Soubhagya K B

Materials Today: Proceedings 25 - Contents lists available at ScienceDirect Materials Today: Proceedings journal homepage: www.elsevier.com/locate/matpr Qualitative analysis of phytochemicals and anti-bacterial screening using ZnS/Mn nanoparticles encapsulated with ethanolic extract of Nyctanthes arbor-tristis Linn. K.B. Soubhagya a,⇑, M. Anilkumar b a b Mar Athanasius College, Kothamangalam 686666, India Union Christian College, Aluva, India a r t i c l e i n f o Article history: Received 17 December 2019 Received in revised form 2 March 2020 Accepted 6 March 2020 Available online 3 April 2020 Keywords: Phytochemicals Antibacterial screening Agar well diffusion method Zone of inhibition ZnS/Mn nanoparticles a b s t r a c t Nyctanthes arbor-tristis Linn. (Nyctanthaceae) is one among the most useful traditional plants in India. It is a valuable medicinal plant used in Ayurveda. Various parts of the plant like fruits, leaves, seeds, flowers, barks and stem contain many phytochemicals and are used for the treatment and management of various diseases. Present investigation deals with the qualitative analysis of phytochemicals and antibacterial screening of dried leaf and flower extracts prepared in ethyl acetate, ethanol and benzene. The effect of ZnS/Mn nano particles coated with the ethanolic extract of leaf and flower was also reported. The qualitative analysis of the leaf and flower extracts revealed the presence of various classes of phytochemicals such as alkaloids, flavonoids, coumarins, phenols, resins, tanins, and steroids. The antibacterial potential of the extracts were evaluated on gram-positive (Bacillus subtilis) and gram-negative (Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas pyogens) bacteria in terms of zone of inhibition using agar well diffusion method.In the anti-bacterial screening, it is evident that flower has more anti-bacterial property than leaf. Among the three solvent extracts used, benzene gave no or poor zone of inhibition while ethyl acetate gave maximum zone of inhibition and the ethanolic extract gave only moderate zone of inhibition. ZnS/Mn nanoparticle encapsulated with ethanolic extracts of leaf and flower was screened for anti-bacterial activity against ethanolic extract. The ZnS/Mn encapsulated extracts gave more zone of inhibition than the extract alone. The results of this study indicates the potential of ZnS/Mn nanoparticles as a strong antibacterial agent which can be useful for antimicrobial applications. Further studies in this aspect has the potential to lead towards a few more astonishing facts towards the anti-biotic property of ZnS/Mn nanoparticles coated with the extracts. Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials. 1. Introduction An impressive number of recent drugs have been isolated from natural sources among which most of them were based on their use in traditional medicines. Various medicinal plants have been used for years in day-to-day life to treat ailments all over the world [1]. Historically, plants have been reported to be inspirational novel drug constituents, as plant derived medicines have made large contributions to human health and well being [2]. Natural ⇑ Corresponding author. products from plants offer new agents for antimicrobial use. A distinct feature of higher plants is their capability to produce a large number of organic chemicals of high structural diversity and has been termed as secondary metabolites [3]. According to world health organization (WHO), any plant which comprises substance that can be used for therapeutic purposes or which are precursors of chemo pharmaceutical semi synthetic new drugs, is referred as medicinal [4]. Medicinal plant would be the significant source of obtaining a variety of drugs as phytochemicals are more specific, biodegradable and are supposed to have fewer side effects. They offer exclusive platform for structural diversity and biological functionality which is a requisite for drug discovery [5]. E-mail address:-(K.B. Soubhagya). https://doi.org/10.1016/j.matpr-/Ó 2019 Elsevier Ltd. All rights reserved. Selection and peer-review under responsibility of the scientific committee of the International Conference on the Science and Technology of Advanced Materials. 337 K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - Nyctanthes arbor-tristis Linn. Is commonly called Night Jasmine due to the strong and pleasant fragrance emitted by flowers during night. The generic name Nyctanthes have been coined from two Greek words, nykhta (night) and anthos (flowers). The specific name arbor-tristis means it loses its brightness during daytime. Nyctanthes is a large shrub or a small tree widely cultivated in tropical and subtropical regions all over the world. The plant has been screened for anti-malarial anti-histaminic, anti-arthritis activities, local anaesthetic, anti-hypnotic, analgesic, anti-ulcer, anti-pyretic, anti-depressant, anti-cancer, anti-larvicidal, anti-allergic, antiviral, immune modulatory, anti-helminthic, antioxidant, antidiuretic, antioxidant and CNS modulatory properties [6]. 1.1. Preliminary phytochemical analysis Phytochemical screening refers to the extraction, screening and identification of medicinally active constituents found in plants [7]. Some of such bio-active substances derived from plants are flavonoids, alkaloids, carotenoids, tannins, anti-oxidants and phenolic compounds. Although the knowledge of how these substances provide medicinal value to humans reflects a relatively recent scientific understanding the use of plants and plant extracts to heal, relieve pain and promote good health dates back before the beginning of medical science. 1.2. Antibacterial screening It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious diseases by letting the agent multiply in a predetermined medium. Because of the failure of available antimicrobials to treat infectious diseases, many researchers have focused on the investigation of natural products as source of new bioactive molecules [8]. 1.3. ZnS/Mn nanoparticles in antibacterial screening Interdisciplinary research has widened the horizons of material research, drawing new inspirations from biological systems. The towering ecological concerns had triggered the researchers to device innovative methods of synthesizing the nano materials in biological systems such as bacteria, fungi and plants, termed as green chemistry approaches [9]. The field of nanotechnology is one of the active area of research in modern material science. Nanoparticles show enhanced properties on specific characteristics such as size, distribution and morphology. Nanoparticles and nanomaterials applications are growing rapidly. Taking advantages of the specific characteristics of NPs such as high surface to volume ratio, homogeneous particles size distribution, possibility of facile surface modification, good stability, and the ease of preparation, these materials offer new solutions in the fields of pharmacy, dentistry, medicine, biology, and material science. Size, shape, size distribution and surface decoration of NPs are the key factors determining their specific properties. Due to the strong antibacterial properties and low toxicity towards mammalian cells of some NPs they have been effectively useful in a wide range of areas including wound dressing, protective clothing, new nanomedicines, antibacterial surfaces, water treatment, food preservation, and cosmetics as biocidal and disinfecting agents. The present study was undertaken to carry out the systematic phytochemical examination of Nyctanthes arbor-tristis Linn. Studies had been conducted to evaluate antibacterial property of leaf and flower extracts. ZnS/Mn nanoparticle had been used to enhance the zone formation in antibacterial screening. 2. Materials and methods 2.1. Collection and authentication of plant material Leaves and flowers of Nyctanthes arbor-tritis Linn. were collected from the botanical garden, Union Christian College, Aluva, Ernakulam District, Kerala, taxonomically identified and herbarium specimen were prepared and deposited in the herbarium of Botany Department. 2.2. Method of extraction The collected N. arbor-tristis Linn. leaves and flowers were dried at 70C for 72h and 40h respectively and was powdered. The pow¯ ders were subjected for extraction by cold percolation method in ethanol, ethyl acetate and benzene. The extracts were filtered separately and the filtrates were stored in refrigerator. 2.3. Preliminary phytochemical analysis Preliminary phytochemicals analysis of the ethanol and ethyl acetate extracts of Nyctanthes arbor-tritis Linn. leaves and flowers were carried out using standard procedures [10,11]. 2.4. Antibacterial activity 2.4.1. Growth and maintenance of microorganisms The bacterial culture of Pseudomonas pyogens, Bacillus subtilis, Proteus vulgaris and Klebsiella pneumoniae were obtained from ATTC, Chandigarh, India. The strains were taken from the stock culture, maintained at 4C in nutrient broth, in the Department of Bot¯ any, Union Christian College, Aluva. 2.4.2. Well diffusion technique The antibacterial activity of the extract was determined by the well diffusion technique [12]. From the stock culture of different species of bacteria, single colonies were taken out. The bacteria were kept in sterile nutrient broth at 37C for 24h in bacteriological ¯ incubator. From the turbid bacterial culture sufficient quantity of bacteria were taken using a sterile swab and prepared lawn cultures. Three wells were made on each petri plate using a cork borer and added ethanol, ethyl acetate and benzene extracts. These petri plates were kept in the incubator at 37C for 24h and observed the ¯ zone of inhibition in the bacterial lawn culture. 2.4.3. Anti-bacterial study using ZnS:Mn nanoparticles ZnS/Mn nanoparticles encapsulated with N. arbor-tristis Linn. leaf and flower extracts prepared by colloidal precipitation method [13] in ethanol medium and anti-bacterial assay was carried out by well diffusion method against four pathogenic strains viz. Table 1 Preliminary phytochemical screening in the ethanolic leaves and flowers. Flavonoids Coumarins Alkaloids Tanins Steroids Saponins Quinines Anthraquinone Phenol Resin Reducing sugar Protein Carbohydrate Leaf Flower + + + + + + + + + + + + + + + + 338 K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - B. subtilis, K. pneumoniae, P. vulgaris and P. pyogens, which were cultured in sterile nutrient broth. Bacterial lawn was made using sterile swabs on the medium. Two wells were made on each agar medium, appropriately separated from each other to avoid overlapping of inhibition zones. To one of the well the ZnS/Mn encapsulated with N. arbor-tristis (leaf and flower separately) and to the other ethanolic extract which is used as the control is added and incubated at 37C in a bacteriological incubator. ¯ 3. Results 3.1. Preliminary phytochemical analysis A variety of constituents belonging to different chemical classes such as terpenes, steroids, tannins, phenols, reducing sugars, flavonoids, proteins etc. were screened in the ethanolic leaves and flowers (Table 1). Table 2 Zone of inhibition formed in antibacterial study using leaf extract. Sl. No. Extracts Zone of inhibition (in cm) Bacillus 1 2 3 Ethanolic extract Ethyl acetate extract Benzene extract 0.9 2 Klebsiella 3.2 Proteus Pseudomonas 2.6 1 3 Fig. 1. Antibacterial screening in leaf extract of N. arbor-tristis Linn. Table 3 Zone of inhibition in antibacterial study using flower extracts. Sl. No. Extracts 1 2 3 Ethanolic extract Ethyl acetate extract Benzene extract Zone of inhibition (in cm) Bacillus Klebsiella Proteus Pseudomonas 2.2 2.4 2.6 3.8 2.5 3.2 2.6 3 K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - 339 Fig. 2. Antibacterial screening in flower extract of N. arbor-tristis Linn. 3.2. Antibacterial screening The anti-bacterial screening of ethanol, benzene and ethyl acetate leaf and flower extracts of N. arbor-tristis Linn., done against four pathogenic bacterial strains, both gram positive and gram negative, such as P. pyogens, K. pneuomoniae, P. vulgaris, and B. subtilis. The ethanol, benzene and ethyl acetate extracts of both leaf and flower exhibited varying degree of inhibitory effect against all pathogenic strains. In anti-bacterial screening of leaf extract, the maximum activity was shown by ethyl acetate extract with a zone of 2cm in bacillus, 3.2cm in K. pneumoniae, 2.6 in Proteus vulgaris and 3cm in P. pyogens. Ethanol extract shown anti-bacterial activity in only B. subtilis and P. pyogens with a zone of inhibition of 0.9cm and 1cm respectively which is less than in ethyl acetate extract. Benzene extract Table 4 Zone of inhibition in antibacterial study using ZnS/Mn nano particles in leaf extract. Sl. No. 1 2 3 4 Bacteria Bacillus Klebsiella Proteus Pseudomonas Zone of inhibition (in cm) Extract used Nanocoated Ethanolic extract - - showed no zone of inhibition in any of the four bacterial lawns (Table 2, Fig. 1). In anti-bacterial screening of flower extracts, activity was shown by both ethyl acetate and ethanol. But maximum zone of inhibition was shown by ethyl acetate extract in all the four bacterial strains as 2.4cm in B. subtilis, 3.8cm in K. pneumonia 3.2cm in Proteus and 3cm in Pseudomonas (Table 3, Fig. 2). 3.3. Anti-bacterial study using ZnS/Mn nanoparticles Ethanolic extracts of leaves and flower showed zone of inhibition but less than that of ethyl acetate extracts of leaves(Table 4, Fig. 3) and flower(Table 5, Fig. 4). Thus, ethanolic extract was tested to enhance zone of inhibition using ZnS/Mn nano particles. Comparison of anti-bacterial screening of ethanolic extracts of leaf and flower against ZnS/Mn nano particles coated with the concentrated ethanolic extracts of leaves and flower gave more zone of inhibition in nanoparticle coated extracts in both of them. 4. Discussion The fruitful prediction of botanical compounds from plant largely dependent on the type of solvent used in the extraction procedure. Traditional healers use primarily water as the solvent but in present studies the plant extracts in organic solvent (ethanol, ethyl 340 K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - Fig. 3. Anti-bacterial study using ZnS-Mn nanoparticles coated with ethanolic extract of N. arbor-tristis leaf and control (ethanolic extract). Table 5 Zone of inhibition in antibacterial study using ZnS/Mn nano particles in flower extract. Sl. No. Bacteria Zone of inhibition (in cm) Extract used Nano-coated Ethanolic extract 1 2 3 4 Bacillus Klebsiella Proteus Pseudomonas - - acetate and benzene) provided more consistent biological activity compared to those extracted in water. These observations can be rationalized in terms of the polarity of the compounds being extracted by each solvent and in addition to their intrinsic bioactivity, by their capability to dissolve or diffuse in the different media used in the assay. It is also quite possible that the test plant material that were less effective in this do not possess antibiotic properties, or the extracts may have contained antimicrobial constituents, just not insufficient concentrations so as to be effective. It is also possible that the active chemical constituents were not soluble in ethanol or water. The drying process may have caused conformational variations to occur in some of the chemical components found in the test plant materials. The quantitative analysis of the leaves and flower extracts of N. arbor-tristis Linn. showed that possess various classes of chemicals such as alkaloids, flavonoids, coumarins, phenols, resin etc.. The presence of these phytochemicals may be the source for its antibacterial property and their wide use in medicines. The occurrence of secondary metabolites like alkaloids, glycosides, saponins, flavonoids and coumarins exhibited as a wide range of immune modulatory substances [14]. These natural products serve as pant defence mechanism against predation by micro-organisms, insects and herbivores [15]. The biological function of alkaloids is very important and is used in analgesic, antispasmodic and bactericidal activities. Flavonoids are water soluble phytochemicals and a vital plant phenolic. They have anti cancerous, anti inflammatory activities and a large effect in lower intestinal tract and heart disease. Flavonoids from these plants provide anti-inflammatory action [16].Tannins draw the tissue closer together and improve their resistance to infection. Tannins are capable of lowering available protein by antagonistic competition and can therefore cause protein deficiency syndrome kwashiorkor. Phenol is responsible for anti-oxidant property, anti-inflammatory, anti-carcinogenic and anti-atherosclerotic activities [17]. Agar well diffusion method was used here in order to get the antibacterial properties of the different extracts against the test organisms, agar well diffusion has been used earlier by [18], for antibacterial assay. In the anti-bacterial screening, it is evident that flower has more anti-bacterial property than leaf. Among the three solvent extracts used ethyl acetate and ethanol gave zone of inhibition while benzene gave no or poor zones. Here, both in leaf and K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - 341 Fig. 4. Anti-bacterial study using ZnS/Mn nanoparticles coated with ethanolic extract of N. arbor-tristis flower and control (ethanolic extract). flower, ethyl acetate gave maximum zone of inhibition. This property of the extract may be attributed to the compounds present in it. The different solvent extracts of test material showed marked antibacterial activity. The variation in the susceptibility of the organism could be attributed to the intrinsic properties of the organism. Advanced studies in this aspect have the potential towards a few more astonishing facts towards the anti-biotic property of its extracts. Anti-bacterial studies using the three solvent extracts of leaf and flower, the ethanolic extract gave only moderate zone of inhibition. ZnS/Mn nanoparticle encapsulated with ethanolic extracts of leaf and flower was screened for anti-bacterial activity. The ZnS/Mn encapsulated extracts gave superior zone of inhibition than the standard. The anti-bacterial result states ZnS/Mn nanoparticles as a strong antibacterial agent which can be useful for antimicrobial applications. controlled and extensive clinical trials. N. arbor-tristis leaf and flower contain a number of important phytochemical constituents which could contribute to medicinally important bioactivities of this plant including antibacterial activity. The data obtained demonstrated that the antimicrobial activity depends on the plant parts, solvent used for extraction and the test organism tested for susceptibility assay. Time has come to make good use of times old knowledge of Nyctanthes arbor-tristis Linn. through modern methodologies of drug development which has given inspiration among researchers in exploring more information about this medicinal plant. CRediT authorship contribution statement K.B. Soubhagya: Conceptualization, Data curation, Writing original draft. M. Anilkumar: Methodology, Supervision, Writing - review & editing. 5. Conclusion Nyctanthes arbor-tristis Linn. is an easily available medicinal and ornamental plant. It is a rich source of biologically active compounds, which would attract the attention of drug discovery groups to discover novel bioactive molecules for safer and effective treatment of various ailments. The widespread use of the plant in traditional system of medicine for varied ailments can be supported by various studies involving its pharmacological evaluations. Potential of this plant in management of various diseases makes it a plant of clinical interest. Though plant has been found to be safe but the powerful curative effects of the plant against particular human ailments need to be verified by more Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] R. Nair, T. Kalariya, S. Chandana, Antibacterial activity of some selected medicinal flora, Turk. J. Biol. -. [2] Z.Y. Astal, A.E.R.A. Ashour, A.A.M. Kerrit, J. Pak. Med. Sci. 21 -. [3] S.A.H. Naqri, M.S.Y. Khan, S.B. Vobora, Fittoterapia 62 -. 342 K.B. Soubhagya, M. Anilkumar / Materials Today: Proceedings 25 - [4] Hassanali. 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