It is widely used as a vegetable and as a spice. Thus it is the soul and the heart of all cuisines. Because of its intense heat produced after its consumption which aids in digestion and intense bitterness makes it the core ingredient of Indian, Thai and Mexican foods. From being tingling and mild in taste, it can be explosively hot. Capsiacin provides intense taste to the person who consumes green chilli. On one hand where it is low in fat and cholesterol, on the other hand it is a rich source of vitamins, minerals and fibres.
BOTANICAL CLASSIFICATION OF GREEN CHILLI:-
Kingdom- Plantae (Plants)
Sub-kingdom- Tracheobionta (vascular plants)
Super-division- Spermatophyta (seed palnts)
Division- Magnoliophta (flowering plants)
Class- Magnoliopsida (Dicotyledons)
Family- Solanaceae (Nightshade family)
Genus- Capsicum L. Peppers
It is a small perennial shrub which is characterized by its greenish-white or whitish Corolla with node having one or more pedicels having fruit of varying shape and sizes. Its pungency varies with its size with smaller being the more pungent than the large fleshy one. It grows quite rapidly with a maturation period of about 2-3 months. Major chilli producing countries include Ghana, China, Mexico and Turkey (MiDA, 2010). It is a vitally important commercial crop. The security agencies use chilli for the making of tear gas. The uses and the advantage of chilli do not end here only, it has vast uses in both commercial and pharmaceutical industries.
Green chilli contain a large amount of vitamin C and beta carotene. When eaten, they releases endorphins that boosts the mood and help in reducing the pain. Being rich in vitamin K, it helps in reducing the chances of osteoporosis.
Genotypic characteristics of chillies are distinguished on the basis of their phenotypic characteristics such as flower colour, shape of the fruit, height of the plant and weight of the fruit etc. The fundamental step in any characterization programme is the use of phenotypic characters in describing and grouping the germplasm. Various studies on green chilli and pepper have shown that the morphological grouping of chillies, being a simple way of detecting difference in the genotypes, is greatly influenced by the environmental conditions and factors and therefore may not be able to distinguish between the closely related organisms. Therefore there needed a method of classifying chillies which would be independent of the environmental factors. Hence it become inevitable to back morphological characterization with molecular DNA marker technology which is proved to be highly objective and independent of the environmental factors. These molecular markers being the powerful tools in complementing phenotypic characterization help in detection of additional sources of genetic diversity present in the gene pool. For studying genetic diversity in capsicum species, various DNA markers like restriction Fragment Length Polymorphism, isozymes, random amplified polymorphic DNA etc have been used. Through the construction of genetic maps, these markers are helpful in determining the genetic variability.
Capsicum species produces fruits that synthesize and accumulate the hot compounds called capsaicinoids in placental tissues. Two biosynthetic pathways are used for the synthesis of capsaicinoids: the first is the phenylpropanoid, which provide the precursor phenylalanine and the second is the branched chain fatty acid pathway which provide the precursor valine or leucine. The genes which are possibly thought to be involved in the capsiacinoid biosynthetic pathways have been identified by the establishment of cDNA libraries and the study of comparative genes expressions in pungent and non pungent chilli pepper fruits. When mammals eat Capsicum fruits, the compound which is responsible for the pungent taste and sensation that occurs within them is the capsiacinoid. In nature, these compounds are synthesized only within the chilli pepper fruit. Capsiacinoid also provide protection against some pathogens. They are formed by condensing the molecule of vanillylamine which is derived from phenylalanine, to a branched fatty acid having 9-11 carbon atoms synthesized from either leucine or valine. However there are many number of capsiacinoid structures that are present but the most predominantly occuring capsiacinoid are CAP and DHCAP which accounts for almost 95% of all capsiacinoids. These both forms differ only by an unsaturated double bond at carbon-9 of the branched chain fatty acids. Capsiacin is hydrophobic, colourless, odourless and crystalline compound with a molar mass of 305.4g/mol. Being hydrophobic, organic solvents are used to solubilize capsiacin for use in tropical preparations and sprays.
Capsaicinoids synthesis occurs in the placental epidermal cells, where they are secreted towards the outer cell wall and will finally accumulate in the structures called BLISTERS, which are located on the placental surface. The pharmacological properties of the capsaicinoids are due to the release of substance P, which is a neuropeptide implicated in pain transmission from the terminals of primary sensory neurones by the action of capsaicinoids. Capsaicin have the ability to inhibit the growth of cancerous cells. It induces apoptosis i.e. cell death in in vitro in human gastric cancer cells. Capsaicin also help in reducing the growth of numerous cell lines of leukaemia by G0 – G1 phase cell cycle arrest and apoptosis.
Capsaicin is measured on the scoville scale and is expressed in terms of scoville heat units i.e. SHUs. The naturally occurring content of capsiacinoids in spices ranges from 0.2mg/g in chilli pepper to 3mg/g in red pepper. A number of pain related effects that can be produced by capsiacin depends largely on the dose and route of its Administration. The consequent effects may be withdrawal of afferent nerve terminals, sensitisation, desensitization or even death when given to neonatal animals. This lipo-solubility of capsiacin shows and explains why an orally eaten capsiacin food is not cured by drinking water and a yoghurt based drink like lassi is able to remove the vanilloid from the mouth.
The specific objectives of this work are:-
1. Extraction of capsaicin from green chillies.
2. Determination of capsaicin as secondary metabolite by using HPLC
3. To check the antimicrobial activity of capsiacin using 4 different strains of bacteria (E.coli,) by Plate diffusion method
4. Extraction of RNA from Capsicum using trizol
5. cDNA synthesis using promega kit
REVIEW OF LITERATURE:
Around the world, the chillies are grown fir their pungent taste, colour, aroma and nutritional value. India being the largest producer of chilli with 36% share in global market and the annual production is about 15 lakh tonnes. Andhra Pradesh, Maharashtra, Gujarat, Orissa, Karnataka and Tamil Nadu are the states in India which are rich in chilli production. Andhra Pradesh contributes 60% of Indias total chilli production followed by Karnataka (15%) which is the second largest producer of chilli in India. Different varieties of chillies have been grown across the country including the native and hybrid forms (Post harvest profile of chilli, 2009; Bhadragoudar and Patil, 2011).
Chillies can be produced in any type of soil but loam or silt loam soils with good water holding capacity and soil pH between 5.5 to 6.8 are best for its growth (Berke et al., 2005; karvy, 2008)
Sr. no Country Percentage
1. India 38%
3. Pakistan 5%
4. Bangladesh 5%
5. Peru 5%
6. Thailand 4%
7. Myanmar 3%
8. Ethiopia 2%
9. Vietnam 2%
10. Others 29%
Figure 1: major country distribution of chili production
Sr.no State Percentage
1. Andhra Pradesh 61%
2. Tamil Nadu 17%
3. Odisha 7%
4. West Bengal 3%
5. Karnataka 3%
6. Gujarat 2%
7. Kerala 1%
8. Others 6%
Figure 2: major state distribution of chilli production
Capsicum is a highly heterogeneous plant with a wide variety and difference in its fruit, flower and pungency (Walsh and Hoot, 2001). Pubescence of stems and leaves varies from glabrous to highly pubescent. It produces bisexual flowers which are developed at the intersection between the leaves and the stem at the point where the stem divides into a fork of branches. The inflorescence varies widely from solitary to 8-10 flowers at one node (Berke, 2000).
Capsicum frutescens is the botanical name of the green chilli. The placental area is the area highly rich in capsiacin. About 60% of the total capsiacin is concentrated in the white pith and the remaining 40% is present in the seeds. The seeds are either white or creamy and are circular and flat in shape. Different parts of the world grow different types of chilli plants as ornamental plants such as Medusha pepper, Thai ornamental, Numex twilight etc.
Research by Eshbaugh (1970), Novak and Betlakh (1971) and Pikersgill (1971) observed breeding behaviour and the connectivity between the various domestic and wild species of capsicum. Cytological studies by Ohta (1962a) and Habib and Cheenaveeraiha (1966) classified various taxas on the basis of their specific karyotypes.
Lopes et al., (1978) after conducting the flavonoid analysis concluded that a great affinity exists between Capsicum annuum and Capsicum frutescens.
Singh and Gupta (1978), said that the more diverse parents, within a normal reachable range, there would be more number of chances to improve the character in the question. Phenotypic and geographical diversity are the major criteria taken into account to isolate the genetically divergent population to get the inbred lines (Rai, 1979).
Forty five lines of green chilli were under Mahalanobis D2 analysis by Singh and Singh (1976) and the lines differ dramatically for nine characters. By analysing the 27 different varieties of chillies by D2 analysis, Mehra and Peter (1980) concluded that fruits per plant contributed maximum towards diversity.
Panda et al., (1986) compared the protein composition of seed of eight taxas of capsicum by using the method of disc electrophoresis and found that the all the protein bands vary among taxas and all the taxas could be separated by seed protein electrophoresis. Mcleod et al., (1979) gave the classification of capsicum species using 15 enzymes over the horizontal starch gel electrophoresis technique.
Wang and Dehua (1987) studied the capsicum germplasm and based on their zymograms, the eight species were divided into four groups i.e. GROUP-A (Capsicum annuum, Capsicum frutescens and Capsicum chinense), GROUP-B (Capsicum chacoense and Capsicum pubescens), GROUO-C (Capsicum praetermissum) and GROUO-D (Capsicum baccatum and Capsicum eximium).
Biotic constraints can cause serious major damage to the chilli production. Amongst them, pests and diseases are reported as the major damage causing constraints. Insects such as fruit borers, cutworms, thrips, mites etc can cause harm to the chilli crop production. Viruses, bacteria, fungi and nematodes can cause serious diseases in plants. Chilli anthracnose is a fungal disease caused by Colletotrichum species affects the yield and causes crop losses. Soil-borne fungi such as Pythium aphanidermatum and Rhizoctania solani can cause damping off. This is much more common in chilli nurseries where seedlings and seeds are destroyed by rotting effect of fungi. Oidiopsis taurica causes defoliation of leaves and therefore results in chilli powdery mildew. Fusarium annuum causes Fusarium wilt which results in death of the plants by wilting and infection to the whole plant. Bacterial wilt and Bacterial leaf spot caused by Ralstonia solanacearum and Xanthomonas vesicatoria respectively are some bacterial diseases in chilli plants (Agrios, 2005; Berke et al., 2008).
Bailey and Jeger (1992) concluded that Colletotrichum species are the most vital plant pathogens that causes anthracnose in wide range of plants such as legumes, perennials, cereals etc. Paul and Behl (1990) reported that Capsicum annuum is highly affected by Colletotrichum species.
Wilt caused by Ralstonia solanacearum (Smith) Yabuuchi et al is a well known spread disease of chilli causing the reduction in the production of chillies in the acidic soils. It was USA who reported this disease for the first time in 1898 (Rolfs 1898) and in India it was reported to be present from 1969, first from Madhya Pradesh.
Deshpande et al., (1988) by using the principal of component analysis, determined the two factors and reported that the one factor accounts for the 39% whereas the other accounts for 24% of the variability. Murthy and Murthy (1962) found that the level of naturally occurring cross pollination in green chilli was about 56-68%.
Cochran and Dempsey (1966) investigated and reached to the conclusion that the progressive decrease occurs in receptivity with every day increase in stigma age from the day of anthesis, when the mean temperature becomes relatively high (>72°F).
Betlach and Novak (1971) estimated that the most perfect time for the emasculation was between 4 to 6 p.m. and temperature is the deciding factor at the time of pollination, which is 18° to 24°C.
Bigotti (1972) reported the normal meiosis with 12 bivalents in almost 28 varieties of Capsicum. Yaqub (1968) concluded the gametophytic incompatibility in green chilli. He also experimented and showed that the almost all strains of Capsicum annuum and Capsicum frutescens tested were self compatible. A number of scientists have demonstrated the anti-inflammatory properties of capsiacin such as the inhibition of the production of IL-6, TNF, nitric oxide and PGE2 which are pro-inflammatory mediators. By inhibiting the enzyme lipoxygenase (LOX), the anti-inflammatory activity of the Capsicum annuum can be detected. When experimented, it showed the higher result in terms of percentage in LOX inhibition by green capsicum, followed by yellow and red capsicum (Monsereenusorn Y, 1980).
The intestinal glucose absorption inhibition can be achieved by the crude extract of chilli. It is responsible for lowering the blood sugar level. Daily consumption of green chilli help in attenuating post-prandial hyperinsulinemia. Baek J.et al., (2013) showed the anti-diabetic activity of chilli against alpha glucosidase, alpha amylase and ACE inhibitors. It has been found to be effective against the growth of prostrate cancer cells inducing the apoptosis of negative-positiveprostrate cancer cell lines.
Capsaicin and its properties have been extensively studied. It have phenol group and is weakly acidic. It is not precipitated usually but can be precipitated by using reagents that precipitate alkaloids. By methylating its phenol group, methyl capsiacin can be formed, which is a derivative of parent capsiacin but have less pungency than the parent compound.
Capsiacin and other compounds related to this class are called capsaicinoids. They are so produced as secondary metabolites by chilli peppers and mostly works as a deterrent against plant eating animals called herbivores.
Pic: capsiacinoid and structure
Presently, Capsiacin is used as a topical ointments to relieve the sensation of peripheral neuropathy such as post-herpetic neuralgia caused by shingles. Here, capsiacin is used in concentration between 0.025% to 0.075%. Capsaicin is now found as a cure for the diabetes by scientists in Toronto, Canada. Here, the capsaicin was injected into the pancreatic sensory nerves in mice with type 1 diabetes because of the researched link between the diabetes and the nerves.
At Howard Medical School, Clifford Woolf, the Richard J. Kitz Professor of Anesthesia Research reported that capsiacin can be used to deter abuse of drugs like OxyContin and Ritalin and some other extended release drugs.
MECHANISM OF ACTION OF CAPSIACIN:-
When capsiacin comes in contact with the sensory neurones, they produces a painful and burning sensation as a result of the chemical reaction between them. Vanilloid receptor subtype 1(VR-1) is a receptor to which the capsiacin binds, which is also a member of vanilloid family. VR-1 is an iron-channel receptor and was first cloned in 1997. This receptor can be activated by heat or by physical abrasion, leading to the permit of captions to cross the cell membrane and inside the cell when activated. The resulting neuron depolarization sends the signal to the brain. With chronic exposure to the capsaicin, neurons are depleted to the neurotransmitters. This causes the reduction in sensation of pain and if the capsiacin is removed, the neurons recover.
Tresh, in 1876, crystallised the capsaicin and Nelson and Dawson in 1919 resolved its molecular structure. It was found that capsiacin exhibits cis/trans Isomerism because the double bond prevents internal rotation. Capsiacin is always found to have the trans form because in the cis form, the longer carbon chain on the other side of double bond will be in close proximity , causing them to repel each other , this steric hindrance does not exists in trans isomer.
Pic: regions of molecules of capsiacin (A) aromatic ring, (B) amide bond and (C) hydrophobic side chain.
Capsiacin agonists are the substances that have the capability to bind to a receptor and elicit a response in the cell. SAR i.e Structure Activity Relationship fir capsiacin agonists have been studied by dividing the capsiacin into three regions- aromatic ring, amide bond and hydrophobic side chain. It is concluded from the studies that the substituents in the 3rd and 4th position of the A- ring are vital for the potent agonist activity. The phenolic H-bond donor or receptor are the basis of the agonist activity. Capsiacinoid are processed in the placenta of chilli by the enzymatic condensation of vannillylamine and varying size fatty acid chains which are elongated by fatty acid synthase. The condensation is caused by capsiacin synthase enzyme (CS) which requires Mg2+, coenzyme A (Co-A), ATP and vanillylamine.