Extraction and Analysis of Antimicrobial Compounds from Onosma Bracteatum Using Response Surface Methodology

ABSTRACT

Background:

Infectious diseases are the world’s leading cause of premature mortality, almost killing 65,000 people every day.

Objective:

The main objective of the study is to find the extraction and analysis of antimicrobial compounds from Onosma Bracteatum using response surface methodology.

Materials and Methods:

ATCC culture samples and antibiotic discs were purchased from PCSIR and scientific markets. All chemicals with reagents of analytical and commercial grades were purchased from Sigma Millipore Chemical Germany, Daejung Chemicals, China, and Unichem England. ONOSMA BRACTEATUM was purchased from the herbal market and its extracts were prepared in the lab.

Results:

The plant extracts; ONO (3.82%) having light green color were arranged and further valued by performing phytochemical analysis of plant extracts. The highest quantity of phenolic content was observed in Ethanolic, Chloroform, and n-hexane extract of fruit with values 97.0 ± 0.91, 39.5 ± 0.55, and 8.1 ± 0.67 μg GAE/mg extract respectively.

Conclusion:

It very well might be deduced from this research that in-vitro investigation of the O. bracteatum plant showed a critical zone of inhibition against Escherichia coli, Bacillus subtilis, Staph aureus, klebsiella pneumonia, and Pseudomonas aeruginosa.

INTRODUCTION

The theranostic approach focuses on the precise targeting of infection at various sites with the integrated design of modular insights in designing antibiotic derivatives. Infectious diseases are the world’s leading cause of premature mortality, almost killing 65,000 people every day. The emergence of antimicrobial resistance has led to the foundation of novel and new antimicrobial therapeutic agents being compatible with emerging antibiotic resistance against severe infections.[1] In recent decades, raising trends on kinetic profiling as well as designing of Nano therapeutic agents is very necessary with synthetic antibiotics employed in the treatment of infectious and communicable diseases, caused by pathogens in animals and humans across the globe. Over the last two decades, some antibiotics have shown clinical significance in fighting various infections.[2] The study of infectious diseases requires robust consideration at the population, individual, and genetic cell levels to understand the pathogenic insights. The hospital sector is the most potent reservoir of nosocomial infections which occur at hospital premises due to exposure to pathogenic and virulent microbes.[3] The specialized and variable mechanisms need IPC measures with good clinical practices and GMPs enforced by the quality management cell in the hospital vicinity. Steward programs and workshops being scheduled and implemented by the International Conference of Harmonization and FDA lead to the foundation of measures, and the outcome-based organizational hierarchy which prevents the prevalence of infection and intensive efforts have been utilized to probe clinically useful antimicrobial medicines.[4] In any healthy demographic population, incentivization of the Research and development section is the most important factor in vaccine development.[5] The biological interconnection of pathogens and hosts has provided the impulse to search for novel pathways from natural products in launching a potent vaccine that could reduce the mortality rate.[6] The main objective of the study is to find the extraction and analysis of antimicrobial compounds from O. Bracteatum using response surface methodology.

MATERIALS AND METHODS

The experimental research was conducted at the University of Lahore. ATCC culture samples and antibiotic discs were purchased from PCSIR and scientific markets. All chemicals with reagents of analytical and commercial grades were purchased from Sigma Millipore Chemical Germany, Daejung Chemicals, China, and Unichem England. O. Bracteatum was purchased from the herbal market, and its extracts were prepared in the lab.

Antioxidant assays

A protocol by Kedare et al. (2011)[7] was followed with slight adjustments. The samples were analyzed to decide their conceivable action against free radicals, which was specified by the staining of the purple DPPH solution. Originally, 30 μl of the stock solution was taken and relocated to the relating wells of a 96-well microplate, at that point 180 μl of DPPH reagent was added to each well containing the corresponding sample. The final amount of the sample was 500 μg/ml. The solution was subjected to incubation at 37 ° C for a time of an hour in the dark. Ascorbate was utilized as a norm (positive control) while DMSO was utilized as a negative control. The absorbance of the sample mixture was estimated at 520 nm utilizing a microplate reader.

Total antioxidant capacity

Phosphomolybdenum was used to assess the total antioxidant capacity of O. bracteatum. Some components including 4 M ammonium molybdate {(NH₄)₆MO₇O₂₄}, 28 M trisodium phosphate {Na₃PO₄} and sulfuric acid {H₂SO₄} and 100 ml of 0.6 M 100 μl with any plant extracts of thin mixed solution was used. The incubation was done at 90 ° for 90 minutes. Absorbance measurement at 640 nm with a microplate reader.

Reducing power estimation

Reducing power was estimated according to the method by Ahmad et al. (2005) Concisely, 0.1ml of O. bracteatum extract (4 mg/ml) to 400 μl of phosphate buffer and 250 μl of potassium ferricyanide 1% [K₃Fe (US) ₆] mixed in Eppendorf tubes. The mixture was then incubated at 50 ° for 20 minutes of trichloroacetic acid to the O. bracteatum extract, which was then centrifuged at 6000 rpm at room temperature for about 10 minutes. The solution of the upper layer (150 ul) from the ground to the plate from the wells of the microtiter with FeCl₃ 0.1% 50 microliter

Absorbance was measured at 645 nm and the absorbance increase of the reaction indicated a greater reducing power. The blank is prepared by adding 100 ul DMSO mixture described above in reaction to a sample test. Ascorbate (1 mg/ml) at final concentrations of 25, 12.5, and 6.25 3.125 mg/ml was used as a standard. A calibration curve using different concentrations of ascorbic acid (−14, 994, and R² = =0.0559×) was obtained.

Antimicrobial characterization

A colony was selected from the stock solution with a sterile wire loop and used to inoculate a 10 ml sterile nutrient broth aliquot and incubated for 24 hours at 37 ° C. The turbidity was checked and adjusted according to the standard of McFarland 0.5 turbidity method. For the preliminary testing of O. bracteatum plant extracts’ anti-bacterial activity, the agar disk diffusion technique was used. The seeding density of the bacterial culture was adjusted according to the test requirement (1 × 10⁶ CFU/ml) and fresh culture medium (100 μl) was used to prepare the lawn on a nutrient agar p. Test (class 5 μl) was applied on filter paper and sterilized properly marked discs were placed on agar discs with the traditional p. Standard antibiotics served the positive control, while the disc impregnated with DMSO served in the negative control. The plates were incubated at 37°C for one day.

RESULTS

The plant extracts; ONO (3.82%) having a light green color were arranged and further valued by performing phytochemical analysis of plant extracts. Several biochemical assays of the crude plant extracts of O. Bracteatum wall (ONO.Cr) were implemented to evaluate the confirmation/absence of various primary and secondary metabolites. The performed calculated results of phytochemical evaluation of plant extracts of ONO.Cr showed the Total flavonoids content (TFC) and Total phenolics content (TPC) of 46.33 ± 1.2 and 43.23 ± 5.67 respectively [Table 1].

Table 1.

The performed calculated results of the phytochemical evaluation of the plant extracts of ONOSMA BRACTEATUM (ONO) positive sign specifies the presence and negative sign specifies the absence of metabolites

	TPC		TFC
ONO.Cr	43.23±5.67		46.33±1.2

Total flavonoids content

Total flavonoid content was determined from all extracts of O. bracteatum and results are expressed as μg quercetin equivalent per mg extract (μg QE/mg extract). The maximum TFC was given by M extract of fruits part with the value of 97.0 ± 02 μg QE/mg extract followed by CF extract having a value of 39.5 ± 0.8 μg QE/mg extract then NH extract having a value of 28.1 ± 0.4 μg QE/mg extract [Figure 1].

Figure 1.

Thin layer chromatography plate of Onosma bracteatum Wall extracts spotted by capillary tube with ethanolic crude extract (a) The samples from left to right were O. bracteatum Wall crude ethanolic extract in different concentrations as discussed above

Total phenolics content

Total phenolic contents were determined from all extracts of Onosma bracteatum and results are expressed as μg gallic acid equivalent per mg extract (μg GAE/mg extract). The highest quantity of phenolic content was observed in ethanolic, chloroform, and n-hexane extract of fruit with values 97.0 ± 0.91, 39.5 ± 0.55, and 8.1 ± 0.67 μg GAE/mg extract, respectively [Figure 2].

Figure 2.

Thin layer chromatographic plate of Onosma bracteatum wall extracts. From left to right: O. bracteatum wall extracts of concentration 1% (1000 mg/ml), 0.5% (500 mg/ml), solvent solution, standard ascorbic acid. From Top to bottom: - O. bracteatum wall extract in various successful solvent systems starting from Water, Ethanol, Hexane

DISCUSSION

Herbal plants are regarded as a comprehensive, accurate, and valuable tool for the treatment of diseases. In the global arcade, many scientists employ complementary approaches, and therapeutic regimens in the formulation and designing of products related to medicinal plants.[8] The phytochemical experts encompass a favorable environment that negates misuse of antibiotics, promotes the ethnobiological relevance of medicinal plants, and creates a bridge between old conventional approaches and new botanical insights. The main aim of our study was to investigate the antibacterial potential of O. bracteatum utilizing Response surface methodology software.[9] Plants are immobile organisms that are attacked by herbivorous organisms and have to face environmental challenges they possess many defense tools like spines and thorns.[10] Polyphenols area assembly of highly hydroxylated phenolic compounds offered in extractive fractions of several plant materials. Polyphenols are an assembly of profoundly hydroxylated phenolic phytocompounds offered in the extractive part of a few plant materials.[11] Poly phenols in plants incorporate hydroxycoumarins, flavonols, flavones, and flavanones or hydroxycinnamate compounds, tannins, anthocyanins, stilbene, aurones and so forth polyphenols are very much recorded to have microbicidal action against a set of pathogenic organisms.[12]

Our outcomes suggest that gram-positive microbes are by and large generally delicate to the therapeutic plants when equated with gram-negative microorganisms. This was predictable with the previous examination on Phytoconstituents.[13] Gram-positive and negative microbes showed differential sensitivity to antibiotics may be because of their morphological disparities while using response surface methodology to test O. bracteatum plant extract.[14]

CONCLUSION

It very well might be deduced from this research that in-vitro investigation of the O. bracteatum plant showed a critical zone of inhibition against Escherichia coli, Bacillus subtilis, Staph aureus, klebsiella pneumonia, and Pseudomonas aeruginosa. These promising outcomes show that these phytoconstituents have extraordinary antibacterial potential and may be enslaved as natural antibiotics for the treatment of different infectious diseases. These bacterial strains and potentiated cocktail of various secondary metabolites against pathogenic and non-pathogenic bacteria prompt new decisions for the treatment of a few diseases. It could be an effective plant to synthesize a very high potency antimycobacterial drug by using response surface methodology with optimized values of conc, temperature, and pH.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.