Phytochemical Analysis and their Antimicrobial Potential Against the Phytopathogens of Brinjal and Chillies Plants

The medicinal plants are laden with numerous effective pharmacological agents that provide an alternative means of therapy to various infections caused by drug resistant bacteria or dreadful diseases like cancer and other physiological disorders. Azadirachta indica commonly known as Neem, belongs to Meliaceae family and it is well known in India and its neighbour countries for more than 200 years as one of the most versatile medicinal plant that has a wide spectrum of biological activity Cunha, (2001); Dhar et al., (1979). The first indication that Neem was being used in medical treatment was about 4500 years ago during the high point of the Indian Harappa culture, one of the greatest civilisations of the ancient world. In fact, neem (A. indica), is the most useful traditional medical plant in India. Every part of the tree has been used as a traditional medicine for household remedy against various human ailments, from antiquity. Besides that, Neem has been extensively used in Ayurveda, Yunani and homoeopathic medicine and has become a cynosure of modern medicine. Neem has shown to be an excellent wound healer. The antiseptic and healing properties of neem make it an excellent first aid for minor cuts and abrasions. The plant has the ability to increase vascular permeability by increasing the blood flow and by helping the body to rapidly create collagen fibers to close wounds (Ellof, 1998; Evans, 2002 and Gislene et al., 2000). Neem also plays a role in treating skin burns. Besides, neem has been reported to have antipyretic compounds that have traditionally been used to reduce fevers. Studies done by Sharma et al., 2011, has demonstrated that the methanol extract from neem leaves exert an antipyretic effect in male rabbits, which is caused by the same virus, as chickenpox, at the surface level if took internally during times of stress. It can inactivate the viruses, and preventing the virus from multiplying sufficiently to cause an outbreak (Dev, 1979; Grayer RJ and Harborne JB 1994). Neem leaves have been used for centuries in traditional Ayurvedic and Siddha medicine for their antibacterial, antifungal, anti-inflammatory, and antioxidant properties. While more scientific research is needed, studies suggest potential benefits for skin health, blood sugar management, and more. Citrus sinensis (sweet orange) has been traditionally used for various ailments, supported by compounds that offer potential benefits for immune support, heart health, and digestion. It is rich in antioxidants like vitamin C, which boosts the immune system, and flavonoids like hesperidin, which may help lower cholesterol and improve heart health. Other uses include aiding in the prevention of kidney stones due to its citrate content, and helping with digestive issues like constipation and diarrhea.  Rose petals have various medicinal uses, including calming anxiety, reducing inflammation, soothing skin irritation, and easing digestive and menstrual issues. They can be consumed as a tea, used topically, or inhaled through aromatherapy, and are rich in antioxidants and vitamins like C and A.  Plants produce several secondary metabolite compounds including alkaloids, cyanogenic 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). The activity of these compounds also depends on the method and solvent used for extraction, its concentration and structure (Ateb DA and Erdo Url OT., 2003). Therefore, the significance of this study is to analysis phytochemicals and antimicrobial activities of the methanol extract and aqueous extract from neem leaf and determine the potential of these extracts against phytopathogens (Harborne JB, 1988 & 1989; Mansfield JW, 2000).

MATERIALS AND METHODS:

Collection of plant material

The fresh Neem (Azadirachta indica) leaves were collected from neem trees at Nexus Research Institute, Guntur. Rose petals and Orange peels or Citrus sinensis were also collected from the Guntur market (Fig. 1). They were washed under running tab water for 5 minutes in order to remove the dust particles stuck on their surface. They were allowed to dry for 10 days at room temperature. The dried material were then blended using dry blender to obtain the powder form for more efficient and effective organic extraction. For aqueous extraction, the disease free and fresh plants were selected. About 10 g of fresh and healthy leaves, peels and flowers were taken and surface sterilized with 0.1% mercuric chloride for 20 seconds. Again the leaves, peels and flower were washed thoroughly with distilled water (Bhandarkar and Khan. 2003; Bhattacharjee et al., 2006).

Fig.1. A: Leaves of Azadirachta indica, B: Petals of Rosa damascena or Rosa chinensis, C: Peels of Citrus sinensis

The dried above plant were ground to powder form. 50 g of each sample was soaked with chloroform and acetone in a closed 500 ml conical flasks and then kept at room temperature for 24 hours. The extraction process was repeated 2 times (extraction for 2 days). Then the extracts were filtered by using Whatman 1filter paper and concentrated at reduced pressure using a rotary evaporator at 40^oC. The dried extracts were kept in the refrigerator at 4°C for further use.

Methanol and Aqueous extraction:

For methanol extraction, about 50g of the dried and powdered materials were taken and added to 100 ml of methanol. The mixtures were sonicated for 30 min, and then left at room temperature overnight. The extracts were filtered over Whatmann No.1 filter paper, and the filtrates were concentrated under reduced pressure to pasty mass. The methanol extract was subjected to chemical tests to screen for the presence of various secondary metabolites and antimicrobial properties (Ramachandra et al.,1993). 10 g of sterilized plant leaves and flower were kept in 20 ml of sterilized distilled water (1:2). Then they were ground well with the help of mortar and pestle. The plants were subjected to centrifugation for 15 min at 1000 rpm. Again, it was filtered through Whatmann No.1 filter paper. The supernatant were collected and the plant extracts of different dilution.

Phytochemical screening: Different qualitative chemical tests can be performed for establishing profile of methanol and aqueous extract for its chemical composition. The following tests were performed on extracts to detect various phytoconstituents present in them.

1. Detection of Alkaloids (Evans, 1997) Solvent free extract, 50 mg is stirred with few ml of dilute hydrochloric acid and filtered. The filtrate is tested carefully with various alkaloidal reagents are,

1. Mayer’s test (Evans, 1997): To a few ml of filtrate, a drop or two of Mayer’s reagent are added by the side of the test tube. A white or creamy precipitate indicates the test as positive.
2. B. Wagner’s test (Wagner, 1998): To a few ml of filtrate, few drops of Wagner’s reagent are added by the side of the test tube. A reddish- brown precipitate confirms the test as positive.

2. Detection of Carbohydrates and Glycosides:

A. Molish’s test: To 2 ml of filtrate, two drops of alcoholic solution of α- naphthol are added, the mixture is shaken well and 1 ml of concentrated sulphuric acid is added slowly along the sides of the test tube and allowed to stand. A violet ring indicates the presence of carbohydrates.

B. Benedict’s test: To 0.5 ml of filtrate, 0.5 ml of Benedict’s reagent is added. The mixture is heated on a boiling water bath for 2 min. A characteristic coloured precipitate indicates the presence of sugar.

C. Borntrager’s test for Glycosides (Evans, 1997): 50 mg of Extract is hydrolysed with concentrated Hydrochloric acid for 2 h on a water bath, filtered. To 2 ml of filtered hydrolysate, 3 ml of chloroform is added and shaken, chloroform layer is separated and 10% ammonia solution is added to it. Pink colour indicates the presence of glycosides.

3. Detection of Saponins (Kokate, 1999): The extract (50 mg) is diluted with distilled water and made up to 20 ml. The suspension is shaken in a graduated cylinder for 15 min. A 2 cm layer of foam indicates the presence of saponins.

4. Detection of Proteins and Amino acids (Ruthmann, 1970): The extract (100 mg) is dissolved in 10 ml of distilled water and filtered through whatmann No.1 filter paper and the filtrate is subjected to tests for proteins and amino acids.

1. Millon’s test (Rasch and Swift, 1960) To 2 ml of filtrate, few drops of Millon’s reagent are added. A white precipitate indicates the presence of proteins.
2. Biuret test (Gahan, 1984) An aliquot of 2 ml filtrate is treated with one drop of 2 % copper sulphate solution. To this, 1 ml of ethanol (95%) is added, followed by excess of potassium hydroxide pellets. Pink colour in the ethanolic layer indicates the presence of proteins.

5. Detection of fixed Oils and Fats (Kokate, 1999)

A. Spot test A small quantity of extract is pressed between two filter papers. Oil stain on the paper indicates the presence of fixed oil.

B. Saponification test A few drops of 0.5 N alcoholic potassium hydroxide solution is added to a small quantity of extract along with a drop of phenolphthalein. The mixture is heated on water bath for 2 h. Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

6. Detection of Phenolic compounds and Tannins: Ferric chloride test (Mace, 1963): The extract (50 mg) is dissolved in 5 ml of distilled water. To this, few drops of neutral 5% ferric chloride solution is added. A dark green colour indicates the presence of phenolic compounds.

7. Detection of Steroids: 2 ml of acetic anhydride was added to 0.5 g of the extract of each with 2 ml of H2SO4. The colour changed from violet to blue or green in some samples indicates the presence of steroids.

8. Detection of Terpenoids: (Salkowski test):  0.2 g of the extract of the plant sample was mixed with 2 ml of chloroform (CHCl3) and concentrated H2SO4 (3ml) was carefully added to form a layer. A reddish-brown colouration in the interface indicates positive results for the presence of terpenoids.

Isolation of Phytopathogens: Fungal pathogens are able to infect various plant parts such as roots, stems, leaves, flowers and fruits, inducing characteristic visible symptoms like spots, blights, anthracnose, wilts, rots etc. Collected infected parts of brinjal and chillies was cut into small pieces. After washing the tissues thoroughly in sterile water, the causal fungi are isolated from plant tissues exhibiting clear symptoms. The infected tissues along with adjacent small unaffected tissue are cut into small pieces (2–5 mm squares) and by using flame-sterilized forceps, they are transferred to sterile petridishes containing 0.1% mercuric chloride solution used for surface sterilization of plant tissues. The plant parts were transferred to CDA plates and Incubated for 5-7days for the complete growth of fungi. The fungal pathogens from brinjal, chillies stems and fruits in which they may be present in the deep-seated tissue have to be isolated by culturing pieces of internal tissues. The infected tissues are thoroughly washed in sterile water and then swabbed with cotton wool dipped into 80% ethanol, followed by exposure to an alcohol flame for a few seconds. The outer layer of tissues are quickly removed by a flame sterilized scalpel. Small pieces from the central core of tissues in the area of the advancing margin of infections are removed by a sterilized scalpel or scissors and sterilized by dipped into 90% alcohol then flaming for a few seconds. The tissues thus sterilizes, are transferred to PDA plates and incubated for 5-7 days.

Assay of antimicrobial activity: Antibacterial activity of plant extracts were carried out against unknown fungal pathogens isolated from brinjal and chillies plants using agar well diffusion method. Initially, the stock cultures of bacteria were revived by inoculating in broth media and grown at 37ºC for 24hrs. The Czapek-Dox agar (CDA) plates were prepared and wells were made in the plate (Mustika et al., 1984). Each plate was inoculated with 24h old cultures (100 μl) and spread evenly on the plate. After 20 min, the wells were filled with methanol and aqueous plant extracts (100µl/ml). The control wells with streptomycin were also prepared. All the plates were incubated at 37ºC for 24-48 h (Zaika, 1988); Arulmozhi et al., 2007; Asaolu et al., 2009.

RESULTS AND DISCUSSION:

The fresh Neem (Azadirachta indica) leaves were collected from neem trees. The leaves were washed under running tab water for 5 minutes in order to remove the dust particles stuck on the leaf surface. The leaves were allowed to dry for 10 days at room temperature. The dried leaves were then blended using dry blender to obtain the powder form of the leaf for more efficient and effective organic extraction. The dried leaves were ground to powder form. 50g of each sample was soaked with chloroform and acetone in a closed 500 ml conical flasks and then kept at room temperature for 24h. The extraction process was repeated 2 times. Then the extracts were filtered by using Whatman 1filter paper. The dried extracts were kept in the refrigerator at 4°C for further use. For methanol extraction, about 50g of the dried and powdered leaves were taken and added to 100 ml of methanol. The mixtures were sonicated for 30 min, and then left at room temperature overnight. The extracts were filtered over Whatmann No.1 filter paper, and the filtrates were concentrated under reduced pressure to pasty mass. The methanol extract was subjected to chemical tests to screen for the presence of various secondary metabolites and antimicrobial properties. 10 g of sterilized plant leaves and flower were kept in 20 ml of sterilized distilled water (1:2). Then they were ground well with the help of mortar and pestle. The six phytopathogens were isolated by using CDA medium from Brinjal and Chillies plants (Fig. 3) and they were coded as NRI-1, NRI-2, NRI-3, NRI-4, NRI-5 and NRI-6. They were purified on CDA slants and used for further studies (Fig. 4).

Phytochemical screening: Different qualitative chemical tests can be performed for establishing profile of methanol and aqueous extract for its chemical composition. The following tests were performed on extracts to detect various phytoconstituents present in them (Fig. 2.1, 2.2).

Table 1: Detection of Phytochemicals

Response (+: Positive / -: Negative)

Fig. 2.1 Phytochemical analysis of Neem leave extracts

(A: Alkaloides test, B: Saponines test, C: Detection of fixed oils and fats, D: Carbohydrates and glycosides, E: Terpenoids)

Fig. 2.2 Phytochemical analysis of Rose petal extracts

(A, B: Phenols, C: Detection of proteins and aminoacids, D: Terpenoids, E: Saponins, F: Gum & Mucilage)

Fig. 3 Isolation of Pathogens (Bacteria and fungi)

Fig. 4 Bacteria and Fungal Phytopathogens (NRI-1 to NRI-6) isolated from Vegetable crops (Brinjal and Chilli)

The most important of these substances include, Plant essential extracts have been used for many resins, mucilages, tannins, gums, phosphorus and calcium thousands of years, in food preservation, for cell growth, replacement and body building pharmaceuticals, alternative medicine and natural. Antimicrobial compounds especially against bacterial Qualitative analysis of phytochemical properties listed in pathogens. In vitro studies in this work showed that the Table 1. plant extracts inhibited bacterial growth but their The antimicrobial activity of many plant extracts effectiveness varied. had been previously reviewed and classified as The medicinal values of the secondary metabolites strong, medium or weak (Zaika). The inhibition are due to the presence of chemical substances that produced by the plant extracts against particular organism produce a definite physiological action on the human depends upon various extrinsic and intrinsic parameters (Kubmarawa et al., 2008; Kraus et al., 1981). Due to variable diffusability in agar medium, therefore Zone of inhibition value has also been computed in this study (Table 2 and figure 5).

Table 2: Antimicrobial activity against phytopathogens collected from Vegetable crops

Fig. 5: Antimicrobial activity of Phytochemicals derived from Azadirachta indica against phytopathogenic fungi NRI-1, NRI-5