Wissenschaftliche Studie, 2016
53 Seiten, Note: 1.5
Table of figures
Table of tables
List of abbreviations
1.2 Scope of the study
1.3 Taxonomical classification
2. Review of literature
2.1 Uses and importance
4. Materials and Methods
4.1 Study area
4.2 Sample collection
4.3 Preparation of extract
4.4 Phytochemical analysis
4.5 Isolation of microorganisms and mass culture
4.6 Antimicrobial activity
4.7 Antimicrobial susceptibility testing
4.8 Statistical analysis
5. Results and discussion
5.1 Phytochemical screening
5.2 Antibacterial susceptibility tests
Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009)
Figure 2. Map of Kerala showing the various sample collection point of Annona reticulate
Figure 3. Differet varieties of custard apples; A) Annona scleroderma, B) Annona diversifolia, C) Annona reticulate, D) Annona cherimola, E) Annona muricata and F) Annona squamosa. Photo courtesy: University of Purdue. 19 https://hort.purdue.edu/newcrop/1492/figs/annona_fig7.gif
Figure 4. Description a) Annona reticulata fruits for sale in shop, b) Annona reticulata fruit with leaves, c) Annona squamosa plant, d) Annona squamosa flower, e) Mature Annona squamosa fruit in tree, f) Annona squamosa fruit crosss section. Photo courtesy: Wikipedia
Figure 5. Description a) leaves of Annona reticulata, b) fruit of Annona reticulata, c) dried and powdered leaves of Annona reticulata, d) dried and powdered fruit of Annona reticulate, e) dried and powdered stem bark of Annona reticulate, f) thimble of soxhlet apparatus loaded with sample
Figure 6. Description a) antimicrobial discs of various plant extracts, b) qualitative analysis of acids, c) qualitative analysis of alkaloids, d) qualitative analysis of betacyanin, e) qualitative analysis of carbohydrates, f) qualitative analysis of cardicglycosides
Figure 7. Description a) qualitative analysis of coumarins, b) qualitative analysis of flavonoids, c) qualitative analysis of glycosides, d) qualitative analysis of phenols, e) qualitative analysis of quinines, f) qualitative analysis of saponins
Figure 8. Description a) qualitative analysis of tannins, b) qualitative analysis of terpenoids
Figure 9. Antibacterial susceptibility tests in E.coli a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts, g) plate with antibiotic discs, h) control plate
Figure 10. Antibacterial susceptibility tests in Pseudomonas ariginosa a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts, g) plate with antibiotic discs, h) control plate
Figure 11. Antibacterial susceptibility tests in Serratia marcescens a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts, g) plate with antibiotic discs, h) control plate
Figure 12. Antibacterial susceptibility tests in Micrococcus luteus a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts, g) plate with antibiotic discs, h) control plate
Figure 13. Antibacterial susceptibility tests in Candida albicans a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts
Figure 14. Antibacterial susceptibility tests in Rhizpous a) 2x chloroform extracts, b) 2x water extracts, c) 4x chloroform extracts, d) 4x water extracts, e) 8x chloroform extracts, f) 8x water extracts
Figure 15. Zone of inhibition of antibiotics (carbencillin, chloramphinicol and kanamycin) among tested microorganisms (E.coli, Pseudomonas aeruginosa, Serratia marcescens, and micrococcus luteus).
Table 1. Different vernacular names of Annona reticulata around the globe and India
Table 2. Details of the plant extracts and solvents used
Table 3. Qualitative analysis of several of phytochemicals present in Annona reticulata plant parts and solvent extracts
Table 4. Zone of inhibition of Annona reticulata plant parts and solvent extracts among tested microorganisms (E.coli, Pseudomonas aeruginosa, Serratia marcescens, micrococcus luteus, Candida albicans and Rhizopus).
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The Annona reticulata and its phytochemical analysis and antimicrobial activities using various solvents
Prem Jose Vazhacharickal, Sajeshkumar N.K, Jiby John Mathew and Clemency Tomy
Annona reticulata belongs to the family Annonaceae, commonly known as honey apple. Qualitative phytochemical analysis of chloroform and water extracts of Annona reticulata fruit, leaf and stem bark was conducted in order to detect the presence of various secondary metabolites using standard procedures. The results of phytochemical screening indicated the presence of secondary metabolites such as tannins, betacyanins, carbohydrates, alkaloids, terpenoids, phenols, quinines, saponins, cardiac glycosides etc. Also the comparative antimicrobial activity of chloroform and water extracts of fruit, leaf and stem bark of Annona reticulata was evaluated against four bacterial species namely, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Micrococcus luteus and two fungal species namely Candida albicans and Rhizopus. Agar well diffusion method and disc diffusion method were selected to check the antimicrobial activities of the extracts. The study revealed that the chloroform extracts of leaf, stem bark and fruit of Annona reticulata has activity against the bacterial strains and fungal strains. Whereas, the water extracts of leaf, fruit and stem bark of Annona reticulata has more activity towards the fungal species. The findings of this study have identified that Annona reticulata extracts acts as a promising source of antimicrobial agent which could be useful in the modern medicine.
Keywords: Anti-oxidant; Annona reticulata; Antimicrobial activity; Potato dextrose agar.
According to the world health organization (WHO) survey 80% populations living in the developing countries rely almost exclusively on traditional medicine for their primary health care needs. Exploration of the chemical constituents of the plants and pharmacological screening may provide us the basis for developing the leads for development of novel agents. In addition, herbs have provided us some important life saving drugs used in armamentarium of modern medicine. However among the estimated 2,50,000-4,00,000 plant species, only 6% have been studied for biological activity and about 15% have been investigated phytochemically (Cragg et al.,1997; Balsndrin et al., 1985; Pandey and Barve 2011). The therapeutic efficacy of many indigenous plants, for various diseases has been described by traditional herbal medicinal practitioners (Gami, 2010; Soni et al., 2011). There are several reasons that people use plants for medication. This includes improvement of health after herbal treatment, low cost of the drugs, non availability of synthetic drugs particularly in the rural areas, where available were either fake or expired drugs and in some cases the people are more accustomed to and comfortable with traditional healing (Audu, 1995).
Various parts of plants such as leaves, fruits, barks, roots and even the seeds are being used for preparation of medicine. Plants produce several secondary metabolite compounds including alkaloids, glycosides, glucosinolates, flavanoids, saponins, steroids and terpenoids to protect themselves from the continuous attack of naturally occurring pathogens, insect pests and environmental stresses (Ebel, 1986). With advances in analytical instrumentation, bioassay techniques and recombinant DNA technology, the scope of using these antimicrobial compounds for disease control was further enhanced. The use of plant extracts with antimicrobial activity offers an economical, safe and easily available alternative method for the management of diseases. In the recent past, several plant species have been screened for antimicrobial activity and extract purified compounds from these plants were found to have a broad spectrum of antimicrobial activity (Grayer and Harborne, 1994).
To ensure reproducible quality of herbal products, authentication of the starting material is essential. According to WHO the macroscopic and microscopic description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such materials and should be carried out before any tests are undertaken.
The members of the Annonaceae family are not foreign in the search for new bioactive compounds as this family has a number of compounds that have been identified for their cytotoxic and pesticidal properties. The family Annonaceae is one of the important families in the tropical lowland forest in the continents of Asia, Africa and America with 130 genera and over 2000 identified species (Kessler, 1993; Wiart, 2000).
Annona reticulata is a highly apparent plant in ayurvedic system of medicine for the treatment of various ailments. The plant is traditionally used for the treatment of epilepsy, dysentery, cardiac problems, worm infestation, constipation, haemorrhage, antibacterial infection, dysuria, fever, and ulcer. It also has anti-fertility, anti-tumour and abortifacient properties. Ethanolic extracts of leaves and stem are reported to have an anti-cancerous activity. The aqueous leaf extract has also been reported to ameliorate hyperthyroidism (Kaleem, 2006). Ripe fruit is sweet, cooling, good tonic and sedative. It enriches the blood, increases muscular strength, and lessens burning sensation, tendency to biliousness and vomiting. Leaf can be used for destroying lice (Morton, 1987). Fragments of the root bark are packed around the gums to relieve toothache. The bark is very astringent and the decoction is taken as a tonic and also as a remedy for diarrhoea and dysentery. The leaf decoction is given as a vermifuge. Crushed leaves or a paste of the flesh may be poulticed on boils, abscesses and ulcers. The unripe dried fruit dried is employed against diarrhoea and dysentery. In severe cases, leaves bark and green fruits are all boiled together for 5 minutes in a litre of water to make an extremely potent decoction (Parvin et al., 2003).
The extracts of plant materials of plants in the family Annonaceae is attributed with medicinal properties due to several secondary metabolite compounds including alkaloids, glycosides, glucosinolates, flavanoids, saponins, steroids, terpenoids and others. The isolation of various compounds done using soxhlet extraction procedure utilizing chloroform and water as extraction solvents. The presence of various metabolites in the extract is analysed through preliminary phytochemical studies of extract using procedures given in standard text (Pollock, 1965). Each extract is screened for the presence of secondary metabolites. Qualitative phytochemical analysis is carried out using method described by Trease and Evans (1989).
The extracts are determined to have secondary metabolites which have antimicrobial activities also. This activity is tested against bacterial strains Escherichia coli (E. coli), Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens and two fungal strains (Candida albicans and ). Both agar well diffusion method and disc diffusion method are used to study the antimicrobial activity of the extracts. Mueller hinton agar (MHA) is used as bacteriological medium and potato dextrose agar (PDA) is used as fungal medium for testing the antimicrobial activity. The activity is evaluated by measuring the inhibition zones formed by the extracts.
Even though other plants of the same family, such as Annona squamosa are studied well enough to characterize its properties, not in the case of Annona reticulata. Pharmacognostical and phytochemical studies of Annona reticulata using three different polar solvents were reported (Sathyanarayana et al., 2013). The chemical composition of extracts of Annona reticulata using gas chromatography- mass specrtophotometry technique were also reported (, 2013). Less volatile substances such as alkaloids, diterpenoids, and acetogenins (Ogunwande et al., 2006) have been identified so far from oil of various parts of the plant. However, the antimicrobial potential of Annona reticulata is not yet been explored properly.
A comparative antimicrobial study using the extracts of leaf, fruit and stem bark of Annona reticulata using chloroform and water as extraction solvents is still not reported. With this in mind, the present work was an attempt to perform the studies on antimicrobial activity of leaf, fruit and stem bark extracts of Annona reticulata using chloroform and water. The objective is to study the presence of secondary metabolites present in the chloroform and water extracts of Annona reticulata leaf, fruit and stem bark and to evaluate its antimicrobial activity against bacterial and fungal strains by agar well diffusion and disc diffusion methods.
The study using the extracts of leaf, fruit and stem bark of Annona reticulata could develop new drug formulations to combat various diseases. Moreover such studies could improve the potential value of the underutilized fruits especially Annona reticulata.
1.3 Taxonomical classification Kingdom: Plantae-- planta, plantes, plants, vegetal Subkingdom: Viridiplantae Division: Tracheophyta – vascular plants, tracheophytes Class: Magnoliopsida -- dicots, dicotyledones, dicotyledons Order: Magnoliales Family: Annonaceae – custard apples Genus: Annona L. Species: Annona reticulate L.
Table 1. Different vernacular names of Annona reticulata around the globe and India.
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Antibiotic resistance has increased substantially in the recent years and is posing an ever increasing therapeutic problem. One of the methods to reduce the resistance to antibiotics is by using antibiotic resistance inhibitors from plants (Kim et al., 1995; Alagesaboopathi, 2011). Plants are known to produce a variety of compounds to protect themselves against a variety of pathogens. It is expected that plant extracts showing target sites other than those used by antibiotics will be active against drug resistant pathogens (Ahmad and Beg, 2001). Medicinal plants have been used as traditional treatments for numerous human diseases for thousands of years and in many parts of the World. Hence, researchers have recently paid attention to safer phytomedicines and biologically active compounds isolated from plant species used in herbal medicines with acceptable therapeutic index for the development of novel drugs (Pavithra et al., 2010). In various indigenous and traditional sources of medicine plants have been extensively used for treatments. Various parts of plants such as the leaves, fruits, the barks, roots and even the seeds are being used for preparation of medicine. Annona spp. is also been extensively used as traditional medicine in various culture. The genus name, ‘Annona’ is from the Latin word ‘anon’, meaning ‘yearly produce’, referring to the production of fruits of the various species in this genus (Pinto et al., 2005).
Annona reticulata Linn is a small deciduous ever green tree in the family Annonaceae is cultivated throughout India for its fruits, different parts of Annona reticulata Linn. used in folkloric medicine for the treatment of various disease (Manoharan et al., 2006). This plant is commonly called Netted custard apple in English and Ramaphalam in Telugu in India (Raj et al., 2009). It is best known for its fruit and the common name custard apple is shared with fruits of several other species in the same genus: Annona cherimola (Mahdeem, 1998) and Annona squamosa (Aluka, 2008); sometimes called wild-sweetsop, bull's heart, bullock's-heart, or ox-heart. The flavour of the fruit is sweet and pleasant, but less popular than that of Annona cherimola.
Annona reticulata is a tree reaching 8 to 10 metres tall with an open, irregular crown. The slender leaves are not hairy, straight and pointed at the apex (in some varieties wrinkled), 10 to 20 centimetres long and 2 to 7 centimetres wide. The yellow-green flowers (clusters of three or four) are 2 to 3 centimetres in diameter, with three long outer petals and three very small inner ones The fruits are variable in shape: heart-shaped, spherical, oblong or irregular. The size ranges from 7 to 12 centimetres depending on the cultivar. When ripe, the fruit is brown or yellowish, with red highlights and a varying degree of reticulation, depending again on the variety. The flesh varies from juicy and very aromatic to hard with a repulsive taste. The flavour is sweet and pleasant, akin to the taste of 'traditional' custard (Mahdeem, 1998).
Possibly a native of the Caribbean and Central America, Annona reticulata is now pantropical and can be found growing upto altitudes of 1,500 metres (4,900 ft) in areas of Central America that have alternating seasons (Mahdeem, 1998). It is cultivated and naturalized in many tropical countries, and also occurs as feral populations in many parts of the world, including Southeast Asia, Taiwan, India, Bangladesh, Pakistan, Australia, and Africa (Aluka, 2008).
Another species in the same genus, Annona squamosa L., is commonly found in deciduous forests, also cultivated wild in various parts of India. Literatures of many research works prove that every parts of Annona squamosa possess medicinal property (Kirtikar and Basu, 1993). One previous study showed that various organisms including Bacillus substilis, Staphylococcus aureus, Staphylococcus epidermidis and Vibrio alginolyticus were effectively inhibited by extracts of Annona squamosa species (Padhi and Panda, 2011).
In another, four different solvent extracts of leaves of Custard apple (Annona squamosa L.) were studied for its antibacterial activity. Agar diffusion method was selected to check antibacterial activity, whereas phytochemical analysis was done by high performance thin layer chromatography (HPTLC) instrument. The screening results showed that highest zone of inhibition was observed in methanol extract against Pseudomomonas aeruginosa (MIC (minimum inhibitory concentration): 130μg/ml) followed by petroleum ether extract against Pseudomomonas aeruginosa (MIC: 165 μg/ml) and methanol extract against E. coli (MIC: 180 μg/ml). Various Studies demonstrates the presence of some phytochemicals (Linalool, Borneol, Eugenol, Farnesol, and Geraniol) in extracts which provide antibacterial activity (Patel and Kumar, 2008).
The antioxidant activity of extracted phenolic compounds from Annona squamosa using methanol was evaluated employing various established in vitro systems (Mariod et al., 2012). Screening of Annona squamosa extracts for antibacterial activity against repiratory tract isolates showed activity against Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumonia and Proteus species. The in vitro evaluation of antibacterial potential of Annona squamosa L. against pathogenic bacteria showed activity against various pathogenic bacteria’s (Aher et al., 2012). Also a study conducted to assess the biological activity of Annona muricata hexane, methanol and chloroform seed extracts showed moderate larvicidal activity (Hoe et al., 2010).
Different pharmacognostical and phytoochemical studies of Annona reticulata Linn. were studied according to the WHO and pharmacopoeial guidelines. Potential bioactivity of Annona reticulata leaf extract and its compound identification by gas chromatography-mass spectrometry (GC-MS) was carried out, which suggested a wide range of clinical applications against various pathogenic bacteria and fungus (Rajini and Jothi Nisha, 2013). According to this study the leaf extract of Annona reticulata posses promising bioactivity.
The antimicrobial activity of the crude chloroform and distilled water extract of Annona reticulata was determined using by agar well diffusion assay and disc diffusion method against four strains of bacterial species, viz., Escherichia coli, Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens and two fungal strains, viz., Candida albicans and Rhizopus.
Escherichia coli is a gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls due to food contamination (Vogt and Dippold, 2005). The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2 (Bentley and Meganathan, 1982), and by preventing the establishment of pathogenic bacteria within the intestine (Hudault et al., 2001). E. coli and related bacteria constitute about 0.1% of gut flora (Eckburg et al., 2005), and faecal–oral transmission is the major route through which pathogenic strains of the bacterium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them ideal indicator organisms to test environmental samples for faecal contamination (Thompson, 2007). E.coli can cause gastroenteritis, urinary tract infections and neonatal meningitis. In some cases, virulent strains are also responsible for haemolytic uremic syndrome, peritonitis, mastitis, septicaemia and pneumonia (Todar, 2007).
Pseudomonas is a genus of rod shaped, gram-negative, one or more polar flagella, providing motility, aerobic, non–spore forming gamma proteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species (Van Eldere, 2003) Pseudomonas aeruginosa is increasingly recognized as an emerging opportunistic pathogen of clinical relevance that can cause urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia and a variety of systemic infections. Several different epidemiological studies indicate antibiotic resistance is increasing in clinical isolates. As a result of their metabolic diversity, ability to grow at low temperatures and ubiquitous nature, many Pseudomonas spp. can cause food spoilage (Pereira and Mohan, 1957)
Micrococcus luteus is a gram-positive, not motile, spherical, saprotrophic bacterium that belongs to the family Micrococcaceae (Madigan and Martinko, 2005). An obligate aerobe, Micrococcus luteus is found in soil, dust, water and air, and as part of the normal flora of the mammalian skin. The bacterium also colonizes the human mouth, mucosae, oropharynx and upper respiratory tract. Micrococcus is generally thought of as harmless bacterium, but there have been rare cases of Micrococcus infections in people with compromised immune systems, as occurs with human immunodeficiency virus (HIV) patients. They have been known to get skin infections or chronic cutaneous infections, which results in pruritic eruptions of the skin in some areas as well as scattered papule lesions with or without central ulcerations (Smith, et al. 1999).
Serratia marcescens is a motile, short rod-shaped, gram-negative, facultative anaerobe bacterium, classified as an opportunistic pathogen that causes nosocomial infections. Serratia marcescens was classified as a human pathogen in the 1960s. It is resistant to many antibiotics traditionally used to treat bacterial infections, such as penicillin and ampicillin (Cappuccino et al., 2005). Most strains are resistant to several antibiotics because of the presence of R-factors (genes coding for antibiotic resistance) on plasmids (Codling et al., 2004). There are many diseases that are associated with Serratia marcescens: sepsis, bacteremia, meningitis and cerebral abscesses, urinary tract infections, osteomyelitis, ocular infections, and endocarditis. Due to the wide range of diseases Serratia marcescens causes, there is not one determining symptom or source of origin (Hejazi and Falkiner, 1997).
Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans (Ryan and Ray, 2004). Systemic fungal infections (fungemias) including those by Candida albicans have emerged as important causes of morbidity and mortality in immune-compromised patients (AIDS; acquired immune deficiency syndrome, cancer chemotherapy, organ/bone marrow transplantation). Candida albicans bio films may form on the surface of implantable medical devices. In addition, hospital-acquired infections by Candida albicans have become a cause of major health concerns. Candida albicans is commensal and a constituent of the normal gut flora comprising microorganisms that live in the human mouth and gastrointestinal tract. Candida albicans lives in 80% of the human population without causing harmful effects, although overgrowth of the fungus results in candidiasis (candidosis). A common form of candidiasis restricted to the mucosal membranes in mouth or vagina is thrush, which is usually easily cured in people who are not immune-compromised (Zadik et al., 2010). To infect host tissue, the usual unicellular yeast-like form of Candida albicans reacts to environmental cues and switches into an invasive, multicellular filamentous form, a phenomenon called dimorphism (Ryan and Ray, 2004)
Rhizopus is a genus of common saprobic fungi on plants and specialized parasites on animals. They are found on a wide variety of organic substrates, including "mature fruits and vegetables" (Kirk, et al., 2008), faeces, jellies, syrups, leather, bread, peanuts and tobacco. Some Rhizopus species are opportunistic agents of human zygomycosis (fungal infection) and can be fatal. Rhizopus infections may also be a complication of diabetic ketoacidosis (Chinn and Diamond, 1982).
The selected bacterial and fungal species were used to determine the antimicrobial activity of the plant extracts. The extract preparation is carried out using soxhlet extractor. A Soxhlet extractor is a piece of laboratory apparatus invented in 1879 by Franz von Soxhlet (Harwood et al., 1989). It was originally designed for the extraction of a lipid from a solid material. However, a Soxhlet extractor is not limited to the extraction of lipids. Typically, a Soxhlet extraction is required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. The solvent is heated to reflux. The solvent vapour travels up a distillation arm, and floods into the chamber housing the thimble of solid. . The desired compounds will dissolve in the warm solvent. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. During each cycle, a portion of the non-volatile compound dissolves in the solvent. After many cycles the desired compound is concentrated in the distillation flask. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled (William, 2007).
The extract preparation of Annona reticulata can be successively done by using chloroform and water as solvents (Chandrashekar and Kulkarni, 2011). Different qualitative chemical tests can be performed for establishing profile of chloroform and aqueous extract for its chemical composition. Different tests were performed on extracts to detect various phytoconstituents present in them (Shalini and Sampathkumar, 2012). The qualitative analysis (Vanitha et al., 2012) includes various tests for carbohydrates, saponins (Kokate, 1999), flavanoids, alkaloids (Evans, 1997), betacyanin, quinines, glycosides, cardiac glycosides, terpenoids, phenols, tannins (Mace, 1963), coumarins and acids (Trease and Evans, 1989).
The concentrated extracts are then checked for its antimicrobial activity. An antimicrobial is an agent that kills microorganisms or inhibits their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against. Antibacterials (antibiotics) are used against bacteria and antifungals are used against fungi. Use of substances with antimicrobial properties is known to have been common practice for at least 2000 years. Ancient Egyptians and ancient Greeks used specific moulds and plant extracts to treat infection (Wainwright, 1989).
Different methods can be utilized to check the antimicrobial activity of the plant extracts. Kirby-Bauer Disk Diffusion Susceptibility Test and Agar well diffusion method were utilized during the experiment.
In 1956, Kirby and his colleagues at the University of Washington (School of Medicine) and the King County Hospital proposed a single disk method for antimicrobial susceptibility testing (Winn, 2006). Interpretation of susceptibility and resistance was based only on the presence or absence of a zone of inhibition surrounding the disk, and two or three different concentrations of the same antimicrobial were tested against the pathogen (Bauer et al., 1959).
In agar well diffusion method of antimicrobial susceptibility test, wells were cut and the extract was poured to the well and was incubated to detect the activity. The antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well (Wong et al., 1999).
The fruit of Annona reticulata is widely used among villages in India. The fruit posses high nutritional value with all essential vitamins and minerals. In addition the fruits of Annona reticulata posses antioxidant, anti-cancer and antihelmintic activity (Chavan et al., 2014).
The current research work is based on the following hypothesis
1) The phytochemical constituents and antimicrobial activities of Annona reticulata vary widely in different plant parts as well as solvent extracts
Kerala state covers an area of 38,863 km2 with a population density of 859 per km2 and spread across 14 districts. The climate is characterized by tropical wet and dry with average annual rainfall amounts to 2,817 ± 406 mm and mean annual temperature is 26.8°C (averages from 1871-2005; Krishnakumar et al ., 2009). Maximum rainfall occurs from June to September mainly due to South West Monsoon and temperatures are highest in May and November (Figure 1).
The leaves, fruit and stem bark of Annona reticulata were collected from the herbal garden of Mar Augusthinose college, Ramapuram, Kottayam District, Kerala and were authentified based on morphometric characters (Figure 2). Care was taken to select healthy plants and for normal organs. The leaf, stem bark and fruit were cut and removed from the plant and were surface sterilized using 70% ethanol. The collected plant parts were shade dried for 10 days and then powdered well using mechanical grinder and stored in air tight bottles.
Soxhlet apparatus was used for extraction purpose. 25 grams of powdered material was taken in a clean cotton cloth bag and tied its ends and loaded in the apparatus for extraction. Poured 150 ml of solvent into 250 ml round bottomed flask. Placed the thimble with the sample in the extractor fitting. A flow of water in from the bottom outlet and out from the top outlet of the condenser was provided for condensation. Solvents used for the extraction were water and chloroform. The heating of the round bottomed flask using a mantle was carried out at a temperature of 100°C for water and 61°C for chloroform. Monitored the heating process. The solvent was brought to the vaporization stage. Allowed the solvent to flush through the sample and regular flushing was maintained. The extraction was carried out for 4 hours for chloroform solvent and 7 hours for water solvent. Extraction time varies with type of solvent used. After extraction, extract was filtered through Whatman filter paper. The extract was then concentrated to a final volume of 10 ml. The filtrate was then stored in air tight bottles in a refrigerator for further use.
Plant materials were collected from a healthy plant and is dried and powdered which was then used for the extraction process. Soxhlet apparatus was used to extract the antimicrobial compounds in Annona reticulata. Extraction is done by using solvents chloroform and water. The extracts are then concentrated by heating to a final volume of 10 ml. 10 μl of extract was calculated as 1x concentration.
Different qualitative chemical tests were performed for establishing profile of chloroform and aqueous extract for its chemical composition. The following tests were performed on extracts to detect various phytoconstituents present in them. Preliminary phytochemical analysis were carried out to test the presence of tannins, flavonoids, terpenoids, alkaloids, phenols, coumarins, reducing sugars, saponins, quinines, betacyanin, glycosides and acids in the extracts following standard protocol.
To 2 ml of plant extracts, 1ml of Molisch’s reagent and few drops of concentrated sulphuric acid were added. Purple colour formation indicated the presence of carbohydrates.
To 1ml of plant extract, 2 ml of 5% ferric chloride was added. Formation of greenish black indicated the presence of tannins.
To 2 ml of plant extract, 2 ml of distilled water was added and shaken for 15minutes lengthwise. Formation of 1 cm layer of foam indicated the presence of saponins.
5 ml of dilute ammonia solution was added to a portion of the aqueous filtrate of plant extract followed by addition of concentrated sulphuric acid. Appearance of yellow colouration indicated the presence of flavonoids.
To 2 ml of plant extract, 2 ml of concentrated hydrochloric acid was added. Then few drops of Mayer’s reagent were added. Presence of green colour indicated the presence of alkaloids.
To 2 ml of plant extract, 1ml of 2N sodium hydroxide was added and heated for 5 minutes at 100ºC. Formation of yellow colour indicated the presence of betacyanin.
To 1ml of extract, 1ml of concentrated sulphuric acid was added. Formation of red colour indicated the presence of quinones.
To 2ml of plant extract, 3ml of chloroform and 10% ammonia solution was added. Pink colour formation indicated the presence of glycosides.
To 0.5ml of extract, 2 ml of glacial acetic acid and few drops of 5% ferric chloride were added. This was under layered with 1 ml of concentrated sulphuric acid. Brown ring formation at the interface indicated the presence of cardiac glycosides.
To 0.5 ml of extract, 2 ml of chloroform was added and concentrated sulphuric acid was added carefully. Red brown colour formation at the interface indicated the presence of terpenoids.
To 1ml of the extract, 2 ml of distilled water followed by few drops of 10% ferric chloride was added. Formation of green colour indicated the presence of phenols.
To 1 ml of extract, 1ml of 10% Sodium hydroxide was added. Formation of yellow colour indicated the presence of coumarins.
1ml of extract was treated with sodium bicarbonate solution. Formation of effervescence indicates the presence of acids.
Bacterial species Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens (gram negative) and Micrococcus luteus (gram positive) and fungal species Candida albicans, Rhizopus and Aspergillus were used for this study. The test microorganisms were obtained from the stock cultures from the Department of Biotechnology, Mar Augusthinose College, Ramapuram, Kottayam District. The bacterial species were subcultured in nutrient agar slants by streaking and the treated tubes were incubated at 37°C for 24 hours. A total of 3 test tubes were used for each test bacterial species. The fungal species were subcultured in potato dextrose agar slants and the treated tubes were incubated at room temperature for 48 hours. A total of 3 test tubes were used for each test fungal species. Mass culture of bacterial species was carried out in nutrient broth and mass culture of fungal species was carried out in fungal broth and the inoculated broth cultures were incubated for appropriate incubation periods.
The antimicrobial activity was tested against bacterial and fungal species. The antimicrobial activity was tested using disc diffusion method and agar cup method. Discs of different concentrations were prepared for disc diffusion method. Sterile susceptibility discs were taken. Pipetted out 20 μl and 40 μl of each plant extract and were loaded on 6 mm sterile discs and allowed to dry. Weighed appropriate grams of MHA (for bacterial species) and PDA (for fungal species) and heated to dissolve the contents. Sterilized the medium in an autoclave at 121°C at 15 psi. Poured about 15-20 ml of the sterilized media to clean and sterilized petri plates. Prepared plates with MHA and PDA were allowed to solidify.
Antimicrobial activities of the extracts were studied by two different methods; Disc diffusion method and Agar cup method.
The method of Bauer et al., 1966 (Kirby-Bauer method) was adopted for testing the efficiency of herbal extracts against the test organisms.
The agar well diffusion method was used to study the antimicrobial activity of the extracts as described by Panda et al., (2009). Mueller Hinton agar was used as bacteriological medium and PDA used as fungal medium.
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Figure 1. Mean monthly rainfall (mm), maximum and minimum temperatures (°C) in Kerala, India (1871-2005; Krishnakumar et al., 2009). Authors own work.
illustration not visible in this excerpt
Figure 2. Map of Kerala showing the various sample collection point of Annona reticulate. Authors own work.
The results were analyzed and descriptive statistics were done using SPSS 12.0 (SPSS Inc., an IBM Company, Chicago, USA) and graphs were generated using Sigma Plot 7 (Systat Software Inc., Chicago, USA).
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