Phytochemical screening of Alstonia scholaris leaf and bark extracts and their antimicrobial activities.

Alstonia sholaris is an evergreen tree commonly found in South East Asia. In traditional medicine pharmacological activities are attributed to the leaves and bark of this plant. The aim of this study is characterizing the chemicals present in A. sholaris leaves and bark extracts and study their antimicrobial activities. Solvent extractions with Soxhlet apparatus of leaves and bark were obtained using hexane, benzene, isopropanol, methanol, and water. The crude extracts were concentrated and screened for qualitative phytochemical analysis and thin layer chromatography, and the antibacterial, antifungal an antiviral activity of crude extracts were measured by in vitro methods. Isopropanol and methanol extracts showed significant antibacterial activity and it was more pronounced against Gram positive than against Gram negative bacteria. Hexane, benzene, isopropanol and methanol fractions of A. scholaris bark and leaf showed activity against Enterobacter cloacae. Isopropanol extract showed maximum activity against selected human pathogenic fungus. In conclusion, the leaves and bark of A. scholaris are rich in phytochemicals with antimicrobial activities against human pathogens, being the isopropanol fraction the one with the highest antibacterial, antifungal, antiviral and anti-mycobacterial activities.


Introduction
Microbes are very important microorganisms of human pathogens, which cause diseases and death. One of the most important therapeutic discoveries of the 20 th century was the antimicrobial agents. However, with the "antibiotic era" barely five decades old, mankind is now faced with the global problem of emerging resistance in virtually all pathogens (1). Resistance has been developed in antivirals, antimycotics and almost all groups of antibiotics (2). This is indeed quite alarming when considering that in 1990, out of the 39.5 million of death in the developing world, 9.2 million were estimated to have been caused by infectious and parasitic diseases and that 98% of death in children in developing countries resulted mostly from infectious diseases (3). Antibiotics are very much prevalent now a day due to indiscriminate use and abuse of antibiotics (1). In human medicine alone, the US Centre for Disease Control and Prevention estimates that approximately one-third of the 150 million prescriptions for antibiotics written each year were not needed. It is of utmost importance to find appropriate solutions associated with drug resis-tance (4).
New antimicrobial agents are needed to treat diseases in humans caused by drug resistant microorganisms. In addition, there is a continuing consumer demand for cosmetic products as well as natural, preservative-free, microbiologically safe foods now days (5)(6)(7)(8)(9).
The antimicrobial properties of extracts from natural sources have been studied for nearly 60 years. During the past 20-25 years, interest in their antimicrobial nature has expanded due to increased resistance of pathogenic microbes to currently employed antimicrobial drugs, and toxicity or adverse host reactions of other anti-infectives (10). The antimicrobial extracts exhibit their activity either by lysis or disruption of the outer membrane of microbes. Others interact with specific internal targets or cause pore formation and leakage (11).
Plants are complex chemical storehouses of undiscovered biodynamic compounds with unrealized potential for use in modern medicine (12)(13)(14)(15)(16). It has long been established that naturally occurring substances in plants have anti-bacterial, anti-viral and anti-fungal activities (17). In India, secondary metabolites or medicinal plants, for centuries, have been used for the treatment of a wide range of ailments, many of which are still in use today and hold favored positions among local traditional practitioners (18).
Alstonia scholaris R.Br. (Apocynaceae) is a tropical evergreen native to South and Southeast Asia. Different plant parts have been used for a wide spectrum of ailments in traditional medicinal systems such as China, India, Thailand, Malaysia, Philippines, Africa and Australia (19,20); however, A. scholaris is traditionally used to treat infectious diseases. Several researchers have evaluated the antimicrobial potential of different parts of A. scholaris such as leaves, stembark, root, and flower to evaluate its antimicrobial potential (21,22) and cytotoxic and antioxidant activities (23). The aim of the present study is characterizing the chemicals present in A. sholaris leaves and bark extracts and investigate their antibacterial, antiviral and antifungal activities.

Collection of samples
Samples of fresh leaves and bark of A. scholaris ( Fig. 1) were collected from the university campus in Kariavattom, Trivandrum (India). The samples were processed by shade drying for 4 days. The sample was finely powdered in a blender, weighed and stored in dry polythene bags. Weight of the powder was taken.

Solvent extraction
The dry powdered material was subjected to successive organic solvent extraction by refluxing in the Soxhlet apparatus each for 12 h. The solvents used were nonpolar to polar consisting of hexane, benzene, isopropanol, methanol, and water. Each fraction was collected when no further elution of compounds was observed. The collected extracts were subjected to vacuum drying and stored in sterile containers in the refrigerator till further analysis.

Phytochemical analysis of plant extracts
Prior to starting of the experiment the phytochemical extracts were dissolved in dimethyl sulfoxide (DMSO) except water extract, which was dissolved in distilled water (24).

Chemical test for proteins and amino acids
Ninhydrin test: the test is used to detect the presence of alpha-amino acids and proteins containing free amino groups. To 200 µL of the extract few drops of ninhydrin reagent was added and boiled over water bath, formation of purple color indicates a positive test.

Chemical test for alkaloids
Wagner's test: to 200 µL of the extract add few drops of Wagner's reagent (dilute iodine solution) to the sides of the tube. Formation of reddish-brown precipitate indicates a positive result.

Chemical tests for steroid and triterpenoid glycosides
Salkovaski test: alcoholic extract of drug was evaporated to dryness and extracted with CHCl 3 , add conc. H 2 SO 4 from sidewall of test tube to the CHCl 3 extract. Formation of yellow colored ring at the junction of two liquids, which turns red after 2 min indicates positive test.

Chemical tests for cardiac glycosides
Keller Killiani test: to 200 µL of the drug add 100 µL of glacial acetic acid containing 1 drop of ferric chloride solution followed by 100 µL of con. H 2 SO 4 . A brown ring at the interface indicates a deoxysugar characteristic of cardienolides. A violet ring may appear below the brown ring, while in acetic acid layer, a greenish ring may form just gradually throughout thin layer.

Test for oils and fats
Spot test: a small quantity of the extract was pressed between two filter papers. Oil stain on the paper indicated the presence of fixed oil and fats.

Chemical tests for phenolic compounds
chloride test: to the mixture of 200 µL of the extract and 2 mL of distilled water, was added a few drops of 5% ferric chloride along the sides of the test tube. A dark green color showed the presences of phenolic compounds.

Phytochemical screening by thin layer chromatography (TLC)
The n-hexane and isopropanol extracts of A. schola ris were subjected to silica gel layer chromatographic (TLC) separation using ready-made TLC plates (silica gel G 60 F 254 , Merck). 1 mg of dried active extracts (nhexane and isopropanol extracts of A. scholaris) was dissolved in isopropanol or n-hexane separately and 10 µL of the respective extract was spotted on TLC plate using capillary tubes. For spotting two extracts (n-hexane and isopropanol) of A. scholaris, TLC plate with a dimension of 4.5 x 10 cm was used. The spotted TLC plates were resolved using n-hexane: chloroform: methanol (5:4:1, v/v) solvent system in a chromatographic chamber. The resolved plates were examined under UV light at 356 nm and photographed without derivatization. Since all bands were not visible, TLC plates were developed by spraying with anisaldehyde-sulphuric acid reagent and heated at 60ºC.

Phytochemical screening by GC-MS analysis
Homogenized A. scholaris bark isopropanol frac-

Nutrient agar media
Nutrient agar plate (Hi-media) was prepared by dissolving nutrient agar (37 g/L) in distilled water. The media were sterilized in an autoclave at 121°C for 15 min and poured in sterile Petri dishes. The Petri dish was dried, kept for 24 h for sterility check up. Sterile plates only were selected for bacterial cultures (25).

Muller Hinton agar (MHA)
Starch was emulsified in a small amount of cold water and then beef infusion, casein hydrolysate and the agar were added. Volume was made up to 1 L with distilled water. All the constituents were dissolved by heating gently at 100°C with agitation. It was filtered and pH adjusted to 7.4. The media was then distributed into stock bottles and autoclaved at 121ºC for 20 min. Autoclaved medium was then poured into sterile flat bottomed petri plates in a laminar flow hood and allowed to solidify and stored in a cold room (4ºC) for later use.
Plates were prepared and wells of 3 mm, 6 mm and 8 mm diameter were cut using a sterile borer. 100 μL of each of the 2 h culture of test bacteria was placed on the nutrient agar. The inoculum was swab bed uniformly over the entire agar surface and allowed to dry for 5 min. 80 μL of various extracts dissolved in DMSO was loaded into the wells. Plates were incubated at 37°C for 24 h. DMSO was used as negative control and streptomycin (10 µg/80 µL) as positive control. At the end of the incubation period, inhibition zones were measured.

MacConkey agar
MacConkey agar plates (Hi-media) were prepared by dissolving MacConkey agar (55.07 g/L) in distilled water. The medium was heated to boiling to dissolve the medium completely sterilized by autoclaving at 121°C for 15 min and poured in to sterile Petri dishes. The Petri dishes were dried, kept for 24 h for sterility check up. Only sterile plates were selected for bacterial cultures.

5% sheep blood agar
Dissolved trypticase soy agar base (Hi-media) and autoclaved. Cool the steriled blood agar base to 45°C to 50°C. Aseptically added 50 ml of sterile defibrinated blood. Mixed thoroughly, to avoid accumulation of air bubbles. Dispensed in to sterile tubes or plates while in liquid.

Antifungal activity of crude extracts
Crude extracts of plants were subjected to fungal studies to detect their fungicidal properties against human pathogens, plant pathogens and industrially important strains of fungi by incorporating crude extracts in the Sabouraud dextrose agar (SDA) media used for fungal culture. The following standard strains of fungi were used for the study:

Bioactivity assays
The crude extract of each plant part was subjected to in vitro methods like antibacterial, antifungal and antiviral activities.

Antibacterial activity of crude extracts by well diffusion method
Crude extracts were tested to detect their antibacterial property against a group of human pathogens by well diffusion method. Stock cultures were maintained at 4°C on slopes of nutrient agar. A pure single colony grown on an agar plate was transferred to 5 mL of peptone water and incubated for 2 h at 37°C.

Antibacterial activity against Enterobacter cloacae dissolvens
For neutralizing activity testing for various solvent extracts, 50 mg each of the various dried plant extracts was dissolved in 1 mL of DMSO. The test organism used was a 4 h young culture containing 10 5 / mL colonies. After overnight incubation of the extract and test organism, a loop full of it was plated on Mac-Conkey agar to check for growth.

Anti-Mycobacterial activity against atypical Mycobacterium
Neutralizing activity of various solvent extracts was tested using 50 mg of the various dried plant extracts, which was dissolved in 1000 µL of DMSO. The test organism used was a 5 days old culture containing 10 5 / mL colonies of Mycobacterium. After overnight incubation of the extract and test organism a loop full of it was plated on 5% sheep blood agar to check for growth. media, 67 g/L) in distilled water The media were sterilized in an autoclave at 121°C and 1.05 kg/cm 2 and poured in to sterile culture tubes (25 mL capacity), 5 mL in each tube. To each tube 0.5 mL of particulate crude extract was added. Contents were mixed well by shaking the tubes and allowed to set to form slants. The slants were kept for sterility check before use. Negative control tubes were treated with solvents only. Fungal culture were inoculated on SDA slopes and incubated at room temperature at 30-32°C for 5 to 7 days. The results were compared with standard fungicide (imidazole). Fungal cultures were inoculated to SDA crude extract slants and kept at room temperature for 5 to 7 days.

In vitro antiviral activity against Hepatitis B virus by neutralization test
HepG2.2.15 cells were cultured in MEM (Hi-media) containing 10% fetal calf serum (FBS) and gentamycin 20 µg/100 mL medium at 37°C in a humidified incubator gassed with 5% CO 2 . 50 mg of the extract was dissolved in 1 mL of DMSO and in the Hepatitis neutralization test 500 µL of the extract containing 25 mg was used in the test. 500 µL of various fractions of plant extracts were added to 500 µL of the MEM medium in which HepG2.2.15 cell line established growth was taken in various tubes and incubated overnight for neutralization to occur. Each of the tubes was tested for quantitating the Hepatitis B surface antigen after 24 h of incubation at room temperature using ELFA test.

Principle of ELFA test (enzyme-linked fluorescent immunoassay)
The solid phase receptacle (SPR) serves as the solid phase as well as the pipetting device for the assay. At each stage of the reaction, it aspirates the reagents in and out, thus preventing any inter-reagent or inter-sample contamination. The reagents for the assay are ready to use and pre-dispensed in the sealed reagent strips. The strip consists of 10 wells covered with a labeled foil seal. The label comprises a bar code, which mainly indicates the assay code, kit lot number, etc. The foil of the first well is perforated to facilitate the introduction of the sample. The last well of each strip is a cuvette in which the fluorometric reading is performed. The wells in the center section of the strip contain various reagents required for the assay. The interior of the SPR is coated during production with monoclonal anti-HBsAg antibody (mouse). Each SPR is identified by the HBS code. All the steps of the assay were performed automatically by the instrument, VIDAS®-Auto immuno analyser (Bio Merieux). The reaction medium is cycled in and out of the SPR several times. After a preliminary washing step, the antigen present in the sample will bind simultaneously to the monoclonal antibody coating the interior of the SPR and to the antibody conjugated with biotin. Unbound sample components are washed away. The antigen bound to the solid phase and to the biotynilated antibody is in contact with streptavidine conjugated with alkaline phosphatase, which will bind with biotin. Another wash step follows and removes unbound components. During the final detection step, the substrate (4-methyl-umbelliferyl phosphate) is cycled in and out of the SPR. The conjugate enzyme catalyzes the hydrolyses of the substrate into a fluorescent product (4-methyl-umbelliferone). The fluorescence of which is measured at 450 nm. The intensity of the fluorescence is proportional to the concentration of antigen present in the sample. At the end of the assay, results are expressed as an index calculated using a standard. The sensitivity of the assay is with 0.12 ng/mL.
Once the assay is completed, the computer analyzes the results automatically. Fluorescence is measured twice in the Reagent strip's reading cuvette for each sample tested. The first reading is a background reading of the substrate cuvette before the SPR is introduced into the substrate. The second reading is taken after incubating the substrate with the enzyme remaining on the interior of the SPR. The relative fluorescence value (RFV) is calculated by subtracting the background reading from the final result. RFV < 0.13 is taken as negative and RFV > 0.13 is considered positive.

Yield from extracts
Fresh leaves of A. scholaris were collected and weighted 350 g and on drying it, approximately 125 g powder was obtained similarly the 460 g of stem bark of the A. scholaris on drying give around 185 g. Yield of different extract is shown in Table 1. The yield in A. sholaris leaf was maximum in water extract (10.3 g) followed by methanol (6.5 g), isopropanol (5. 5 g), hexane (4.8 g) and least in benzene (3.2 g). In A. scholaris stem bark the yield was maximum in water and least in benzene extraction. The nature of the crude extracts is shown in Table 2.

Phytochemical screening
The results of qualitative phytochemical screening of hexane, benzene, isopropanol, methanol and water extracts of A. scholaris leaf and bark revealed the presence of alkaloids, carbohydrates, tannins, terpenoids, saponins, flavonoids, steroids and fixed oils and fats as mentioned in Table 3. Figure 2 shows the class of compounds present in hexane and isopropanol extracts of A. scholaris bark.  Table 4 represents the active principles with their molecular formula, molecular weight (MW), retention time, probability percentage, total percentage content and fragment peak of compounds identified in A. scholaris. The prevailing compounds were hexadecanoic acid methyl ester, 1,4-benzenedicarboxylic acid dimethyl ester, and dodecanoic acid methyl ester.

Antibacterial activity of crude extracts
Results of antibacterial activity with different solvent extracts of A. scholaris leaf and bark are presented in Table 5 (nutrient agar) and Table 6 (Muller Hinton agar).
Results showed that except isopropanol and methanol extract none of the other extracts showed no activity against the selected strains. On nutrient agar, bark extracted with isopropanol, methanol, exhibited comparatively strong activity against most of the gram positive and MDR stains. The isopropanol extracts had activity against Proteus spp, Shigella spp, Salmonella typhi, Pseudomonas aeruginosa, E.coli, Klebsiella spp Sal monella paratyphi A, Staphylococcus aureus and MDR strains of E.coli and Klebsiella spp. The methanol extract of bark showed antibacterial activity against all of selected organism except Shigella spp.
Hexane, benzene, isopropanol and methanol fractions of A. scholaris bark and leaf showed activity against Enterobacter cloacae (Table 7). Water extract was not active.

Antifungal activity of crude extracts
The antifungal potential of different extracts of leaf of A. scholaris are presented in Table 8. The hexane, benzene, isopropanol, methanol and water extracts of leaf strongly inhibited the growth of fungus include human pathogenic strains. It was observed that the isopropanol, methanol and water extract of leaf inhibited the growth of P. marneffei, Cryptococcus and Candida. Hexane, benzene and water extract inhibited Aspergil lus niger. The water and hexane extract showed inhibition against the growth of Rhizopus spp.
Hexane, benzene, isopropanol and methanol fractions of A. scholaris bark shows activity against atypical Mycobacterium (Table 9). Isopropanol and methanol fractions of A. scholaris leaf as well as benzene, isopropanol shows activity. Hexane and benzene fraction of A. scholaris leaf hexane fraction was not active.

Anti-Hepatitis B virus of crude extracts
Hexane, benzene and isopropanol fractions of A. scholaris leaf and bark showed activity against Hepatitis B virus (Table 10). Methanol fractions of A. scholaris bark also is active whereas methanol and water of leaf A. scholaris does not inhibit the growth of the Hepatitis B virus.
Previous studies on A. scholaris have revealed the antibacterial and antioxidant properties (21,22,33,(35)(36)(37)(38)(39). Khyade and Vaikos (33) and Misra,Pratyush (21) supported the same activity of the methanol extract of A. scholaris leaf against the human pathogens. As the present study, several other studies have also reported similar antibacterial potential of the leaf methanolic extract (21,33). Molly, Shekhar (22) tested hexane, chloroform, butanol, ethyl acetate and water fractions of methanol extracts of A. scholaris leaf and bark for antibacterial activity observing that fractions of leaf extract had pronounced antibacterial activity against methicillin resistant S. aureus and Providence stuartii. Moreover, in this study antibacterial activity of these extracts was tested against several Gram positive and Gram negative bacterial strains and was found to reside maximum in the butanol and ethyl acetate fractions of methanol extract of leaf and bark. Cowan (40) reported that the differences in the observed activities of the various extracts may be due to varying degree of solubility of the active constituents in the four solvents used. It has been documented that different solvents have diverse solubility capacities for different phytochemical constituents.
The leaves and bark from A. scholaris are rich in phytochemicals with antimicrobial activities against human pathogens. On the basis of the results obtained in the present study it can be concluded that the isopropanol fraction of A. scholaris have antibacterial, antifungal, antiviral, and anti-mycobacterial activities. The present study may serve to be the basis of new studies to found new antimicrobials agents to be used in phytotheraphy or development of new drugs.

Conflicts of Interest
The authors declare no conflict of interest.