Phytochemical Screening and Antibacterial Activity of Solanum Trilobatum L. and Azadirachta Indica A
Gopalakrishnan B1, Selvam R2, Lakshmanan G3
1,2,3Bharath Institute Of Higher Education And Research, Selaiyur, Chennai
Medicinal plants play a pivotal role in curing various human ailments. The incidence in the frequency of diseases due to pathogenic microorganisms has increased alarmingly due to the development of resistance to existing drugs and the present scenario necessitate the continuous search for new classes of antimicrobial agents preferably from natural resources. Phytochemicals are ecologically derived secondary metabolites synthesized by the plants from the primary metabolites such as carbohydrates, lipids and amino acids to protect them against environmental challenges such as UV- irradiation, extreme cold, drought, microbial attack, wound, sugar and nutrient deficiency. They often contribute to the unique odor, taste and color in plants. Diseases due to pathogenic microbes pose a great burden on human health and they have been correlated with socioeconomic, environmental, ecological factors. The threat due to infectious diseases is further intensified by the continued emergence of new and multidrug resistant microorganisms.
Pathogenic Microorganisms, Antimicrobial, Phytochemicals, Multidrug resistant.
Nature has bestowed on us a very rich botanical wealth. Large number of diverse plants grows in different parts of the country as well as in the world. World health organization (WHO) lists more than 21,000 species of plants as medicinal plants. Medicinal plants form the backbone of traditional system of medicine in India. India is the largest producer of medicinal herbs and it is therefore known as the “Botanical garden of the world”. Medicinal plants are used as pharmaceuticals, neutraceuticals, cosmetics and food supplements. The drugs are derived from plant or from leaves, stem, bark, flower and seed. Some drugs are prepared from excretory plant products such as gums, resins and latex. Medicinal plants represents a rich source from which anti microbial agents may be obtained. Plants are used medicinally as a source of many potent and powerful drugs ( Richard, 2014).
Subsequent to the serendipitous discovery of penicillin in the year 1928, antibiotics have been recognized as the only means of effective to control the pathogenesis of microorganisms.
Along with the usage of new antibiotics as therapeutics, there is a developing threat of drug- resistance among the pathogenic microorganisms worldwide. Recent advances in the field of medicinal chemistry lead to the discovery of isolating the active phytochemicals from various parts of the medicinal plants for treating human infectious diseases (Bennett ,1994; Chang, 2013).
Numerous structural analogs of plant secondary metabolites have been successfully generated and widely used for their pharmacological actions (Rao,2002). In present study two medicinal plants have been selected to find out phytochemicals and antibacterial activity. They are Solanum trilobatum L. (Solanaceae) and Azadirachta indica A. (Meliaceae). All infectious diseases either newly emerging or remerging represent a continued threat to humanity. Some pathogens, after a period of quiescent, are capable of acquiring features that enable them to reemerge their original or new hosts, usually in increasingly alarming proportions.
2. Materials And Methods Collections And Preparation Of Plants Extract
Fresh plants of Solanum trilobatum L. (Solanaceae) and Azadirachta indica A. were collected in and around Chennai. Fresh plant materials were washed under running tap water, dried under shade, then ground to fine powder and stored in air tight bottles.
5g of dried plant material was soaked in 100ml of water for 48 hrs with intermittent shaking.
Then the extract was filtered through filter paper or 8 layers muslin cloth. Then the filtrate was collected. This filtrate was used for phytochemical screening and TLC. The filtrate was pooled and concentrated using vacuum evaporator. Then the concentrate was evaporated to dryness at 45°C. Methanol and ethanol extracts were prepared in the same way as aqueous extract. All extracts were dissolved in Dimethyl sulphoxide and methanol (1 :1 ) mixture and used for assay of antibacterial activity.
The leaf extracts were tested for possible antibacterial activity in the disc diffusion method using the following clinical isolates - Staphylococcus aureus, Streptococcus mutans (Gram positive), Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa ( Gram negative).
Qualitative Phytochemical Analysis
Phytochemical screening was carried out on Solanum Trilobatum L and Azadirachta Indica A using standard procedures to identify the constituents by the method of Trease and Evans (1989) and Harborne (1973).
Qualitative Analysis Of Thin Layer Chromatography Tlc Plate Preparation
An inorganic substance such as silica gel is used as a stationary phase. Silica gel is made in to slurry using sodium acetate buffer. The prepared slurry is then poured on to the glass plate and spread evenly as thin flim. The plates are then dried at 80°C by keeping in an oven for 2 hours.
The sample is applied using a micropipette as a spot leaving 2 or 2.5cm from the edge of the glass plate and dried. Care must be taken not to scrap the thin layer of the stationary phase while applying the sample. The plate is then placed in a chamber containing the solvent mixture (mobile phase) and developed by ascending chromatography.
Preparation Of Inoculum
A loopful of culture is transferred to 10ml tryptic soy broth and the tubes were incubated at 37○C.
Turbidity is measured visually according to Mc Farland standard. Antibacterial activity assay was done by the method by Bauer et al 1966 and Mueller and Hinton 1941.
3. Results And Discussion
The demand for medicinal plants is increasing worldwide due to growing recognition of natural products, being non-toxic, and more efficacies, easily available at affordable prices. Throughout
the world, many traditional plants have been found successful for the treatment of several primary and secondary health complications. Further, most of the marketed medicines are distillations, combinations, reproductions or variations of substances that exist in nature (Ravishankar, 2000; Ravishankar, 2000).
The qualitative analysis of phytochemical screening of aqueous, methanol and ethanol leaves extract of Solanum trilobatum L. and Azadirachta indica A. Phytochemicals such as tannin, phlobatannin, saponin, flavonoid, cardiac glycoside, steroid, terpenoid, carbohydrate and amino acid was found to be present in all the leaves extract of Solanum trilobatum L. and Azadirachta indica A in table I. Table II depicts the colour of the spots of secondary metabolites which are separated by thin layer chromatography. Based on the chemical nature, the secondary metabolites are mainly classified into alkaloids, steroids, saponins, tannins, lectins, pectins, terpenoids, anthraquinones, flavonoids, glycosides and phenolic compounds (Mouhssen,2013;
Amenu, 2014; Dias, 2012). Interestingly, these secondary metabolites are known to bring out significant pharmacological and beneficial effects to alleviate chronic diseases such as cancer, diabetes and cardiovascular diseases due to their antioxidant, anti-inflammatory and regulatory actions.
Table 1: Phytochemical Screening Of Aqueous, Methanol And Ethanol Leaves Extracts Of Solanum Trilobatum L. And Azadirachta Indica a.
SOLANUM TRILOBATUM L AZADIRACHTA INDICA A
AQUEOUS METHANOL ETHANOL AQUEOUS METHANOL ETHANOL
Tannin + + + + + +
Phlobatannin + + + + + +
Saponin + + + + + +
Flavinoid + + + + + +
Steroid + + + + + +
Terpenoid + + + + + +
Cardiac glycoside + + + + + +
Carbohydrate + + + + + +
Aminoacid + + + + + +
Table 2: TLC - Separation Of Phytochemicals Of Aqueous, Methanol And Ethanol Leaves Extracts Of Solanum Trilobatum L. And Azadirachta Indica A.
NAME OF THE TEST
Solanum Trilobatum L. Azadirachta Indica A
COLOUR OF THE SPOT(OBSERVED)
COLOUR OF THE SPOT(OBSERVED)
Alkaloids Dragendorff’s test Green Brown
Flavonoids Aluminium chloride
Glycosides Diphenylamine test Green Green
Phenols Folin-ciocalteu test Blue Blue
Saponins Iodine test Brown Greenish yellow
Sterols Vanillin test Brown, Pink Green, violet, pink,
purple Greenish brown
Table –3: In Vitro Antibacterial Activity Of Aqueous, Methanol And Ethanol Leaves Extracts Of Solanum Trilobatum L.
AQUEOUS METHANOL ETHANOL
CONC (10mg) Zone of inhibition(mm)
Staphylococcus aureus 11 9 11 13 10 9 32 38
Klebsiella pneumonia 17 7 7 7 7 7 30 35
Streptococcus mutans 9 9 10 11 9 12 24 27
Escherichia coli 7 7 10 7 12 7 26 29
Pseudomonas aeruginosa 7 8 8 7 9 9 28 30
Table 4: In Vitro Antibacterial Activity Of Aqueous, Methanol And Ethanol Leaves Extracts Of Azadirachta Indica A
ORGANISMS AQUEOUS METHANOL ETHANOL
CONTROL Amoxicillin CONC
CONC (10mg) Zone of inhibition(mm)
8 8 10 11 9 10 32 38
7 7 8 9 7 8 30 35
8 8 9 10 10 9 24 27
Escherichia coli 8 9 9 10 9 9 26 29
7 8 7 9 8 9 28 30
Figure 1 IN VITRO Antibacterial activity of aqueous, methanol and ethanol leaves extracts of Solanum Trilobatum L.
Figure 2IN VITRO Antibacterial activity of aqueous, methanol and ethanol leaves extracts of Azadirachta Indica A
Figure3: TLC Separation of aqueous, ethanol, methanol extract of Solanum Trilobatum
Figure 6-TLC Separation of aqueous, ethanol, methanol extract of Azadirachta Indica A
A B C
A B C
The antimicrobial property may be due to their ability to complex with extracellular and insoluble proteins or to complex with bacterial cell walls. Lipophilic flavonoids may also disrupt microbial membrane. It also exhibits anti oxidant activity, reduces the risk of cardiovascular disease. A steroid represents a large group of compounds which exist in nature. It acts as an insulator against nerve impulse which discharges electrical charges (Kirtikar, 2001; Joshi, 2000) Terpenoids are derived from 5 carbon isoprene unit. Terpenoids are active against bacteria, fungi, viruses and protozoa (Pradeepa, 2014). The mechanism of action may involve the disruption of microbial membrane. Cardiac glycosides are secondary metabolites composed of two structural features, sugar (glycosides) and non sugar (aglycone- steroid moieties). It inhibits Na+ - K+ pump and thus it improves cardiac output and reduces distention of heart. It has been reported that carbohydrate and amino acids form complexes with other substances and thus act on the cell membrane of bacteria (Shahzad, 2009; Shokeen, 2009). This showed their importance in exhibiting antibacterial activity.
Separation of phytochemicals by thin layer chromatography was carried out to support antibacterial studies. Aqueous extract of Solanum trilobatum L. was found to have maximum inhibitory activity against klebsiella pneumoniae. S.aureus was found to be moderately inhibited by all the extract. Aqueous, methanol and ethanol extract showed minimum inhibitory activity against E.coli and pseudomonas.
Methanol extract of Azadirachta showed maximum inhibitory activity against Solanum Trilobatum. All the microorganisms are minimally inhibited by the aqueous methanol and ethanol extracts. The antibacterial activity of the plants may be due to the presence of phytochemical constituents
The results of the present study has evidenced that the ethanolic extract of Solanum trilobatum L.
and Azadirachta indica A leaves contains biologically active secondary metabolites in addition to significant amounts of essential trace elements. The leaves may be considered as an important source for the screening of natural lead molecules. The synergistic effect of both the organic and inorganic compounds present in the Solanum trilobatum L. and Azadirachta indica A leaves can play a chief role in antibacterial activity and preventing free radical mediated diseases such as diabetes and cancer. Above all, the present study provides a scientific rationale for the use of Solanum trilobatum L. and Azadirachta indica A leaves in the traditional medicinal system for the treatment of various human ailments. The antibacterial activity may be due to the presence of the active constituents in the plant extracts.
The authors grateful to The Dean, Bharath Institute of Higher Education and Research, Selaiyur, Chennai – 600 073 for providing the laboratory facilities to carry out this work.
Authors have declared that no competing interests exist Authors’ Contributions
All the Authors have equal contributions in designing, executing and preparation of manuscript.
 Richard JF, Yitzhak T, Antibiotics and Bacterial Resistance in the 21st Century, Perspectives in Medicinal Chemistry, 6, 2014, 25–64.
 Chang CL, Chen YC, Chen HM, Yang NS, Yang WC. Natural cures for type 1 diabetes: a review of phytochemicals, biological actions, and clinical potential, Curr Med Chem, 20(7), 2013, 899-907.
 Bennett RN, Wallsgrove RM, Secondary metabolites in plant defence mechanisms, New Phytol., 127, 1994, 617- 33.
 Rao SR, Ravishankar GA, Plant cell cultures: chemical factories of secondary metabolites.,Biotechnol Adv., 20, 2002,101-53.
 Ravishankar GA, Venkataraman LV, Food applications of plant cell cultures, Curr Sci, 57, 1990, 381-3.
 Ravishankar GA, Rao SR, Biotechnological production of phytopharmaceuticals, J Biochem. Mol. Biol Biophys., 4, 2000, 73-102.
 Mouhssen Lahlou, The Success of Natural Products in Drug Discovery, Pharmacology
& Pharmacy., 4, 2013, 17-31.
 Amenu D, Antimicrobial Activity of Medicinal Plant Extracts and Their Synergistic Effect on Some Selected Pathogens, American Journal of Ethnomedicine., 2014, 018- 029.
 Dias DA, Urban S, Roessner U, A historical overview of natural products in drug discovery, Metabolites, 2012,16,2(2), 303-36.
 Harborne JB.Phytochemical methods, Chapman and Hall Int., New York,1998; Third Edition
 Kokate CK, Purohit AP and Gokhale SB, Pharmacognosy, Thirty-ninth edn, Nirali Prakasham, Pune, 2007;97-132, 607-611.
 Bauer RW, Kirby MDK, Sherris JC and Turck M. Antibiotic susceptibility testing by standard single disc diffusion method. Am J. Clinical Pathol. 1966, 45:493-496.
 Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd eds. Lalit Mohan Basu, Allahabad, India, 2001, p.1492.
 Joshi SG. Text book of medicinal plants. New Delhi-Oxford: IBH Publishing Co;
2000. p. 11
 Pradeepa S, Subramanian S, Kaviyarasan V. Evaluation of antimicrobial activity of Pithecellobium dulce pod pulp extract. Asian J Pharm Clin Res. 2014, 7(1):32-37.
 Shahzad K, Ahmad R, Nawaz S, Saeed S, Iqbal Z. Comparative antimicrobial activity of Aloe vera gel on microorganisms of public health significance. Pharmacologyonline.
 Shokeen P, Bala M, Tandon V. Evaluation of the activity of 16 medicinal plants against Neisseria gonorrhoeae. Int J Antimicrob Agents. 2009,33(1):86-91