PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL STUDY OF SELECTED HERBAL SPECIES EXTRACTS

Transcription

1 142 PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL STUDY OF SELECTED HERBAL SPECIES EXTRACTS Waseem Ahmad, Ajay Singh and Anshu Kumar Department of Chemistry, Uttaranchal University, Dehradun , (U. K.), India Abstract The aim of the present study was to evaluate the phytochemical constitution and antimicrobial study of chloroform and hexane extract of three medicinally important herbal species namely Cucuma longa, Zingiber officiale, Allium sativum utilized in our routine diet. Qualitative analysis of phytochemical constituents viz. tannins, phlobatannins, saponins, flavonoids, steroids, alkaloids, quinones, coumarin, terpenoids and cardiac glycosides and quantitative analysis of total phenolics, alkaloids, saponins and flavonoids was performed by the well-known tests protocol available in the literature. The phytochemical screening revealed the extract richness alkaloid, flavonoid, saponin, terpenoid, and steroid. Chloroform and hexane extract of selected herbal species were tested against Proteus mirabilis, Listeria monocytogenes, Clostridium absonum, Aspergillus niger Aspergillus fumigates, Arthrogrophais cuboida of bacteria and fungus strain by the agar disc diffusion method. Keywords: Herbel species, Antimicrobial activity, phytochemical constitution. Introduction Nature is a most important source of medicinal plant. Today a large number of various medicinal plant and other herbal products are present in market, which contain phytoconstituents (Wadood et al, 2013). In recent year the medicinal plant and herbal products are used in developing countries because these plants derived drugs have been reported to be safe and without side-effects (Bansod et al, 2008). In India thousand of species have medicinal value and the applied of various parts of several medicinal plants to cure specific aliments. Great number of these plants are very important in modern pharmaceutical industry (Suresh et al., 2013). They used as therapeutic agents and raw material for the manufacture of modern and traditional medicine. These plants are important source of bioactive substance (Afroz et al., 2012). Medicinal plant is useful for curing as well as healing of human diseases because of the presence of bioactive compound. Today there is growing awareness in chemical composition of plant based medicines (Wadood et al 2013; Yadav et al 2011). Several bioactive constituents have been isolated and studied for

2 143 pharmacological activity. During the last two decades, the pharmaceutical industry has made huge investment in pharmacological and chemical researches all over the world in an attempt to discover much more potent drugs, rather, a few new drugs (Thilagavathi et al;2015). Considering the vast potentiality of plants as sources for antimicrobial drugs with reference to antibacterial and antifungal agents, a systematic investigation was undertaken to screen the local flora for antibacterial and antifungal activity from Cucuma longa, Zingiber officiale and Allium sativum. The Cucuma longa Turmeric (Curcuma longa L.) is a medicinal plant extensively used in Ayurvedha, Unani and Siddha medicine as home remedy for various diseases. C. longa L., botanically related to ginger belongs to the Zingiberaceae family (Hitesh et al (2011). Turmeric rhizome is used as a food additive (spice), preservative and colouring agent in Asian countries, including China and South East Asia. It is also considered as auspicious and is a part of religious rituals. In old Hindu medicine, it is extensively used for the treatment of sprains and swelling caused by injury (Lotempio et al 2005). In recent times, traditional Indian medicine uses turmeric powder for the treatment of biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis (Manimegalai et al 2011). The Zingiber officiale is extensively employed in medicine for the management of different diseased conditions like nausea, vomiting, motion sickness, gastrointestinal ulcers, diabetes, fever, arterial tension, rheumatoid arthritis, dry mouth/ xerostomia, cancer, migraine headache, sore throat, minor respiratory ailments. Zingiber offcinale Roscoe, commonly known as Ginger, belongs to Zingiberaceae family (Ravindran and Babu, 2004; Maldonado, 2014). The Allium sativum (Garlic) is one of the edible plant which have produced a lot of interest throughout human history as a therapeutic panacea (Ross et al., 2000). Garlic is one of the most widelyresearched medicinal plants and they are typical odor and antibacterial activity depends upon allicin produced by enzymatic activity of allinase. Garlic has been described to reduce blood lipids and they have anticancer effects (Johnson et al., 2008). Material and methods Collection of Plant Samples Collection of our raw materials cum starting materials for the extraction by using soxhlet assembly. The material which are basically used for the extraction is ginger, garlic and turmeric. They having many useful advantages and application in the field of medicinal

3 144 values, food & processing as a color enhancer, as an ingredient, as a pigments, as a flavor enhancer, taste enhancer, and having several important useful application in different field of research and development. The collection of ginger, garlic and turmeric is to be done from the field of these crops and the collected sample stands not fully-matured at the sample assembly time. The collected samples were stored at room temperature then go for the next step such as preparation and other influences. Samples preparation The collected sample is washed through tap water for removal any type of physical impurities cum contamination then removal of bark by using of knife clean all sample and washed again for removal of bark impurities. Crush the sample in the mortar continuously after complete crushing now kept in oven at C up to complete removal of moisture from the sample. The sample is held at room temperature for maintain the higher temperature of sample now sample is ready for extraction with different types of solvents such as chloroform, hexane with the ratio 1:10 and set the sample and solvent in the soxhlet assembly for 6-8hour at boiling temperature of superior solvents after complete extraction of given sample. Phytochemical screening The concentrated extracts was subjected to different chemical tests for the detection of different phytoconstituents using standard methods (Harborne et al, 1998.; Sofowora 1993) (i)test for saponins Saponins were tested by dissolving one half gram (0.5 g) of the crude extract in a test tube containing 3 ml of hot distilled water and then the mixture was shaken vigorously for one minute and persistent foaming observed indicated the presence of saponins. (ii) Test for flavonoids One half gram (0.5 g) of the crude extract was dissolved in methanol and 2 ml of concentrated hydrochloric acid added. A spatula full of magnesium turnings was added and the mixture observed for effervescence. A brick red colouration observed indicated the presence flavonoids. (iii) Test for steroids About one half gram (0.5 g) of the crude extract was dissolved in 0.5mL dichloromethane to give a dilute solution and then 0.5 ml of acetic anhydride added, followed by three drops of concentrated sulphuric acid. A blue-green colouration indicated the presence of steroids. (iv)test for tannins

4 145 One half gram (0.5 g) of the crude extract was dissolved and added to a tube containing 20 ml of boiling distilled water and then boiled for an hour. A few drops of ferric chloride was added and allowed to stand for proper colour development. A blue-black colouration indicated the presence of tannins. (v)test for Alkaloids Alkaloids are basic nitrogenous compounds with definite physiological and pharmacological activity. Alkaloid solution produces white yellowish precipitate when a few drops of Mayer s reagents are added. Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer s reagent. The alcoholic extract was evaporated to dryness and the residue was heated on a boiling water bath with 2% hydrochloric acid. After cooling, the mixture was filtered and treated with a few drops of Mayer's reagent. The samples were then observed for the presence of turbidity or yellow precipitation. (vi) Test for carbohydrate 2ml of Felhing A and Felhing B reagents were mixed together in equal volume. These reagent are added in crude extract and gently boiled.the bottom of the test tube a brick red precipitate is appeared and indicate the presence of reducing sugar. Test microorganism for antimicrobial studies. Bacterial cultures of Proteus Mirabilis, Listeria Monocytogenes, Clostridium absonum and fungal cultures of Apergillus Niger, Aspergillus fumigates, Arthrogrophais cuboida were obtained from culture collection center department of biotechnology of Uttaranchal University Dehradun. The bacteria were maintained on nutrient broth (NB) at 37 C and funguses were maintained on Potato dextrose agar (PDA) at 28 C. Anti-bacterial Activity- The anti-bacterial activity of leaf and flower extract of Euphorbia hirta was tested by disc diffusion method. The test microorganisms were seeded in to respective medium by spread plate method 10 µl of bacteria in mueller Hinton agar. After solidification the filter paper discs (5 mm in diameter) impregnated with the extracts were placed on test organism seeded organism plates. Concern solvent is used as negative control. The antibacterial assay plates were incubated at 37 C for 24h. The diameters of the inhibition zones were measured in mm. Antifungal Activity: The antifungal activity was tested by disc diffusion method. Mueller Hinton agar (for fungus) plates were inoculated with each fungal culture by point inoculation. The filter paper discs (5 mm in diameter) impregnated with 100 μg ml -1 concentrations of the each extracts were placed on test organism seeded plates Blank disc impregnated with

5 146 concern solvent followed by drying off was used negative control. The activity was determined after 72 h of incubation at 28 C. The diameters of the inhibition zones were measured in mm. Results and discussion The natural spices plants are playing vital role in having beneficial therapeutic effects in traditional Indian system of medicines. Studies on different medicinal plants are gaining consensus in recent years in India and Abroad. In the present study of three different extracts of ginger, garlic, turmeric were subjected to be qualitative and quantitative phytochemical analysis to explore its anti-microbial activity for its medicinal applications. The percentage yields of extracts and the phytochemical constituents of the plants are shown in Tables 1 and 2 respectively. The highest yield of extract was found when extraction was done with chloroform and the lowest in Hexane. Table 1. Percentage yield of extracts under different solvents Plant samples Chloroform extract Hexane extract Ginger Garlic Turmeric 22 15

6 147 The presence of antimicrobial activities in the particular parts in species can be due to the presence of compounds such as alkaloids, saponins, glycosides, steroids, flavonoids etc. Chloroform and Hexane are used as a solvent source for extraction of phytochemicals. So bit is very important to study the Phytochemical analysis of the isolated extract of selected herbal species. The preliminary phytochemical study of the Chloroform and Hexane extracts of selected herbal species revealed the presence of alkaloids, flavonoids, steroids and terpenoids. The results of phytochemical analysis of tested natural spices are given in the Tables 2, 3 and 4. Table 2. Phytochemical analysis of Ginger Phytochemical constituents Chloroform Hexane 1.Steroids Alkaloids Terpenoids Phenolics Tannins - 6.Flavonoids Saponins - - Table 3. Phytochemical analysis of Garlic

7 148 Phytochemical Chloroform Hexane constituents 1.Steroids Alkaloids Terpenoids Phenolics Tannins - 6.Flavonoids Saponins - - Table 4. Phytochemical analysis of Turmeric Phytochemical Chloroform Hexane constituents 1.Steroids Alkaloids Terpenoids Phenolics Tannins - 6.Flavonoids Saponins - - The antimicrobial activities of selected natural herbal spices, Garlic, Turmeric and Ginger are show in the Tables are 5, 6, 7, 8, 9, and 10 given below. Table 5. Diameter of zones of inhibition (mm) of garlic extracts against the bacteria Clostridium absonum 22mm 24mm

8 149 Proteus Mirabilis 27mm 26mm Listeria Monocytogenes 20mm 22mm Table 6. Diameter of zones of inhibition (mm) of garlic extracts against the Fungi Apergillus Niger 21mm 23mm Aspergillus fumigates 25mm 21mm Arthrogrophaiscuboida 22mm 24mm Table 7. Diameter of zones of inhibition (mm) of Turmeric extracts against the bacteria Clostridium absonum 24mm 23mm Proteus Mirabilis 22mm 21mm Listeria Monocytogenes 25mm 24mm Table 8. Diameter of zones of inhibition (mm) of Turmeric extracts against the fungi Apergillus Niger 23mm 20mm Aspergillus fumigates 22mm 21mm Arthrogrophaiscuboida 26mm 24mm Table 9. Diameter of zones of inhibition (mm) of Ginger extracts against the bacteria Clostridium absonum 25mm 24mm Proteus Mirabilis 21mm 22mm Listeria Monocytogenes 22mm 20mm Table 10. Diameter of zones of inhibition (mm) of Ginger extracts against the fungi

9 150 Apergillus Niger 23mm 25mm Aspergillus fumigates 20mm 22mm Arthrogrophaiscuboida 21mm 24mm In this study, we found that the extract of selected herbal species contain large amount of alkaloids and flavanoids along with terpenoids, saponins, tannins and carbohydrate in small ration so these species can be used as an important source of phytochemical and antimicrobial activity. On the basis of our antimicrobial study we find that our selected herbal species shows significant antibacterial and antifungal activity against selected gram positive strains. and fungal strains. So the study our results clearly indicate that we may use our selected herbal species as potent antibacterial and antifungal agent of natural origin. Further work will give emphasis to the isolation and characterization of active principles responsible for bioefficacy and bioactivity. References A. Wadood, M. Ghufran, S. B.Jamal, M. Naeem, A. Khan, R. Ghaffar & Ashad, Molecules, 2013, 2, H. Rahmati, S. Salehi, A. Malekpour & F. Farhangi, International Journal of Molecular Medicine and Advance Science, 2015, 11, Harborne. Chapman and Hall London, 1998, 1, Hitesh K. Dhamija and Ankit S. Chauhan. Der Pharmacia Sinica, 2011, 2, Johnson, M., Maridass, M., Irudayaraj, V. Ethnobotanical Leaflets, 2008, 12, J.B Sofowora A, Medicinal plants and Traditional medicine in Africa. 2nd Edition, John Wily and Sons, 1993, 6-56 Lotempio MM, Verma MS, Steel HL, Ramamurthy B, Ohen AN and Wary MB. Clin.Cancer. Res. 2005, 11, Manimegalai V, T. Selvaraj and V. Ambikapathy. Advances in Applied Science Research, 2011, 2, Maldonado, A., Contribución al estudio de la materia médica peruana. In: Proceedings of the Anales de la Facultad de Medicina, 1920,

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