 away from the truth? (Al-Anaam, Verse: 95)
       And We cause therein the grain to grow and grapes and cloverplants
 (i.e.green fodder for the cattle) and olives and date-palms(Surah Abasa, Verses: 27-
 29).
       And whatsoever He has created for you on the earth of varying colours [and
 qualities from vegetation and fruits (botanical life) and from animals (zoological
 life)] Verily! In this is a sign for people who remember. (An-Nahl, Verse: 13)
        In Islamic tradition, the first Muslim physician is believed to have been
Prophet Mohammad (Sull-Allah-ho-Alaihe-Wasallum) himself, as a significant
number of Hadiths (His sayings) concerning medicine are attributed to him. Several
Sahaba (his companions who has seen him) are said to have been successfully treated
of certain diseases by following his medical advice of. The three methods of healing
known to have been mentioned by him were honey, fire cupping and cauterization,
though he was generally opposed to the use of cauterization unless it "suits the
ailment" and Mohammad (Sull-Allah-ho-Alaihe-Wasallum) also appears to have
been the first to suggest that there is always a cause and a cure for every disease
(Deuraseh, 2003; Borchardt, 2002):
       Make use of medical treatment, for Allah has not made a disease without
appointing a remedy for it, with the exception of one disease, namely old age Sunan
Abi Dawood, 28:3846.


        There are many sayings of Prophet Mohammad (Sull-Allah-ho-Alaihe-
Wasallum) describing medicinal uses of the plants like olives and dates used by
Arabs that time (Farooqi, 1998; Ghaznavi, 1991).
        The belief that there is a cure for every disease encouraged early Muslims to
engage in biomedical research and seek out a cure for every disease known to them.
The works of ancient Greek and Roman physicians Hippocrates (father of medicine),

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     Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

Dioscorides, Soranus, Celsus and Galen had a lasting impact on Islamic medicine
(Saad et al., 2005). From the 9th century, Muslim physicians soon began making
many of their own significant advances and contributions to medicine, including all
the fields of medicines and natural sciences. Responding to circumstances of time
and place, Islamic physicians and scholars developed a large and complex medical
literature exploring and synthesizing the theory and practice of medicine. Muslim
physicians set up the earliest dedicated hospitals in the modern sense which were
establishments where the ill were welcomed and cared for by qualified staff, and
which were clearly distinguished from the ancient healing temples, which were more
concerned with isolating the sick and the mad (insane) from society "rather than to
offer them any way to a true cure (Morelon and Rashed, 1996).


1.2. TAXONOMY OF PLANTS
       Biological classification or scientific classification, is a method by which
taxonomists group and categorize plants by biological type, such as genus or species.
The classification of plants by grouping data according to morphological similarities
is probably the oldest and most widely-used of all the approaches (Quinlan, 1993).
However many approaches evolved over time towards the taxonomy of plants.




           Scheme 1: Approaches towards the classification of plants


1.2.1. CAUSES OF TAXONOMIC COMPLEXITY
       Morphological variants of a species results because of different factors. In
addition, many species exhibit considerable genetic variation, both florally and
vegetatively. This variation may occur in different populations of the same species,
or may characterize different infraspecific categories of a species. Sometimes, the

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

character may be genetically controlled in one species, but phenotypically plastic in
another. Quaternary climatic changes have had a profound impact on speciation,
structuring of genetic diversity and the shaping of the present-day distributions of
plant and animal taxa (Avise, 2000; Hewitt, 1996, 2000, 2004; Vuilleumier, 1971).
Oscillations of population sizes, bottle necks, founder events and other population
historical events associated with climatic shifts have further contributed to
differentiation among regional population groups. As a combined effect of range
shifts and population differentiation, divergent lineages have occasionally formed
contact zones, leading to reticulate speciation by means of hybridization and
polyploidization (Grant, 1981; Stebbins, 1984). Polyploid speciation has long been
recognized as an important process in plant evolution (Müntzing, 1936; Stebbins
1950; Grant, 1981). Recent genomic studies have made it clear that angiosperms
possess genomes with considerable gene redundancy, indicating that ―most (if not
all) plants have undergone one or more episodes of polyploidization‖ (Soltis et al.,
2003).


1.3. CHEMOTAXONOMY
         Chemotaxonomy is also called chemosystematics or biochemical systematics.
The science of chemical taxonomy is used on the classification of plants on the basis
of their chemical constituents which are deeply concerned with the molecular
characteristics. The method of chemical taxonomy is simple in principal and is based
on the investigations of the distribution of chemical compounds or groups of
biosynthetically related compounds in series of related plants. Different plants
sometimes contain substances which although belong to different chemical
compounds appear to be biosynthetically analogous. Such plants may contain similar
enzyme systems, and the compounds produced by such enzymes are indicative of the
relationships that exist between the plants. However, the chemotaxonomic studies
include the investigation of the patterns of the compounds existing in plant. Climatic
conditions have a major influence on the distribution of plants containing certain
substances e.g. fats, volatile oils, alkaloids, flavonoids etc. It is well known that for
tropics, and perhaps for all climates, the chemical products are highly organized.
According to Reichert (1919), it is possible to identify many plants by their starch
grains. Stress has been given on the importance of β-Cyanins and β-Xanthins in plant


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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

taxonomy. β-Cyanins are commonly met within the families of order centrospermae.
The other chemicals are also found specifically in particular orders or families of
flowering plants e.g. Isoquinoline (Alkaloids) is found in the families of Ranales;
retanone in the families of leguminales, biflavonoils in casuarinas equisetifolia
(casuarinaceae). The presence of such chemicals in different groups of plants has
great taxonomic significance (Stuessy, 2008). Taxonomic studies for various plant
taxa by using different parameters have been successfully carried out in Pakistan
including that of cereals (Ashraf et al., 2003), Legumes (Ahmad et al., 2007) and
Maize (Nawaz and Ashraf, 2007).


1.3.1. APPLICATION OF CHEMOTAXONOMY (Stuessy, 2008)
       There are a few angiospermic taxa which are characterized by specific
compounds of general occurrence. For example leaving aside the family of
caryophyllaceae, the rest of the families such as chenopodiaceae, amaranthaceae,
aizoacaceae etc. of the taxon caryophyllales (centrospermea) contain β-cyanin a
colored substance but differs from anthocyanins. It appears that, with the exception
caryophyllaceae, these families are closely related and therefore caryophyllaceae
may be isolated. β-cyanin also occurs in cactaceae and therefore, the members of
caryophyllales are phylogenetically related. There are certain other chemical
connections between cactaceae and members of caryophyllales e.g. common
presence of isoquinole alkaloids in Salsola of Chenopodiaceae and cactaceae.
Another example that may be cited is in the family crucifereae, where unsaturated
acid erucic acid is prominent and also in Tropaelum erucic acid is present; it
indicates the relationship between Geraniales and Rhocadales. In umbellifereae and
Araliaceae petroselinic acid (a structural isomer of Oleic acid) occurs and these two
families are related and belong to the same order. The other examples are from
Magnoliales Ranals taxa, where it is shown that magnoliaceae, lauraceae,
Ranulculaceae, Annoraceae, the alkaloid isoquinoline is present, this supports that
these families are loosely related. On the other hand Asclepiadaceae and
Gentianaceae are allied due to the common occurrence of pyridine. The lilliaceae and
Amarylldeceae are closely associated and this is supported by the presence of
Isoquinoline in both. A number of citations regarding chemotaxonomy and
secondary metabolites are given in Chapter 2.



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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION



1.4. FAMILY SOLANACEAE
       The Solanaceae, to which the genus Solanum belongs, is a cosmopolitan
family which is widely distributed throughout tropical and temperate regions of the
world, with centers of diversity occurring in Central and South America and
Australia. It is composed of approximately 84 genera and 3000 species. The name of
the family comes from the Latin word Solanum, meaning "the nightshade plant", but
the further etymology of that word is unclear; it has been suggested it originates from
the Latin verb ‗solari‘ meaning "to soothe". This would presumably refer to alleged
soothing pharmacological properties of some of the psychoactive species found in
the family. It is more likely, however, that the name comes from the perceived
resemblance that some of the flowers bear to the sun and its rays, and in fact a
species of S. nigrum Complex (Solanum retroflexum) is known as the sunberry. The
family is also informally known as the nightshade or potato family (Yasin, 1985).


1.4.1. Importance of Solanaceae
       The Solanaceae family is characteristically ethnobotanical and is an important
source of food, spice and medicine. The family includes variety of plants like
important vegetables and fruits as well as some poisonous plants (some plants have
both edible and toxic parts). Most common plants of this family are the Datura or
Jimson weed, eggplant, mandrake, deadly nightshade or belladonna, capsicum
(paprika, chili pepper), potato, tomato, Petunia, Schizanthus and Lycium species.
       It includes many species cultivated for their edible fruits or tubers, such as the
tomato, potato, aubergine/eggplant and chilli pepper. The most important species of
this family for the global diet is the potato (S. tuberosum) whose carbohydrate-rich
tubers have been a staple food in many times and places, and which is one of the
most grown crops today. In many genera of this, the fruits are the economically
desirable item, for example, tomatoes (S. lycopersicum L.), eggplants (S. melongena
L.), and peppers (Capsicum sp.).
       It also contains tobacco (Nicotiana tabacum L.), one of the most harmful yet
economically important plants in the world. It has a documented record of causing
heart, lung, and circulatory problems as well as cancer and other health problems
(Tso, 1977; Zitnak, 1977). Paradoxically, while we usually think of members of the


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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


family as essential and familiar foods, many other members of the Solanaceae are
famed for their alkaloid content and have been used throughout history for their
medicinal, poisonous, psychotropic effects; examples include tobacco, jimson weed,
henbane, and belladonna. Solanaceae species are often rich in alkaloids that can
range in their toxicity to humans and animals from mildly irritating to fatal in small
quantities (Edmonds and Chweya, 1997). Several solanaceous plants and products
are highly poisonous, such as deadly black nightshade, Atropa belladonna L., and
Jimsonweed, Atropa stramonium L (Heiser,1987; Kingsbury, 1968). Tropane
alkaloids that have always played a significant role in ethno medicine as well as
orthodox medicine were also extracted from the genera Datura, Brugmansia, and
Atropa of the Solanaceae family. Plants in the drug family, Solanaceae (nightshades)
are an important causative factor in arthritis in sensitive people (Childers and
Margoles, 1993). Some drugs from the Solanaceae are widely used in medicine, such
as scopolamine, atropine, hyoscyamine, and belladonna (Lewis, 1977).
       Common ornamental plants such as Petunia, Schizanthus, Salpiglossis,
Lycium and Browallia are members of this family. Tobacco, petunia, tomato and
potato are used as model experimental organisms in examining fundamental
biological questions in cell, molecular and genetic studies.
       Alkaloids and steroids in the Solanaceae are reported extensively in the
literature. By examining the biosynthetic routes leading to different alkaloids, the
pathways can be visualized as a spiral from which the various compounds can be
derived. Arrangement of the genera of Solanaceae according to their chemical
contents in relationship to this spiral supports traditional classifications of the family,
but the Anthocercidoideae and Atropoideae must be recognized as new subfamilies
due to their biochemical synthesis syndromes. Similarly, Solaninae and Physalinae
must be accepted as separate subtribes of tribe Solaneae because of their differing
and exclusive steroid synthesis. Acnistus and Dunalia must be allied with Jaborosa in
tribe Jaboroseae (Tetenyi, 1987).




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.5. GENUS SOLANUM
       Within Solanaceae family, Solanum constitutes the largest, variable and most
complex genus. It is consists of annual and perennial plants, forbs, vines, sub-shrubs,
shrubs, and small trees. They often have attractive fruit and flowers. Together with
many other plants of both poisonous and medicinal value Solanum constitutes the
largest and most complex genus of the family. It is composed of more than 1500
species, many of which are also economically important throughout their
cosmopolitan distribution (Ganapathi and Rao, 1986; Edmonds and Chweya, 1997).
Solanum is one of the most commercially and economically important genra of
Solanaceae which had been extensively studied. Fig. 1 show a plot of number of
papers published in different years depicting its popularity.


                               Solanum sp.




Fig. 1: Popularity of Solanum sp. over time: [Plots of numbers of papers mentioning
Solanum sp. (filled column histogram and left hand axis scale) and line of best fit,
1926 to 2006 (complete line, with equation and % variation accounted for, in box on
the left hand side); Plots of a proportional micro index, derived from numbers of
papers mentioning Solanum sp. as a proportion (scaled by multiplying by one
million) of the total number of papers published for that year (broken line frequency
polygon and right hand scale) and line of best fit, 1926 to 2006 (broken line, with
equation and % variation accounted for, in broken line box on the right hand side)].
(Courtesy of Australian New Crops Web Site visited on 20-05-2009)



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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION



1.6. SOLANUM NIGRUM: TAXONOMIC COMPLICATIONS
       Solanum nigrum is the most variable species of the genus Solanum. The
species related to S. nigrum have been reclassified innumerable times. Characters
used by later taxonomists to separate and describe additional taxa often differed very
slightly from those given for species by earlier workers. These Solanum species
display varying amounts of phenotypic variation, particularly in their vegetative
features such as plant habit, leaf size and form, and stem winging. In addition,
senescence is often accompanied by smaller and fewer flowers and fruits (Ganapathi
and Rao, 1986).
       S. nigrum was first delimited in four taxa with polynomials by Dillenius.
Linnaeus subsequently modified Dillenius‘s work, describing these in six varieties
under the binomial S. nigrum (Edmonds and Chweya, 1997). Since then, the plants
morphologically related to S. nigrum have been reclassified many times. Over 300
post-Linnean specific and infraspecific names have now been published, and
synonymy is extensive within the section. However, no satisfactory revision of the
whole section has yet been devised. The boundaries between many of the species are
still ill-defined, with many of the ‗new‘ taxa proving to be no more than slight
morphological variants of those already described. The situation is further
complicated by the researchers who either treated different members of the section as
varieties of S. nigrum or considered them as different species on the basis of
morphological differences (e.g. Edmonds and Chweya, 1997; Schilling and
Andersen, 1990; Stebbins and Paddock, 1949; Symon, 1970). These Solanum species
display varying amounts of phenotypic variation, particularly in their vegetative
features such as plant habit, leaf size and form, and stem winging. In addition,
senescence is often accompanied by smaller and fewer flowers and fruits than usual.
Natural hybridization is probably more widespread in this section than generally
supposed. It is now named as Solanum nigrum Complex because it is composed of
a large number (about 30) of morphologically distinct taxa (Schilling and Andersen,
1990). Only during the revision of Solanum section Solanum appear in 1979 for
Flora Europaea 3, drawn turned out that in Europe two different forms of the species
coexist. The most widespread form was considered subspecies S. nigrum ssp.




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

nigrum, the second, rarely encountered species as S. nigrum ssp. schultesii classified
(Edmonds and Chweya, 1997).


       Deadly nightshade, identified as S. nigrum, causes belladonna poisoning
(Hubbs, 1947), with symptoms including widely dilated pupils (characteristic of the
atropine group, but either unexpressed, or expressed very mildly, in poisonings by
plants whose major poisoning principle is of the solanine group). Deadly nightshade
is identified as S. nigrum, but black nightshade is botanically unidentified and gives a
strong cat's-eye test for atropine (Case, 1955). According to a research at University
of Pennsylvania, unripe berries are said to be more toxic than ripe berries. Berries are
more toxic than leaves which, in turn, are more toxic than stems or roots. Overall
plant glycoalkaloid content is often higher in the autumn than in the spring. These
problems clearly state the importance of proper identification and detailed
composition analysis of each taxa of S. nigrum Complex.


       The taxa of S. nigrum Complex are difficult to distinguish because
       1. They are morphologically similar.
       2. These species are all highly phenotypically plastic.




       Three taxa belonging to S. nigrum Complex viz.: S. americanum Mill., S.
nigrum L. and S. villosum Mill. had been reported in Pakistan (Schilling and
Andersen, 1990). S. chenopodioides Lam. and S. retroflexum Dunal are two other
species that were found growing wild in and around Botanic Garden, GC University,
Lahore. Morphologically S. nigrum is different from S. villosum in the respect that
the former has black matured berries with peduncles longer than pedicels while latter
has orange/orange-red matured berries and peduncles shorter than or equal to the
pedicels. Classification of S. nigrum and S. villosum as varieties or distinct species
started taxonomic controversy between Linnaeus and Miller (Edmonds and Chweya,
1997). Though S. americanum Mill., S. chenopodioides Lam. and S. retroflexum
Dunal have morphological resemblance with S. nigrum, yet no chemotaxonomic
relationship has so far been established due to lack of a comprehensive study of their
chemical composition.



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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION



1.7. BOTANICAL ASPECTS OF THE INVESTIGATED TAXA


       The five locally available taxa investigated were:


   1. Solanum americanum
   2. Solanum chenopodioides
   3. Solanum nigrum
   4. Solanum retroflexum
   5. Solanum villosum


        These Solanum species display varying amounts of phenotypic variation,
particularly in their vegetative features such as plant habit, leaf size and form, and
stem winging. In addition, senescence is often accompanied by smaller and fewer
flowers and fruits than usual, while the gene for anthocyanin pigmentation in flowers
seems to be dependent on light intensity and temperature for its expression, in some
species. It is therefore often difficult to define the limits within which such features
are genetically fixed (Baylis, 1958; Henderson, 1974; Edmonds, 1977).


        Natural hybridization is probably more widespread in this section than
generally supposed. Though this is probably followed by subsequent genetic
breakdown in F1 or F2 generations (Edmonds, 1977), it may also be followed by
back-crossing to the parental species. This would result in morphogenetically
complex population variation: the collection of specimens from such populations
would explain some of the difficulties encountered in the morphological
differentiation of these species in the herbarium (Edmonds, 1979).


        A brief overview of their individual botanical aspects (Edmonds and
Chweya, 1997; Ganapathi and Rao, 1986; Karschon and Horowitz, 1985; Schilling
and Anderson, 1992) is given below:




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.7.1. SOLANUM AMERICANUM MILLER
1.7.1.1. Morphological Features
        S. americanum (Fig. 2), the American nightshade, is an herbaceous
flowering plant native to the Americas. It grows up to 1-1.5 m tall and is an annual or
short-lived perennial. Stem with edentate to inconspicuously dentate ridges. Leaves
ovate-lanceolate to lanceolate, lower surfaces glabrescent to moderately or densely
pilose; margins entire to sinuate, rarely sinuate-dentate. Inflorescence simple,
umbellate cymes, 3 to 6 flowered. Berries globose, black, rarely dark green, with
shiny opaque cuticles, falling from calyces when ripe.


1.7.1.2. Distribution:
       Its habitat is Rocky or dry open woods, thickets, shores or openings, often on
cultivated or waste ground. This plant is native to the Americas, from the south and
west of the United States south to Paraguay and Peru; it also occurs in Hawaii, where
it is considered possibly indigenous or may be a Polynesian introduction. It is used as
a medicinal in Cameroon, Kenya, Hawaii, Panama, Sierra Leone, and Tanzania, and
as a wild or cultivated pot herb in Cameroon, Ghana, Guatemala, Kenya,
Madagascar, Mauritius, Hawaii and other Pacific Islands, Nigeria, Papua New
Guinea, Peru, Sierra Leone, the Seychelles, South Africa, Tanzania, and Uganda.


1.7.1.3. Vernacular names:
 Argentina: Arachichu
 Australia: Glossy nightshade
 New Zealand: Small-flowered nightshade




                              Fig. 2: S. americanum

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.7.2. SOLANUM CHENOPODIOIDES LAM.
1.7.2.1. Morphological Features
       S. chenopodioides (Fig. 3), is a species of nightshade known by the common
name forked nightshade. It is native to South America. Plants sprawling herbs to 1m
high, often grayish-green in color. Stem usually smooth with edentate ridges. Leaves
elliptic to lanceolate, obtuse to acute, distinct lobes usually absent. Inflorescence
simple, umbellate cymes, 4 to 6 flowered. Berries globose to broadly ovoid, purple,
with dull opaque cuticles.


1.7.2.2. Distribution:
       It is native to Southern America and is present in Brazil, Argentina and
Uruguay. It is also naturalized in Australia, Europe, New Zealand, South Africa, &
United States. It grows in gullies and creeks; widespread in coastal districts, west to
Mt Tomah area. It is usually found on waste land, but appears to be fairly tolerant
and may turn up in most situations.


1.7.2.3. Vernacular names
 Australia: White tip nightshade
 Brazil: Liaghe
 New Zealand: Velvety nightshade




                                Fig. 3: S. chenopodioides




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.7.3. SOLANUM NIGRUM L.
1.7.3.1. Morphological Features
       S. nigrum (Fig. 4), or The Black nightshade is a fairly common plant, found
in many wooded areas, as well as disturbed habitats. It has a height of 30-120 cm
(12-48"). Herb, shrubs or small trees, often prickly or hairy, berries in small hanging
clusters, remaining on plants or falling from calyces when ripe. Flowers rarely
solitary. Stem decumbent to erect. Leaves ovate, ovate-lanceolate, and ovate-
rhombic to lanceolate. Berries usually broadly ovoid, dull purple to blackish or
yellowish-green, remaining on plants or falling from calyces when ripe.


1.7.3.2. Distribution:
       The Black Nightshade is an annual plant, common and generally distributed
in the South of England, less abundant in the North and somewhat infrequent in
Scotland. It is one of the most cosmopolitan of wild plants, extending almost over the
whole globe. It is sometimes called the Garden Nightshade, because it so often
occurs in cultivated ground.


1.7.3.3. Vernacular names:
 Australia: Black or black berry nightshade
 Ethiopia: ―Dime people eat‖
 Europe: Black Nightshade, annual nightshade, common nightshade
 South Africa: Nightshade




                                Fig. 4: S. nigrum

                                                                                     15
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.7.4. SOLANUM RETROFLEXUM DUNAL
1.7.4.1. Morphological Features
S. retroflexum (Fig. 5), is spreading, erect, pubescent herb upto 70 cm tall. Stem
with smooth or inconspicuously dentate ridges. Leaves rhomboidal to ovate-
lanceolate, broad, margins usually deeply lobed with 3 to 5(7) obliquely triangular
lobes on each margin. Inflorescence simple, umbellate erect cymes, 3 to 6(7)-
flowered; Berries spherical, purple, dull with opaque cuticles, falling from calyces
when ripe.


1.7.4.2. Distribution:
It is widely distributed in Northeast Tropical Africa in Ethiopia, Somalia, Sudan and
In East Tropical Africa in Tanzania. In West Tropical Africa, Mauritania; Nigeria;
Sierra Leone, in South Tropical Africa, Angola; Malawi; Mozambique; Zambia;
Zimbabwe and also in Southern Africa, Botswana; Lesotho; Namibia; South Africa
Cape Province, Natal, Orange Free State, Transva; Swaziland. It is sparingly
cultivated in North America & naturalized in South Australia


1.7.4.3. Vernacular names
North America: Sunberry, Wonderberry
South Africa: Nastergal




                             Fig. 5: S. retroflexum




                                                                                   16
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.7.5. SOLANUM VILLOSUM MILLER
1.7.5.1. Morphological Features
       S. villosum (Fig. 6), is sub glabrous to villous annuals, up to 50 cm high.
Stem rounded to angled, almost glabrous to pubescent with appressed hairs. Leaves
rhombic to ovatelanceolate. Inflorescence simple, umbellate to slightly lax solitary
cymes, 3 to 5 lowered, rarely 10 flowered. Berries usually longer than wide,
occasionally globose, red, orange or yellow.


1.7.5.2. Distribution:
       S. villosum is believed to have originated in Eurasia, and is sometimes
considered to have a southern European origin. It is widespread, but absent in
Central and South America, and New Guinea. It has been introduced in North
America and Australia. In Africa it is recorded from Tunisia, Algeria and South
Africa, and from many countries of tropical Africa, e.g. Burundi, Sudan, Ethiopia,
Somalia, Kenya, Uganda, Tanzania, Zambia and Angola. It can grow on a wide
range of soils, but prefers soils that are rich in organic matter and land covered with
ash of recently burnt vegetation. In the wild along the edges of agricultural fields.


1.7.5.3. Vernacular names:
Great Britain: Red-fruited nightshade
Kenya: Soiyot-Ap-Poinet
Uganda: Eswiga




                              Fig. 6: S. villosum

                                                                                        17
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


1.7.6. KEY TO THE INVESTIGATED TAXA OF S. NIGRUM COMPLEX
S. americanum Miller: Plants glabrescent to moderately pilose with appressed
eglandular hairs; flowers small, fruiting pedicels usually erecto-patent; berries
spherical, black and usually shiny when mature; seeds 1-1.5 mm long.


S. chenopodioides Lam.: Plants somewhat tomentose; umbellate cyme inflorescence
with 4,6 sometimes 8 flowers; fruiting peduncles strongly deflexed from the base;
berries globose to ovoid, purple with dull opaque cuticle.


S. nigrum L.: Plants subglabrous to pubescent usually with appressed, eglandular-
headed multicellular hairs; berries black.


S. retroflexum Dunal: Plants pubescent with appressed, eglandular-headed
multicellular hairs; flowers white with distinct purple vein to outer surface of petals;
berries usually spherical, purple with opaque cuticles.


S. villosum Miller: Plants villous, covered with glandular headed and often patent
multicellular hairs; stems usually terete, with smooth ridges; berries red, orange or
yellow.


1.8. IMPORTANCE OF S. NIGRUM COMPLEX
1.8.1. MEDICINAL USES
          Many varieties of S. nigrum Complex have been reported to have medicinal
properties although most of the reported work does not throw light on the taxonomy
of varieties used. S. nigrum is antiperiodic, antiphlogistic, diaphoretic, diuretic,
emollient, febrifuge, narcotic, purgative and sedative (Emboden, 1979; Lust, 1983;
Grieve, 1985; Singh and Kachroo, 1985).
          The leaves, stems and roots are used externally as a poultice; wash etc. in the
treatment of cancerous sores, boils, leucoderma and wounds (Lust, 1983). Extracts of
the plant are analgesic, antispasmodic, anti-inflammatory and vasodilator. The plant
has been used in the manufacture of locally analgesic ointments.




                                                                                       18
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

       The juice of the fruit has been used as an analgesic for toothaches. S. nigrum
mixed with other herbal medicines has hepatoprotective effect in cirrhotic patients,
anti-oxidative and immunomodulating properties of the commonest herbs it also
protects against hepatitis B virus infection.
       In Hawaii plants conspecific with S. americanum are used in disorders of the
respiratory tract, skin eruptions, cuts, wounds and trachoma. While in the Mauritius,
a poultice of the plant is used to relieve abdominal pain and Inflammation of the
urinary bladder (Grieve, 1985). In Tanzania, ground and soaked leaves of S. villosum
were reportedly placed on swellings and fruit juice squeezed into sore eye
(Ganapathi and Rao, 1986).


1.8.2. SOURCE OF ALKALOIDS
       The berries of Solanum contain the alkaloid solasodine. It is a glycol which
is used by pharmaceutical companies for the preparation of many important drugs. It
is a nitrogen analogue of diosyenin and is a good source of sapogenin. Sapogenin is
used as a base for the preparation of cortisone and allied product. The synthetic
substituents are not known for these drugs; hence the importance of this source thus
increases. Cortisone, a steroidal harmone prepared from solasodine is found to be
effected in treatment of acute stages of rheumatoid arthritis, chronic cases of asthma,
leukemia (Panday, 2004).


1.8.3. NUTRITIONAL VALUE
       Several studies have been conducted to investigate the nutritive value of the
vegetable black nightshades. The leaves can provide appreciable amounts of protein
and amino acids, minerals including calcium, iron and phosphorus, vitamins A and
C, fat and fiber, as well as appreciable amounts of methionine, an amino acid scarce
in other vegetables (Fawusi, 1983).
       They are also widely used in pies and preserves, and sometimes as a
substitute for raisins in plum puddings, particularly in North America. They can also
make a delightful jam, with the ‗Wonderberry‘ making an excellent preserve for tea
with bread and butter. Leaves and tender shoots are widely used as vegetables
throughout the world and have provided a food source since early times.




                                                                                     19
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


       S. nigrum being used as an ancient famine plant and the Chinese S. villosum
are frequently eaten as fruits, particularly in parts of Africa. However, S.
chenopodioides does not seem to be utilized as a food source, with the only reference
to its use as ‗spinach‘-plant being Natal in South Africa. S. retroflexum is believed
to be the ‗Sunberry‘ promoted by the plant breeder Luther Burbank as ―a new food
plant from a poisonous family‖ at the beginning of this century in North America
(Edmonds and Chweya, 1997).


1.8.4. COMMERCIAL VALUE
       It was a widespread practice in Argentina, S. chenopodioides together with
wool waste was used as manure. It has also been extensively spread on light
agricultural sandy soils and has been a major source of South American adventives
which have become established as weeds in Europe. Both the leaves and berries are
used as a source of dyes. Leaves are macerated to extract a dye used to color sisal
baskets (Khanna and Rathore, 1977). The black berries of S. americanum are
reportedly used as a source of ink. Seeds of fresh fruit rubbed on cheeks to remove
freckles and improve the complexion.
       The species are apparently used as fodder and browse by various animals,
especially in Africa. Herbarium records showed that plants tentatively identified as S.
villosum are eaten by sheep and goats in the Sudan, and by bush-buck and browsed
by goats (Grieve, 1985).
       The plants of both S. americanum and S. nigrum persisting as weeds of
cultivation in Australia were known to be alternative hosts for insects attacking crops
such as tobacco, for plant viruses transmitted by insects, and for pathogenic bacteria
attacking commercial strains of ginger (Lust, 1983).
       This specie group has been found to be effective in removing PCB's from the
soil and detoxifying them. The plant is more effective in doing this if it is infected
with the bacterial parasite Agrobacterium tumefaciens (Moerman, 1998).




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.9. SECONDARY METABOLITES
         The term ―secondary metabolites‖ indicates compounds that are not required
for plant growth and development but presumed to function in communication or
defence (Luckner, 1990). Secondary metabolites, on the basis of their properties and
functions, can be classified into different groups like Alkaloids, Flavonoids, Tannins,
and Saponins etc (Mann, 1986).


1.9.1. ALKALOIDS:
         The name ―Alkaloids‖ is derived from the fact that these compounds in
general behave similar to alkali bases (e.g. NaOH) in that they neutralize acids. Thus
early workers coined the term alkaloid from ―Alkali‖ and ―Oid‖ meaning ―Alkali-
like‖ (Mann, 1986). Most recently W. Pelletier suggested the following new
definition for an alkaloid:
         ―An alkaloid is a cyclic organic compound containing nitrogen in a negative
oxidation state, which is of limited distribution among living organisms‖ (Bhat et al.,
2005).
         Alkaloids are a naturally occurring group of compounds containing
heterocyclic nitrogen, having a more or less distinctly basic character, and a complex
molecular structure. They possess recognizable physiological and pharmaceutical
activity (Wilson and Gisvold, 1956). The most recent comprehensive survey had
shown that a total of 1932 individual compounds have been reported as alkaloids by
the investigators who isolated them from 158 botanical families (Swain, 1960). Not
withstanding the many valuable synthetic medicinal and antibiotic agents that have
been added to the list of weapons against diseases, the alkaloids still constitutes an
indispensable and most potent group of substances for the treatment and migration of
functional disturbances and relief from suffering (William and Schubert, 1961).


1.9.1.1. Studies on Alkaloids
         The history of alkaloid chemistry, in structural terms, began in 1804, when
Sertürner (the Paderborn apothecary) discovered the so-called principium
somniferum in opium (Trommsdorf, 1805), which he reported the following year in
the Journal der Pharmacie (Sertürner, 1805). The attention of scientists, however,
was aroused only twelve years later by a publication appearing in the Annalen der



                                                                                     21
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

Physik (Sertürner, 1817; Schmitz, 1983). There, Sertürner named his principium
somniferum for the first time "morphium" (after Morpheus, the son or servant of
sleep and creater of dream states in Ovid; altered to "morphinium" by the French
physicist Gay-Lussac). Often, a very great deal of time would pass between the
isolation of an alkaloid and the determination of both its structure and absolute
configuration. In the case of strychnine, 138 years passed by and for morphine 150
years (Hesse, 2002). Today, it is usual to determine the structure of a substance in the
year of its isolation, especially when it seems to possess pharmacological properties
as promising as those of strychnine and morphine.
         In true alkaloids the basic units of biogenesis are amino acids. The non-
nitrogen containing rings or side chains are derived from terpene units and/or acetate,
while methionine is responsible for the addition of methyl groups to nitrogen atoms.
Alkaloids are basic and form water-soluble salts. Most alkaloids are well-defined
crystalline substances that react with acids to form salts. In plants they may exist in
the free state, as salts or as N-oxides. The different criteria currently used for the
classification of 243 alkaloids are biogenesis, structural relationship, biological
origin     and   spectroscopic/spectrometric   properties   (chromophores      in   UV
spectroscopy, ring systems in mass spectrometry) (Hesse, 2002). Based on amino
acid precursor, alkaloids can be further subdivided. The principal precursors are
ornithine, lysine, nicotinic acid, tyrosine, tryptophan, anthranilic acid and histidine.
Ornithine gives rise to pyrrolidine and trypane alkaloids, lysine to piperidine,
quinolizidine and indolizidine alkaloids and nicotinic acid to pyridine alkaloids.
Tyrosine          produces         phenylethylamines,           tetrahydroisoquinoline,
benzyltetrahydroisoquinoline,             phenethylisoquinoline,              terpenoid
tetrahydroisoquinoline and Amaryllidaceae alkaloids. Tryptophan gives rise to β-
carboline, terpenoid indole, quinoline, pyrroloindole and ergot alkaloids. Anthranilic
acid acts as a precursor to quinazoline, quinoline and acridine alkaloids, while
histidine gives imidazole derivates (Dewick, 2002).
         Concerning the distribution of the main secondary metabolites in Rubiaceae
(Robbrecht, 1988), indole alkaloids were the chemotaxonomic markers more
intensely studied so far, aiming the establishment of phyllogenetic correlations
between secondary metabolites and taxonomic data. Our chemical studies revealed
several interesting correlations among tribes and subfamilies of Rubiaceae due to
their structural variability and restrict distribution (Bolzani et al., 2001). In

                                                                                      22
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

Rubiaceae, the occurrence and distribution of iridoids, indole alkaloids and
anthraquinones has provided valuable chemosystematic clues (Young et al., 1996).
       Terpenes, sterols and saponins are often found in association with alkaloids
or as their precursors.
       Saponins are glycoside compounds often referred to as a ―Natural Detergent‖
because of their foamy texture. They are mainly of triterpenoidal type, being the
oleanolic acid and the hedagenin in the main constituents. They are found in many
plants and get their name from soapwort plant (Saponaria), the root of which was
used historically as a soap (Latin sapo means soap). They are shown to be
anticarcinogenic and antioxidant (Fink and Fusion, 1918).
       The name terpene is derived from English word ―Turpentine‖. The terpenes
are generally colorless liquids, which are lighter than water and boil in 140-190 0C
temperature range. They are insoluble in water, highly refractive and optically active
and rotate the plane of polarized light. The terpenes are unsaturated hydrocarbons,
which have distinct architectural and chemical relation to simple isoprene molecule
C5H8. They have the molecular formula C10H16, thus are constituted by two isoprene
units combines by head to tail union (Pinder, 1960).




Following are some important classes of alkaloids:




                          Scheme 2: Classification of Alkaloids




                                                                                    23
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.9.1.2. Solanum Alkaloids
       Steroidal glycoalkaloids derived from a cholestane skeleton are mainly found
in the Solanaceae family and therefore termed Solanum glycoalkaloids. Besides S.
nigrum complex, there are also other plants in the Solanaceae that contain
glycoalkaloids: e.g. potato (Solanum tuberosum), tomato (Lycopersicon esculentum
Mill.) and eggplant (Solanum melongena L.). Examples: Solanidine, Solanine,
Chaconine, Solasodine, (Solanum Alkaloids) Veratramine, Muldamine; Samandarin
(Fire Salamander Alkaloids)




               Fig. 7: General Structure of Steroidal Alkaloids


1.9.1.3. Bioactive Properties of Alkaloids
     The isoquinoline alkaloid emetine obtained from the underground part of
Cephaelis ipecacuanha, and related species, has been used for many years as
amoebicidal drug as well as for the treatment of abscesses due to the spread of
Escherichia histolytica infections. Another important drug of plant origin with a long
history of use is quinine. This alkaloid occurs naturally in the bark of Cinchona tree.
Apart from its continual usefulness in the treatment of malaria, it can also used to
relieve nocturnal leg cramps. Currently widely prescribed drugs are analogs of
quinine such as chloroquinine .Some strains of malarial parasites have become
resistant to quinines,therefore, antimalarial drugs with novel mode of action are
required. Similarly, higher plants have made important contributions in the areas
beyond anti-infective, such as cancer therapies. Early examples include the anti-
leukaemic alkaloids, vinblastine and vincristine, which were both obtained from the
Madagascan periwinkle (de-Pavia, 2003). Other cancer therapeutic agents include
taxol, homoharringtonine and several derivatives of camptothein. For example, a
well- known benzyl isoquinoline alkaloid, papaverine, has been shown to have a
potent inhibitory effect on the replication of several viruses including
cytomegalovirus, measles and HIV. Most recently, three new atropisomeric naphthyl


                                                                                     24
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

isoquinoline alkaloid dimmers, michellamines A, B, and C were isolated from a
newly described species tropical liana Ancistrocladus korupensis from the rain forest
of Cameroon. The three compounds showed potential anti-HIV with michellamine B
being the most patent and abundant member of the series. These compounds were
capable of complete inhibition of the cytopathic effects of HIV-1 and HIV-2 on
human lymphoblastoid target cell in vitro (Robert, 1985).
     Toxicity: Glycoalkaloids are toxic compounds. An official safety or acceptable
limit of total glycoalkaloid content for human consumption in tubers is 200 mg/kg
fresh weight (fw) (1000mg/kg dry weight, dw) or 1 mg/kg body weight (bw).
Glycoalkaloid contents below this guideline are not thought to represent health risks
for humans. However, lower acceptable levels for different plant types have been
recommended because of the variation in glycoalkaloid concentrations between years
and growth locations. According to estimates based on poisoning cases reported, a
toxic dose of glycoalkaloids in human consumption can vary between 2–5 mg/kg bw
and a lethal dose about 3–6 mg/kg bw (Morris & Lee 1984) or it can be even as low
as from 1 to 2 mg/kg bw (reviewed in Friedman & McDonald 1997). Symptoms of
poisoning caused by glycoalkaloids have been reported to be gastrointestinal, causing
vomiting, diarrhoea, abdominal pain, neurological resulting in restlessness,
confusion, delirium, stuporose, drowsiness, hallucination, and others such as nausea,
malaise and skin lesions (van Gelder 1991). It is worth noting that glycoalkaloids are
stable and are not destroyed by cooking, except during frying where a minor
reduction in glycoalkaloid levels has been reported (Bushway & Ponnampalam
1981). Glycoalkaloids are toxic compounds for all mammals. However, animals are
generally less susceptible to glycoalkaloids than humans. The effects of
glycoalkaloids on animals have been reviewed by Morris and Lee (1984) and
Friedman and McDonald (1997). Their major toxic properties are due to:
       i)       The ability of glycoalkaloids to bind with membrane 3β-hydroxy
                sterols and to disrupt membrane function.
       ii)      The ability to inhibit acetylcholinesterase.
In producing toxic effects, glycoalkaloids may act synergistically, i.e. a mixture of
glycoalkaloids has greater/different toxicity than could be expected from their
individual effects.
     To be concluded, different glycoalkaloids and aglycones possess various,
specific biological activities and toxicity depends on their structural characteristics.

                                                                                      25
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.9.2. FLAVONOIDS
       The term flavonoids refer to a class of plant secondary metabolite. They are
the largest group of naturally occurring phenolic compounds, which occur in
different plants parts both in the free state and as glycosides (Harborne, 1974).
       The term flavonoids has been derived from a Latin word ―FLAVUS‖
meaning yellow as a large no of flavonoids are yellow in color. Flavonoids are also
known as plant pigment or co pigment. The presence of these pigments is responsible
for color and combination of colors exhibited by bark, leaves, flower, fruits and
seeds of plants. Flavonoids are commonly referred to as bioflavonoid in the media;
the terms are largely equivalent and interchangeable for most flavonoids are
biological in origin (Ray Sahelian, 2005).
       They are polyphenolic compounds with general structure possessing 15
carbon atoms, two benzene rings joined by a linear three carbon chain. The skeleton
below can be represented as the C6 - C3 - C6 system.




                                    (a)                                    (b)
Fig. 8: Basic Structure of Flavonoids: (a) Carbon skeleton (b) Chromane ring

       The chemical structure is based on a C15 skeleton with a chromane ring
bearing a second aromatic ring B in position 2, 3 or is replaced, in a few cases, where
the six-membered heterocyclic ring C occurs in an isomeric open form or by a five
membered ring.


The major classes of Flavonoids are
   Flavone
   Flavonol
   Flavanone
   Flavanol
   Isoflavone
   Anthocyanin



                                                                                     26
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


1.9.2.1. Studies on Flavonoids
    Flavonoids first came into the spotlight in the 1930‘s when Szent-Gyorgyi and
his colleagues extracted two flavonoids from citrus fruit. They discovered that a
crude form of vitamin C which contained a flavonoids fraction worked better for
treating bleeding gums than did a more refined form of vitamin C. They investigated
the effects of the flavonoids and found they decreased the fragility and permeability
of human capillaries. This is why flavonoids were then called "vitamin P" [P for
permeability] (Ray Sahelian, 2005).


1.9.2.2. Solanum Flavonoids
       Many glycosides of quercetin, kaempferol and myricetin had been reported
from various Solanum species (da Silva et al., 2003). Schilling (1984) isolated 10
flavonoids from leaf extract of 11 species belonging to the section Solanum;
including coumarins (such as scopolrtin). Flavonols and Anthocyanidins for S.
scabrum in Nigeria (Gbile and Adesine, 1984) and the anthocyanin pigments were
also found in European samples of this species (Francis and Harborne 1966).
       Quercetin is the most commonly occurring flavonol aglycone detected in S.
nigrum Complex. It forms many glycosides like quercitrin, isoquercitrin and rutin
together with rhamnose and glucose as sugar moieties attached in different patterns.




                                                        OH




                              Fig. 9: Quercetin


1.9.2.3. Bioactive Properties of Flavonoids
       Research suggests that flavonoids may have diverse benefits including
antioxidant, antiviral, anti-allergic, antimicrobial, anti-platelet, anti-inflammatory,
and anti-tumor effects. In vivo research has demonstrated that quercetin can increase



                                                                                       27
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


the anti-tumor activity of cisplatin and busulfan and can be used in conjunction with
doxorubicin and etoposide without interfering with their therapeutic action.
       Flavonoids are some of the most powerful and effective antioxidant
compounds available to humans and since we are unable to produce flavonoids
ourselves, we must get them from the food we eat and from supplements. Flavonoids
exert these antioxidant effects by neutralizing all types of oxidizing radicals (Robak
and Gryglewski, 1988) including the superoxide (Husain et al., 1987) and hydroxyl
radicals (So et al., 1996) and by chelation. Certain flavonoids may even have
antihistamine, memory and mood enhancing properties. Most flavonoids have anti-
germ activity. Immuno-Shield is an immune system product formulated by Dr.
Sahelian that has flavonoids and several immune herbs and nutrients (Ray Sahelian,
2005). While they are not considered essential nutrients, some flavonoids support
health by strengthening capillaries and other connective tissue, and some function as
antihistaminic and antiviral agents. Rutin and several other flavonoids may also
protect blood vessels.
       Flavonoids have been referred to as ―nature‘s biological response modifiers‖
because of strong experimental evidence of their inherent ability to modify the
body‘s reaction to allergens, viruses and carcinogens. They show antimicrobial and
anticancer activity (Bhat et al., 2005). Quercetin is found to be the most active of the
Flavonoids and many medicinal plants owe much of their activity to their high
quercetin content. It has demonstrated significant anti-inflammatory activity because
of direct inhibition of several initial processes of inflammation. For example,
Quercetin inhibits both the production and release of histamine and other allergic,
inflammatory mediators. In addition, it exerts potent antioxidant activity and vitamin
C –sparing action. It can be found in the herbal products based on Hawthorn, which
are used for acute symptoms of Congestive Heart Failure. It has been reported to be
used for the treatment of Diabetes, Peptic ulcer & Edema Allergies, Atherosclerosis
& Cataracts Capillary fragility & Hay fever (So et al., 1981).




                                                                                      28
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION



1.9.3. EPICUTICULAR WAX
       The term ‗wax‘ is derived from the Anglo-Saxon word ‗Weax‘ which was
applied to the natural material obtained from the honeycomb of the bee in beeswax.
When similar substances were found in plants, they were also called ‗Weax‘ or Wax.
Wax can be defined as ‗a substance belonging to a specific group of organic
thermoplastics (as a rule, opaque or translucent but not transparent) with melting
point between 50o and 90oC, exceptionally high to relatively low viscosity liquids or
semisolids or solids which do not exhibit thread spinning phenomenon, compatible
with other waxes, forming pastes of gels with organic or non-polar solvents, having
water repellent properties, imparting gloss, and possessing-in principle-illuminating
power. Chemically waxes are esters of fatty acids and monohydric fatty alcohols.
These are widely distributed in nature with commercially important representatives
in each of the following classification, viz. animal (and insect), vegetable and
mineral (Thorpe, 1937).


        ―The cuticle of terrestrial vascular plants and some bryophytes is covered
with a complex hydrophobic mixture of lipids, usually called epicuticular waxes.‖


        Self-assembly processes of wax molecules lead to crystalline three-
dimensional micro- and nano structures that emerge from an underlying wax film.
Wax crystals form a hydrophobic water-repellent surface due to their chemistry and
micro structure. Such surfaces often display a self-cleaning property, called the
‗lotus-effect‘, by increased water repellency and reduced adhesion of the
contaminating particles (Barthlott, and Neinhuis, 1997). Epicuticular waxes form thin
films or thick crustsand often superimposed three-dimensional structures on an
underlying wax film (Kolattukudy, 1980; Barthlott, 1990; Bianchi, 1995; Barthlott et
al., 1998). The varying shape of the wax crystals is determined by their chemical
composition. In some types of crystals, the ultrastructure is determined by one
predominating wax component (Jeffree et al., 1975; Jeffree et al., 1976).
Nevertheless, due to their small size, the chemical composition of an individual wax
crystal is still hypothetical




                                                                                    29
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


1.9.3.1. Studies on Epicuticular Waxes
       The first person to describe waxes on plant surfaces, using light microscopy,
and Term them ‗kristalloids‘ (crystalloids) was De Bary (1871). More recently, the
crystalline Nature of the wax of many species has been verified by X-ray powder
Diffractograms and electron diffraction (Reynhardt, 1997; Reynhardt and Riederer,
1988, 1994; Meusel et al., 1994, 1999, 2000).
       The epicuticular waxes had often been considered as a potential character for
chemotaxonomy of the plants in general (Evans et al., 1975; Griffiths et al., 1999;
Tsydendambaev et al., 2004).


1.9.3.2. Solanum Wax
Compounds found in the Epicuticular waxes (Osske and Schreiber, 1965; Jewers et
al., 1969; Croteau and Fagerson, 1971; Streibl et al., 1974; Meusel et al., 2000)
include:
    Hydrocarbons                 Ketones                  Aldehydes
    Fatty acids                  Alcohols                 Esters
    Sterols                      Flavonoids               terpenes


       Hanna et al. (1996), although, had reported the presence of some fatty acids
such as Palmitic, Stearic, Linolenic acids and Squalene without specifying the taxon
of S. nigrum Complex, yet no detailed chemotaxonomic study based upon chemical
constituents of epicuticular waxes of the complex has been undertaken.


1.9.3.3. Uses of Epicuticular Waxes
       Epicuticular waxes form the outermost boundary layer of the plant,
representing a multifunctional interface between plant and environment. A major
function is to serve as a barrier against uncontrolled water loss (Schönherr, 1976,
1982). In some cases waxes cause an increase in the reflection of solar radiation
(Barnes and Cardoso-Vilhena, 1996; Holmes and Keiller, 2002).




                                                                                  30
    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

1.10. BIOLOGICAL EVALUATION
1.10.1. ANTIMICROBIALS
       An antimicrobial is a substance that kills or inhibits the growth of
microorganisms whereas Antimicrobial drugs either kill microbes (microbicidal) or
prevent the growth of microbes (microbistatic). Antibiotics are now considered to be
the specific chemical products or the derivatives of such products often produced
from the microorganisms and having an inhibitory action against certain kind of
microorganisms (Nandkarni, 1954). In today's common usage, the term antibiotic is
used to refer to almost any drug that cures a bacterial infection.
       The history of antimicrobials begins with the observations of Pasteur and
Joubert, who discovered that one type of bacteria could prevent the growth of
another. They did not know at that time that the reason one bacterium failed to grow
was that the other bacterium was producing an antibiotic. There are several plants
that are the source of antibiotics medicine, which are successively used against
several diseases caused by bacteria and fungi. Antimicrobials include not just
antibiotics, but synthetically formed compounds as well. The discovery of
antimicrobials like penicillin and tetracycline paved the way for better health for
millions around the world. Before 1941, the year penicillin was discovered, no true
cure for gonorrhea, strep throat, or pneumonia existed. Patients with infected wounds
often had to have a wounded limb removed, or face death from infection. Now, most
of these infections can be easily cured with a short course of antimicrobials.
Main classes of antimicrobials include:
    Antibiotics
    Antivirals
    Antifungals
    Antiparasitics
    Non-pharmaceutical antimicrobials
       However, the future effectiveness of antimicrobial therapy is somewhat in
doubt. Microorganisms, especially bacteria, are becoming resistant to more and more
antimicrobial agents. Bacteria found in hospitals appear to be especially resilient, and
are causing increasing difficulty for the sickest patients–those in the hospital.
Currently, bacterial resistance is combated by the discovery of new drugs. However,
microorganisms are becoming resistant more quickly than new drugs are being



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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

found. Thus, future research in antimicrobial therapy may focus on finding how to
overcome resistance to antimicrobials, or how to treat infections with alternative
means.
   When people from the remote communities get an infectious disease, they are
usually treated by traditional healers because of their expertise in such procedures as
making diagnoses, treating wounds, setting bones and making herbal medicines.
Traditional healers claim that their medicine is cheaper and more effective than
modern medicine. Patients of these communities have a reduced risk to get infectious
diseases from resistant pathogens than people from urban areas treated with
traditional antibiotics. However, if they are treated in a hospital the chance of
contracting a nosocomial infection is increased (Ospina et al., 2002). One way to
prevent antibiotic resistance of pathogenic species is by using new compounds that
are not based on existing synthetic antimicrobial agents (Shah, 2005). Traditional
healers claim that some medicinal plants such as bixa spp. and bidens spp. are more
efficient to treat infectious diseases than synthetic antibiotics. It is necessary to
evaluate, in a scientific base, the potential use of folk medicine for the treatment of
infectious diseases produced by common pathogens. Medicinal plants might
represent an alternative treatment in non-severe cases of infectious diseases. They
can also be a possible source for new potent antibiotics to which pathogen strains are
not resistant (Fabricant and Farnsworth, 2001).


1.10.2. ANTIOXIDANTS
         Antioxidants are substances that may protect our cells against the effects of
free radicals. Free radicals are molecules produced when our body breaks down food
in oxidation reactions, or by environmental exposures like tobacco smoke and
radiation. Chain reactions are stated that damage cells and may play a role in heart
disease, cancer and other diseases (Medline plus, 2009). Antioxidants terminate these
chain reactions by removing free radical intermediates, and inhibit other oxidation
reactions by being oxidized themselves. As a result, antioxidants are often reducing
agents such as thiols, ascorbic acid or polyphenols (Sies, 1997).
         Although oxidation reactions are crucial for life, they can also be damaging;
hence, plants and animals maintain complex systems of multiple types of
antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such
as catalase, superoxide dismutase and various peroxidases. Low levels of

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

antioxidants, or inhibition of the antioxidant enzymes, causes oxidative stress and
may damage or kill cells. While the body has its defenses against oxidative stress,
these defenses are thought to become less effective with aging as oxidative stress
becomes greater. Research suggests there is involvement of the resulting free radicals
in a number of degenerative diseases associated with aging, such as cancer,
cardiovascular disease, cognitive impairment, Alzheimer‘s disease, immune
dysfunction, cataracts, and macular degeneration. Certain conditions, such as chronic
diseases and aging, can tip the balance in favor of free radical formation, which can
contribute to ill effects on health.
        Consumption of antioxidants is thought to provide protection against
oxidative damage and contribute positive health benefits. For example, the
carotenoids Lutein and Zeaxanthin engage in antioxidant activities that have been
shown to increase macular pigment density in the eye. Whether this will prevent or
reverse the progression of macular degeneration remains to be determined. An
increasing body of evidence suggests beneficial effects of the antioxidants present in
grapes, cocoa, blueberries, and teas on cardiovascular health, Alzheimer‘s disease,
and even reduction of the risk of some cancers (Allemann and Baumann, 2008).
        Antioxidants are present in foods as vitamins, minerals and carotenoids
among others. Many antioxidants are often identified in food by their distinctive
colors—the deep red of cherries and of tomatoes; the orange of carrots; the yellow of
corn, mangos, and saffron; and the blue-purple of blueberries, blackberries, and
grapes. The most well-known components of food with antioxidant activities are
vitamins A, C, and E; β-carotene; the mineral selenium; and more recently, the
compound lycopene (Allemann and Baumann, 2008).
        The increasing interest in naturally occurring antioxidants (polyphenols,
vitamins) is attributed to their capability of scavenging free radicals that are formed
in various biochemical processes. The reactive oxygen species like superoxide anion,
hydrogen peroxide, and hydroxyl radicals cause an extensive oxidative damage to
biomolecules such as nucleic acids, proteins, and lipids. These highly unstable
radicals have been found to be related to oxidative stress-related diseases like
cardiovascular diseases, cancer, inflammatory disorders, neurological degeneration
(Parkinson's and Alzheimer's diseases), premature ageing, etc.
        Polyphenols (cinnamic acid derivatives, flavonols, anthocyanins) and
vitamins are present in vegetables, fruits, berries, and herbs, which are the main

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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION

source of natural antioxidants in our daily diet. The basic structure of polyphenols is
composed of one or more phenolic rings that are substituted with several hydroxyl
groups and these are highly correlated with their strong antioxidant activity.
Vitamins are structurally a heterogeneous group of compounds, which are essential
in the diet for the maintenance of healthy growth and development. In general,
vitamins are divided into two main categories, fat and water-soluble ones.
       Among vegetables, tomato (Solanum lycopersicum), eggplant (Solanum
melongena), chilli pepper (Capsicum annuum), and potato (Solanum tuberosum),
which belong to the Solanaceae family, are important for their richness in healthy
components due to which they are also widely consumed. Tomato is rich in phenolic
compounds (flavonoids, flavones, cinnamic acid derivatives), phytoalexins, protease
inhibitors, glycoalkaloids, and carotenoids, but especially in lycopene and [beta]-
carotene. In addition, vitamins C, E, and A have been determined in tomato. The
main polyphenols found in eggplant are phenolic acids (chlorogenic acid, caffeic
acid, p-coumaric p acid), but this vegetable is poor in provitamin A and vitamin E.
However, the presence of vitamins C and B in eggplant has been established. It is
also rich in anthocyanins like nasunin and delphinidin conjugates. Chilli pepper has
been reported to contain flavones (luteolin, quercetin), flavonols (myricetin,
quercetin), and capsaicinoids. Of phenolic compounds, chlorogenic and caffeic acid,
catechins, and also glycoalkaloids have been reported to be the main compounds
present in potato. Vitamin C has been also determined in potato (Sies, 1997).
       In the late 19th and early 20th century, extensive study was devoted to the
uses of antioxidants in important industrial processes, such as the prevention of metal
corrosion, the vulcanization of rubber, and the polymerization of fuels in the fouling
of internal combustion engines (Matill, 1947).
       Early research on the role of antioxidants in biology focused on their use in
preventing the oxidation of unsaturated fats, which is the cause of rancidity.
Antioxidant activity could be measured simply by placing the fat in a closed
container with oxygen and measuring the rate of oxygen consumption. However, it
was the identification of vitamins A, C, and E as antioxidants that revolutionized the
field and led to the realization of the importance of antioxidants in the biochemistry
of living organisms (Jacob, 1996; Knight, 1998).




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION




1.11. AIMS AND OBJECTIVES


       There are various approaches to the taxonomic studies of the plants based on
the structural, cytological and chemical constituents. The ancient classification of the
plants was mainly carried out on the comparative morphological and anatomical
concepts of the natural plant flora. However with the rapid progress in the isolation,
purification, identification, elucidation of structure and the configuration of natural
plant products, the phytochemists and ethnobotanists believe that it is possible to
characterize and classify the plants on the basis of their chemical constituents. The
chemical constituents are formed within the plants by definite biosynthetic pathways
aided by the specific enzymes.


       Linnaeus classified S. nigrum in 2 varieties viz.: S. nigrum Var. nigrum (with
black berries) and S. nigrum Var. villosum (with orange red berries). But the other
taxonomists like Lamarck and Miller classified each of these as a separate species
(Edmonds and Chweya, 1997). Since there is a controversy to assign S. villosum a
separate species of the genus Solanum or a variety of S. nigrum, therefore it needed
further clarification. S. nigrum is now named as S. nigrum Complex because it is
composed of about 30 morphologically different taxa Meanwhile, in addition to the
above mentioned two, three more morphological variants of S. nigrum viz.: S.
americanum, S. chenopodioides and S. retroflexum were reported in the Botanic
Garden of GC University Lahore, Pakistan. So the present study was designed on the
chemotaxonomic evaluation of all these five taxa of S. nigrum Complex found in
Pakistan. The purpose was to confirm their taxonomic status on the basis of their
secondary metabolites, as they are more or less morphologically similar.




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties
Chap.1: INTRODUCTION


       So far there had been very little and insufficient chemical analysis reported
with respect to the three lately mentioned taxa in literature. Hence our specific aims
and objectives for these studies were:


 [1]. Phytochemical analysis to study the profiles of secondary metabolites in the
     five locally available taxa;
 [2]. Chemotaxonomic studies of all these taxa in order to eliminate/reduce the
     criticism/confusion on their taxonomic status;
 [3]. And finally the ethnopharmacological studies of the biologically active
     components of these taxa.




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    Chemotaxonomical Characterization of Solanum nigrum and its Varieties

								
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