Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1): 25-31 Research article DOI: https://doi.org/10.54796/njb.v10i1.227 ©NJB, BSN 25 Phytochemical and Antimicrobial Screening of Bark Extract of Shorea robusta (Sal) Bijay Kumar Shrestha , Bidhya Dhungana , Jenish Shakya , Romika Shrestha , Sujata Chauhan Department of Microbiology, Central Campus of Technology, Tribhuvan University, Hattisar, Dharan, Nepal Received: 10 Oct 2020; Revised: 29 Dec 2021; Accepted: 21 Jul 2022; Published online: 30 Jul 2022 Abstract Different parts of Shorea robusta (Sal) are being used in ancestral and ayurvedic medicines and are known to cure health ailments. The different phytochemicals present in S. robusta is known to possess antimicrobial property. The different botanical parts of this plant have been used in ayurvedic medicines to cure certain infectious diseases. The main aim of this study was to screen phytochemicals and antimicrobial activity of bark extract of S. robusta. Literatures were collected through books, journals and further additional information were collected from residents and traditional ayurvedic practitioners. The ethanolic bark extract of S. robusta was obtained through 70% ethanol in rotatory shaker for 72 hours at 37 ℃ and then the crude extract was dried, preserved and analyzed for phytochemical analysis and antimicrobial activity. The phytochemical screening of ethanolic extract of bark of S. robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, steroids, anthraquinone and absence of phlobatannins, terpenoids, starch and proteins. The extract of S. robusta on Staphylococcus aureus exhibited clear zone of inhibition of 21mm at minimum inhibitory concentration (MIC) of 2 mg/mL while on Escherichia coli exhibited clear zone of inhibition of 9 mm at MIC of 4 mg/mL. The antimicrobial activity may be conferred due to the presence of plant phytochemicals. S. robusta bark extract exhibiting significant minimum inhibitory concentration and antimicrobial activity indicates the efficacy of this plant to be considered for discovering and extracting new antimicrobial products against the pathogens. These findings need further support for appropriate formulation of the drug and its therapeutic use in clinical settings. Keywords: Antimicrobial screening, Bark extract, Minimum Inhibitory concentration, pathogens, phytochemical profile, Shorea robusta Corresponding author, e-mail: interfacebj@gmail.com Introduction Shorea robusta is a deciduous large tree, exceptionally reaching a height of 50 m. [1]. S. robusta has always been a tree of medical, cultural and economic importance and is known by common name ‘Sal’. S. robusta belongs to family Dipterocarpaceae and is reported to possess antimicrobial properties [2]. The entire tree of S. robusta is used for different purposes such as timber in house construction, firewood, leaves for making leaf-plates and cups [3]. The different chemical composition of S. robusta plant such as Asiatic acid, triterpenic acid, tannic acid and phenol is known to possess antimicrobial property [4]. The different parts of S. robusta like bark, flower, resins are known to possess pharmacological action to treat diarrheal diseases, Diabetes mellitus and bacterial infections etc. [5]. Staphylococcus aureus is a pathogenic superbug that causes bloodstream infection, tissue infections and often associated with infective endocarditis [6]. Whereas, Escherichia coli is prominent cause of gastroenteritis, urine infection and bloodstream infections [7]. These two are the most prevalent and common cause of human infections that induce clinical mastitis and need to be correctly cured by appropriate antimicrobials for preventing and controlling emerging drug resistance and nosocomial infections [8]. Since the pathogens are emerging with drug resistance, the need for research in antimicrobial products from medicinal plants can address the issue brought by drug resistant strains in clinical settings [9, 10]. The number of infections caused by emerging drug resistant pathogens grow daily and the hospitalized patients with immunocompromised condition are more prone to severe infections [11]. There is a line of antibiotics on market, but bacterial species shows resistance to most antimicrobials used in the clinical treatment [12]. It has been documented that the bark of S. robusta is traditionally used as astringent, acrid, cooling, anthelminthic, anodyne, constipating, and urinary astringent, union promoter depurative and tonic [13]. They are useful in vitiated conditions of cough, ulcers, wounds, bacterial infections diarrhea, dysentery, Nepal Journal of Biotechnology Publisher: Biotechnology Society of Nepal ISSN (Online): 2467-9313 Journal Homepage: https://nepjb.com/index.php/NJB ISSN (Print): 2091-1130 mailto:interfacebj@gmail.com https://orcid.org/0000-0002-6542-829X mailto:interfacebj@gmail.com https://orcid.org/0000-0002-5390-9889 https://orcid.org/0000-0002-9397-4511 https://orcid.org/0000-0002-4925-9297 https://orcid.org/0000-0002-7528-3792 Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):2 5 - 3 1 Shrestha et al. ©NJB, BSN 26 gonorrhea, leucorrhea, pruritus, leprosy, cough, and anemia [14]. Therefore, present study was aimed to screen and evaluate phytochemicals and antibacterial activity of crude ethanolic extracts of S. robusta bark extract against S. aureus and E. coli. Material and Methodology Research Design This study was conducted from February 2017 to April 2017 at Microbiology Laboratory of Central Campus of Technology, Dharan. Bark of S. robusta was collected from Bijaypur hill forest of Dharan-14 which extends at an altitude of 390 meters (Latitude 26° 49′ 12″ N and Longitude: 87° 18′ 0″ E). The selected plants were firstly identified from herbarium collection of Postgraduate Campus Biratnagar, Nepal. The different botanical information was collected by field study, research articles and books. Medicinal, cultural and economic information about the plants was obtained from experienced traditional ayurvedic practitioners and local individuals. All the information about plants, medicinal values and uses were documented. About 500 grams bark samples were collected from the forest and were brought in Microbiology lab of Central Campus of Technology, Dharan. Microorganisms used The bacterial strains used in this research were S. aureus (25923) and E. coli (CFT073) strain. These microbial cultures were requested and received from Microbiology Department of Central Campus of Technology, Dharan. The growth media used were Mannitol Salt agar (MSA) (HiMedia, India) for S. aureus and MacConkey agar (MAC) (HiMedia, India) for E. coli. These selective growth medias were used for recovering the bacterial strains from preserved culture for further study. The isolated bacterial strains from selective media were further subcultured in brainheart infusion broth media (HiMedia, India). Extract Preparation The extraction methodology was carried out according to Agrawal and Paridhavi (2012) [15]. The bark of S. robusta was chopped and dried under shade at room temperature of 25 ℃ for two weeks. The dried bark of S. robusta was powdered using mortar and pestle at room temperature. About 20 grams of the powdered plants was extracted with 400 mL of 70% ethanol in rotatory shaker for 72 hours at 37 ℃. The obtained extract was concentrated and dried by evaporation in hot air oven at 60 ℃. Stock solutions of 32 mg/ mL was prepared in 10% sterile dimethyl sulfoxide (DMSO). The stock solution was stored at 4 °C until use. Crude powder extract or aqueous suspension of ethanolic dried extract was used in phytochemical screening. Phytochemical Assays Screening of the phytochemical constituents was carried out for detection of documented chemical constituents of bark extract as described by Thilagavathi et al., (2015) [17] and Harborne (1998) [18]. Test for Alkaloids In a test tube, 2 mL ethanolic bark extract was inoculated with 2-3 drops of HCl (dilute hydrochloric acid). To this suspension about 1 mL of Dragendorff’s reagent was added. The presence of alkaloids is indicated by the appearance of orange to red precipitate. Test for flavonoids In a test tube, 4 mL ethanolic bark extract was suspended with 1.5 mL methanol. The solution was gently heated to warm with addition of magnesium with 4 drops of Conc. HCL (Concentrated Hydrochloric acid). Development of color change is indicative for presence of flavonoids. Test for Phlobatannins Bark extract sample was boiled with 1% aqueous hydrochloric acid. Suspension of red precipitate is evidence for the Phlobatannins. Test for tannins About 2 mL of ethanolic bark extract was treated with 2- 3 drops of 10% lead acetate. Tannins are indicated by the development of white precipitate. Test for steroids About 10 mL of chloroform was taken in a test tube and 2 mL of ethanolic bark extract was added to it. This suspension was treated with 1 mL of acetic anhydride and then 2 mL of concentrated sulphuric acid. The presence of steroids is indicated by the development of blue green color at the junction. Test for Anthraquinone In a test tube, dilute Sulphuric acid and 1 mL of diluted ammonia were added to 5 mL of the ethanolic bark extract. Development of pink color indicates the existence of anthraquinone. Test for terpenoids About 10 mL of chloroform was added to 2 mL of ethanolic bark extract. The resulting suspension was inoculated with 1 mL of acetic anhydride and 2 mL of concentrated sulphuric acid. The existence of terpenoids is indicated by development of red, pink or violet color at the junction. Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):2 5 - 3 1 Shrestha et al. ©NJB, BSN 27 Test for Starch Benedict’s test: One litre of Benedict’s solution was be prepared from 100 gm of anhydrous sodium carbonate, 173 gm of sodium citrate and 17.3 gm of copper (II) sulfate pentahydrate. In a test tube, 0.5 mL of Benedict’s reagent was added to 0.5 mL of the ethanolic bark extract. The suspension was heated on water bath at 100 ºC for 2 minutes. The existence of starch is indicated by the development of red color precipitate. Test for proteins Ninhydrin Test: About 1 mL of the ethanolic bark extract was treated with 2-3 drops of Ninhydrin agent and heated in a boiling water bath. The presence of proteins is indicated by the appearance of purple blue color. Antimicrobial Assay Antibacterial tests were carried out by well diffusion method as described by Aneja (2009) [16]. For antibacterial bioassay, the fresh bacterial inoculum with standard turbidity of 0.5 McFarland standard for microorganism was arranged in Mueller Hinton broth (HiMedia, India) and about 100 µL culture was seeded over the Mueller Hinton agar (HiMedia, India). Mueller Hinton agar (MHA) with 2% NaCl was seeded by S. aureus culture and MHA without 2% NaCl was seeded by E. coli culture. With the cork borer no. 6, the wells of about 6mm were created in the media plates. The different test concentrations ranging from, 0.0625-16 mg/mL of bark extract was developed in 10% DMSO solution. About 50 µL aliquot of extracts with different concentrations were inoculated into the wells of MHA plates seeded by the S. aureus strains. Similarly, about 50 µL aliquot of extract with different concentrations were inoculated into the wells of MHA plates seeded by the E. coli strains. The inoculated culture medias were allowed to incubate at 37 ℃ for 24 hours. About 50 µL of 10% sterile DMSO solution was used for the negative control and penicillin (10 µg) and Gentamicin (10 µg) was used as the positive control. After the incubation, the plates were observed for the halo zone around the well. The halo zone around the well represented zone of inhibition which was measured and documented. The experiments were performed for three times and the mean zone of inhibition was computed. Minimum Inhibitory Concentration The minimum inhibitory concentration (MIC) of plant extracts was studied using 96-well microtitre plates as explained by CLSI (2012) [19]. The 96-well plates were prepared from 95 µL aliquot of Mueller Hinton Broth (MHB) (HiMedia, India) suspended in wells. In each well, 5 µL bacterial culture of 0.5 McFarland standard, prepared in MHB medium was inoculated. About 100 µL of stock extract was suspended in first well and so was the same volume of suspension serially diluted to achieve two-fold dilution ranging from 16-0.0625 mg/mL. For negative control DMSO solution was used. The microtitre plates were covered with sterile lid and incubated at 37 °C exactly for 24 hours. The minimum concentration of the extract sample, that inhibited growth of tested organism after overnight incubation, was determined as MIC. Quality Control Complete aseptic condition was maintained during media preparation, sample collection, sample processing. Reagents and culture media were regularly monitored for their manufacture and expiry date and proper storage. Laboratory equipment like incubator, refrigerator, autoclave and hot air oven were regularly monitored for their efficiency. The temperature of the incubator and refrigerator was monitored every day. Contamination of biological samples was prevented by performing the work in laminar flow cabinet. Data analysis The information collected was documented and the data would be analyzed by Microsoft Excel 2010. Results Phytochemical Screening The phytochemical screening of ethanolic extract of bark of Shorea robusta indicated presence of phytochemicals like, alkaloids, flavonoids, tannins, steroids, anthraquinone and absence of phlobatannins terpenoids, starch and proteins (Table 1). Antimicrobial assay and MIC The ethanolic bark extract was tested for antimicrobial activity against both bacteria S. aureus (25923) and E. coli (CFT073). The antimicrobial assay with zone of inhibition with positive controls are shown in Table 2. Table 1. Phytochemicals screening of bark of Shorea robusta Phytochemicals Results Intensity of color Alkaloids Present +++ Flavonoids Present ++ Phlobatannins Absent - Tannins Present ++ Steroids Present + Anthraquinone Present ++ Terpenoids Absent - Starch Absent - Proteins Absent - Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):2 5 - 3 1 Shrestha et al. ©NJB, BSN 28 The ethanolic bark extract of S. robusta on S. aureus exhibited clear zone of inhibition of 21 mm at MIC of 2 mg/mL. The ethanolic bark extract of S. robusta on E. coli exhibited clear zone of inhibition of 9 mm at MIC of 4 mg/mL (Figure 1 and 2). The ethanolic bark extract of S. robusta expressed suppressive activity on both S. aureus (25923) and E. coli (CFT073). The findings of this study provide significant in vitro antimicrobial activity of ethanolic extract of bark of S. robusta against S. aureus and E. coli. Figure 1. Antimicrobial Screening of bark extract of S. robusta against S. aureus Figure 2. Antimicrobial Screening of bark extract of S. robusta against E. coli Figure 3: Antimicrobial susceptibility test by S. robusta extract. Medicinal information The medicinal, cultural, and economic information was collected from Ayurvedic practitioners and locals are included in Table 3. Table 3. The medicinal, cultural, and economic information of S. robusta Plant Parts Medicinal use Other cultural and economic use S. robusta Bark Enhance immunity power, treat typhoid, diarrhea, ulcer. Used in incense stick Leaves Reduce obesity, inhibit pain. Biodegradable leaf plates and cups Resins Lower Fever, skin disorder, diarrhea. Used in incense stick Discussion Nepal has always stood a nation of natural biodiversity rich in natural vegetation that includes herbs, shrubs at different climate and altitude [20]. Many of such herbs have been used as traditional medicines by local people including some even been market as ayurvedic medicine [21]. Nowadays, pharmacology industries are also seemed interested in incorporating natural drugs since because of modern drugs imposing many health side effects with growing incidence of antibiotic resistance. The S. robusta which is one of the species of plant found in Nepal has been known to be best timber producing tree [22]. Its many parts have been used by local people for medicinal and cultural purposes [23]. In this study the phytochemical screening of ethanolic extract of S. robusta bark showed the presence of alkaloids, flavonoids, anthraquinone tannins and steroids whereas phytochemicals like terpenoids, starch, phlobatannins and proteins were absent. This result Table 2. Antimicrobial assay of positive controls Microorganisms Type of Bacteria Antibiotic Zone of inhibition in mm S. aureus Gram + Penicillin G (10µg)- 30 mm E. coli Gram - Gentamicin (10 µg) – 17 mm 0 2 4 6 8 10 12 14 16 1 2 3 4 5 6 7 8 9 16 8 4 2 1 0.5 0.25 0.125 0.0625 14 11 9 0 0 0 0 0 0 MIC (mg/mL ) Zone of Inhibition (mm) 0 5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 16 8 4 2 1 0.5 0.25 0.125 0.0625 26 24 23 21 0 MIC (mg/mL ) Zone of Inhibition (mm) Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):2 5 - 3 1 Shrestha et al. ©NJB, BSN 29 coincides with the study performed by [2, 24] which showed the presence of common phytochemicals. Similarly, the study performed by [3] showed the absence of anthraquinone which has shown similarity with this present study. There are other many factors that affect phytochemical composition such as geographical condition, climatic condition, collection procedure, storage condition, etc. [25, 26, 27]. The effectiveness of the plant extract against selected bacterial strains may be due to the collective antimicrobial action of different phytochemical constituents [28]. The botanical biomolecules like flavonoids, alkaloids and variety of other phenolic constituents have been identified with antimicrobial properties [29]. The phytochemical screening of ethanolic extract of bark of S. robusta showed the presence of flavonoids, which is an antioxidant compound having antimicrobial property to suppress both certain species of Gram-positive and Gram-negative bacteria [30]. Studies have forwarded different antimicrobial mechanism of phytochemicals. Some phytochemicals are supposed to suppress growth and development of microorganism, bacterial cell membrane disarrangement, halting microbial metabolism and modifying microbial genetic expression [31]. Investigators have proved antibacterial activity of S. robusta extracts against bacterial pathogens [32]. In one study, S. robusta extract exhibited antibacterial activity against different clinical pathogens [2]. In agreement with this study even in the present study the ethanolic extract of bark of S. robusta exhibited significant antimicrobial activity with significant level of Minimum inhibitory concentration against both bacterial pathogens. These results clearly suggest its antimicrobial efficacy in treating the infection caused by those pathogens. Plant extract for natural antibiotic could be a strong potent drug against many pathogenic species and could be drug of choice against emerging drug resistant strains [33]. The extended study for purification, activation and therapeutic uses of plant extracts should be conducted to examine its effective antimicrobial role against pathogenic microorganisms. If being effective the nature’s best metabolite could be effective in treating infections caused by antibiotic drug resistance pathogens that have become one of the major therapeutic challenges in clinical settings. The indigenous community of developing nations are still using the plant based traditional drugs for treating many diseases. Resins, leaves, flower and bark extracts of S. robusta has been identified with rich medicinal importance [34]. Potent antimicrobial drugs extracted from S. robusta may be safe for treating many infections and inflammations. In some Asian nations the research on antimicrobial and immunomodulatory effect of the plant has been carried out and even they have come up with excellent results on its antimicrobial and immunological property [35]. These findings will support for scientific research on this plant for pharmacological importance against nosocomial and community acquired pathogens. With new emerging infectious diseases people have been depending upon allopathic, homeopathic and ayurvedic medicines. If proper research could be carried out, then this botanical species could be used for excellent antimicrobial and immunomodulatory medicine. In Nepal, the study has been limited to Taxonomic study and conservation. The bark of S. robusta is not available like that of other plant products. It is the tree which requires long tenure for its proper growth and development [36]. Therefore, scientific tissue culture, conservation and promotion can be achieved through community forest and commercial forestry. Where in context of Nepal the scientific screening and study of S. robusta based drugs are not adequately performed and analyzed, thus this study will help to provide lead to explore more about the pharmacological importance of S. robusta. Conclusion Ethanolic extract of S. robusta bark extract displayed acceptable inhibitory activity against both S. aureus and E. coli. The antimicrobial activity may be conferred due to the presence of plant phytochemicals like alkaloids, tannins, anthraquinone etc. Further development of the plant-based drugs could bring sustained drug release and would help to reduce side effects of synthetic drugs. In-vivo studies of these plant-based extracts are required for therapeutic application. Authors Contribution BKS and BD designed the study, participated in sample collection, extraction and processing. BKS and JS participated in literature review, sample extraction, sample processing quality control, data analysis and result interpretation. RS and SC participated in drafting and proofreading the manuscript. All five authors participated in drafting the manuscript and approved for publication. Competing Interests There is not any competing interest. Funding Self-financed Nepal J Biotechnol. 2 0 2 2 J u l ; 1 0 (1):2 5 - 3 1 Shrestha et al. ©NJB, BSN 30 Acknowledgements Authors want to thank the Microbiology Department of Central Campus of Technology, Hattisar, Dharan for laboratory support. Authors extend sincere thanks to the residents, traditional ayurvedic medicine practitioners and herbarium collection of Postgraduate Campus Biratnagar, Nepal for immense support and response. Ethical Approval and Consent Not applicable. Abbreviations MSA: Mannitol Salt Agar MAC: MacConkey agar MHA: Mueller Hinton Agar MHB: Mueller Hinton Broth CLSI: Clinical & Laboratory Standards Institute DMSO: Dimethyl sulfoxide MIC: Minimum Inhibitory Concentration CFU/mL: Colony Forming Unit NaCl: Sodium Chloride References 1. Tah J, Mukherjee AK. Ecological Hindrances for Establishment of Mass Population of Sal (Shorea robusta) in Forest Gardens Overtaking. Asian Journal of Applied Science and Technology.2018;2(2):857-872. 2. 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