INTRODUCTION:

In recent decades the multidrug-resistant bacteria have developed dramatically and caused many serious infections that are not treatable by antibiotics¹. The use of herbal-based antibacterial compound could be an alternative method to treat resistant bacterial infections². Herbal based drugs are mostly used nowadays due to their efficacy, quality and safety and they are also rich in natural complex chemicals³.

Both infectious and chronic diseases can be treated with the various phytochemicals present in medicinal plants and these plants are used as a traditional medicine in developing countries⁴. Glycosides, tannins, volatile oils and alkaloids are the secondary metabolites with significant bioactivities that can be used as a therapeutic agent and these metabolites are synthesized and accumulates in plants⁵. As the natural products are readily available at low cost with no adverse side effects, the plant-based therapeutics are demand in developed and developing countries. The broad use of synthetic antibiotics can be replaced with the medicinal herbal products as they are good anti-microbial agent⁶.

Medicinal and nutritive properties are present in the flower crops and ornamentals which are used for decorative purposes⁷. An ornament plant Delonix regia is used in the treatment of diabetes in Bangladesh folk medicine. Delonix regia, Catharanthus roseus and Nerium oleander are some of the ornamental plants with medicinal properties⁸. Amaltas, asoca, bauhinia, kadamba, palash are the ornamental tree species used as the traditional medicine in ayurvedic system⁷. GAP31 from jacalin and MAP30 from bittermelon is the lectins with antibacterial, antiviral and antifungal functions. Bougainvillea plant leaves are simple ovate-acuminate and alternate with various colors of flowers belongs to Nyctinaginacea family and this thorny woody plant is commonly known as a paper flower, the glory of the garden^9,10. In Mandsaur, the traditional practitioners use stem decoction to treat hepatitis and dried flowers decoction for leucorrhoea and blood vessels¹¹. The leaves part of Bougainvillea glabra are mostly used for therapeutic purposes¹¹. Antihyperglycemic activity, antidiarrheal activity, anti-ulcer, insecticidal activity, anti-microbial activity are the pharmacological properties reported in the leaves of Bougainvillea glabra choicy¹⁰. Steroid, cardiac glycosides, phlobatanin, tannins, flavonoids, terpenoid and saponin are the phytochemical constituents present in the B. glabra leaves¹².

Polyalthia longifolia is a tall evergreen plant commonly known as Ashoka, Asaphala, Indian Fire tree, Indian mast tree, Buddha tree and green champa and the plant belongs to the family Annonaceae^3,6,13. Helminthiasis, fever, skin diseases, diabetes, rheumatism, menorrhagia, scorpion sting and hypertension are cured with different parts of the versatile plant P. longifolia in the traditional system of medicine^6,13.The phytochemical studies of P. longifolia revealed the presence of sesquiterpenoids, quercetin, bulbocapnin, a-sitosterol, stigmasterol campest, enihalimane diterpenes, flavonoids and alkaloids. Anti hyperglycaemic, cytotoxic, hypoglycemic, hypertensive and antimicrobial actions are reported in leaves and bark of P. longifolia^14.

Ixora coccinea Linn. is a flowering shrub belongs to family Rubiaceae and commonly known as red ixora, burning love, the flame of the woods, scarlet ixora, flame of the forest, jungle flame, jungle of geranium and vetchi in Ayurveda^15,16. In the traditional medicine, skin diseases, sprains, chronic ulcers, hypertension, menstrual irregularities and various types of infections are treated with diverse parts of this plant¹⁷. A decoction of roots is used in the treatment of anorexia, nausea and hiccups and the roots are used externally for treating chronic ulcers, sores and eczema¹⁵. To clarify the urine, a decoction of I. coccinea Linn. root is used in Indo – china and for healing contusions, eczema, boils and sprains, poultice fresh stems and leaves are used¹⁵. Dysentery, hemoptysis, dysmenorrheal, catarrhal bronchitis and leucorrhoea can be treated which flowers and roots of Ixora coccinea Linn., whereas leaves are used to cure diarrhea¹⁸. Anti-inflammatory, anti-oxidant, chemoprotective, cytotoxic, antitumor, antimicrobial, anti-nociceptive, antimitotic and hepatoprotective activities are observed in Ixora coccinea Linn^15,17. Flavonoids, tannins, saponins, alkaloids and steroids are the phytochemical constituents present in the I. coccinea Linn. flowers extract and preliminary screening revealed the absence of gum in it¹⁹.

The family Apocynaceae includes a very common evergreen Plumeria rubra, extensively distributed over all southern India²⁰. Frangipani is the commonly referred name for Plumeria in India and in Malaysia, it is known as kemboja²¹. Traditionally, Plumeria rubra fruit used as abortifacient and for curing toothache and tooth decay, the latex of this plant was used. Blenorrhagia, drastic and purgative can be treated with root bark of P. rubra and pectoral syrup is made using this aromatic flowers²². P. rubra is used traditionally for treating typhoid, dysentery and diarrhea⁵. Hypolipidemic and antioxidant activity was found in flavone glycoside, which was isolated from Plumeria rubra²¹. The phytochemical analysis of different extracts of Plumeria species showed the presence of saponins, steroids, tannins, carbonyls, flavonoids, phlobatannis, glycosides, terpenoids, reducing sugars and alkaloids. In swiss albino mice, the ethanolic leaves extract of Plumeria rubra showed anticancer activity towards the Ehrlich Ascites Carcinoma (EAC)²¹.

Euphorbia milli is an exotic, flowering, decorative and ornamental plant belongs to the family Euphorbiaceae^{23, 24}. Christ thrown, christ plant is the commonly referred name for this flowering plant. It is native to Madagascar and Philippines and extensively distributed in India. The most effective latex in Euphorbia species was from E. milli which has a toxic effect for mammals²³. Mollusks can be controlled by using E. milli latex due to its embryofetal-toxicity and in humans, cattle and sheep this is used as the traditional medicine to cure schistosomiasis²⁴. The medicinal properties of Euphorbia are antitussive, antimicrobial, antioxidant, antispasmodic, anticancer, anticonvulsant, antinociceptive, antifungal, antitumor, antiarthritis, antidiabetic, anti-eczema and anti-inflammatory²³.

MATERIAL AND METHODS:

Collection of Plant Material:

The fresh leaves of Bougainvillea glabra, Polyalthia longifolia, Ixora coccinea Linn., Plumeria rubra and Euphorbia milli were collected from VIT, Vellore. The leaves were washed using distilled water to remove unwanted dust particles and shade dried for two weeks.

Extraction Method:

The shade dried leaves were pulverized into powdered form. About 15grams of leaf powder was dissolved in 100mL of Petroleum ether and kept in the rotatory shaker for 24 hours at room temperature. After 24 h the extract was filtered through Whatman No. 1 filter paper to obtain particle-free crude extracts. The same procedure was followed for the other solvents methanol and the crude extracts obtained were used for further investigation. The extraction process was done with two different solvents non-polar petroleum ether and highly polar methanol.

Phytochemical Screening:

The qualitative phytochemical screening was done using three different extracts of dried leaf powder. Using the standard protocol, the presence of alkaloids, saponins, phenolics, tannins, flavonoids, anthraquinone glycoside, sterol and triterpenes were analyzed²⁵.

Determination of Antimicrobial Activity:

The antifungal and antibacterial activity was determined by the agar well diffusion method. Mueller-Hinton agar for antibacterial and Potato Dextrose Agar for antifungal were used to study the inhibitory effect of plant extracts. Bacterial test pathogens include two gram-positive bacteria (Bacillus cereus MTCC0430, Staphylococcus aureus MTCC3160) and three gram-negative bacteria (Escherichia coli MTCC1687, Protease mirabilis MTCC3310, Klebsiella pneumonia MTCC7028). Wells were made in the agar using a sterile cork borer. The antimicrobial activity of the crude extract was studied by loading 100μL of crude extract at different concentrations (2.5, 5, 10mg) in the wells. Agar plates were kept at room temperature for 30 min, then incubated at 37°C for 24 h to study antibacterial activity and for antifungal, plates incubated at 28ºC for 24- 48 h. After the incubation period, the zone of inhibition around the sample was observed. The inhibition zone was measured in term of millimeters to determine the antibacterial activity of samples^26,27.

Silica Gel Column Chromatography:

Preparation and Treatment of the Column:

About 50g of Silica (60-120, HiMedia) was heated in the hot air oven at 105°C for 30 min. Silica was suspended in 100mL of petroleum ether and by wet packing method, silica was packed into the column. For uniform distribution of adsorbent, slight air pressure has to be applied while packing the column and covered with petroleum ether dipped cotton. About 1.5grams of crude extract of P. longifolia was mixed with silica and loaded on the packed column. 250mL of eluent (Pet ether: acetone 9:1) was used to wash the column at the rate of 1mL/min. The active fractions were collected and subjected to the preparatory TLC method and separated pure compound was used for further studies²⁸.

Thin Layer Chromatography:

TLC is a technique widely used to separate the compounds present in the samples. Silica-coated TLC sheets were used for separation and these plates were kept at 100ºC for 1h in a hot air oven. The sample was prepared by dissolving crude extracts in acetone. The solvent system was prepared with petroleum ether and acetone in the ratio of 9:1. Using the capillary tube, samples were spotted in the TLC sheet (Merck, India) at 1cm above the edge and allowed to dry for a few seconds. Then, the plates were placed in the Chromo tank and allowed to run. The plates were removed from the solvent system and visualized under visible light, UV 365 and 254nm. Deep Vision-TLC Chamber was used to visualize the separated bands²⁹.

Gas Chromatography-Mass Spectrometry Analysis:

The petroleum ether extract of Polyalthia longifolia was subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The GC-MS analysis was carried out on PerkinElmer Clarus 680 equipped with mass spectrometer Clarus 600. The column used was elite - 5MS capillary column (30m, 0.25mmID, 250μm df). The initial temperature of GC oven was maintained at 60°C for 2 min, ramp 10°C/min−300°C then the oven was controlled at 300°C temp for6 min. The carrier gas used for the analysis was Helium at a constant flow rate of 1 mL/ min and mass transfer line. 240°C was maintained as the source temperature and for spectral analysis Turbo mass version 5.4.2 software was used. The spectrums obtained for the sample components was compared with the spectrum of known components in NIST-2008 library and structure of the sample compounds was determined³⁰.

Fourier-Transform Infrared Spectroscopy:

Perkin Elmer Spectrophotometer system, UK was used for FTIR analysis of the plant crude extracts. FTIR analysis was done to identify the functional groups present in the compounds. The chemical bonds in the molecule are determined by interpreting the infrared absorption spectrum. The chemical bonds present in the sample absorb a specific wavelength of light which can be seen in the annotated spectrum. The dried crude extract of plant leaf powder was used for FTIR instrumentation analysis³¹.

In Silico Docking Analysis:

The 3D structure of ligands molecule PHDC ligand was drawn using ChemSketch-11 and 3D structures of the macromolecules were downloaded from the RCSB PDB website (http://www.rcsb.org/pdb) with the following PDB ID: 1ILE, 3B39, 3VMR, 4FGG, 1FFY, 1AD1, 2INR and 3G75 and prepared by removing all non-amino acid moieties in the structure, using PyMOL software. AutoDock 4.2.1 was used to perform the protein-ligand dockings and PyMOL was used to view the docking results obtained from AutoDock ³².

RESULTS AND DISCUSSIONS:

Phytochemical Screening:

Preliminary phytochemical screening for Bougainvillea glabra, Polyalthia longifolia, Ixora coccinea Linn., Plumeria rubra and Euphorbia milli has revealed the presence of different kind of chemical groups. The results for phytochemical studies of different plants are presented in Table.1. Bougainvillea glabra contains phytoconsistuents such as flavonoids, saponins, alkaloids, and phenols. Both P.longifolia and P. rubra showed positivity for the existence of flavonoids, tannins, saponins, phenols, anthraquinone glycosides and alkaloids. I. coccinea Linn. possess all those phytoconstituents including flavonoids, alkaloids, phenols, anthraquinone glycosides, tannins, and saponins. Phytocompounds such as alkaloids, phenols, and flavonoids are observed to be present in almost all investigated plants.

Assay for Antifungal Activity:

The antifungal activity of all test fractions was tested against Aspergillus niger and Aspergillus flavus and inhibition zone was measured. A. niger MTCC3323 and A.flavus MTCC2799 was not susceptible to most of the extracts. Antifungal activity was observed in three plants B. glabra, I. coccinea Linn and P. rubra. Among those, the maximum activity was found in the pet ether extract of P. rubra with a 17mm inhibition zone at 100mg/mL concentration towards A. niger. The highest inhibition zone of 15mm for A. flavus was observed for pet ether extract of Bougainvillea glabra. Antifungal activity of B. glabra crude extract was also reported by Joshny J et al., (2013) and they found the minimum fungicidal concentration at a concentration of 400mg/mL against Candida albicans³³.

Assay of Antibacterial Activity:

Antibacterial activity of all extracts was tested against two Gram-positive and two Gram-negative bacterial strains at different concentrations of 2.5,5 and 10mg. The test was performed in triplicates and the results for antibacterial assay of plant extracts were reported in Table.2A and Table.3B. Bacillus cereus, Staphylococcus aureus and Protease mirabilis are susceptible to most of the pet ether and methanol extracts. Among five plants, P. longifolia was showed remarkable prominent activity against gram-positive bacteria (B. cereus, S. aureus) and against gram-negative bacteria (E. coli, P. mirabilis, K. pneumonia). The pet ether extract showed significant activity even at lower concentrations and the zone of inhibition was clearly shown in Figure.1. At the concentration of 100µg, the extract showed 25, 21, 30, 13 and 18mm zone of inhibition against B. cereus, E. coli, S. aureus, K. pneumonia and P. mirabilis respectively. Sampath M et al., (2013) reported the antibacterial activity of ethanolic extract of P. longifolia leaves against P. aeruginosa, P. vulgaris, B. Subtillis, E. coli, K. aerogenes, Serratia marcescent, Salmonella typhi, Shigella flexneri and Streptococcus pyogenes with the zone of diameter 15±0.99, 14±1.18, 17±2.87, 16±0.56, 17±0.20, 18±0.21, 20±1.45, 16±0.56 and 22±.51mm respectively¹⁴.

A previous study conducted by Anupam Ghosh et al., (2008) also revealed the highest antibacterial activity of methanol and hot aqueous extract of P.longifolia towards K. pneumonia with the inhibition zone of 3.03±0.09 and 3.23±0.20cm at the specific concentration of 30µl of 2000µg/mL³⁴. The pet ether extract of Polyalthia longifolia showed maximum antibacterial activity against all the test pathogen which was nearly equal to the standard antibiotic streptomycin and the extract was further selected for compound separation.

Table 2. A: Antibacterial efficiency of plants petroleum ether extracts

Plants extracts

Concentration

(mg)

Inhibition zone in mm

Gram Positive

Gram negative

B.cereus

S.aureus

E. coli

P.mirabilis

K. pneumonia

B. glabra

2.5

P. longifolia

2.5

I. coccinea Linn.

2.5

P. rubra

2.5

E. milli

2.5

Streptomycin

‘-’ Inhibition zone was not observed

Table 2.B: Antibacterial activity of Methanolic plants extracts

Plant extracts

Concentration

(mg)

Inhibition zone in mm

Gram positive

Gram negative

B.cereus

S.aureus

E. coli

P.mirabilis

K. pneumoniae

B. glabra

2.5

P. longifolia

2.5

I.coccinea Linn.

2.5

P. rubra

2.5

E. milli

2.5

Streptomycin

10mg

Figure. 1: Zone of inhibition of the plant extract against tested bacterial strains

Silica Gel Column Chromatography:

The pure compounds were separated from the crude extract using the solvent system of petroleum ether: acetone (9:1) in TLC. The purity of compound separated was observed in TLC as single bands as shown in Figure. 2 (A). About 15 mg of pure compound was obtained from pet ether extract of P.longifolia by silica gel column chromatography method and subjected to NMR, FT-IR and GC-MS analysis. The spectrum for the pure compound was shown in Figure.2 (B). FT-IR spectrum revealed the presence of the following functional groups: C-H (3020.53) (744.52), C-O (1215.15) The FT-IR spectrum recorded for the active compound is given in Figure.2 (C).

The results from the mass spectrum analysis shown that the pure compound had a molecular weight of 316.35 g/mol and displayed in Figure.2 (D). The chemical structure of the pure compound 3,3’-(propane-2,2-diyl)bis(3,4,5,6,7,8-hexahydro-1H-isochromene) – PHDC was shown in Figure.2 (E). The compound separated was identified as 3,3’-(propane-2,2-diyl)bis (3,4,5,6,7,8-hexahydro-1H-isochromene) by using these results and the molecular formula for the compound is C₂₁H₃₂O_2.

In Vitro Antibacterial Assay for PHDC:

In vitro antibacterial assay was performed with two bacterial strains Protease mirabilis (MTCC3310) and Klebsiella pneumonia (MTCC7028) to study the antibacterial activity of PHDC extracted from Polyalthia longifolia. PHDC showed antibacterial effect towards P. mirabilis and K. pneumonia at the concentration of 5mg with an inhibition zone of 15 and 10 mm was recorded in Table.3. This clearly proved that the novel compound PHDC extracted was also responsible for the antibacterial activity of Polyalthia longifolia.

Table.3: Zone of inhibition for PHDC in mm

Bacteria	PHDC		Streptomycin 5mg
Bacteria	2.5mg	5mg	Streptomycin 5mg
*Protease mirabilis*	13	15	20
*Klebsiella pneumonia*	-	10	15

Figure. 2: Spectroscopic characterization and identification of PHDC (A) TLC of Pet ether extract and pure compound (a) Crude extract and (b) Pure compound (B)gas chromatogram (C)FTIR (D)mass spectrum (E) Chemical structure for pure compound PHDC

In Silico Docking Analysis:

In the in silico molecular docking, the interaction between PHDC and target proteins was studied. PHDC demonstrated the highest affinity towards Isoleucyl tRNA synthetase protein with the free binding energy of- 8.7Kcal/Mol and this interaction could be the possible mode of its antibacterial activity. The Auto Dock analysis of PHDC with the chosen protein targets are given in Table. 4.

Figure 3: Interactions between PHDC and Isoleucyl tRNA Synthetase (PyMOL)

Table.4: In silico docking of PHDC with drug target proteins

Target Proteins	PDB ID	Binding energy (Kcal/Mol)	No. of Hydrogen bonds
Isoleucyl tRNA Synthetase	1ILE	-8.7	-
DNAG	3B39	-8.0	7
Dihydropteroate synthetase	3VMR	-7.9	15
DNA Gyrase	4FGG	-7.5	23
Transglycosylase	1FFY	-7.3	9
PBP1b	1AD1	-7.2	1
DNA Topoisomerase 4	2INR	-7.1	10
DHFR	3G75	-6.6	1

CONCLUSION:

The extensive and improper usage of antibiotics leads to the emergence of drug resistant bacterial infections. Plant-based drugs could be a promising alternative medicine to treat such infections. Numerous phytoconstituents are reported to possess diverse therapeutic properties like antioxidants. antibacterial, antifungal and antiviral. In the present study, the petroleum ether extract of P. longifolia showed prominent antibacterial activity against all test pathogens such as Bacillus cereus, S. aureus (gram-positive bacteria) and E. coli, P. mirabilis and K. pneumonia (gram-negative bacteria) and this antibacterial efficiency was found to be nearly equal to the inhibition of standard antibiotic streptomycin. Antibacterial activity guided extraction of PHDC from P. longifolia crude extract can lead to the discovery of a new drugs for treating antibacterial infections and disease. In silico docking, analysis revealed the high affinity of PHDC towards Isoleucyl tRNA Synthetase. PHDC extracted from pet ether of P. longifolia has the ability to demonstrate significant antibacterial activity in in-vitro condition and thus it can be recommended to be used as the natural antibacterial agent to treat diseases caused by antibiotic-resistant bacteria.

ACKNOWLEDGMENT:

The authors thank Vellore Institute of Technology for providing lab facility to carry out this research work.

CONFLICTS OF INTEREST:

No conflicts of interest.

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Received on 18.04.2020 Modified on 27.05.2020

Research J. Pharm. and Tech. 2021; 14(4):2101-2107.

DOI: 10.52711/0974-360X.2021.00372

Phytochemical constituents	*Bougainvillea glabra*	*Polyalthia longifolia*	*Ixora coccinea* Linn.	*Plumeria rubra*	*Euphorbia milli*
Flavonoids	+	+	+	+	+
Tannins	-	+	+	+	+
Saponins	+	+	+	+	-
Phenols	+	+	+	+	+
Anthraquinone Glycosides	-	+	+	+	+
Terpenoids	-	-	+	-	-
Alkaloids	+	+	+	+	+