In Vitro Antimicrobial Bioassays, DPPH Radical Scavenging Activity, and FTIR Spectroscopy Analysis of Heliotropium bacciferum

The present study deals with the antimicrobial, antioxidant, and functional group analysis of Heliotropium bacciferum extracts. Disc diffusion susceptibility method was followed for antimicrobial assessment. Noteworthy antimicrobial activities were recorded by various plant extracts against antibiotic resistant microorganisms. Plant flower extracts antioxidant activity was investigated against 2, 2-diphenyl-1-picryl hydrazyl radical by ultraviolet spectrophotometer (517 nm). Plant extracts displayed noteworthy radical scavenging activities at all concentrations (25–225 μg/mL). Notable activities were recorded by crude, chloroform and ethyl acetate extracts up to 88.27% at 225 μg/mL concentration. Compounds functional groups were examined by Fourier transform infrared spectroscopic studies. Alkanes, alkenes, alkyl halides, amines, carboxylic acids, amides, esters, alcohols, phenols, nitrocompounds, and aromatic compounds were identified by FTIR analysis. Thin layer chromatography bioautography was carried out for all plant extracts. Different bands were separated by various solvent systems. The results of the current study justify the use of Heliotropium bacciferum in traditional remedial herbal medicines.


Introduction
Plants are the foremost sources of traditional medicines with a huge variety of bioactive components, which are effective against various diseases. Plants biological activities are attributed to these bioactive components. Medicinal plants which are the rich sources of antifungal and antibacterial agents are used as basis of effective beneficial drugs in many countries [1]. Fungi, bacteria, viruses, and other microorganisms are potentially pathogenic to humans and animals. Worldwide, antibiotic resistant bacteria epidemics have been reported in hospitals. Therefore, discovery of novel antimicrobial agents to fight such diseases becomes very significant and indispensable [2]. Medicinal plants are the richest sources of these microbial agents. In traditional systems of modern medicines, plants are the richest resources of drugs, food supplements, folk medicines, nutraceuticals, and chemical permitted for synthetic drugs. Plant potential as source of novel drugs is still largely unfamiliar. In phytochemical perspective, only a slight percentage of plant has been explored [3]. Worldwide, antimicrobial agent's resistance has amplified and caused considerable mortality and morbidity. Bacteria have genetic capability to transmit and gain resistance to therapeutically active drugs. In developing countries, elevated cost of drug treatment has contributed to eminent frequency of opportunistic and chronic diseases. To manage these infections, there is an essential search for novel agents with less toxicity and larger antibacterial activity [4][5][6][7]. Heliotropium genus belongs to Boraginaceae family consists of about 100 genera and 2000 species [8]. Flavonoids and polyphenols distribution in Boraginaceae family has different pharmaceutical activities such as antibacterial, antioxidant, anti-inflammatory, antiviral, and hepatoprotecting [9]. Heliotropium bacciferum is a potent source 2 BioMed Research International of various phytochemicals and reported significant Diphenyl picryl hydrazyl (DPPH) radical scavenging activities [10]. It is a wealthy source of pyrrolizidine alkaloids, which have antimicrobial, antihyperlipidemic, antidiabetic, and antitumor properties [11]. Previously reported study revealed that the aerial parts of Heliotropium bacciferum have significant antibacterial and antifungal effects. All tested plant extracts exhibited significant activities against different bacterial and fungal strains. The result against various microorganisms divulged the curative potential of the plant Heliotropium bacciferum [12].
There is no reported data on the antimicrobial activities of individual parts of the plant. Therefore, the present research was designed to screen the antibacterial, antifungal (leaves, flowers, and stem), and antioxidant (flower) assays of Heliotropium bacciferum extracts. Investigation of bioactive compounds functional groups and thin layer chromatography bioautography was also the key focus of the present study.

Plant Collection and
Authentication. Heliotropium bacciferum was collected from Karak, KPK, Pakistan. Sample washing and cleansing were accomplished by deionized water for further processing. Plant parts (leaves, flowers, and stem) were separated, dried at room temperature, and crushed into coarse powder. Herbarium staff of Plant Sciences, University of Peshawar, authenticates the plant species and kept the plant species in the laboratory for further processing.

Chemicals and Reagents.
Analytical and HPLC grade chemicals and reagents were used for experimental screening. Methanol, n-hexane, chloroform, ethyl acetate, and n-butanol were used for plant constituent's extraction. Solvent purification was accomplished by dehydrating agents (Na 2 SO 4 and MgSO 4 ).

Antibacterial
Assay. Antibacterial assay of leaves, flowers, and stem extracts of Heliotropium bacciferum was investigated by disc diffusion susceptibility method [14]. Seven bacterial species, that is, Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538), Bacillus cereus (ATCC 7722), Pseudomonas aeruginosa (ATCC 9721), Klebsiella pneumoniae (ATCC 6824), Proteus mirabilis (ATCC 7103), and Erwinia carotovora (ATCC 8452) were used for antibacterial bioassay. The solvents used for antibacterial evaluation were purified by dehydrating agents such as Na 2 SO 4 and MgSO 4 . Fractional distillation was also carried out for further solvent purification. Plant extract stock solutions (1 mg/mL) were prepared in dimethyl sulfoxide (DMSO). Nutrient agar media (2.8 g/100 mL) were used for microbe's culturing and growth, while nutrient broth (1.3 g/100 mL) was used for microorganism's standardization. Standardized microbial cultures (50 L) with glass spreader were inoculated on each nutrient agar plate in a laminar flow hood for microbial growth and incubated at 37 ∘ C for 24 hrs. The first streaked cultures were inoculated and incubated again. The second streaked microbial cultures were inoculated in nutrient broth (20-25 mL) and incubated for 18 hrs at 37 ∘ C in shaking water bath (200 rpm). Sterilized nutrient broth dilution was accomplished for standardization of microbial cultures and compared with 0.5 McFarland turbidity standard. Whatman filter paper discs (5 mm in diameter) were placed on solidified agar media with the help of sterilized forceps. Plant extracts (15 g/disc) of leaves, flower, and stem were applied to media plates and incubated at 37 ∘ C for 24 hours. As negative control, DMSO (5%) was used, while ampicillin antibiotic (8 g/disc) was used as a positive control. The zone of inhibition (mm) was then measured for each plant extract.

Antifungal Assay.
Agar well diffusion technique was used for the assessment of antifungal bioassay. Five fungal strains, that is, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, and Aspergillus fumigatus, were used for this activity. Fungal strains were cultured on Sabouraud's dextrose agar (SDA) media for 3-5 days at 28 ∘ C. Nutrient broth media (28 g/1000 mL) were prepared in distilled water for the refreshment of fungal strains. Sterilized SDA plates were taken and 6 mm diameter sterile cork borer was used to bore wells in the agar media. Plant extracts (15 g/well) were then added into each well. Plates were allowed to stand for 1 hour at 37 ∘ C for extract diffusion into agar and incubated at 28 ∘ C for 24 hrs. DMSO (5%) was taken as negative control, while clotrimazole antibiotic (8 g/well) was used as positive control. The zone of inhibition (mm) was then measured for each plant extract.
2.6. DPPH Radical Scavenging Activity. Antioxidant activity of plant flower extracts was investigated against 2, 2-diphenyl-1-picryl hydrazyl radical by ultraviolet spectrophotometer (517 nm). The methodology of Ahmad et al. [10] was used for the activity. Plant extracts stock solution (1000 mg/mL) was prepared and diluted (25,75,125,175, and 225 g/mL) with the respective solvents. Ascorbic acid was used as control for comparison. The same five dilutions were also prepared for control. DPPH solution was prepared at concentration of 0.003 g/100 mL. All plant extracts were then treated with DPPH solution. Spectrophotometer was used for absorbance calculation at 517 nm after 30 mints. Absorbance decline by DPPH solution was used as an indication for high antioxidant activity. The percent antioxidant activity was calculated by the given formula: and Aspergillus fumigatus were about 5 × 10 5 , 3 × 10 4 , 3 × 10 6 , 2 × 10 3 , and 2 × 10 5 cells per milliliter, respectively. Bacterial and fungal suspensions were sprayed on TLC chromatograms. Laminar flow hood was used for bacterial processing and biosafety cabinet was used for fungal processing. TLC plates were then kept in dark (100% relative humidity) at 35 ∘ C for a night. The plates were sprayed with p-iodonitrotetrazolium (2 mg/mL) violet and incubated overnight.

Statistical Analysis.
All values were presented as the mean ± standard error of mean and analyzed for Two-Way ANOVA and One-Way ANOVA. Statistical analysis was carried out on GraphPad PRISM 6.

Antibacterial Activity. The antibacterial activity of
Heliotropium bacciferum leaves, flowers, and stem extracts was recorded against various microorganisms. All plant extracts exhibit a range of inhibitory potentials (Table 1)         extracts (72.36%) were slightly active and revealed notable activity at higher concentrations (225 g/mL) ( Table 3).

Thin Layer Chromatography (TLC) Bioautography.
Thin layer chromatography bioautography technique is used for bioactive components isolation on TLC plates which link these compounds with the biological activities. Bioautography name is used due to the connection of TLC with the biological activities especially antimicrobial activities. Bands visualization was accomplished by ultraviolet light at 305 and 368 nm. values, inhibition of microorganism's growth, and the active bands were found out by TLC   values. Stem extracts active compounds were also found at different values against P. mirabilis and E. carotovora (Table 10). Various values were found to be against different fungal species by plant leaves, flowers,   [15].
In the present study, the results signified that the plant extracts inhibited the growth of different microorganisms, therefore showing that the plant extracts contained substances which can inhibit the growth of different microbes. Different researchers have also shown that the plant extracts inhibit the growth of diverse microorganisms [16]. A study reported that plant extracts antibacterial potential is mainly due to the presence of different phytochemicals [17]. Many observed that the antimicrobial effects of plant extracts are due to the presence of various secondary metabolites [16]. Traditionally, plant extracts are used in the treatment of sore, boils in the ear, wound healing, diarrhea, and control dysentery [18]. Plant extracts divulged noteworthy activities against Pseudomonas aeruginosa and Staphylococcus aureus, which explore their use in the cure of bores, open wounds, and sores [19]. Staphylococcus aureus infections healing has become problematic as it has developed several mechanisms to become resistant to nearly all notorious antibiotics [20,21]. Antibacterial activity of plant extracts against Escherichia coli justifies their use in the treatment of diarrhea and dysentery. Escherichia coli is the major reason of diarrhea and in humans other diverse diarrhoeagenic infections [22]. Phenolic compounds due to its peroxidation inhibition and scavenging of oxygen radical are vital for antioxidant activity. These compounds are significant for the treatment of cancer, cardiovascular disorders, inflammatory diseases, and aging [23]. Some phytochemicals such as flavonoids, anthocyanin, catechin, coumarin, isoflavones, flavones, isocatechin, and lignans are responsible for radical scavenging potential [24]. Previous study reported the presence of different bioactive constituents such as alkanes, carboxylic acids, aldehydes, ethers, alcohols, ketones, and amindes by Fourier transform infrared spectroscopic study [10]. Fourier transform infrared spectroscopy has been revealed to be a significant mean for classification and differentiation of intimately relevant microbial species, plants, and other diverse organisms [25][26][27]. Fourier transform infrared spectrophotometric assessment showed that alkyl halides and alkanes prevalent in plant extracts contained elevated number of functional groups, which were found more significant against Staphylococcus aureus, Candida albicans, and Escherichia coli [28]. Chemical constituents of plants could yield pharmaceutically significant drugs [29].

Conclusion
Results of the present study revealed noteworthy antimicrobial and antioxidant activities of different parts of Heliotropium bacciferum. Plant extracts have immense potential as antibacterial and antifungal compounds against antibiotic resistant microorganisms. Therefore, they can be used in the cure of infectious diseases caused by resistant microorganisms. Alkanes, alkenes, alkyl halides, amines, carboxylic acids, amides, esters, alcohols, phenols, nitrocompounds, and aromatic compounds were identified by FTIR spectroscopic analysis. The results of this evaluation give evidence that Heliotropium bacciferum might be a promising source of phytocompounds which can be isolated and analyzed for pharmacological activities, in vitro and in vivo bioassays.