Evaluation of the Antibacterial Potential of Rhododendron arboretum Flower Extract against Pathogenic Bacteria

Rhododendron was drived from the Greek word ‘rhodo’ means ‘rose’ and ‘dendron’ means ‘tree’ which belong to family Ericaceae [1] and was first described by Carl Linnaeus in 1837[19]. It is the National flower of Nepal (Laligurans), state flower of Sikkim and Himachal Pradesh (Burans), and also the state tree of Uttrakhand[2]. First Rhododendron was coming from Southeastern Asia. The tree species of R.arboreum was discovered by Captin Hardwicke in 1799. It was arrived in india 1811. In 1821 Don was introduced two species i.e. R.anthopogen and R.setosum from Asia. The first hybrid species of Rhododendron was introduced from China. Worldwide, around 1200 species of Rhododendron have been estimated among which China has the highest number of species that is 571 species of total species in the world, of which 404 endemics [17]. In India, there are about 80 species, 10 subspecies and 14 varieties. The existing record indicate that 98% of the Indian species are found in the Himalayan region [18]. The native places are Bhutan, China, India, Nepal and Pakistan [3]. In India, it is mainly found in J&K, H.P. and Uttrakhand. In Himachal Pradesh, it is found in Chamba,Kangra, Kullu, Shimla, Mandi, Kinnaur and Sirmour districts. India is the major center for research of natural products and safer alternatives of medicine. Indian Himalayan Region (IHR) is one of the major hot-spots for medicinal plant species and Himachal is one of the bioresource rich and full of potential opportunities [4]. According to WHO (2000), 65% of the world^’s population integrate the medicinal plant for treatment and 80% of the Indian population used plant product for treating many diseases [20]. Infectious diseases are the number one among all causes of death. About 50-75% of the hospitals deaths are reported due toinfectious diseases caused by microorganism [21]. In developing countries, traditional medicine is one of the primary healthcare systems [22].  Many herbs are used in the traditional medicine and their curvature potentials are well documented [23]. Rhododendron arboreum is an evergreen plant and widely distributed in Himalyas from Kashmir to Nagaland at an altitude ranging from 1500-5500 meter [5]. About 65% of newly drugs developed between 1981 to 2005 which were depends upon natural herbs and specially play a important role area of life threatening disease [24]. The natural products provide various elements which are useful for molecular diversity and some biological functionality, which leads to novel drug delivery. In ancient days, bacterial resistance to antibiotics has become a serious therapeutic problem. The rare of new antibiotics are produced slowly. In the present time aqueous and alcoholic extracts methods were subjected for antibacterial activity against gram+ve bacteria (Mycobacterium bacillus) and gram –vebacteria (Pseudomonas salmonella) [6]. Flower of R.arboreum contain phenols, saponins, xanthprotein, steroids, tannins and courmarin. More plant rich in secondary metabolities which have been found in vitro to have antibacterial properties [25]. It also contains minerals such as manganese, iron, zinc, copper, sodium, chromium, nickel, lead and arsenic. Minerals play a vital role in maintaining certain physicochemical processes which are essential for life. Manganese, copper, selenium, zinc, iron are important cofactor found in the structure of certain enzymes and are indispensable in numerous biochemical pathway. Sodium is important in maintain the osmotic balance between cells and interstitial fluid [16]. Traditionallythe R.arboreum is used for the treatment of various diseases like diarrhea,headache[7]. Some experiments shows that R.arboreum hasphytochemical activity which is used to cure human diseases like diabetis[8], inflammation,bacterial and fungal infection [9].
       
           
       

Table 1: Scientific Classification Rhododendron arboretum

• Collection: The Rhododendron arboreum flowers were collected from area ofdistt. Mandi, Himachal Pradesh, India and used for the treatment of antibacterial diseases.
• Processing of Flower Material: Flower of R.arboreum were collected from respective plant, washed thoroughly under distilled water. After that the flower were cut into smaller pieces for quick drying. Cleaned flowers were shade dried for 15-20 days. The dried plant material were crushed into fine powder with the help of pestle mortar. Finally the fine powder was stored in air tight container at room temperature [10].
• Extraction Methods:

The extraction of Rhododendron arboreum flower was done with solvent-solvent extraction method. It is divided into two types:

1. Aqueous extraction method: Take 5g powered form of flower extract in a beaker and add sufficient amount of water in which flower powder immersed completely. After that chloroform was added as a preservative and placed aside for 72 hours with alternate stirring. Filter the material and get brown colour extract and this extract dried in desicator.
2. Alcoholic extraction method: Take 5g powered form of flower extract and packed into the soxlet apparatus. The material was subjected to continous hot percolation for 8-9 hours with 175ml methanol. The obtained extact was semi-solid and dried in desicator[13].

Determination of Antibacterial Activity:

1.Test organism2.Antibacterial screening

1. Test organism: The pure cultures of bacteria maintained in the microbiology laboratory were used for the microbiological work. The test orgamisms were maintained on agar medium [6]. The antibacterial activity of R.arboreum flower extract was assessed against to bacterial species gram+ ve and gram-ve bacteria. The gram+ve bacteria were Mycobacterium bacillus and Lactobacillus. The gram-ve bacteria were Pseudomonas salmonella.

2. Antibacterial screening: It can be done by various methods. The methods are:

(a) Agar disc diffusion method

(b) Broth or agar dilution method

(c) Broth microdilution method

(d) Broth macrodilution method

Agar disc diffusion method: This method developed in 1940 and in present days this method is used in many clinical microbiology laboratories for antibacterial testing. Mycobacterium bacillus, Lactobacillus and Pseudomonas salmonella using specific culture media, various incubation condition for inhibition zone [11].

PROCEDURE:

• The nutrient agar media was prepared in the conical flask and sterilized into the autoclave.
• Add the culture media into two different petri plates in the aseptic condition or in laminar hood and left for solidify.
• In first perti plate, add the gram+ve bacteria (Mycobacterium bacillus, Lactobacillus) and solidify for 5 minutes.
• In the second petri plate, add the gram-ve bacteria (Pseudomonas salmonella) and also solidify for 5 minutes.
• In both petri plates, pore with the help of borer.
• After that add alcoholic or aqueous extract of R.arboreum flower in pores of the petri plates and keep the plates into the incubator at 30⁰C to 35⁰C.
• After 24 to 48 hours of incubation, if the microorganism is susceptible for plant extract, zone of inhibition was visible.
• Measure the diameter of the clear zone [12].

Agar disc diffusion method is not appropriate to determine the minimum inhibitory concentration (MIC). It is impossible to determinquantative amount of antimicrobial agent diffuse into the agar media [14]. The advantages of disc diffusion method are low cost, simplicity, ablity to test various microorganisms and antimicrobial agents [15].

Prepration of extracts:

% Inhibition of growth of pathogenic bacterial at different concentration of methanol, acetone, chloroform and petroleum ether extract of R. arboretum: