PROJECT PROPOSAL TOPIC: Morphological analysis, phytochemical analysis and Silica Gel Chromatographic Study of phenolic compounds in Vegetable African Nightshades. BY Abu, Richard A. UR201400186 DEPARTMENT OF BIOLOGICAL SCIENCES FEDERAL UNIVERSITY, WUKARI SUPERVISED BY MR. EKONG, N.J EVALUATION OF PHYLOGENETIC RELATIONSHIP THAT EXIST AMONG SELECTED AFRICAN NIGHTSHADES ( scabrum Mill., L. and Solanum villosum Mill.).

INTRODUCTION

African indigenous vegetables (AIVs) are important nutrient-rich foods consumed locally and in the sub-Saharan Africa region, with many also utilized for their medicinal properties (Keding G. et al 2007). Such AIVs, also called traditional

African vegetables, are collected from the wild or cultivated to a limited extent and consumed or marketed, serving as an important income generating opportunity for the typical small-scale farmer, especially in such economically limited regions

(Weinberg K. et al 2004). Adapted to the local environment, AIVs often provide more sustainable production than exotic or introduced crops such as European vegetables (Mal B. 2007). Efforts are being made to increase the farming and marketing of AIVs in an attempt to alleviate hunger and improve nutrition, and to increase farmer’s income, improving the local and regional economy (Mal B.

2007).

African nightshades are among the most popular and as such high priority African traditional vegetables. They represent a wide group of botanically and genetically related belonging to approximately 30 species in the Solanum genus of the

Solanaceae family, and are diversely referred to as garden huckleberries, vegetable nightshades, edible nightshades, garden nightshades, common nightshades, ‘S. nigrum complex’, or ‘S. nigrum’ and related species (Yang R-Y et al 2013).

Despite their frequently reported nutritional attributes, Solanum species are also well known to contain toxic alkaloids, such as glycosides of solasodine and solanidine (Milner SE et al 2011). This safety concern is associated with the edible

African Solanum nightshade species, as these compounds are known to be present in the fruits (Carle R 1981) and have limited the promotion of their cultivation and marketing.

African nightshades are several species of plants in the section Solanum of the genus Solanum that are commonly consumed as leafy vegetables and herbs (Gaya,

A. S.; et al 2007). African nightshades are grown in both high and lowland areas in

West and East Africa, particularly in Nigeria and Cameroon. There is a large variation in diversity of the African nightshades, which have many nutritional and medicinal benefits, (Abukutsta-Onyango et al., 2013) even though the family of nightshade is commonly known as comprising dangerous weeds or poisonous plants. Species known as African nightshade include Solanum scabrum , Solanum villosum , Solanum nigrum , and Solanum americanum ( Drescher, A. W et al

2009). Other common names for African nightshade are Black nightshade and

Narrow-leaved nightshade. (Biovision 2018) Local names of African nightshade include mnavu ( Swahili ), managu ( Kisii), namasaka ( Luhya ), osuga ( Luo ), isoiyot ( Kipsigis ), kitulu ( Kamba), ormomoi ( Maa ), ndunda ( Taita ), and nsugga ( Luganda ) (Biovision 2018). African nightshade is an erect dicot with many branches, growing 0.5 to 1.0 m high. [4] The has thin, oval leaves which are ~15 cm in length and purplish in colour. The plant has numerous flowers that are black or purple and round berries, which are about 0.75 cm in diameter, having small, flat, yellowish seeds. The berries of this plant can be black or orange, depending on the species. (Biovision

2018) There are many diversities in African nightshades related to growth patterns, leaf sizes, tastes (bitterness) flowering time, colour, as well as nutritional and nutraceutical value, along with quantities and composition of anti-nutrient factors

(Gaya A.S et al 2007)

Historically incorrect nomenclature, due to phenotypic plasticity, has created confusion in the of African nightshades. For instance, S. scabrum and S. nigrum are not clearly distinguished in different parts of the world (Olet, Heun, &

Lye, 2005), and S. nodiflorum is used interchangeably with S. americanum in

African accessions (Manoko, van den Berg, Feron, van der Weerden, & Mariani,

2007) by both consumers and taxonomists. Identification and classification of new and previously described taxa is still being undertaken (Manoko, van der Weerden, van den Berg, & Mariani, 2012), and many more taxa remain unexplored. Key research tools for studying the genetic diversity of plants include morphological traits, biochemical analysis, and molecular markers (Xiang, 2000). In particular, molecular markers promise to be an effective tool in resolving the taxonomic relationships within the S. nigrum complex. This review highlights recent research on the genetic diversity of African nightshades, their cytological variability and biochemical composition, in line with important fundamentals in crop variety development.

The taxonomic and nomenclature of African nightshades is complex due to extensive synonymy, frequent occurance of spontaneous inter-specific hybrids, existence of polyploidy series, phenotypic plasticity, inconsistent use of many local names and discordant genetic variation (Edmond and Chweya 1997).

Taxonomic complexity associated with African nightshades has led to considerable confusion regarding the identification of popular nightshades vegetables. For example, most communities where these plants are cultivated and or consumed call them by a single name and grow more than one species together without any knowledge of their morphological differences.

In light of these, the current study will sought to investigate the taxonomic relationship using the silica gel chromatography as well as the phytochemical and physiochemical constituents of three species in the nightshades group of the genera solanum (Solanoceae).

STATEMENT OF THE PROBLEM Africa is richly endowed with plant genetic resources, with many well-adapted indigenous food crops that have long been grown on the continent. These crops play an important role in the food security of many resource poor farming families, and have potential value as a genetic resource for the global community [1, 2].

Hence it is sad that African researchers, policy-makers and farmers have neglected the potential of these crops in reducing food insecurity and poverty. Leafy vegetables, including several African Indigenous Vegetables (AIVs), are highly valued in the typical African diet as accompaniment to carbohydrate-based staples

[3, 4].

The morphology, taxonomy and nomenclature of African nightshades is complex due to extensive synonymy, frequent occurrence of spontaneous inter-specific hybrids, existence of polyploidy series, phenotypic plasticity, inconsistent use of many local names and discordant genetic variation (Edmond and Chweya 1997).

Taxonomic complexity associated with African nightshades has led to considerable confusion regarding the identification of popular nightshades vegetables. For example, most communities where these plants are cultivated and or consumed call them by a single name and grow more than one species together without any knowledge of their morphological differences. In light of this, the current study will sought to investigate the taxonomic relationship using the silica gel chromatography as well as the morphological and phytochemical constituents of three species in the nightshades group of the genera solanum (Solanoceae).

AIMS AND OBJECTIVES

The aim of this study is to analyze the morphological parameters, phytochemical constituents and silica gel chromatographic study of African Nightshades (S. scabrum Mill.; Solanum nigrum L.; and Solanum villosum Mill.). The specific objectives of this research are;

 To determine the morphological parameters of the selected species of the

nightshades group of (Solanum scabrum Mill.; Solanum nigrum

L.; and Solanum villosum Mill.)

 To analyze the phytochemical constituents present in the selected species of

the nightshades group of solanum section Solanaceae (Solanum scabrum

Mill.; Solanum nigrum L.; and Solanum villosum Mill.)

 To determine the taxonomic relationship between the selected species using

Silica Gel chromatography study.

 To help unwind complexities that exist among members of the nightshades

groups of Solanum section solanaceae. JUSTIFICATION

The findings from this research will provide information on the taxonomic relationships that exist among African nightshades, provide information on the phytochemicals contained in the plants and also educate cultivators and consumers in the area on the differences that exist among the plants and possible importance and risk associated with the consumption of the plants.

MATERIALS AND METHODS

Study Area

Plant material

The plant material, Solanum scabrum, Solanum nigrum, Solanum villosum were collected from Obudu Local Govt. Area of Cross River State between latitude

6033’N and 904’E, Kurumi Local Govt. Area of Taraba State between latitude

7050’N and 9046’E. Fresh specimen were collected and pressed for taxonomic identification while others were air dried at room temperature for extraction purposes and other studies, the fruits were also collected in field fresh and seeds extracted for planting.

Phytochemical screening Phytochemical examinations were carried out for all the extracts as per the standard methods.

1. Detection of alkaloids: Extracts were dissolved individually in dilute

Hydrochloric acid and filtered.

Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric

Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.

Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium

Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids.

Dragendroff’s Test: Filtrates were treated with Dragendroff’s reagent (solution of

Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids.

2. Detection of carbohydrates: Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates.

Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates. Benedict’s test: Filtrates were treated with Benedict’s reagent and heated gently.

Orange red precipitate indicates the presence of reducing sugars.

Fehling’s Test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars.

3. Detection of glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides.

Modified Borntrager’s Test: Extracts were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides.

Legal’s Test: Extracts were treated with sodium nitropruside in pyridine and sodium hydroxide. Formation of pink to blood red colour indicates the presence of cardiac glycosides.

4. Detection of saponins Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

Foam Test: 0.5g of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins.

5. Detection of phytosterols

Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand.

Appearance of golden yellow colour indicates the presence of triterpenes.

Libermann Burchard’s test: Extracts were treated with chloroform and filtered.

The filtrates were treated with few drops of acetic anhydride, boiled and cooled.

Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.

6. Detection of phenols

Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

7. Detection of tannins Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins.

8. Detection of flavonoids

Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

Lead acetate Test: Extracts were treated with few drops of lead acetate solution.

Formation of yellow colour precipitate indicates the presence of flavonoids.

9. Detection of proteins and aminoacids

Xanthoproteic Test: The extracts were treated with few drops of conc. Nitric acid.

Formation of yellow colour indicates the presence of proteins.

10. Detection of diterpenes

Copper acetate Test: Extracts were dissolved in water and treated with 3-4 drops of copper acetate solution. Formation of emerald green colour indicates the presence of diterpenes Ninhydrin Test: To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid.

Silica Gel Chromatographic study

A chromatographic plate was prepared with silica gel. 0.1 ml methanolic extract was applied at the starting point of the plate. It was then dipped in the solvent TCA

(toluene-chloroform-acetone) and allowed to develop chromatogram. The chromatogram was first treated with ammonia vapour, then with iodine vapour and finally with 1% leads acetate as recommended by Block et al. (1953) to distinguish the spots. Ammonia vapour gave distinct colour under visible and UV light in case of some phenolic spots. The spots of other phenolic compounds became apparent after treatment with iodine vapour and lead acetate. The visible spots were traced on a transparent paper. The RF (relative distance) of each spot was used as a basis for comparison and specification of various phenolic compounds obtained. On the basis of colour and position, spots assumed to be identical in two or more species were assigned the same number. The chromatographic results were subjected to numerical taxonomic treatment as an aid to establish phenolic relationship in the selected species of the Nightshades group of Solanum section Solanoceae.

Analysis of phytochemical data The method adopted by Ellison et al. (1962) was followed to make the suitable comparisons in the form of qualitative relationships. Species were compared on the basis of their biochemical affinities. Values of paired affinity (PA), group affinity

(GA) and Isolation value (IV) was calculated as follows:

GA = Total PA +100

LITERATURE

AFRICAN NIGHTSHADES

African nightshades are several species of plants in the section Solanum of the family Solanaceae that are commonly consumed as leafy vegetables and herbs

(Gaya, A.S et al 2007). African nightshades are grown in both high and lowland areas in West and East Africa , particularly in Nigeria and Cameroon . There is a large variation in diversity of the African nightshades, which have many nutritional and medicinal benefits (Abukusta-Onyango, M.O et al 2013), even though the family of nightshade is commonly known as comprising dangerous weeds or poisonous plants . Species known as African nightshade include Solanum scabrum

, Solanum villosum , Solanum nigrum , and Solanum americanum (Drescher, A.W et al 2009).

African nightshades comprise several species of the genus Solanum in the section

Solanum, also referred to as the Solanum nigrum complex. The genus Solanum is the largest and most diverse genus in the family

Solanaceae, and species belonging to African nightshades have been assigned to the section Solanum. They are mostly used as leafy vegetables and herbs in most parts of West and East Africa (Ojiewo, C.O et al 2013). The most commonly grown species are S. scabrum, S. villosum, and S. americanum were –preferences differ with different regions. Unlike other major vegetable crops in the genus

Solanum, such as potato, tomato, and eggplant,

African nightshades are not widely utilised outside West and East Africa, as they are considered poisonous, or weeds, in many parts of the world. With the realisation of their rich nutritional value, their low input requirements for growth and cultivation, and their potential in nutrition security, they are emerging as an important food crop in sub-Saharan Africa to generate secure income, primarily for resource poor, rural and peri-urban, mainly female populations (Poczai &

Hyvönen, 2011)(Weinberger, K. et al 2011). Their cultivation has therefore been promoted by governmental and non-governmental institutions in the last few years.

Although some cultivars were developed by public and private seed companies, working in collaboration with research institutions, most farmers rely on low- quality seed lots from local markets, or self-harvested seeds, leading to low and varying yields. Therefore, vegetable nightshades have attracted the interest of the research community, and are among the top priority African indigenous vegetables identified for cultivar improvement and development to increase their market share.

Solanum villosum Mill.

Vernacular names

Red-fruited nightshade, hairy nightshade (En). Morelle jaune (Fr). Mnavu (Sw). Origin and geographic distribution

Solanum 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. in Central Africa from

Burundi, in East Africa from Sudan, Ethiopia, Somalia, Kenya, Uganda and

Tanzania, and in southern Africa from Zambia and Angola. In West Africa

Solanum villosum has been recorded only from Nigeria. However, its distribution is incompletely known, and it may occur in many other countries. Its use as vegetable is most popular in East Africa.

Uses

Leaves and young shoots of sparsely hairy types of Solanum villosum are used as a leafy vegetable. The young leaves are boiled with water and are sometimes fried.

In Tanzania young shoots and leaves are picked, chopped and then boiled or, especially in urban areas, fried with onions and tomatoes and sometimes mixed with meat or fish. In the Mara region in Tanzania whole young plants are used as a vegetable. In Kenya the Luo and Pokot people prepare this vegetable together with less bitter vegetables such as amaranths, whereas other cultural groups mix it with meat, spider plant ( Cleome gynandra L.), bitter leaf (Vernonia spp.) or cowpea leaves. Nandi women in Eldoret, Kenya use milk to boil the vegetable. In Uganda the young shoots and leaves are mixed with groundnut paste, wrapped in banana leaves and steamed. Other people prefer to boil the vegetable, drain off the remaining water and add sesame ( Sesamum indicum L.) to make it a thick and more tasty sauce. In some instances, it is chopped, washed and fried either directly or after boiling it in sour milk. Groundnuts or sesame paste can be added to this preparation.

The ripe fruits are eaten in Ethiopia, Kenya, Uganda and Tanzania, where orange, yellow and red fruited types are found. Solanum villosum also forms an important part of traditional medicine in Africa. In Kenya, unripe fruits are used to soothe toothache. They are also squeezed on babies’ gums to ease pain during teething.

Leaves are used to treat stomachache and extracts from leaves and fruits are used to treat tonsillitis. Maasai boil the roots in milk and give it to children as a tonic. In

Tanzania, Sukuma people apply leaves to swellings, whereas the fruit juice is used to calm sore eyes. Banyankore and Banyoro people in Uganda believe that addition of Solanum villosum leaves to the diet contributes to the treatment of fever associated with hypertension. Pregnant women in most parts of Kenya are encouraged to eat boiled Solanum leaves; people believe that they will then give birth to dark-eyed and smooth-skinned babies. It is further believed that children who eat Solanum vegetables cooked with milk, groundnuts or sesame rarely develop marasmus or kwashiorkor. Leaves of Solanum villosum are used as fodder for goats and sheep in Sudan, and for cattle and goats in Kenya.

Production and international trade

Properties

The composition of Solanum villosum leaves is probably comparable with that of other dark green leafy vegetables. Two alkaloids have been isolated from green fruits of Solanum villosum, diosgenin and solasodine, but the amount of alkaloids is lower than in Solanum americanum Mill.

Description

Annual or short-lived perennial herb up to 50(–60) cm tall, much branched, unarmed; stem rounded to angled, almost glabrous to pubescent with appressed hairs. Leaves arranged spirally, simple; stipules absent; petiole 0.5–1 cm long; blade ovate to elliptical, up to 4(–8) cm × 3(–6) cm, cuneate at base and decurrent along the petiole, acute at apex, entire to sinuately or coarsely toothed, sparsely pubescent. Inflorescence an extra-axillary, umbel-like cyme, 3–5(–7)-flowered; peduncle 4–7 mm long, elongating up to 2 cm in fruit. Flowers bisexual, regular, 5- merous; pedicel 4–6(–10) mm long, becoming deflexed in fruit; calyx cup-shaped,

1–2 mm in diameter, lobes obtuse to acute or acuminate, deflexed in fruit; corolla stellate, (4–)5–8(–10) mm in diameter, white with basal yellow-green star, lobes ovate-oblong, c. 3 mm long; stamens inserted on corolla throat, filaments 2 mm long, with hairs below, anthers connivent, 1.5–2.5 mm long, opening by terminal pores; ovary superior, globose, c. 1.5 mm in diameter, style (3–)4–5 mm long, hairy at base, stigma capitate, pale green. Fruit a globose berry 6–8(–10) mm in diameter, red, orange or yellow when ripe, many-seeded. Seeds discoid, c. 1 mm long. Seedling with epigeal germination.

Other botanical information

Solanum villosum belongs to the subgenus Solanum and section Solanum, formerly known as section Maurella , or section or subsection Morella . Currently about 30 species are included in this section, of which 10–12 are known to occur in Africa.

Research is still needed to better understand the species and their diversity within section Solanum. In herbaria in Africa several members of the section are lumped under Solanum nigrum. Solanum villosum has yellow, orange or red fruits whereas

Solanum nigrum L. has black or greenish fruits when ripe. Several botanists erroneously grouped taxa with black fruits together with yellow to red-fruited taxa in Solanum nigrum. Two subspecies of Solanum villosum have been distinguished: subsp. villosum and subsp. miniatum (Bernh. ex Willd.) Edmonds, based on hair density and presence or absence of glands on the hairs and whether the stem is rounded and smooth or angled with toothed ridges. The less hairy subsp. miniatum is preferred as a vegetable. However, many authors do not recognize these subspecies.

Growth and development

Under optimum conditions of moisture and temperature, the seeds of Solanum villosum germinate within seven days. Growth of seedlings is fast and flowering starts after 5–8 weeks. Under stress, flowering can start even earlier. Vegetative growth slows down with flowering as a result of competition. Solanum villosum is self-pollinating and self-compatible and sets fruit easily under favourable environmental conditions. It continues flowering even when it has started fruit set, resulting in plants bearing mature fruits on the lower branches, young ones in the middle and flowers in the top part. Fruits remain on the plant and drop only when over-ripe. They are attractive to birds, rodents, lizards and rabbits, but also cattle and even humans are partly responsible for seed dispersal. Seeds can pass through the digestive system of animals without being damaged.

Ecology

Solanum villosum occurs from sea-level to about 2400 m altitude, but it does not tolerate night frost. The optimum temperature is probably between 20–30°C. It performs well during the rainy season or when irrigated regularly, and is not resistant to drought. An annual rainfall of 500–1200 mm is suitable. Solanum villosum 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, it is found in disturbed areas and along the edges of agricultural fields.

Propagation and planting

Solanum villosum is mainly propagated by seed. The 1000-seed weight is about

1.0 g. Stem cuttings have also been used for propagation. Farmers collect seed from their farm or buy it at the market. Seeds from fresh fruits that have been carefully dried germinate well. In home gardens direct sowing is common; commercial farmers often use nurseries. In nurseries the seed is mixed with sand or ash, or sometimes both, to facilitate uniform sowing. The beds are prepared by loosening the soil by hand hoe after application of decomposed manure. The beds are covered with grasses which are burnt to sterilize the soil. This also adds potash to the soil. Seed is broadcast or sown in furrows 15–20 cm apart. The seeds are covered with a thin layer of soil, but sometimes farmers prefer to leave them open so that they germinate earlier. Especially during the dry season, the beds are covered with tall grasses to maintain soil moisture. The grass is removed when the seedlings are about 3 cm tall. Nurseries require watering twice a day and careful weeding. Pest management is also important at this stage. Seedlings are transplanted when they have 6 true leaves into well-prepared and irrigated fields at a spacing of 25–30 cm × 30–40 cm. Solanum villosum is sometimes intercropped with other crops, e.g. maize. In this case, the seeds are direct sown, normally 3–10 per hole. If the plants have enough space, direct sowing results in taller plants with larger leaves and branches than a transplanted crop, thus producing more dry matter. For once-over harvesting, usually done when enough land is available for raising several successive crops, a dense spacing of 10 cm × 10 cm is practiced.

Solanum scabrum Mill.

Vernacular names

African nightshade, black nightshade, garden huckleberry (En). Morelle de Guinée, morelle noire (Fr). Erva moura (Po). Mnavu (Sw).

Origin and geographic distribution

Solanum scabrum occurs as a cultivated vegetable from Liberia to Ethiopia, and south to Mozambique and South Africa. It is very common in lowland as well as highland regions in West and East Africa. It is also reported from Réunion and may well occur on other Indian Ocean islands, where its status needs to be confirmed. The wide range of diversity of Solanum scabrum found especially in

Nigeria and Cameroon suggests that its origin is likely to be in the warm humid forest belt of West and Central Africa. Outside Africa, Solanum scabrum can be found in Europe, Asia (India, China and the Philippines), Australia, New Zealand,

North America and the Caribbean.

Uses

Leaves and fresh shoots of Solanum scabrum are widely used as a cooked vegetable. They are served with corn ‘fufu’, plantains, sweet potatoes, potatoes, yams, maize or pounded cocoyams.

Solanum scabrum is popular in Côte d’Ivoire (known as ‘fouet’), Benin

(‘ogomoh’), Nigeria (‘ogunmo’ or ‘odu’) and Cameroon (‘osan’ or ‘zom’). As it has a bitter taste, some people prefer not to use salt. Contrary to what is reported in older literature, fruits of Solanum scabrum are not eaten in Africa. Reports on its edible fruit from South Africa probably refer to

Solanum retroflexum Dunal, and from North America, Asia, Australia and New

Zealand refer to types or cultivar-groups that do not occur in Africa. In south- western Nigeria the inflorescence with buds, flowers and small fruits is normally removed before cooking; it can be very bitter in taste but this is appreciated by elderly people who may add them to their soup. Bitterness is reduced by discarding the cooking water and replacing it with fresh water. The cooking water may be very dark, which is not appreciated. Some people add milk or salt to further reduce the bitterness. Solanum scabrum is widely used as medicinal plant. Leaf extracts are used to treat diarrhoea in children and certain eye infections and jaundice. In East Africa the raw fruit is chewed and swallowed to treat stomach ulcers or stomach-ache.

Infusions of leaves and seeds are rubbed onto the gums of children who have developed crooked teeth. In the literature many other medicinal uses for Solanum species with black fruits have been recorded, but it is not likely that these refer to

Solanum scabrum .

Solanum scabrum is used as fodder for cattle and goats. Both the leaves and fruits are a source of dyes. The anthocyanin pigments in the purple to black fruits are used as a dye or as a kind of ink.

Properties

The composition of 100 g edible portion of African nightshade leaves is: water

87.8 g, energy 163 kJ (39 kcal), protein 3.2 g, fat 1.0 g, carbohydrate 6.4 g, fibre

2.2 g, Ca 200 mg, P 54 mg, Fe 0.3 mg, β-carotene 3.7 mg, ascorbic acid 24 mg

(Leung, W.-T.W., Busson, F. & Jardin, C., 1968). The dry matter content varies greatly, from 6–18 % depending on plant age, soil moisture and fertilizing. The protein is rich in methionine.

Green fruits contain comparatively high amounts of the glycoalkaloid solanine and the less poisonous solanidine. The initial effect of solanine poisoning includes diarrhoea and vomiting, and frequent consumption of this compound may lead to accumulation in the liver, causing dizziness, mental confusion and loss of speech, and it can even result in blindness. The leaves contain only low levels of these alkaloids, which are probably associated with its bitter taste. Unfortunately, heating or frying will not reduce the toxic effects of solanine and solanidine. The acceptable limit for these alkaloids is 20 mg per 100 g fresh weight of the edible portion. Most research stations in Africa have no facilities to analyse these alkaloids and are thus not able to screen accessions for this important characteristic. The degree of bitterness is easier to establish, and research is currently ongoing to determine how the glycoalkaloids relate to bitterness.

Description

Annual or short-lived perennial herb, erect and widely spreading, up to 100(–150) cm tall, unarmed; stem rounded or narrowly winged with more or less toothed wings, glabrous or sparsely pubescent, young stem more or less pubescent with short, simple hairs. Leaves arranged spirally, sometimes almost opposite, simple; stipules absent; petiole 2–10 cm long; blade rhomboid to ovate-lanceolate, up to

4.5–22 cm × 3–16 cm, cuneate at base and decurrent along the petiole, acute to acuminate at apex, sometimes obtuse, entire to sinuate or slightly toothed, glabrous or sparsely pubescent. Inflorescence an extra-axillary, umbel-like cyme, 3–10(–

12)-flowered; peduncle 1–2.5 cm long, elongating up to 4 cm in fruit. Flowers bisexual, regular, 5-merous; pedicel 4–9 mm long, elongating to 12 mm in fruit, erect or nodding; calyx cup-shaped, 2–4.5 mm long, lobes triangular, becoming reflexed in fruit; corolla stellate, 7–16 mm in diameter, white or flushed purple with basal yellow-green star, lobes ovate-elliptical, 3–6 mm long; stamens inserted on corolla throat, filaments c. 1 mm long, with hairs on inner side, anthers connivent, 2–3 mm long, usually brown-yellow, opening by terminal pores; ovary superior, conical to ovoid, c. 1.5 mm long, style 3–4.5 mm long, hairy in the lower part, stigma capitate, pale green. Fruit a globose berry 10–16 mm in diameter, glossy deeply purple to purplish black at maturity, many-seeded. Seeds discoid, 2–

3 mm long, creamy coloured, often tinged with purple. Seedling with epigeal germination; hypocotyl 4–5 mm long; cotyledons leafy, elliptical, 4–6 mm × 2–3 mm.

Other botanical information

Solanum scabrum belongs to the subgenus Solanum and section Solanum, formerly known as section Maurella , or section or subsection Morella . Currently about 30 species are included in this section of which 10–12 are known to occur in Africa.

Research is still needed to better understand the species within section Solanum and their diversity. In Africa the name Solanum nigrum is often used for almost all species of section Solanum with blackish fruits, including Solanum scabrum . This confusion is probably aggravated by the use of vernacular names whereby one name can apply to several species, or several names to the same species. Solanum scabrum is often confused with Solanum americanum, but more slender stems, narrower leaves and smaller flowers and fruits distinguish the latter.

Growth and development

Seed germination can be problematic because of low vigour caused by improper seed extraction and therefore inadequate removal of sugars and germination inhibitors present in the fruit. Other causes of problematic germination are that seeds are not dried and stored properly, or that the seed is dormant. The seeds can remain viable for several years when kept dry and cool. After seed emergence, growth is fast. The first flowers appear 8–11 weeks after sowing. Flowering occurs earlier when the seeds are sown directly than when seedlings are transplanted. The plant continues to produce new flowers for several months. The flowers are mainly self-pollinated. Solanum scabrum has low levels of out-crossing, which is mainly done by honeybees, bumble bees and black syrphid flies.

Ecology

The optimum temperature for seed germination is 15–30°C and for growth it is 20–

30°C. Solanum scabrum grows from sea-level to well over 2000 m but does not tolerate night frost. The rainfall during the growing season should be at least 500 mm; it grows well under conditions with much higher rainfall but then becomes susceptible to leaf diseases. It prefers fertile soils, with high nitrogen content and rich in organic matter. Sandy loams to friable clay soils with a pH of 6.0–6.5 are suitable. The plants tolerate some shade, but grow better when exposed to full sun as long as they have adequate access to water.

Propagation and planting

Propagation of Solanum scabrum is by seed and, less commonly, by cuttings. Most farmers produce their own seed and some buy their seed or seedlings from specialized producers. For a subsistence crop seeds are sown directly at the beginning of the rainy season. There are about 1000 seeds per g. A few (3–10) seeds are used per hole when sown among other crops in an intercropping system.

The strongest plants are kept and the others removed as the first harvest or for transplanting. Direct sowing during the wet season results in taller plants and, when there is adequate room, in more and larger leaves and branches and higher dry matter content than with transplanting. Sowing in nurseries and transplanting is normally practised when the crop is cultivated commercially. The seed can be mixed with ash, sand, soil, or dry poultry manure before broadcasting to spread the fine seeds evenly. The nursery requires manure for a good emergence of seedlings.

Seeds are sown in lines 10–20 cm apart or seeds are broadcast. The soil of the nursery bed should be loosened to facilitate rooting. After sowing, the beds should be covered with a thin layer of soil, which also helps to prevent ants from carrying off the seeds. Sometimes the weed vegetation in the field is burnt to provide a layer of ash that is rich in nutrients, especially potash, and also to kill soil-borne pathogens and weeds. Transplanting takes place 4–6 weeks later, depending on prevailing temperatures, when the seedlings are at least 6–8 cm tall and have 5–6 true leaves, but are not more than 15 cm tall to avoid weak and thin plants. The seedlings are selected for their strength and freedom from diseases and planted late in the afternoon or early in the morning. Adequate water is needed just before and immediately after transplanting since roots are sensitive to drought. When propagation by cuttings is practised, cuttings of 20–30 cm long are taken from the main stem and are trimmed before they are inserted into the soil. The spacing is 40 cm × 40 cm or even 40 cm × 60 cm, considering that plants may reach 1 m in height (if not trimmed). The advantage of this propagation method is that the first harvest can start early (3–4 weeks after planting). However, the total yield is lower than from transplanted seedlings or from plants sown directly. Usually farmers use sole cropping. The spacing may differ, depending on cultivar and season. It is usually wider during the rainy season, when ventilation is required to reduce the incidence of diseases. Spacing is normally between 15–25 cm × 15–40 cm. A wider spacing is used when the crop is to be kept for a long period, encouraging stronger branches and an extended harvest period for which additional fertilizing is needed. Branching is stronger at a wider spacing, making up for the lower number of plants. Close planting is mainly used when the growing season is expected to be short or with once-over harvesting.

Solanum nigrum L.

Solanum nigrum ( European black nightshade ) is a species in the Solanum genus, native to Eurasia and introduced in the Americas , Australasia, and

South Africa . It is also known as black nightshade . Parts of this plant can be toxic to livestock and humans. Nonetheless, ripe berries and cooked leaves of edible strains are used as food in some locales, and plant parts are used as a traditional medicine . A tendency exists in literature to incorrectly refer to many of the other

"black nightshade" species as " Solanum nigrum ". [1] Solanum nigrum has been recorded from deposits of the Paleolithic and Mesolithic era of ancient Britain and it is suggested by the botanist and ecologist Edward Salisbury that it was part of the native flora there before Neolithic agriculture emerged. [2] The species was mentioned by Pliny the Elder in the first century AD and by the great herbalists , including Dioscorides . [3] In 1753, Carl Linnaeus described six varieties of Solanum nigrum in Species Plantarum . [4]

Description Black nightshade is a common herb or short-lived perennial shrub, found in many wooded areas, as well as disturbed habitats. It reaches a height of 30 to 120 cm (12 to 47 in), leaves 4.0 to 7.5 cm (1.6 to 3.0 in) long and 2 to 5 cm (1 to 2 in) wide; ovate to heart-shaped, with wavy or large-toothed edges; both surfaces hairy or hairless; petiole 1 to 3 cm (0.5 to 1 in) long with a winged upper portion. The flowers have greenish to whitish, recurved when aged and surround prominent bright yellow anthers. The berry is mostly 6 to 8 mm (0.24 to 0.31 in) in diam., dull black or purple-black. [5] In India, another strain is found with berries that turn red when ripe. [6] Sometimes S. nigrum is confused for the more toxic deadly nightshade , Atropa belladonna, in a different Solanaceae genus altogether.

A comparison of the fruit shows that the black nightshade berries grow in bunches, the deadly nightshade berries grow individually.

Taxonomy

The S. nigrum species is a highly variable taxon with many varieties and forms described. [7] The recognized subspecies are: [3]

1. S. nigrum L. subsp. nigrum — glabrous to slightly hairy with appressed non- glandular hairs

2. S. nigrum L. subsp. schultesii (Opiz) Wessley — densely hairy with patent, glandular hairs The Solanum nigrum complex — also known as

Solanum L. section Solanum — is the group of black nightshade species characterized by their lack of prickles and stellate hairs, their white flowers, and their green or black fruits arranged in an umbelliform fashion. [7] The Solanum species in this group can be taxonomically confused, more so by intermediate forms and hybridization between the species. [3] Some of the major species within the S. nigrum complex are: S. nigrum , S. americanum, S. douglasii, S. opacum , S. ptychanthum , S.retroflexum , S. sarrachoides , S. scabrum , and S. villosum .

Toxicity

Leaves, flowers and fruit of S. nigrum Solanine levels in S. nigrum can be toxic .

Children have died from poisoning after eating unripe berries. [8] However, the plant is rarely fatal, [9] with ripe berries causing symptoms of mild abdominal pains, vomiting, and diarrhea . [8]

Poisoning symptoms are typically delayed for 6 to 12 hours after ingestion. [10]

Initial symptoms of toxicity include fever , sweating, vomiting , abdominal pain, diarrhea, confusion, and drowsiness . [11] Death from ingesting large amounts of the plant results from cardiac arrhythmias and respiratory failure . [11] Livestock have also been poisoned from nitrate toxicity by grazing the leaves of S. nigrum . [3] All kinds of animals can be poisoned after ingesting nightshade, including cattle, sheep, poultry, and swine. [8] However, in central Spain, the great bustard ( Otis tarda ) may act as a seed disperser of European black nightshade ( Solanum nigrum ).

[12] Black nightshade is highly variable, and poisonous plant experts advise to avoid eating the berries unless they are a known edible strain.

[13] The toxin levels may also be affected by the plant's growing conditions. [3]

The toxins in S. nigrum are most concentrated in the unripe green berries, and immature fruit should be treated as toxic. [10][11][14] Most cases of suspected poisoning are due to consumption of leaves or unripe fruit.

There are ethnobotanical accounts of S. nigrum leaves and shoots being boiled as a vegetable with the cooking water being discarded and replaced several times to remove toxins. [3]

Uses

Ripe berries of the "Red Makoi" variety of S. nigrum are edible

Some of the uses ascribed to S. nigrum in literature may actually apply to other black nightshade species within the same species complex, and proper species identification is essential for food and medicinal uses (See

Taxonomy section). [1][7] S. nigrum has been widely used as a food since early times, and the fruit was recorded as a famine food in 15th-century China . [15] Despite toxicity issues with some forms, the ripe berries and boiled leaves of edible strains are eaten. The thoroughly boiled leaves — although strong and slightly bitter flavoured — are used like spinach as horta and in fataya pies and quiches. The ripe black berries are described as sweet and salty, with hints of liquorice and melon. [16]

In India, the berries are casually grown and eaten, but not cultivated for commercial use. In South India, the leaves and berries are routinely consumed as food after cooking with tamarind, onion, and cumin seeds. [17] The berries are referred to as "fragrant tomato". Although not very popular across much of its growing region, the fruit and dish are common in Tamil Nadu (in Tamil), [18]

Kerala, southern Andhra Pradesh, and southern Karnataka.

In Ghana , the unripe green berries are called kwaansusuaa or abedru , and are used in preparing various soups and stews, including the popular palm nut soup commonly eaten with banku or fufu '. [21]

In South Africa , the very ripe and hand-selected fruit ( nastergal in Afrikaans and umsobo in Zulu) is cooked into a beautiful but quite runny purple jam. [22]

Medicinal usage The plant has a long history of medicinal usage, dating back to ancient Greece. "...

In the fourteenth century, we hear of the plant under the name of Petty Morel being used for canker and with Horehound and wine taken for dropsy." [28] It was a traditional European medicine used as a strong sudorific, analgesic and sedative with powerful narcotic properties, but was considered a "somewhat dangerous remedy". [28][29] Internal use has fallen out of favor in Western herbalism due to its variable chemistry and toxicity, but it is used topically as a treatment for herpes zoster.

[30][31][32][33]

S. nigrum is an important ingredient in traditional Indian medicines. Infusions are used in dysentery, stomach complaints, and fever . [34] The juice of the plant is used on ulcers and other skin diseases. [34] The fruits are used as a tonic, laxative, appetite stimulant, and for treating asthma and "excessive thirst". [34] Traditionally the plant was used to treat tuberculosis. [35] It is known as peddakasha pandla koora in the Telangana region. This plant's leaves are used to treat mouth ulcers that happen during winter periods of Tamil Nadu, India. Apart from its use as a home remedy for mouth ulcers, is used in cooking like spinach. In North India, the boiled extracts of leaves and berries are also used to alleviate liver-related ailments, including jaundice. In Assam, the juice from its roots is used against asthma and whooping cough. [36] S. nigrum is a widely used plant in oriental medicine where it is considered to be antitumorigenic, antioxidant, anti-inflammatory, hepatoprotective, diuretic , and antipyretic. [37] Chinese experiments confirm that the plant inhibits growth of cervical carcinoma in mice. [38]

Solanum nigrum is known to contain solasodine (a steroidal glycoalkaloid that can be used to make 16-DPA progenitor); a possible commercial source could be via cultivating the hairy roots of this plant. [39][40]

Cultivation

Black nightshade is cultivated as a food crop on several continents, including

Africa and North America. The leaves of cultivated strains are eaten after cooking.

[16] A garden form with fruit 1.27 cm (0.50 in) diam. is occasionally cultivated.

[41]

Taxonomy and genetic diversity of African nightshades

African nightshades are angiosperms belonging to the Solanaceae family. They are herbaceous annual plants, growing up to a height of 1.5 m, in upright to decumbent growth habits. They show phenotypic plasticity, and variations are observed in different populations of the same species (Manoko et al., 2007). There have been a number of studies to resolve taxonomic complexities regarding this section in

Africa. Bukenya and Hall (1988) and Bukenya and Carasco (1995) made taxonomic studies of plant material from Ghana and Uganda, respectively, using herbarium material. They developed a key for the morphological description of the genus Solanum, following the classification of D’Arcy (1972). In their study, eight species belonging to the Solanum nigrum complex, namely S. nigrum L., S. americanum, S. scabrum Mill., S. sarrachoides Sendtn., S. villosum

Mill., S. grossedentatum A. Rich., S. florulentum Bitter, and S. tarderemotum

Bitter, were differentiated, based on descriptive morphological traits. In order to evaluate the genetic diversity and biogeographic relationships, multilocus markers have been used in different studies. Jacoby, Labuschagne, and Viljoen (2003) used amplified fragment length polymorphism (AFLP) to study the genetic relationships of eight species of African nightshades sampled in South Africa (Table 1). The study involved 14 accessions, which were analysed using three EcoR I/Mse I primer combinations, generating 359 markers, of which 222 (62%) were polymorphic. Binary data was used to generate a dendrogram using NCSS 2000

(Number Cruncher Statistical Systems), and dissimilarity was expressed as

Euclidean genetic distances. S. villosum formed a sub-cluster with S. retroflexum, with a dissimilarity coefficient of 0.51, while S. scabrum clustered separately with the diploid S. americanum, with a dissimilarity coefficient of 0.52. The phylogenetic grouping, however, did not provide information about the variability within species, since only one accession was used for every species, except for S. retroflexum, which showed low variability. Additionally, the authors did not apply any statistical test estimating the support of their clustering, for example using bootstrapping.

Dehmer and Hammer (2004) also used AFLP to study taxonomic status and geographic provenance from a gene bank collection involving 44 accessions representing five species from different origins. The two EcoR I/Mse I primer combinations used generated a total of 523 polymorphic bands. Genetic similarities, according to Dice, were used to construct a phenogram using the unweighted pair group with arithmetic mean method that generated four major clusters from the five species. The three accessions from S. physalifolium were nested within the S. nigrum and S. villosum clusters. In comparison to the other species examined, S. americanum was placed in a separate cluster, with only 43% genetic similarity to the other species, but also showed the lowest infraspecific similarity (49%) with two well supported subgroups from Cuba and Central

America, respectively. S. scabrum and S. nigrum were the most closely related species, with 68% genetic similarity, and are related to the S. villosum cluster, with

62% genetic similarity. All three species were well separated, with high bootstrap support in independent clusters.

Geographic provenances were only supported with bootstrap values above 60% for the S. villosum and S. americanum accessions. Those of S. scabrum and S. nigrum were inconclusive, due to a small number of accessions and unknown origins, respectively. In their study on genetic diversity of the section Solanum using AFLP markers, Manoko et al. (2007) were able to separate S. americanum and S. nodiflorum, which had been treated as one species by earlier studies using morphological characterisation. The study also identified a new species from

Tanzania, which was later described as Solanum umalilaense (Manoko et al.,

2012).

The hexaploid S. scabrum and S. nigrum accessions did not cluster according to their geographical provenance, and showed low genetic differentiation in both neighbour-joining and maximum-parsimony phylogenetic trees, according to

Manoko, van den Berg, Feron, van der Weerden, and Mariani (2008). The analysis did not reveal a clear distinction between wild and cultivated germplasm; however, using 448 AFLP markers, they were able to reliably differentiate the 80 S. scabrum accessions from the 41 S. nigrum and 15 S. opacum genotypes, with high Jacknife support. The study of Olet, Lye, and Heun (2011) also detected a high variability within S. villosum, showing 60% polymorphism, and within S. americanum, showing 64% polymorphism.

For S. villosum, the previous differentiation into subspecies could not be confirmed by the AFLP data. The neighbour-joining dendrogram generated from 510 AFLP markers (481 polymorphic) generated six main clusters from the eight species used in the study Low variability was observed in S. scabrum (10% polymorphism), and genotypes from this species were placed in a separate main cluster. The S. scabrum accessions did not cluster according to their geographical origin, even when they originated from different continents. The 40 S. villosum and the 42 S. americanum accessions could also be grouped into two large clusters; however, the structure of the dendrogram was not supported by bootstrap or jack- knife analysis. The random amplified polymorphic DNA (RAPD) technique was used by Poczai, Mátyás, Taller, and Szabó (2010) to investigate the relationship between S. scabrum, S. nigrum, S. retroflexum, and S. villosum on 13 accessions provided by theGeorgikon Botanical Garden

(Hungary), for which the original sampling provenance was not indicated. All four species could be clearly separated from each other using the 252 RAPD markers with high bootstrap support. The authors stated that the morphological traits in S. scabrum exhibited high variations, which were not reflected by the molecular data, but a dendrogram constructed from the morphological data showed the same clustering as the dendrogram from the RAPD data; however, all morphological parameters were probably measured only from one individual per accession in two years. Van Biljon, Labuschagne, and Koen (2010) used microsatellite markers in five species of the S. nigrum complex found in South Africa (S. americanum, S. burbankii, S. chenopodioides, S. retroflexum, andS. scabrum), and in crosses between them. Because only seven out of

29 SSR primer pairs detected polymorphisms between the accessions, the estimated genetic distances between the species might not be very reliable, and bootstrap analyses were again not presented. Two subspecies have been proposed for S. scabrum, on the basis of morphological data (Manoko et al., 2008; Olet et al., 2005; Poczai et al., 2010), with subsp. scabrum measuring up to 2m in height, having a larger number of leaves and a smaller number of fruits, and subsp. laevis being less than

1 m in height, with fewer leaves and a larger number of fruits. But this division has not been supported by molecular data. The observed morphological differences could be due to selection by farmers, since in Kenya, S. scabrum is the most cultivated leafy vegetable in the section Solanum.

From the molecular data presented in the different publications, it could be concluded that S. nigrum and S. scabrum are more closely related to each other than to S. villosum, whereas S. americanum seems to be more distantly related.

The four species could be separated from each other into different clusters in all mentioned publications. A further sub-clustering within a species could only be shown for S. americanum and S. villosum, by Dehmer and Hammer (2004).

Overall, it is hard to draw general conclusions from the different studies, because the overlap in identical species and countries of origin is low. Furthermore, many dendrograms are not statistically supported, either by bootstrap or jack-knife analyses, resulting in limited information content.