Detection of Antioxidant and Antimicrobial Components from Crude Extracts of Hibiscus Arnottianus A. Gray flowers using HPTLC-Bioautographic Technique

Abstract

Hibiscus arnottianus belonging to family, malvaceae, is a herb used in traditional medicine for the treatment of inflammation and renal diseases. Methanolic extract of Hibiscus arnottianus flower was fractionated using different solvents of increasing polarity like petroleum ether, ethylacetate, methanol and water. The preliminary phytochemical screening showed the presence of carbohydrates, steroids, flavonoids, tannin. Methanolic extract of hibiscus arnottianus flower was screened for their antimicrobial and antioxidant activity. The test microorganisms includ Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa. It was concluded that Hibiscus arnottianus flowers extract was well effective against all these microorganisms. HPTLC-DPPH assays were used to identify the antioxidant activity of flower extract. This activity was estimated by HPTLC bioautographic method by using the mobile phase Chloroform:Ethyl Acetate:Methanol:Water (2.8:5.2:1.6:0.4 v/v/v/v). There was a successful chromatogram developed and detection of a band in the sample was observed. Later, the band showing antioxidant characteristic property was identified by DPPH Assay.

Introduction

I. INTRODUCTION

      Hibiscus arnottianus, commonly known as "White Hibiscus," is a species of flowering plant native to Hawaii. It belongs to the family Malvaceae. The plant is characterized by its large, showy white flowers with prominent yellow stamens. Hibiscus arnottianus is endemic to Hawaii. Hibiscus arnottianus A. grey, is an evergreen shrub or small tree generally 3-9 meters in height. The leaves are ovate 5 to 10 centimeters long and 4-7.5 centimeter broad, thin, green and slightly shiny on both surfaces, leafstalks are 2-4 centimeter, stipules are long pointed, shedding early. The faintly fragrant flowers have five white petals 8 to 11 cm long, 2.5 to 3.5 cm wide, with the calyx being 2.5 to 3 centimeters long. Anthers are arranged along the upper third of the white staminal column 10 to 14 cm long. This subspecies is distinguished from the other native Hawaiian members of its genus by its white petals and white staminal column [1]. Hibiscus has a long history of traditional use for various health purposes, hibiscus tea is often included in weight loss and management programs due to its diuretic properties and potential to inhibit the absorption of carbohydrate. Hibiscus flowers contain antioxidants, such as flavones like quercetin-3,5-diglucoside, quercetin-3,7-diglucoside, cyanidin-3,5-diglucoside and kaempeferol-3-xylosyl glucoside which can help combat oxidative stress and reduce inflammation in the body [2]. This antioxidant activity may contribute to various health benefits. The literatures indicates that hibiscus tea may help lower levels of LDL cholesterol and triglycerides, potentially reducing the risk of heart disease. Hibiscus extracts have been studied for their potential hepatoprotective effects, meaning they may help to protect the lever from damage caused by toxins or disease. Hibiscus arnottianus flowers are traditionally used in the treatment of various diseases for long time but no systematic phytochemical studies are reported for this plant. In recent years, thin layer chromatography-bioautography method has been widely and efficiently used for the screening and quantification of antioxidant compounds. Therefore, present investigation was planned to study the preliminary phytochemical detection and identification of antioxidant and antibacterial components in the flower of Hibiscus arnottianus using 2,2-dipheny-1-picryl-hydrazyl (DPPH)- HPTLC bioautographic method [3-7].

II. MATERIALS AND METHODS

A. Plant material

The flower of Hibiscus arnottianus were collected from Bandra, Mumbai district, Maharashtra in September 2019. The plant   was authenticated from blatter herbarium in St. Xaviers college, Mumbai, Maharashtra, India. The flower of the plant was shaded dried, powdered and passed through 70 mm mesh sieve and stored in an airtight container for further use.

B. Preparation of extract

The shade dried flower powder, about 1.0 g, was weighted and sonicated with 10 cm3 of methanol at room temperature for 1 hr. the extract was filtered through whattman no.1 filter paper then extract was stored at 4°C for further use.

C. Proximate analysis

Determination of extractive values, loss on drying, total ash value, water soluble ash value and acid-insoluble ash value were performed by using standard procedures [9]. Data were presented in table no.2.

D. Preliminary phytochemical screening

Preliminary phytochemical studies of various solvent extracts of H. arnottianus flowers were carried out by performing qualitative chemical test as per standard procedure [10]. The results are mentioned in table no.1

E. Estimation of phytoconstituents

Total alkaloid, total phenol, total flavonoid and total tannin contents were carried out by standard procedures [8]. Results were noted.

F. DPPH-HPTLC Bioautographic Assay

Chromatography was performed on 10 x 4 cm HPTLC plates coated with silica gel 60 F254 (Merk HX94930454). Samples (5µl) were applied as a band using Linomat 5 applicator (CAMAG, Switzerland). The plates were developed to a distance of 70 mm in an twin trough developing chamber (CAMAG) with chloroform-ethyl acetate-methanol-water (2.8:5.2:1.6:0.4, v/v/v/v) as developing solvent. The saturation time was 5 min. the plate was then dried for 3 min. After development, the HPTLC plate was immersed in 0.03% (m/v) DPPH methanolic solution. After derivatization, the plates were air-dried. Documentation of the chromatograms was carried out under 254 nm and white light using a CAMAG Scanner 3.

G. Radical Scavenging Assay

Radical scavenging activity of flower extract against DPPH was determined by using standard procedure [11]. DPPH react with antioxidant compound, which can donate hydrogen and reduce DPPH. The change in colour (from deep violet to light yellow) was measured at 517 nm on a UV visible light spectrophotometer. Radical scavenging activity was calculated by the following formula.

% Inhibition = [(A0 – A1)/ A0] x 100

A0 = Absorbance of blank sample

A1 = Absorbance of test extract sample

H. HPTLC-Antimicrobial Bioautographic Assay

The HPTLC-bioautographic method was employed for the determination of antibacterial activities of the methanolic flower extract of Hibiscus arnottianus. The extract was applied on 10 x 4 cm HPTLC silica gel 60 F254 (Merk HX94930454) plates using Linomat 5 applicator (CAMAG, Switzerland). The plates were developed in Twin trough chamber containing respective mobile phases. The developed plate was dried and to obtain well separated bands. The developed silica plates were placed into sterile petri plates. The culture of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa were grown at 37°C at 24 hours. was mixed with Muller & Hinton agar medium and poured onto the sterile petri plate containing developed silica gel plate such that it will form a layer over the TLC plates. The plate was incubated at 370C for over 24 hours. After incubation the plates were observed by pouring INT (2-(-4-iodophenyl-)-3-(-4-nitrophenyl)-5-phenyl-2H-tetrazolium) dye in sterile conditions and further incubation of plates for 20 mins. For location and visualization of antibacterial substances, tetrazolium salts were usually used, which are converted by the dehydrogenases of living microorganisms to intensely colored formazan. After the treatment with INT dye the clear inhibition zones of active components will appear on the plate in comparison to the colored background.

III. RESULT AND DISCUSSION

A. Preliminary phytochemical screening

Different solvents may yield extracts with varying compositions and properties hence various solvents like ethyl acetate, petroleum ether, methanol and water were used to prepare extracts of H. arnottianus. In present study the extractive values of petroleum ether, ethyl acetate, methanol and aqueous were found to be 33%, 32.4%, 42.8% and 24% w/w respectively. Preliminary phytochemical screening of various extracts of H. arnottianus flower reveals the presence of alkaloids, carbohydrates, amino acids, flavonoids, saponin, tannin. Amongst the various solvents methanol extract showed the presence of most of the secondary metabolites (Table no.1). The screening of phytoconstituents of H. arnottianus flower extract was performed on silica gel 60 F254 HPTLC plates. The Rf values of separated components were 0.014, 0.082, 0.321, 0.479, 0.0719, 0.761, 0.901, 0968 and 0.990 (Fig. 1). Possible components present in methanolic extracts were seen by TLC followed with exposure to iodine, dragondroff reagent, p-anisaldehyde-sulfuric acid and 10% ferric chloride (Fig.3).

IV. AKNOWLEDEMENT

I sincerely thank to Principal and to the Head, Department of Chemistry Government of Maharashtra, Ismail Yusuf College of Arts, Science and Commerce, Jogeshwari (East), Mumbai 400 060, India for providing me the research laboratory facilities and constant encouragement throughout my work.

Conclusion

The flowers of H. arnottianus were proven to have both antioxidant and antimicrobial activity and it has been confirmed through HPTLC guided identification. These findings will be useful towards further isolation of polyphenols and flavonoids from methanolic extract of H. arnottianus flower.

References

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Copyright

Copyright © 2024 Sangeeta S. Zinje, Dr. Ramesh S. Yamgar. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.