INTRODUCTION:

The worldwide epidemic of antibiotic resistance has impacted all areas of medicine. In recent years, extensively drug resistant (XDR) organisms have increased many fold¹. According to the European Centre for Disease Control (ECDC) and Centre for Disease Control and Prevention (CDC) Atlanta, extensively drug resistant (XDR) was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories². The emergence of extensively drug resistant has become a major global problem as there are fewer or sometimes no effective antimicrobial agents available for infections caused by these bacteria. This situation poses a major threat to patient’s treatment and increased economic loss to the patient and nation³.

The rise of drug-resistant virulent strains of Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA) posses serious concern in the treatment and control of extensively drug resistant (XDR)⁴. Many studies have shown that the clinical isolates of Methicillin Resistant Staphylococcus aureus (MRSA) has a unique ability to quickly respond to each new antibiotic. It demonstrates the development of resistance mechanism against commonly used antibiotics of groups, aminoglycosides, macrolides, fluoroquinolones, chloramphenicol and tetracycline until the latest, linezolid and daptomycin^5,6. Considering the rapid adaptation of Staphylococci to neutralize antibiotics and limited number of new antimicrobial agents in the development has left fewer effective antibiotics to treat these often life-threatening infections. Therefore, we decided to study resistance-modifying agents (RMAs) of plant Datura inoxia to find an alternate of synthetic antibiotics. The use of phytochemical products and plant extracts is an increasingly active research topic⁷. Datura inoxia is well known for its medicinal properties due to the presence of various phytochemicals that exhibit extraordinary array of antimicrobial activity against drug resistant pathogens⁸. Other Datura species also has potential to treat many infectious diseases and was considered as antispasmodic, anodyne, antiseptic, antipyretic, antiphlogistic, antidiarrheal, antiproliferative, anthelminthic, antimicrobial against Klebsiella pneumoniae and Staphylococcus aureus⁹ also beneficial in various skin diseases, leukoderma, insanity, bronchitis, catarrh, heart disease, ulcers and jaundice^{10,11, 12}. In present study, an attempt was made to isolate and characterize antibacterial compound from hexane leaf extract of Datura inoxia and pharmacological preparation made from identified compound. The choice of the plant and solvent is based on results of our previous work⁷. The work was carried out in laboratories of Amity university Uttar Pradesh Lucknow, Ram Manohar Lohia Institute of Medical Sciences (RMLIMS), Lucknow and Central Drug Research Institute -Sophisticated Analytical Instrument Facility (CDRI-SAIF) for experiments.

MATERIALS AND METHODS:

Sample Collection and Preparation of Plant Extracts:

Datura inoxia was collected from nearby area of Amity University Uttar Pradesh, Lucknow Campus. The plant leaves were washed thoroughly with tap water followed by distilled water to remove the dust particles and allowed to air dry at room temperature on laboratory bench. The dried plant leaves were pulverized with the help of liquid nitrogen and stored at -20°C till further use. For preparing extract, 10gm of the pulverized sample was mixed with 100ml hexane (Merck, India) and kept for 48 hrs. at room temperature. The extract obtained was filtered and concentrated. The final concentration was maintained as 100mg/ml by dissolving the crude extract in 10% DMSO (dimethyl sulfoxide) for bioassay analysis¹³.

Phytochemical Screening:

Phytochemical analysis was performed to determine the existence of biologically active components in extract such as alkaloids, carbohydrates, glycosides, saponins, phytosterols, phenols, tannins, flavonoids, proteins, and diterpenes. The extract was vaporized to dryness and the residues left were then identified for the presence of different phytochemicals through standard methods with some modificatios¹⁴.

Test Organisms and Bacterial Inoculum Preparation:

Staphylococcus aureus ATCC 11632 was taken for the study and was procured from HiMedia Laboratory Pvt. Ltd. and maintained by sub culturing on blood agar (HiMedia Laboratory Pvt. Ltd.) and MacConkey’s agar (HiMedia Laboratory Pvt. Ltd.) plates. To prepare bacterial inoculum, pure isolate of bacteria was diluted in a test tube containing 0.9% normal saline solution and compared with the 0.5 McFarland turbidity standard (TULIP DIAGNOSTICS (P) LTD.), which is equivalent to 1.5 x 10⁸CFU/ml.

Antimicrobial Activity Assay:

The agar well diffusion assay¹⁵ was carried out on Muller Hinton Agar (Hi Media Laboratory Pvt. Ltd.) plates to assess antibacterial activity of crude extract, purified extract, and formulation from purified extract. Wells of 6mm size were dug with the help of a sterile cork borer and 100µl of the hexane extract was loaded followed by incubation at 37⁰c for 24 h. Antibacterial activity of the extracts was calculated in terms of zone of inhibition (diameter in mm). Determinations were done in triplicates and mean variance obtained. Standard antibiotic (Gentamicin) was used as positive control, while DMSO (10%) was used as negative control to compare the antibacterial activity of the extracts.

Fractionation of Extract through Column Chromatography:

Crude hexane extract of Datura inoxia was further fractioned through column chromatography. Column was filled with silica gel (60-120 mesh) which acts as a stationary phase and packed by passing eluent (hexane) acting as a mobile phase. Packed column was then added with the sample and the collection of fractions was done by passing solvent at a flow rate of 1ml/minute until silica gel appears colorless. Final concentration of the collected fractions was maintained as 1000µg/ml by dissolving them in 10% DMSO. Antibacterial activity of each isolated fraction was evaluated by measuring the zone of inhibition through agar well diffusion method.

Minimum Inhibitory Concentration (MIC):

MIC of the most active fraction was evaluated by two-fold macro broth dilution method^8,
16.The growth media was mixed with the different concentrations of the tested fraction and 100ul of the most susceptible bacterial inoculum having desired concentration of 5x10⁵ was then inoculated to all the tubes. The mixtures were then incubated at 37ºC for 24 hr. After incubation, the maximum dilution which inhibited bacterial growth was regarded as the MIC value of the tested fraction. The tube containing bacterial inoculum without the tested fraction was used as the growth control, whereas the tube without bacterial inoculum was taken as the sterility control.

HPLC and ESI -MS Analysis:

Reverse phase HPLC and ESI -MS analysis was done in CDRI-SAIF, Lucknow. Analysis was carried using HPLC Water -515 with PDA. Sample was analyzed using three channels PDA286.9 for run time of 30.0 minutes. Injection volume was 10ul using water and Methanol as solvent system. Data acquisition and computation were carried out with empower 2 software built 2154 SPs.

The Electrospray mass spectra was recorded on ABSCIEX 4000QTRAP.The sample was dissolved in organic solvent and introduced in to the ESI Source through Infusion at 10 µl/min. The Ion spray Voltage was set at 5.5kv. The MS scan was done in Positive mode for 5 min and spectra print out were averaged at top in TIC. Data generated as mass chromatogram. Y-axis showing relative intensity and represented as count per second (cps) and X axis representing mass to charge ratio (m/z).

Compound identification and Structure Prediction:

The constituents were identified after comparison with those available in the computer library (NIST version 2005) and documented. Interpretation of mass spectrum was conducted using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the compound was ascertained. Structure of identified compound was obtained by Corina software. Canonical smile notation is converted to 3D structure and visualized by visualization software.

Pharmacological Preparation:

Herbal formulation (gel) using purified hexane extract was prepared at Amity Institute of Pharmacy, Lucknow campus. Carbopol 940 and propyl paraben were procured from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Olive oil was purchased from CDH, New Delhi. Triethanolamine, propylene glycol, Tween 80, Span 80 and PEG 400 were purchased from s d fine-chem limited, Mumbai. Methyl paraben was procured from RENKEM, New Delhi. Double distilled water was used for all the preparation. All the chemicals were of LR grade and used as received from supplier without any modifications.

Preparation of Carbopol Gel:

Carbopol gel was prepared by swelling carbopol 940 (1.1%) for 24 hr in double distilled water and in mixture of water and alcohol (50:50, 75:25 and 90:10). Added 5% of propylene glycol and 0.2% polyethylene glycol (PEG 400). Finally the solution was neutralized and pH was adjusted between 6.5-6.8 with sufficient quantity of triethanolamine Formulation was optimized for carbopol concentration (0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.2 and 1.3 w/w) and quantity of triethanolamine (sufficient to reach desired pH).

Preparation of Emulgel and Emulsion Containing Purified Extract:

The extract was dried using rotavapor (I Ka RV 10 Digital) at 50ºC temperature and 100rpm and mixed with olive oil. To prepare oil phase for emulsion (o/w), optimized quantity (0.9 % w/w) of span 80 was added to the olive oil containing purified extract. Aqueous phase was prepared by dissolving tween 80 (0.1% w/w), methyl paraben (0.03%) and propyl paraben (0.01%) in water (q.s to 5ml). Emulsion was then prepared by addition of oil phase into aqueous phase. Dispersion of oil was accomplished by ultra-sonication at 50% amplitude for 20 minutes. Sonication was performed in pulsed mode with a pulse of 20 second at 10 seconds interval. Emulgel was prepared by mixing emulsion and carbopol gel in 1:1 ratio. The emulgel formulation containing fraction 5, prepared by mixing Carbopol gel and emulsion in 1:1 ratio was evaluated for transparency, pH, loss on drying, density, appearance, spreadibility and extrudability. Weight before and after drying and density was measured to ensure batch to batch uniformity. pH of the optimized formulation was determined by digital pH meter (Elico) and was found to be 6.7 and it was maintained during storage (3 months) between 6.5-6.8. Spreadability of developed formulation was determined by the method used by Arvouet-Grand et al.,1994¹⁷, 1g of sample was pressed between 20x20cm horizontal plates, the upper of which weighed 125g. The spread diameter was measured after 1min of application of pressure. Optimized formulation showed semi stiff nature with spread diameter between 42-45mm.

RESULTS AND DISCUSSION:

Phytochemical analysis was done for hexane extract of leaves of Datura inoxia in crude and purified extract-fraction 5. Sign “+++” shows significant presence of phytocompounds while “++” represents moderate, “+” represent mild, while “–” represents absence of phytochemical (Table 1). Glycosides and Diterpenes are present in significant amount in both crude and purified extract, but more in purified fraction. Flavonoids and Tannins also shown its mild presence in crude extract whereas absent in purified. Small trace of Saponins is also present in purified extract (Table 1). Glycosides, Diterpenes, Flavonoids and Tannins are secondary metabolites which could only dissolves in organic solvent. Presence of glycosides terpenoids, and saponins by phytochemical analysis is accordance of our other results i.e. presence of triacylglycerol-a lipid (Fig 6,7) in purified extract. The different phytochemicals detected are known to act differently against various human pathogens. Glycosides are the condensation products of sugars with different varieties of organic hydroxyl or thiol compounds¹⁸. They exert antibacterial activity by inducing pore-like structures in the membrane¹⁹. Terpenes are the essential oils responsible for the fragrance of plants. They occur in the form of diterpenes, triterpenes, tetraterpenes, hemiterpenes and sesquiterpenes. When the compound comprises of an extra element, typically oxygen, they are called as terpenoids²⁰. Their modes of antibacterial action are not completely defined but is supposed to cause membrane degradation²¹. Flavonoids are the group of polyphenols commonly distributed among the plant flora. They are produced by plants in response to microbial infections. They act on bacteria by forming multiplexes with extracellular and soluble proteins and with bacterial cell walls, thus inducing disruption of cell membrane²². Tannins are a group of polymeric phenolic chemical constituents responsible for tanning leather. They are present in all the plant parts such as bark, leaves, wood, root and fruits²³. They exert antibacterial effect by inactivating microbial adhesins, enzymes and cell envelope transport proteins²⁴. Saponins are amphipathic glycosides which exerts soap-like behavior when shaken in aqueous solutions²⁵. They inhibit bacteria by attaching with cell membranes to produce changes in cell structure, which results in cell death²².

Table 1: phytochemical analysis of hexane extract of leaves of Datura innoxia in crude and purified extract (fraction 5).

S. No.	TEST NAME	Crude	Purified fraction 5
1	Alkaloids	-	-
2	Carbohydrates	-	-
3	Glycosides	++	+++
4	Saponins	-	+
5	Phytosterols	-	-
6	Phenol	-	-
7	Tannins	+	-
8	Flavonoids	++	-
9	Proteins	-	-
10	Diterpenes	++	+++

The antibacterial potential of crude extract Datura inoxia leaf were observed using agar well diffusion method by measuring the diameter (mm) of zone of inhibition against the standard strain of Staphylococcus aureus -ATCC 11632 (Fig 1). Zone of inhibition (ZOI) is considered mild effect of extract if 0 - 10mm, moderate if 10 – 20mm, significant >20mm. DMSO used as Negative Control while Gentamicin used as Positive Control. Results showed that crude Hexane extract have a significant antibacterial property with the zone of inhibition measured as 21mm.This result encouraged us for identification of compound present in extract so we have done Colum chromatography (gel permeation) of crude extract in order to get purified fractions. Total eight fractions were collected and were tested against staphylococcus aureus for their inhibitory potential. ZOI (Fig 2) showed the variability in the inhibitory effect of each fraction. Fraction 5 (23mm) was the most effective followed by fraction 4 (21mm), fraction 6 (14mm), fraction 3 (11mm), fraction 1(15m) and fraction 7 (10 mm). However, fraction 2 (0.0mm) and fraction 8 (0.0mm) showed no inhibitory effect.

Fraction 5 was further subjected for HPLC, Compound identification by ESI-MS, MIC value and for pharmacological preparation in gel form, due to its greater inhibitory activity. (fig 2,3). Formulation with extract shown moderate effect whereas no antibacterial property was observed by placebo (fig 3). A similar study was reported with the leaf extract of Datura stramonium ²⁶. In other similar experiments, Fatima et al., 2015²⁷ and Tandon et al.,2016⁸, reported the mild inhibitory activity of hexane leaf extract of Datura inoxia against Staphylococcus aureus ATCC-6538 and Staphylococcus aureus ATCC-25923.In all experiment DMSO (fig 1,2) not shown any antibacterial activity, whereas Gentamycin shown significant antibacterial properties.

Fig 1: Bar diagram of Antibacterial Activity of Crude Hexane Extract of Datura inoxia against Staphylococcus aureus. Error bars indicate mean±SD of triplicate samples. Gentamycin and crude extract showing significant antibacterial properties while DMSO does not have any.

Fig 2: Bar diagram of Antibacterial Activity of purified fractions of Hexane Extract of Datura inoxia against Staphylococcus aureus Error bars indicate mean±SD of triplicate samples. Antibacterial activity of Datura inoxia calculated on the basis of their zone of inhibition (mm) against S. aureus. Zone of inhibition (ZOI) is considered mild if 0 - 10 mm, moderate if 10 – 20 mm, significant >20mm. Maximum fractions showing antibacterial properties except F2 and F8. Fraction F5 showing maximum ZOI. DMSO used as Negative Control, Gentamicin as Positive Control.

After fractionation, the phytochemicals may act more synergistically to produce greater inhibitory effect. The extracts were found to be less potent than standard antibiotic tested. The differences between the activities of the extracts and the standard drug may be due to the mixture of compounds present in the extracts compared to the pure compound contained in the standard antibiotics²⁸.

Fraction 5 (23mm) was found to be the most effective, so minimum inhibitory concentration (MIC) was conducted for this fraction. MIC is defined as the lowest concentration of antimicrobial agents which causes reduction in bacterial growth¹⁶. In present study, results of broth macro-dilution method showed that MIC of Hexane fraction 5 of Datura inoxia against Staphylococcus aureus was 250µg/ml. This was indicated by the broth showing no apparent growth in higher dilution (Table 2). A similar study was reported with the leaf extract of ethanolic extract of Datura inoxia leaf⁸. Here high MIC is due to presence of some other compound with triacylglycerol, which is showing antagonistic effect. Our phytochemical analysis (Table 1) also showing presence of more than one compound in purified extract. Further purification may provide a better MIC.

Table 2: MIC of hexane Fraction 5 of Datura inoxia against Staphylococcus aureus. + sign Clear broth, indicating no growth, - Turbidity in the broth, indicating growth

Tested Organisms	Concentration (µg/ml)
Staphylococcus aureus	1000	500	250	125	62.5	31.3
Staphylococcus aureus	+	+	+	-	-	-

Herbal formulation (gel) was prepared using purified hexane extract, fraction 5. All the formulations were found to be opaque having yellow colour and smooth in nature (Table 3). Using different parameters, physical evaluation of herbal formulation was done and obtained the results such as pH- 6.5 to 6.8, Loss on drying- 96.9%, with excellent spreadibility (Table.3). The formulation was found to have extrudibility when allowed to extrude from lacquered aluminium collapsible tube. Herbal gels can facilitate the absorption of poorly absorbed drug by increasing the contact time of the drug with the skin²⁹. It is an attractive route for local and systemic treatment. The delivery of drugs into the skin is recognized as an effective means of therapy for local dermatologic diseases. It can penetrate deeper into skin and hence give better absorption³⁰. In general, they are believed to be more effective and less toxic than conventional formulations due to the bilayer composition and structure³¹. Staphylococcus aureus is well known to cause various skin infections, this pharmacological study will aid in developing effective treatment therapy. Gel (with and without extract) is also tested for its antibacterial efficiency.

Table 3: Physical Evaluation of Herbal Formulation (Gel)based on different parameter like pH, Loss on drying, Loss on drying, Appearance, Spreadibility. Extrudibility.

S. No.	Evaluation Parameter	Result
1	pH	6.5-6.8
2	Loss on drying	96.9%
3	Loss on density	0.9867 unit
4	Appearance	Yellow colour
5	Spreadibility	≤50 (semi stiff)
6	Extrudibility	Excellent

Fig 3: Antibacterial Activity of Formulation prepared from fraction 5 (purified hexane extract of Datura inoxia) against Staphylococcus aureus.

HPLC analysis for fraction 5 was done at 286.9 nm (Fig.4), where X axis was plotted for time taken to flow in minutes and Y axis shows AU and the total run time was 30 minutes. In Fig.4, multiple components in purified fraction no-5 were observed. At retention time (RT) 11.703, one peak was very prominent showing height 137025 with % area of 60.73(3132560), with other 11 minor peaks. The result clearly shows that along with major compounds there are presence of other compounds in traces. Mass chromatogram generated by ESI-MS showing Y-axis for relative intensity and represented as count per second (cps) and X axis representing mass to charge ratio (m/z). Data for ESI-MS (Fig 5) is fragmented mass of all components present into fraction 5. Intensity was calculated in cps from 0.0 to 3.0e6. Different compounds were found in range between 100-1000 m/z value. ESI -MS analysis result showed prominent ions with m/z value of 288.2, 301.1, 316.5, 370.3 502.2, 950.8, 123.0, 202. Highest peak was found at 288.2m/z. Compound identification was done using bioinformatics software. Mass spectra analysis of compound (Fig 6) is identified by using mass bank database. Input file is prepared by using data from mass spectrometry result (Fig 5.,6). File is uploaded in mass bank analysis tool and results are obtained by matching list of masses.

CONCLUSION:

It believed that the modern allopathic system of medicine is known to produce serious side-effects, whereas medicinal agents derived from plants have some advantages such as often fewer side effects, better patient tolerance, relatively less expensive, acceptance due to long history of use and being renewable in nature. Our study provides a good support for the use of plant-based Triacylglycerol and its genins in treating various infectious diseases caused due to Staphylococcus aureus. Other extracts of Datura inoxia should also be explored for various therapeutic properties. As compounds obtained from natural sources have complex structure, we can hypothesize that these agents may have lesser cases of bacterial resistance and can be the answer to many health problems. Although antibiotics were first isolated from fungi and bacteria but over the years more and more synthetic antibiotics are flowing in market. So, instead of developing synthetic antibacterial therapy, we should explore natural antibiotics. It would not only take us closer to nature, but several new healthier ways of living a disease-free life will be discovered. Future prospect of the present research work is to identify antibacterial mechanisms of action of Triacylglycerol followed by its in vivo and in vitro cytotoxic effects.

ABBREVIATIONS:

HPLC: High Pressure Liquid Chromatography,

ESI-MS: Electrospray ionization - Mass Spectrometer,

NIST: National Institute of Standard and Technology,

XDR: Extensively Drug Resistant,

MRSA: Methicillin Resistant Staphylococcus aureus,

DMSO: Dimethyl sulfoxide,

MIC: Minimum Inhibitory Concentration,

ZOI: Zone of Inhibition,

PDA: Photometric Diode Array.

ACKNOWLEDGEMENT:

we are sincerely thankful to Pro vice chancellor, Amity university Uttar Pradesh Lucknow, Director Amity Institute of Biotechnology Lucknow, Director RMLIMS, Director CDRI-SAIF, India for providing necessary facility to carry out research work and Dr Ruchi Yadav for helping in bioinformatics work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 07.04.2020 Modified on 11.06.2020

Research J. Pharm. and Tech. 2020; 13(11):5235-5243.

DOI: 10.5958/0974-360X.2020.00916.6