Applications of Plant Sources for Medicine

1.0 INTRODUCTION

There is a global increase in utilizing drug from plant sources which projected to be sixty billion around the universe (Barnes et al., 2004). Medicines from plants are efficient in curing diseases. The traditional systems of medicine namely Ayurveda, Siddha and Unani are well established in India and are widely acknowledged to be effective and safe without any side effects (Farnsworth, 1998).

Recent years has seen the vast increase in medicinal plants because of its non chemical origin and exhibit remarkable efficacy in the treatment of various ailments (Siddhiqui et al., 1995). A vast number of plants are known to occupy various places in earth. Traditional medicine has been improved in developing countries as an alternative solution to health problems. There is a need for development of new drugs of plant origin against human diseases due to the resistance of pathogens against existing drugs (Govil, 2002).

The World Health Organization (WHO) has compiled a file of twenty thousand curative plants used around the globe. A large number of species have local uses within the country or spread over several countries in a region. The medicinal virtues of the raw materials including chemical contents and composition of the plants merited inclusion in national pharmacopoeias and official formularies in different countries (Amar jyothi Das, 2012).

Treatment of various diseases was carried out with the use of compounds obtained from medicinal plants (Akerele, 1988). The plants need more attention due to their important role in primary healthcare delivery system for improvement of people’s health (Hamayun, 2003). It is an essential component of human healthcare especially for the rural communities who solely rely on forest plants for food, shelter, energy and medicine. The medicinal values of plants lie in some chemical substances that produce a definite physiological action on human body (Chalchat, 1998).

Essential oils have potential application in medical procedures and in the cosmetic, food and pharmaceutical industries (Tongnuanchan and Benjakul, 2014). Essential oils are concentrated liquids of complex mixtures of bioactive compounds and can be extracted from several plant parts. Essential oil which contains several bioactive compounds was the good source (Thorpe, 2007). Inspite rapid fragmentation of natural habitats is greatly narrowing the distribution of the plant and increasing the risk of losing genetic diversity (George, 1984).

1.1 In vitro micropropagation

In vitro cell culture was developed by Gottlieb Haberlandt (1962) from various plant species like Laminum purpureum, Eicchornia crassiples and cultured in glucose enriched Knop’s salt solution. In vitro tissue culture is performed aseptically to produce plants that are similar to plants grown in environmental conditions. Plants are produced either from cells or from whole plant under in vitro conditions this technique is most effective in development of agricultural plant species (Thorpe, 2007). In vitro techniques have been successfully applied to solve the problems of conventional micropropagation in a large number of medicinal plants. In vitro propagation refers to true to type propagation of selected genotypes under laboratory conditions. New plants are generated by different explants such as single cells, protoplasts, pieces of leaves, roots and node on basal media with necessary nutrients. Conservation through vegetative propagation is time consuming however, tissue culture offers an alternative tool for rapid multiplication of disease free propagules in a short period (Muralidharan, 1997).

According to red list of threatened species, 113 endangered plant species, 44 plant species are critically endangered and 87 vulnerable species (IUCN, 2000). The development of plant tissue culture technology holds great promise for conservation and enhancement of valuable medicinal plants. It has many advantages over conventional methods of propagation, which suffers from several limitations (Jeyachandran, 2012).

Tissue culture technique is developed in the improvement of the growth conditions of the plants and the facilitation of international germplasm exchange, genetic transformation, production of cryopreservation and production of secondary metabolites (Alderete, 2006). The compounds obtained from in vitro propagated plants are varied from the wild variety. Instead the structural organization of the plants was not modified by micropropagation  and the  medicinal effects of plant materials typically result from the combinations of secondary metabolites such as Tannins, Alkaloids, Carbohydrates, Steroids, and Phenol compounds, Flavonoids, Resins, Fatty acids, Gums which are capable of producing definite physiological action on human body (Joshi, 2009).

To improve production of high quality plants, micropropagation is required. It is carried out using a selective media consisting of growth hormones that helps in development of morphological characters from naturally grown plant parts (Debergh and Read, 1991).

Preliminary phytochemical and Gas Chromatograph and Mass Spectrometry (GC-MS) analysis of secondary metabolites are advantage for the micropropagation of medicinal plants and it has been reported in many plant species (Singh and Chaturvedi, 2010).

In vitro micropropagation technique is advantageous than conventional methods since it does not depend on seasonal changes, production in mass quantities, characteristic specific clone production, preserving of endangered plant species (Saini and Jaiwal, 2000). Conservation planners have sought objectives and quantitative criteria for setting priorities among the elements of biodiversity to be protected (Williams, 2002).

The micropropagation is known to produce plants that are genetically identical to parent plant. Less variation was observed in the secondary metabolites content than their wild variety (Yamada et al., 1991).

The aim of producing exact replicas of the original plant selected for its desirable characters and considerable progress has been made in the recent years in micropropagation of many plant species (Wang and Hu 1982). Today there is an enormous resurgence of interest in all herbal products and a rediscovery of the traditional use of medicinal herbs, includes the discovery of new useful compounds produced by natural plant populations in very small quantities or compounds that may not be produced by the adult plants which are available in cultures and the biochemical characterization and identification of active compound (Banerjee and Shrivastava, 2006).

The main industrial goal of the plant tissue culture is to produce a large number of plants in a month instead of years (Chopra et al., 1956).                MS (Murashige and Skoog) medium is foremost medium for growth compared to B5 and SH medium. Cytokinins are growth hormones generally used in tissue culture to stimulate cell division and affects shoot and root morphogenesis. Reduction in growth of primary root and density of lateral root is caused by cytokinins (Laplaze, 2007).

The developed tissue culture technique will increase the scarce knowledge about the in vitro response of this native germplasm with potential relevance as an endangered plant species, it is a necessary step in many experiments like clonal propagation, formation of virus free plants and genetic transformation. The entries of virus in the wild variety the plant will be change their genetic nature and compound synthesis.  The large scale growth of plants in liquid culture with bioreactors for production of valuable compounds derived secondary metabolites and recombinant proteins used as biopharmaceuticals (Georgieva et al., 1996).

1.2 Molecular characterization

Genetic studies are fundamental for the management and conservation of valuable medicinal plants. The use of molecular markers is a powerful tool in the genetic study of populations. The molecular marker approach for identification of plant varieties of genotypes seems to be more effective than traditional morphological markers. Since it allows direct access to the hereditary material and makes it possible to understand the relationships connecting individuals (Williams, 1990). Identification of plant species, phylogenetic analysis, population studies and genetic linkage mapping is carried out by molecular markers based on Polymerase Chain Reaction (PCR) (Paterson et al., 1991). Several DNA markers have been successfully employed to assess the genomic stability in regenerated plants including those with no obvious phenotypic alternations (Rahman and Rajora, 2001).

1.2.1 Random amplified polymorphic DNA (RAPD)

Randomly Amplified Polymorphic DNA (RAPD) analysis has been known to detect genetic diversity, genetic profiling, plant species conservation and random locations of the genome along with the cost effectiveness, technical simplicity and does not require template DNA for prior sequence information (Yu and Nguyen, 1994).

RAPD markers were then used for the linkage analysis of some phenotypical characters in plants, such as fruit skin color and comparison of genetic stability of the conserved plants and the controls (Inoue et al., 2006). RAPD Polymerase Chain Reaction method is also faster and more sensitive than biochemical methods. The advantages of RAPD technique is to analyze number of samples economically, the DNA printing is very specific which forms independent ontogeny expression. Besides, all genomes can be analyzed using unlimited number of markers (Titin Purnaningsih, 2013).

RAPD is a simple, foremost, rapid and simple assay marker that requires low quantities of template DNA, and randomly spread throughout the genome. These markers provide good genome coverage (Perez et al., 1998).

1.2.2 Inter Simple Sequence Repeat (ISSR)

ISSR is a fast and highly reliable genotyping technique, amplification of primer requires single sequence repeat motif and is based on variation in the regions between adjacent inversely oriented microsatellites. The technique is exceptionally informative, extremely reproducible and exhibits tremendous detecting polymorphism level (Pathak and Dhawan, 2012).

ISSR markers make use of longer primers (15-30 mers) as compared to RAPD primers (10 mers), that allow the following use of high annealing temperature that leads to higher stringency. In several plant species, RAPD and ISSR markers have known to be efficient to detect genetic fidelity. They have been competent in naming phylogenetic bond between Bacopa monnieri (L.) (Martin et al, 2004).

1.3 Qualitative and Quantitative analysis

Discovery of useful drugs primarily involve phytochemicals. Pharmaceutical companies have found interest in producing new drugs with the aid of phytochemical analysis of the plants and are commercially important (Wadood et al., 2013).

The potential of higher plant species, limited percentage has been investigated of their phytochemical analysis and fraction submitted to biological and pharmacological screening is even smaller. Hence any phytochemical analysis of medicinal plant will reveal only a very narrow spectrum of it components. Therapeutic agents require pharmacological screening of either natural or synthetic compounds. Random screening as tool in discovering new biologically active molecules has been most productive in the area of antibiotics (Gerhartz et al., 1986). Phytochemical constituents of plants are Alkaloids, Steroids, Tannins, Glycosides, Volatile oils, Resins, Phenols Flavonoids, Tannins and Carbohydrates are deposited in their specific parts like leaves, bark, seeds, fruits and root. The beneficial therapeutic effects of plant materials occurs from the mutualism between these secondary products and plants and finds potential use in medicine and pharmacological applications (Tonthubthimthong et al., 2001).

The Gas Chromatography and Mass Spectroscopy is the universally used technique for the identification and quantification of the unknown organic compounds in a complex mixture that can be determined by interpretation and also by matching the spectra with reference spectra. The volatile compound of plants is composed of biogenically derived hydrocarbons and oxygenated materials (Ronald Hites, 1997).  Extracts from natural products, comprising of fruits, vegetables and medicinal herbs are known to be effective against various human diseases    (Wu et al., 2002). Radical scavenging is the process of reaction that takes place between phytochemical compounds and free radical, the gained electron gets delocalized over the phenolic antioxidant and the aromatic nucleus prevents the extension of the free radical chain reaction. Instead polyphenolic compounds inhibit oxidation through a variety of mechanisms (Cuvelier et al., 1992).Plant metabolism function depends on primary and secondary metabolites. Primary metabolites consist of Chlorophylls, Carbohydrates, Amino acids and Proteins, while secondary metabolites is composed of Saponins, Alkaloids, Flavonoids, Steroids and Tannins (Kumar et al., 2009).

1.4 Antioxidant

The antioxidant is used in industrial chemicals to prevent oxidation, and in foods that contain natural chemicals, body tissue that has beneficial health effects. Antioxidants molecules inhibit oxidation that can produce oxidative damage inducing free radicals, prevents oxidative stress, lipid free radical, cellular and tissue damage (Gulcin, 2009). In recent years, the importance of natural antioxidants from plants has significantly increased (Sarikurkar, 2011). Antioxidant compounds such as Phenolic acids, Polyphenols and Flavonoids are known to scavenge free radicals like peroxide, hydroperoxide or lipid peroxyl and thus prevent the oxidative mechanisms that lead to degenerative diseases (Valentao et al., 2002).

Medicinal plants have been extensively studied for their antioxidant activity. Traditionally herbs have been used in nutrition, medicine, flavoring, beverages and cosmetics. Intakes of antioxidant rich fresh fruit, vegetables and tea have been found to prevent cancer and cardiovascular diseases        (Willcox et al., 2004). In vitro experiments carried on antioxidant compounds in higher plants inhibited free radicals and reactive oxygen species and exhibited protection against oxidation damage. These compounds have related structures to synthetic antioxidants and thus can be inferred as potential antioxidants activity (Ali et al., 2008).

1.5 In vitro cytotoxicity assay

In 1952, A. E. Moore, L. Sabachewsky and Toolan established the Human Epithelial type-2 (HEp-2) cell line by injection with epidermoid carcinoma tissue from the larynx of 56 year old male that produced tumours in irradiated cortisonized weanling rats. It could support the growth of 10 of 14 arboviruses, measles virus, and it has been in use for experimental studies in rats for tumor production, hamsters, mice, embryonated eggs and terminal cancer patient volunteers (Toolan, 1954).

Cytotoxicity analysis offers a vital means of selecting compounds for consideration in drug discovery. The selection of drug using a specific viability or cytotoxicity assessment technology could be influenced by explicit research goals. For in vitro human cell culture methods, if a compound interferes with cellular integration, if it considerably modifies cellular morphology, alters cell growth rate or causes cell death, then the compound is termed as cytotoxic (George et al., 2012).

1.6 In vitro anti fungal activity

The increasing incidence and prevalence of fungal infection in developing countries is attributed to immune compromised state such as use of anticancer drugs, immunosuppressive agents, HIV-positivity and etc., most infections of skin and its appendages, the hair and nail are caused by a homogenous group of keratinophilic fungi called the dermatophytes        (Pfaller et al., 1994). Dermatophytes are a group of molds that are related morphologically and physiologically between them, causing infections in humans and animals (De Vroey, 1985).  At least one-fifth of the world populations suffers from mycoses infection. Dermatophyte infections, particularly those involving the skin and mucosal surfaces constitute a serious problems in developing countries and are directly connected with the skin (Tinea corporis) fungal infections such as Feet (Tinea pedis), Hair (Tinea capitis), Nails (Tinea unguium),  Groin (Tinea cruris) because of the prevailing moisture and temperature conditions (Martin and Kobayashi, 1993). Tinea corporis was the most common dermatophytes. Dermatophytes are known to spread by direct contact from people, animals and soil and indirectly from fomites (Barry L Hainer, 2003).

Dermatophytosis, also known as zoonosis, have created  health hazardous due to the close contact between human, particularly children, and animals such as dogs, cats, birds and small rodent and pocket pets. The clinical symptoms may not pose a serious threat, however effective treatment is costly and time consuming since the increasing resistance incidence in known fungal pathogens to the currently available antibiotics has become apparent (Alexander and Perfect, 1997).

There is an increase in the risk of infection by dermatophytes due to factors related to the trend of living in communities, animal contact, and the usage of antineo plastic and antibiotics drugs. Clotrimazole, Miconazole, Fluconazole, Itraconazole and Ketoconazole (Imidazole group) are very effective drugs but they could be harmful to human health (Shahi et al., 1999). Drug resistant strains have caused failures such as host toxicity of available polyenes and fungi static mode of action azoles in current antifungal therapy (Baker and David Rogers, 2006).

1.6.1 Resistant fungus

Treatments to fungal infections have failed due to development of resistant strains. There is need to develop antifungal drugs using in vitro methodology against dermatophytes that are resistant. Clinical and Laboratory Standards Institute (CLSI) have previously carried out micro assays, agar dilution, E test and colorimetric micro dilution, these methodologies could not be carried in many laboratories.

A recent study comparing colorimetric broth microdilution testing of fluconazole with reference broth macrodilution and broth microdilution methods demonstrated excellent agreement among the three methods but broth microdilution method performed according to NCCLS guidelines demonstrated good agreement compared with broth macrodilution method (Diamond, 1991). Antifungal susceptibility testing is becoming more important in the clinical laboratory because the frequency of serious fungal infections, especially yeast are known to cause mucosal infections and are also known to cause male and female genital infections in immunocompromised patients, has increased significantly in recent years (Banerjee et al., 1991)

Cleome is known to be the largest genus that comprises 180 to 200 herbaceous annual species and shrubs distributed widely in tropical and subtropical regions. It has major diversity of 150 species in tropical regions, (Raghavan, 2006). It is also known as spider flower and mountain bee plant. Medicinally used as leaf paste on headache, skin diseases and also as food for pregnant women in African countries. Preparation of black paint is done by boiling Cleome leaves (Sungwarl and Supanee, 2006).

In extreme conditions, C. gynandra are eaten as vegetable. Oil from Cleome chelidonii seeds has been used as insect repellent. Cleome arabica contains higher amount of flavonoids (Rukmini, 1978). The seeds of Cleome viscosa are reported to have nutritive value while the juice of leaves is applied to the skin as counter irritant and whole plants are used too many biological properties such as PRSV melon virus WMV-2, ZYMV melon viruses and skin disease (Berger, 2005).

The leaves and stems of Cleome rutidosperma, along with other nine commonly used medicinal plants of Nigeria and suggested that these medicinal plants could potentially be used as raw materials in drug formulation      (Edeoga et al., 2003). The leaves and flower of Cleome viscosa was used for antimicrobial and antifungal activity against bacterial and fungal organisms that are pathogenic. This plant has been used to raise the formation of blood during childbirth. They are useful in treating vomiting, diphtheria, and stomach disorders. The plant could be intercropped with cabbage to reduce diamondback moth as well as thrip attacks (Silue, 2009).

Hence, the focus of the present study is in vitro micropropagation of Cleome species, Molecular characterization of micropropagated plants with wild plants. Further carryout the pharmacological activities of                 Cleome rutidosperma and Cleome viscosa.

4.1 Collection, identification and authentication of Cleome rutidosperma

and Cleome viscosa

4.1.1 Description of Cleome rutidosperma DC.

Common Name : Fringed spider flower or Purple Cleome

Vernacular Name : Seru walai

Synonyms  : Cleome ciliate, Cleome burmannii, Cleome thyrsiflora,

  Cleome guineensis

Systematic classification

Kingdom  : Plantae

Subkingdom  : Tracheobionta

Infrakingdom  : Streptophyta

Superdivision : Embryophyta

Division  : Magnoliophyta

Subdivision  : Spermatophytina

Class   : Dicotyledonae

Order   : Brassicales

Family  : Cleomaceae

Genus   : Cleome

Species  : rutidosperma

4.1.1.1  Origin and Distribution

Cleome genus comprises 150 to 200 species, the majority of species found in tropical America, whereas about 55 are known from tropical Africa. Cleome rutidosperma is native to tropical Africa and has been introduced and become naturalized in tropical and subtropical regions of Asia, the Americas and the West Indies. Cleome rutidosperma is a low growing herb belonging to the family Cleomaceae, it is a common herb that grows as a weed in disturbed and rural habitats, principally in areas with humid and hot environmental conditions. It is often found as a weed of disturbed ground, roadsides, gardens, crops abandoned lands, and has also been found as an epiphyte on trees, stone walls and cliff faces. This species is included in the global compendium of weeds (Randall, 2012), where it is considered to have moderate economic impacts in a wide range of crops, due to its scrambling habit that smothers and stunts young crop plants. C. rutidosperma has been listed as invasive in China, Malaysia, India, Thailand, Vietnam, Australia, and the Domican republic (Waterhouse and Mitchell, 1998).

4.1.1.2  Taxonomical Description

C. rutidosperma is an annual herb, up to 70 cm tall, widely branched, erect or sometimes spreading. Alternate leaves 3 palmatisect, rhomboid-elliptic to lanceolate leaflets, generally asymmetric, the central 0.5-6 x 0.2-2.5 cm, the lateral smaller, acute to acuminate at the apex, cuneate at the base, ciliolate serrulate margins, conspicuous nerves specially prominent below, petiole up to 7cm. Inflorescence racemes, solitary to few flowered, lax and not clearly demarcated, very short or up to 20 cm. long; bracts usually similar in size to the leaves. Flowers in the axil of leaf-like trifoliate bracts,  pedicels in flower up to 2.5 cm, in fruit to 3.5 cm. Narrowly lanceolate sepals 4, 2-5 x ca 0.5 mm, sub glabrous to sparsely pubescent, often with glandular hairs. Petals 4, white, pink, lilac, violet or blue, narrowed into a basal claw, oblanceolate to elliptic lamina, apiculate. Stamens 6, filaments 5-9 mm, anthers 1-3 mm. Ovary linear, cylindrical, 3-10 mm long, with a gynophore 1.5 mm,  glabrous or with some short hairs and sessile glands,  very short style,  capitate or truncate stigma, papillose (Figure 4.1, 4.2 and 4.3). Capsule 2.5 -7.5 x 0.2-0.5 cm, on a gynophore to 13 mm long, linear-ellipsoid, sometimes slightly torulose, glabrous or glabrescent, beak 2-8 mm, valves with prominent longitudinal anastomosing nerves. Subglobose seeds, slightly laterally compressed, up to 2 mm in diameter, with longitudinal striations and prominent transverse ridges, glabrous, reddish-brown, dark brown or black, whitish elaiosome present  (Widespread, 1972).

4.1.1.3  Medicinal Importance

The whole plant of C. rutidosperma has medicinal value and is known to have a numerous pharmacological effects like antipyretic (prevent fever), antimicrobial, free radical scavenging activities and antiplasmodial activities (Bose et al., 2005). The plant is frequently used in traditional medicine      (Akah and Nwambie, 1993). Leaf sap is applied in Ghana, Gabon and DR Congo to cure earache and deafness. In Ghana a leaf extract is used to treat irritated skin and in Nigeria it is used to treat convulsions. Pollen of this species was found present in honey from Malaysia. In Malaysia planting of                 C. rutidosperma around field edges considered as part of an insect control programmed (Maishihah and Kiew, 1989).

4.1.2 Description of Cleome viscosa L.

Common Name : Asian spider flower

Vernacular Name : Naikkaduku

Synonyms  : Cleome acutifolia, Sinapistrum viscosum, Arivela

                                   viscosa.

Systematic classification

Kingdom  : Plantae

Subkingdom  : Tracheobionta

Infrakingdom  : Streptophyta

Superdivision : Embryophyta

Division  : Magnoliophyta

Subdivision  : Spermatophytina

Class   : Dicotyledonae

Order   : Brassicales

Family  : Cleomaceae

Genus   : Cleome

Species  : viscosa

4.1.2.1  Origin and Distribution

Cleome viscosa occurs in northern tropical Africa, from Cape Verde and Senegal to Egypt, it is absent in southern Africa, inspite present in Madagascar and other Indian Ocean islands. Cleome viscosa is widespread in peninsular Arabia, Asia, Australia and tropical America. Cleome viscosa occurs in marshland and found in both under seasonal dry and humid conditions. Cleome viscosa its autecology and provided information regarding its distribution and abundance which is helpful for its cultivation on a commercial scale       (Jansen, 2004).

4.1.2.2  Taxonomical Description

An annual, sticky herb with a strong penetrating odor and clothed with glandular and simple hairs. Leaves are digitately compound, with 3 to 5 leaflets. Leaflets are obovate, elliptic-oblong, very variable in size, often 2-4 cm long, and petiole up to 5 cm. Racemes elongated, up to 30 cm long, with corymbose flowers at the top and elongated mature fruits below bracteates. Flowers 10 – 15 mm across, yellowish, pedicels 6 – 20 mm long, bracts foliaceous. Sepals oblong lanceolate, 3 – 4 mm long, 1 – 2 mm wide and glandular pubescent. Petals 8 – 15 mm long, 2 – 4 mm broad, oblong –  obovate. Stamens 10 – 12, not exceeding the petals, gynophore absent (Figure 4.4, 4.5 and 4.6) (Vaidyaratnam, 2010).

4.1.2.3  Medicinal Importance

The C. viscosa leaves, flowers, seeds and roots of the plant are widely used in traditional and folkloric systems of medicine as an anti inflammatory, antimicrobial, antipyretic, anthelmintic, immunomodulatory and hepatoprotective activities. The leaves and root are particularly useful for treating skin disease (Mali, 2010). C. viscosa leaves and young shoots used as vegetables. The whole herb was used to treat fungal infections, cough, bronchitis and cardiac disorders (Kirtikar and Basu 1975). Seeds have served as raw materials for the extraction of Coumarinolignoids, a valuable chemical entity needed by pharmaceuticals industries for liver diseases and immunomodulation (Chattopadhyay et al., 2004). The analgesic, antipyretic and anti-diarrheal activities of the extract of C. viscosa and it was noted that the fresh leaves are used for jaundice, seeds and leaf are used to treat viral infections (Devi et al., 2003).

4.1.3 Collection of plants

Cleome rutidosperma DC. and Cleome viscosa L. were collected from natural habitat of Sirumailur village (The latitude 13.0938995 and longitude 80.292356 are the geocoordinate of the Sirumailur), Kanchipuram district, Tamil Nadu.

4.1.3.1  Identification plants

The plants was identified and authenticated by Botanical Survey of India, Coimbatore, India, and voucher specimens were deposited.

4.2. In vitro micropropagation of Cleome rutidosperma and Cleome viscosa

with different plant growth regulators of different concentration and

combination.

4.2.1 Source of explants

Node and shoot tips were used as explants from healthy wild plants of    C. rutidosperma and C. viscosa. The explants were cut into 5 mm in length and used for in vitro micropropagation studies.

4.2.2 Preparation of stock solutions

The plant tissue culture media formulation described as Toshio Murashige and Folke Skoog (1962) experiment referred as MS medium was selected as the optimal culture medium.

MS medium stock solutions prepared for plant tissue culture as follows;

Macronutrients – Ammonium nitrate 1650mg, Potassium nitrate 1900mg, Calcium chloride 440mg, Magnesium sulfate 370mg and Potassium phosphate 170mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.1).

Micronutrients – Potassium iodide 0.83mg, Boric acid 6.2mg, Manganous sulfate 22.3mg, Zinc sulphate 8.6mg, Sodium molybdate 0.25mg, Copper sulfate 0.025mg, Cobaltous chloride 0.025mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.2).

Iron source – 27.85mg of Ferrous Sulphate and 37.30mg of Disodium ethylenediaminetetraacetic acid dihydrate were heated separately and dissolved in one liter sterile distilled water later, and transfer in to sterile storage bottle (Table – 4.3).

The organic supplements – Myo – inositol 100mg, Nicotinic acid 50mg, Pyridoxine HCl 50mg, and Thiamine HCl 50mg, and Glycine 200mg were dissolved in one liter sterile distilled water and transfer in to sterile storage bottle (Table – 4.4).

Carbon source – 3% sucrose was added into the one liter solutions. The pH was adjusted to 5.6 – 5.8. The preparation was then gelled with 0.8% w/v agar (Table – 4.5).

These stock solutions were prepared and stored at 4⁰C until further use.

Table: Composition of revised Murashige and Skoog medium:

Table – 4.1: Major inorganic nutrients stock solution

Table – 4.2: Minor inorganic nutrients stock solution

Table – 4.3: Iron source stock solution

Table – 4.4: Organic supplements stock solution

Table – 4.5: Carbone source

4.2.3 Growth regulators

Plant growth regulators (PGRs) such as cytokinins 6-Benzyl amino purine (BAP), Kinetin (KIN) and auxins Indole-3-acetic acid (lAA), a-Naphthalene acetic acid (NAA),  2,4-Dichlorophenoxy acetic acid (2,4-D), Indole-3-butyric acid (IBA) at different concentrations and in combinations used for the study. Each hormone was dissolved in respective solvents and used for micropropagation (Table – 4.6).

4.2.4 Preparation of plant growth regulators

The Preparation of plant growth regulators stock solutions were as follows.

Cytokinins such as Kinetin 100mg was dissolved in 20ml of 0.1N NaOH and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration. The same concentration of                       6-benzyl amino purine (BAP) at 1.0mg/1.0ml concentration was prepared and stored at 4^ºC.

Auxins such as Indole-3-acedic acid (IAA) (100 mg), Indole-3-butyric acid (IBA) (100mg) and α-Naphthaline acedic acid (NAA) (100mg) were dissolved separately in 20ml of 0.1N NaOH and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration and stored at 4^ºC.

100mg of 2,4-Dichlorophenoxy acetic acid (2,4D) was dissolved separately in 20ml of 70% ethanol and diluted with 980ml of distilled water to make up to 1.0mg/1.0ml concentration and  stored at 4^ºC

All these hormones were added in to MS medium before the sterilization.

Table – 4.6: Solubility of plant growth regulators                             (cytokinins and auxins)

Hormones Name	Empirical formula	Solubility
6 – Benzyl amino purine (BAP) (Himedia PCT 0802)	C12H11N5	0.1N NaOH
Kinetin (Kin) (Himedia PCT 0806)	C10H9N5O	0.1N NaOH
2,4-Dichlorophenoxy acetic acid (2,4D) (Himedia RM 515)	C8H6O3Cl	70% ethanol
Indole-3 acetic acid (IAA) (Himedia PCT 0803)	C10H9NO2	0.1N NaOH
α-Naphthalene acetic acid (NAA) (Himedia PCT 0809)	C12H10O2	0.1N NaOH
Indole-3 butyric acid (IBA) (Himedia PCT 0804)	C12H13NO2	0.1N NaOH

4.2.5 Potential of Hydrogen (pH) adjustment

pH was adjusted using a pH meter. 1N NaOH was added to neutralize the solution. This neutralization can be expressed as follows:

HCl + NaOH → NaCl + H₂O

The above hydrochloric acid 1N (HCl) was neutralized with caustic (NaOH) and yields Sodium chloride (NaCl) and distilled water. NaCl being very soluble in distilled water, remains dissolved in solution, and very little solids are generated.

4.2.6 Preparation of tissue culture medium

An aliquot of the stored stock solution was used for micropropagation. To make up one liter of MS medium, 50ml of macronutrients, 5ml of micronutrients, 5ml of iron stock solution and 5ml of organic supplements were dissolved in 935ml of sterile distilled water. Further 3% sucrose was added into the one liter solutions. The pH was adjusted to 5.6 – 5.8. The preparation was then gelled with 0.8% w/v agar. The MS medium was sterilized by autoclaving at 15psi (121^ºC) for 15 min. After sterilization the 20ml of medium was transferred to each culture tubes (25×150mm).

4.2.7 Experimental design

As per the experimental designs the plant growth regulators were added into the respective MS medium before solidification.  After addition the test tubes were sterilized by autoclaving at 121^ºC for 15 min. The sterilized test tubes were kept in the laminar air flow for solidification and the sterile MS medium culture tubes with plant growth regulators were used for explant cultures (Table – 4.7).

The explants were inoculated into following combination and various concentrations

BAP, KIN and 2,4D

BAP, KIN and NAA

BAP, NAA and IAA

BAP and NAA

IBA, IAA and BAP

4.2.8 Sterilization of explants

The explants nodes and shoot tips were excised from the field grown healthy and vigorously growing mother plants of Cleome rutidosperma and Cleome viscosa. These explants were washed under running tap water to remove all the adhering dust particles and microbes from the surface. The explants were then washed with few drops of Tween 20 (Himedia TC 287) solution for five minutes and rinsed for five minutes in sterile distilled water. The explants were then treated with Bavistin (0.1 % w/v) for another ten minutes to remove the fungal contamination and then washed properly to remove the fungicide.

The explants were further sterilized in the laminar air flow chamber with shoot tip and nodal segments were treated with 0.1% HgCl₂              (Himedia GRM 1383) for four minutes. The explants were then thoroughly washed (5 washings) with sterilized distilled water to remove the traces of HgCl₂ solution (Muthusamy Govarthanan et al., 2015).

4.2.9 Inoculation of explants

The ultra clean air blow through High efficiency particulate air (HEPA) filters, being free from fungal and bacterial contaminants are required for sterile transfer of explants. The explants inoculations were made under the laminar air flow chamber with the sterile precautions.

In the experiments, different explants from node and shoot were cultured on the MS medium supplemented with various concentrations and combination of plant growth regulators (PGRs) were used in both explants of Cleome rutidosperma and Cleome viscosa.

The explants were inoculated with one explant per tube. It was usual procedure to flame the mouth of the test tube after uncapping and before recapping the tubes to reduce contamination. The selection was made on the basis of extensive proliferation of shoots formation. The growth of the shoots was observed in terms of increase in biomass (Archana Sharma, 2013).

4.2.10 Cultural conditions

After inoculation, the culture tubes were transferred to the culture room which was initially sterilized by washing all of its walls and floor with a detergent solution followed by wiping with lyzol solution. The cleaning of culture room was done at regular intervals. Initially culture tubes containing the plant material were incubated in complete darkness for 72 hours till they got established. Subsequently, they were exposed to 18 hours of photo period provided by cool white florescent tube lights. The culture room was maintained at temperature around 25 ± 2°C. Observations were made from 3^rd day up to 28 days. After 4 weeks of inoculation sub culturing of the explants was done.

4.2.11 Rooting

Individual elongated shoots (3cm length) of well developed from the experiment were transferred to MS medium containing 3% (w/v) sucrose, 0.8% (w/v) agar and different concentrations (0.5 to 3.0 mg/l) and combination of auxins such as IBA + IAA + BAP for node and shoot tips were induction. Rooting was observed from 20 days. In vitro plant was well developed rooted shoots. Data were recorded on number and length of roots                       (Figure 4.7 and 4.8).

4.2.12 Hardening

The in vitro raised plantlets developed from explants of nodal and shoot tips from well rooted plants were removed from the culture tubes and were washed in tap water to remove the agar. The plantlets were transferred to pots containing sand: soil (1:1) and cocopit mixture covered with transparent plastic bags with small holes for air circulation. The plants were placed in the shade with low illumination strength, high humidity 90 ± 5% and temperature          26 ± 4°C. After four weeks, the seedling was transferred to wild field conditions with highest light intensity (765.7umol m^-2 s^-1) temperature 21 to 28°C and humidity 65 to 80%.

4.3 Study the molecular genetic variation between wild and  micropropagated plantlets using Random Amplified Polymorphic               DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analysis.

4.3.1 Isolation of genomic DNA

The DNA of wild and micropropagated plants of C. rutidosperma and C. viscosa plant extracted from 1g of freeze-dried powder. The samples were frozen in liquid nitrogen.  The mini extraction was done based on the cetyl trimethyl ammonium bromide (CTAB) according to Voigtet et al., (1999) method. The finely grounded plant materials were transferred to sterile centrifuge tube. Pre-warmed at 65^ºC DNA extraction buffer [100 mM Tris HCl (pH 8.0), 1.4M NaCl, 50mM EDTA (pH 8.0) and 2% CTAB] was added to this, mixed well and incubated in a water bath at 65^ºC with gentle shaking for one hour. After incubation an equal volume of chloroform: isoamyl alcohol        (24:1 v/v) was added gently to denature proteins and centrifuged at 10000 rpm at 25^ºC for 10 min. The aqueous phase was transferred to a new sterile tube and DNA was precipitated with 0.6 volume of cold isopropanol and 0.1 volume of 3M sodium acetate (pH 5.2) and centrifuged at 15000 rpm for 25 min at 25^ºC. The supernatant was poured off and the pellet was washed twice with 70% cold ethanol and dried at room temperature. The pellets were air dried and suspended on TE buffer (pH 8.0).

4.3.2 Purification of Genomic DNA

The extraction of total genomic DNA from the C. rutidosperma and        C. viscosa isolates as per the above procedure was followed by RNase treatment. Genomic DNA was resuspended in 100µl of TE buffer and added 2μl of RNase and incubated at 37^ºC for one hour. After incubation the sample was re-extracted with phenol: chloroform: isoamylalcohol (25:24:1) solution and RNA free DNA was precipitated with 0.1 M sodium acetate solution along with 100% chilled ethanol and centrifuged at 12000rpm for 15min at 40ºC, washed with 70% ethanol, air dried and dissolved in TE buffer. The quality and quantity of DNA was analyzed by spectrophotometrically and in 1% agarose gel. The DNA isolates produced clear sharp bands, indicating high-quality and purity of DNA was calculated by the absorbance ratio of A260/A280nm       (Table 4.8).

Table – 4.8: Yield and purity of DNA isolated from the wild and micropropagated plants of Cleome species

4.3.3 RAPD and ISSR analysis

RAPD analysis was performed by polymerase chain reaction (PCR) conditions and fifteen random primers were used for genomic DNA identification. The number of primers which present strong band resolution were chosen for the study viz., OPA 01, OPA 02, OPA 03, OPA 04, OPA 05, OPA 06, OPA 10, OPA 19, OPA 20, OPC 01, OPE 05, OPE 06, OPE 07, OPAM 01, OPS 18 and three ISSR primers namely ISSR 1, ISSR 2, ISSR 3 were used the PCR amplification was carried out with 25ng of genomic DNA, 2.5 mM MgCl₂, 1µl Taq polymerase, 1x PCR buffer, 1µl ISSR primer and 0.2 mM dNTPs mix. The PCR cycle was as follows: 5 minute at 95ºC, 30 seconds at 32ºC and one minute at 72ºC for 32 rounds. The extension period was 8 minutes at 72ºC. The PCR products of both RAPD and ISSR reaction were resolved on 1.5% agarose gel in 1× TAE buffer stained with ethidium bromide (8µl/100ml) and electrophoresis was carried out at 75 volts for 1.5 hours and visualized under UV-Transilluminator. The gel was photographed with a gel documentation system.

4.3.4 Scoring and Data analysis

The image of the gel electrophoresis was documented through Bio-Profile Bio-1D gel documentation system. The polymorphic bands were scored and analyzed by UPGMA cluster analysis protocol and computed in silico into similarity matrix using NTSYSpc (Numerical Taxonomy and Multivariate Analysis System genome arranged in tandem repeats with each Biostastistics, version 2.11W). The SIMQUAL (similarity for qualitative data) program was used to calculate the Jaccard coefficients. The RAPD patterns of each isolate was evaluated, transcribed spacer regions have been used assigning character state “1” to indicate the presence of  band in the gel and “0” for its absence in the gel. A data matrix was formed which was used to calculate the Jaccard similarity coefficient for each pair wise comparison. Jaccard coefficients were clustered to produce dendrogram using the (Sequential, Hierarchical, Agglomerative, and Nested cluster methods) SHAN clustering program and selecting the un-weighted pair-group methods with arithmetic average (UPGMA) algorithm in NTSYSpc.

4.3.5 Solvent extraction of plant materials

4.3.5.1  Extraction from wild plants

The preliminary phytochemical and GC – MS analysis was done with ethanol (Polar) and chloroform (Non polar) solvent.

Collected fresh plants of C. rutidosperma and C. viscosa were washed thoroughly in sterile distilled water to remove external debris. The plants were shade dried at room temperature, for about a month and ground well into powder with an electric blender. One gram of plant powder was drenched in a 10ml of ethanol and chloroform for 4 hours and sonicated in an Ultrasonic Sonicator at 20 pulses for 25 minutes later centrifuged at 5000 rpm for 20 minutes and the supernatant was stored at 4^oC until further use.

4.3.5.2  Extraction from micropropagated plants

The harvested in vitro plants were washed thoroughly in sterile distilled water to remove external MS medium. Dried plant material was grinded in a pestle and mortar. One gram of plant powder was drenched in a 10ml of ethanol and chloroform for 4 hours and sonicated in an Ultrasonic Sonicator at 20 pulses for 25 minutes later centrifuged at 5000 rpm for 20 minutes and the supernatant was stored at 4^oC until further use (Singh and Tiwari, 2012).

4.4 Analysis the preliminary phytochemical and Gas Chromatograph and Mass Spectrometry (GC-MS) of wild and micropropagated plants

4.4.1 Preliminary phytochemical screening

The qualitative chemical tests were performed for establishing profile of ethanol and chloroform extracts of Cleome rutidosperma and Cleome viscosa.Qualitative phytochemical analysis was done according to Johansen (1940).

4.4.1.1 Test for Alkaloids

Wagner’s reagent

Iodine (2gm) and potassium iodide (6gm) was dissolved in 5ml of distilled water and made up to 100ml with distilled water.

2ml of extracts were dissolved in diluted HCl (258ml HCl and one liter DH₂O) and filtered. Few drops of Wagner’s reagent were added by the side of the test tube. A reddish brown precipitate would confirm the test as positive.

4.4.1.2  Test for Carbohydrates

Molish’s reagent

The 2ml extracts were diluted in 5ml of distilled water and filtered. 2ml of filtrate, two drops of Molish’s reagent were added, the mixture was shaken well and 2ml of concentrated sulphuric acid was added slowly along the sides of the test tube and allowed to stand. A violet ring at the junction would indicate the presence of carbohydrates.

4.4.1.3  Test for Saponins

Frothing test

2ml dilute extracts and 2ml distilled water was added in semi micro test tube. The test tube was shaken vigorously and left for three minutes. Formation of honeycomb like froth will indicate the presence of Saponins.

4.4.1.4 Test for Steroids

Salkowski test

0.5g of extracts, 3ml of chloroform (CHCl₃) and 2ml of concentrated sulphuric acid (H₂SO₄) was added and shaken well. Red brown and acid layer greenish yellow fluorescent appeared. This would confirm the presence of steroids.

4.4.1.5 Test for Terpenoids

Salkowski test

1ml of extracts was diluted with 3ml of chloroform and concentrated H₂SO₄ was added to form a layer. A reddish brown colouration of the interface is indicated the presence of Terpenoids.

4.4.1.6 Test for Flavonoids

Sodium hydroxide test

2ml of extracts were treated with few drops of 20% sodium hydroxide solution. Formation of intense yellow color will indicate the presence of flavonoids.

4.4.1.7 Test for Phenols

Ferric Chloride Test

1ml of the extracts were added in 2ml of distilled water then warmed followed by few drops of 10% aqueous ferric chloride. Formation of black color would indicate the presence of phenols.

4.4.1.8 Test for Amino acids and Proteins

Ninhydrin test

Few drops of 1% Ninhydrin (10mg of ninhydrin in 200ml of acetone) was added into 2ml of extracts and kept in the boiling water for three minutes. Formation of purple color will indicate the presence of amino acids and blue color would indicate the presence of proteins.

4.4.1.9 Test for Tannins

Braymers Test

1ml of extracts were diluted in a 10ml of distilled water and then filtered. Few drops of 10% alcoholic FeCl₃ solution were added. Formation of dark blue or greenish black will indicate the presence of tannins.

4.4.1.10 Test for Cardiac glycosides

Keller-Kilianis Test

5ml extracts were added into 2ml of glacial acetic acid. Few drops of 1% FeCl₃ and conc. H₂SO₄ were added into test tubes. Reddish brown color appears at junction of the two liquid layers and upper layer appears violet ring would confirm the presence of glycosides.

Concentrate H₂SO₄ Test

5ml extracts were mixed with 2ml glacial acetic acid, One drop of 5% FeCl₃ and conc. H₂So₄ were added into test tubes. Brown ring appears, indicating the presence of glycosides.

4.4.1.11 Test for Quinones

Few drops of concentrated hydrochloric acid (HCl) were added into 1ml of extracts. Formation of red color will indicate the presence of quinines.

4.4.1.12 Test for Coumarins

1ml of 10% NaOH was mixed with 1ml of extracts. Formation of yellow color indicated the presence of Coumarins.

4.4.1.13 Test for Oxalate

Few drops of ethanolic glacial acid added into 3ml of extracts. A greenish black coloration indicates the presence of oxalate.

4.4.2. Gas Chromatography and Mass Spectrometry analysis

GC-MS analysis of the ethanol and chloroform extract of                      C. rutidosperma and C. viscosa was performed in a Perkin-Elmer GC Clarus 500 system comprising an AOC-20i auto-sampler and a Gas Chromatograph interfaced to a Mass Spectrometer (GC-MS) equipped with a Elite-5MS (5% diphenyl/95% dimethyl poly siloxane) fused a capillary column (30 × 0.25 μm ID × 0.25 μm df). For GC-MS recognition, an electron ionization system was operated in electron impact mode with ionization energy of 70 eV. Helium gas (99.99%) was used as a carrier gas at a constant flow rate of 1 ml per minutes, and an injection volume of 2μl was employed (a split ratio of 10:1). The injector temperature was maintained at 250°C, the ion-source temperature was 200°C, the oven temperature was programmed from 110°C (isothermal for three minutes), with an increase of 10°C/minutes to 200°C, then 5°C/minutes to 280°C, ending with a 10 minutes isothermal at 280°C. Mass spectra were taken at 70 eV, a scan interval of 0.5s and fragments from 45 to 450 Da. The solvent delay was 0 to 3 minutes, and the total GC-MS running time was 37 minutes. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The mass-detector used in this analysis was Turbo Mass Gold Perkin Elmer, and the software adopted to handle mass spectra and chromatograms was a Turbo-Mass version-5.2.

4.4.2.1. Identification of compounds

Interpretation on mass spectrum GC-MS was conducted using the database of National Institute Standard and Technology (NIST) having 62,000 patterns. The spectrum of the known component was compared with the spectrum of the known components stored in the NIST library.

4.5     In vitro antioxidant activity by 2,2-diphenyl-1-picryl-hydrazyl

hydrate (DPPH) free radicals scavenging assay

The free radical scavenging ability of the wild and in vitro plants ethanol and chloroform extracts of C. rutidosperma and C. viscosa was estimated by   2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radicals scavenging assay described by Von Gadow et al., (1997).

4.5.1 DPPH radical scavenging assay

2ml of 6 ×10^-5 M methanolic solution of DPPH were added to 50μl of ethanolic solution (1mg/ml) of the sample and five concentrations with a range of 100, 50, 25, 12.5 and 6.25μg/ml were chosen for the study. Absorbance measurements commenced instantly. The absorbance reduced at 515nm and recorded in a spectrophotometer for 16 minutes at room temperature. Methanolic solutions of positive control (Ascorbic acid) were tested at 1mg/ml concentration. The scavenging effect (decrease of absorbance at 515nm) was plotted against the time and the percentage of DPPH radical scavenging ability of the sample was calculated from the absorbance value at the end of 16 minutes in duration as follows: The percentage inhibition of the DPPH radical by the samples was calculated according to the formula of Yen and Duh (1994).

IP = [(AC (0) – AA (t) / AC (0))] × 100

IP  = Percentage of inhibition

AC (0) = absorbance of the control at t = 0 min.

AA (t) = absorbance of the antioxidants at t = 16 min.

4.6.1 Media and Cell line

Human epithelial type 2 (HEp-2) cell line was procured from National Centre for Cell Sciences (NCCS), Pune and cultured in a Minimal Essential Media supplemented with 10% Fetal bovine serum, Penicillin, Streptomycin      Amphotericin-B, L-Glutamine and Sodium bicarbonate. HEPES (2-[4-(2-Hydroxyethyl]1-piperzinyl ethane sulphonic acid) buffer in a humidified atmosphere of 5% CO₂ at 37°C.

4.6.2 Sub culturing of cell line

The HEp-2 cells attained a confluent monolayer separation of the cell line was carried out using Phosphate buffered saline (PBS) and TPVG (Trypsin, Phosphate buffered saline, Versene (EDTA), Glucose) under room temperature. The medium over the cell line was gently poured out from the culture flask. The confluent cell line washed gently with 2ml of PBS in order to remove the remaining serum from the cell line. 4ml of TPVG solution was delivered over the cell line by a micropipette.

The culture flask incubated at 37°C for four minutes until cell line becomes opaque. At this stage cells had undergone rounding without detachment which was observed under Inverted Tissue Culture Microscope. The TPVG solution applied over the cell line carefully pipette out from the culture flask. Then 5ml of 5% FBS growth medium was added into the flask and the content was aspirated several times with a pasteur pipette in order to suspend and separate the cell line into individual cells (Figure 4.9).

4.6.3 Cell counting

Cells were counted using a haemocytometer. A drop of cell culture was placed in the space between the chamber and the glass cover. Each grid was focused under the microscope and the cells were counted as per the formula.

The total count from 4 sets of 16 corner = (cells / ml x 10⁴) x 4 squares from one haemocytometer grid. Divide the count by 4:2. Then multiply by 2 to adjust for the 1:2 dilutions in trypan blue. These two steps are equivalent to dividing the cell count by 2.

4.6.4 In vitro cytotoxicity assay on HEp-2 cell line

A cytotoxicity property of ethanol and chloroform extracts of                           C. rutidosperma and C. viscosa was carried out in the tissue culture 96 well microtitre plates. After enumeration of the number of viable cells                     (1 × 10⁵ cells) were plated in 100µl of medium in 96 well plates. This assay was performed according to the procedure reported by Mosmann, 1983:    Good et al., 1966. After 24 hours incubation at 5% CO₂ atmosphere the confluence of monolayer cell was confirmed by observation under Inverted Tissue Culture Microscope (Figure – 4.10).

The plant extracts were dissolved in DMSO and serially diluted with 2% MEM and five concentrations with a range of 100, 50, 25, 12.5 and 6.25μg/1ml were chosen for the study. DMSO concentration was kept less than 0.1% in all the samples. Prepared dilutions were added to different wells, and cells were incubated at 37^ºC for 48 hrs. Control groups added the same amount of DMSO. The triplicate setup was maintained.

4.6.5 In vitro cytotoxicity assay by MTT method

5mg of MTT was dissolved in 1ml of PBS (Phosphate Buffer Saline). The medium was removed from the 96 well plates and 20µl of prepared MTT solution was added into the respective wells and the plate were incubated for four hours at 37˚C. The supernatant was removed and 100µl of DMSO was added to solubilize the formed formazan. The plates were gently shaken for     5 minutes, the colour reaction was measured at 540nm using Microplate reader (Biotek, USA). The optimal density (O.D) values of the experiments and control were observed.

4.7 Antifungal activity of wild and micropropagated plants were  carried out against the dermatophytic fungi and compared with               positive control

4.7.1   Fungal strains

The standard fungal strains were procured from Department of Microbiology, King Institute of Preventive Medicine and Research institute, Government of Tamil Nadu, Guindy, Chennai, India. A total nine dermatophytes strains, namely Candida albicans (MTCC 854), Candida tropicalis (MTCC 461), Aspergillus niger (MTCC 281), Aspergillus flavus (MTCC 277), Aspergillus fumigatus (MTCC 343), Epidermophyton floccosum (MTCC 7880), Microsporum gypseum (MTCC 2819), Microsporum canis (MTCC 2820) and penicillium (MTCC 1995) was maintained as slant culture in Sabouraud dextrose agar (SDA) medium with 0.05% chloramphenicol by incubated at Candida sp.  37°C for 24 hours and dermatophytes at 30°C for 72 hours (Figure – 4.11).

4.7.2 Fungal inoculum preparation of culture media

Stock inoculum suspensions of the fungi were prepared from 7 to 10 day old cultures grown on SDA at 30°C. Mature colonies were enclosed with 5ml of sterile saline (0.85%) by scraping the surface with the tip of a sterile swab. The mixture of conidia and hyphal fragments was transferred to sterile tube. Solid particles were permitted to sediment for 15 to 20 minutes at room temperature. The superior suspension was transferred to a new sterile tube and diluted with a vortex mixer for 15 sec. Each fungal suspension was diluted with 1:100 ratio in RPMI medium. The final spore suspension (1 × 10⁶ CFU/ml) was prepared through 0.5 McFarland Standard (Parvaneh Adimia, 2013).

4.7.3 Measurement of fungal turbidity by McFarland Standard

The turbidity of the fungal supernatants was analyzed using 0.5 McFarland Standard and measured by UV – visible spectrophotometer (Shimadzu – Model UV 1650 PC) at a wavelength of 530nm             (Sundararaj, 1997).

4.7.4 Culture media for antifungal study

Roswell Park Memorial Institute (RPMI 1640) (with L – Glutamine, 2.0 grams per litre Glucose and 0.165M per litre MOPS buffer without Sodium bicarbonate) (Himedia AL200A) was used for in vitro antifungal assay.

4.7.5 Standard antifungal drugs

The standard antifungal agents namely Clotrimazole (Sigma C6019), Itraconazole (SigmaI6657) and Amphotericin B (Himedia – TC019) were dissolved in DMSO 1mg/ml. Fluconazole (Himedia – CMS8387) was dissolved in saline 1mg/ml. The final concentration was prepared with two fold dilution methods.

4.7.6 Minimum Inhibitory Concentration (MIC) by microdilution assay

4.7.6.1 In vitro antifungal assay

The principle of the assay was to estimate the lowest concentration of the extracts that inhibited visible growth and the absorbent was determined.

The in vitro susceptibility of the targeted fungi using plants extracts was carried out by adapting Clinical and Laboratory Standards Institute (CLSI) broth micro dilution method (M-38A) for yeast and filamentous fungi. The antifungal activity of wild and micropropagated plant extracts were performed in sterile flat bottom 96 well micro plates. The ethanol and chloroform extracts were solubilized in dimethyl sulfoxide (DMSO) at 1mg/1ml concentration by two fold serial dilutions.

A volume of 100μl fungal suspension along with RPMI-1640 broth medium was added into each well and 100, 50, 25, 12.5, and 6.25μg/ml of each plant extracts were added into respective wells. 100μl of fungal suspension      RPMI-1640 broth medium and 100μl standard antifungal drugs were added in to control wells. The filamentous fungus experimental setup incubated at appropriate temperature 30°C for 24 hours and the Candida sp. experimental setup incubated at appropriate temperature 37°C for 24 hours (Rajarajan et al., 2002). The visibility of the lowest concentration of the extracts inhibited the growth was recorded and the optimal density (O.D) value of the each well was determined using an automatic microplate reader (Biotek USA) at 540nm (Figure – 4.12). The triplicate setup was maintained.

4.7.7 Minimum Fungicide Concentration (MFC)

The fungicidal activity of ethanol and chloroform extracts of                  C. rutidosperma and C. viscosa was determined by recovery plate method. This technique was performed to determine the antifungicidal activity which was carried out to confirm complete absence of growth of fungi from the incubated broth of 96 wells in the visibly turbid free clear wells. The experimental broth of 20μl from each MIC well was subcultured on Sabouraud Dextrose Agar (SDA) plate. The plates were incubated at appropriate temperature (Candida sp. at 37°C and filamentous at 30°C). Absence of colony formation was presumed as fungicidal activity of the extract at a particular concentration in the well. The 100% inhibitory effect was considered to the MFC value and the results were compared to that of standard fungus positive controls.

The in vitro micropropagation, antioxidant, cytotoxicity assay and in vitro antifungal experimental results were expressed as mean ± standard deviation (SD) of triplicate measurement value. All the experiments data were statistically analyzed to test the significance by using Statistical Package for the Social Sciences (SPSS) version 18.0 software.