Analysis of Phytochemical content and Antioxidant activity of Mycelia sterilia isolated from Boerhaavia diffusa

 

Sugashini Settu¹, K.P.G Uma Anitha², Sathiavelu Arunachalam¹

¹VIT School of Agricultural Innovations and Advanced Learning, Vellore Institute of Technology,

Vellore-14, Tamil Nadu, India.

²School of Bioscience and Technology, Vellore Institute of Technology, Vellore-14, Tamil Nadu, India.

 

ABSTRACT:

Fungal endophytes are living asymptomatically inside the tissue of higher plants are of rising interest as they are the promising resource of bioactive agents. The current study shows that mycelia sterilia isolated from Boerhaavia diffusa would be a potent phytochemical and antioxidant source. The preliminary phytochemical analysis revealed the presence of different secondary metabolites and all the fungal extracts exhibited antioxidant activity. There was a positive association between the amount of phenol content and the antioxidant content of the fungal extracts. Further research needs to be done on isolating the active compound accountable for this free radical scavenging activity and biophysical evaluation will be carried out in the future.

 

 

 

INTRODUCTION: 

Fungal endophytes are an interesting class of microorganisms interdependent with plant tissues and widely considered as plant defensive mutualisms. Endophytic fungi spend the wholly or partially throughout their life-cycle by colonizing within the healthy part of the host, without causing any noticeable manifestation of the disease. Nowadays researchers are focusing on isolating novel compounds from fungal endophytes with pharmacological significance as they are undiscovered to a larger extent.¹ Most of the fungi isolated from medicinal plants are found to have pharmaceutical applications and it is expected that above one million endophytic fungi exist in the world.² Dreyfuss and Chapela (1994) and Hawksworth and Rossman (1997) estimated that on this earth there were about 1.5 million fungal species in which 70,000- 100,000 or 5% are currently known.^3,4

 

Tertiary butylhydroquinone, butylated hydroxytoluene and butylated hydroxyanisole are some of the artificial antioxidant compounds generally used in processed foodstuff. The use of these artificial antioxidants leads to potent health hazards and noxiousness, particularly potentially carcinogenic effects. Hence, it is important to find out a new source of antioxidants from the natural origin to replace synthetic antioxidants and also to permit them in foods and pharmaceutical preparation which are safe and inexpensive.⁵

 

Natural products can serve as a source for the biologically active compounds obtained from plants, microbes or animals which has the potentiality in medicine development.⁶ Antioxidant activity exhibited by the compounds present in the endophytic fungi acts as a significant role against damages caused by reactive oxygen species (ROSs) and oxygen-derived free radicals, which gives away to a diversity of therapeutic effects like DNA damages, cellular degeneration and carcinogenesis.^7,3 Several biological reactions in our body or from exogenous factors that generate free radicals are the reactive oxygen species. Several types of degenerative diseases such as inflammatory diseases raised blood pressure, diabetes, ischemia, arteriosclerotic vascular disease, heart disease, and degenerative nerve diseases causes due to the damage of membrane fatty acids, polypeptides, Deoxyribonucleic acid and enzymes by free radicals which causes oxidative stress.^3,8

 

Approximately, on this earth, there are nearly 300,000 plant species and all of these plants serve as a host to endophytic microbes.^9,3 Boerhaavia diffusa L. belonging to the Nyctaginaceae family is a perennial creeping herb commonly known as 'Punarnava' in the Indian system of medicine as it has high medicinal value.^10,11 In Ayurvedic medicine, Boerhaavia diffusa has gained considerable attention based on widespread use for treating icterus, hepatic disease, dropsy, hypouresis, spelled anemia, retinal disorders etc.^12,13 The leaves are utilized for treating icterus, hepatic disease, low blood pressure, nyctalopia and also act as the anti-venom.¹² In present study, in vitro antioxidant, total phenol content and preliminary phytochemical studies of mycelia sterilia isolated from Boerhaavia diffusa were investigated.

 

MATERIALS AND METHODS:

Sample collection:

Plant materials were collected from fully matured Boerhaavia diffusa from Brahmapuram, Vellore, Tamilnadu, India. With the help of a sterile scalpel, the disease-free stem part was excised and collected in a sterile bag for further use.¹⁴

 

Isolation and extraction of endophytes:

The collected stem part was washed thoroughly in sterile water and cut into small pieces in sterile conditions. The surface sterilization was done by soaking the stem part in 1% sodium hypochlorite for 2 mins and 70% ethanol for 30 sec and rinsed in demineralized water. After drying, the small pieces of stem part were placed in potato dextrose agar medium supplemented with an antibiotic (Streptomycin 3mg/100ml) and the plates were incubated at room temperature until the fungal growth was initiated. After incubation, the actively growing pure cultures agar block was placed in Erlenmeyer flasks containing PDB (Potato Dextrose Broth) for mass cultivation of endophytic fungi. After the incubation period, the fungal cultures were filtered under the sterile condition to remove the mycelia mat and the fungal broth was taken in a separating funnel along with the equal volume of the respective solvents like DCM and butanol and methanol for fungal mat extraction. Then the content was shaken vigorously and kept aside, the cell mass gets isolated and the solvent was collected in a beaker and allowed to dry. The obtained fungal crude extracts were used for further studies.⁶

 

Identification of fungi:

Endophytic fungi were identified by studying their cultural characteristics, the formation of spore and mycelium. Slides were prepared by tease mount method using Lactophenol cotton blue reagent and observed under 40×, 100×.¹⁵

 

Preliminary qualitative phytochemical analysis:

The qualitative phytochemical screening was carried out to examine the presence of secondary metabolites present in the fungal crude extracts using standard procedure.^16,20

 

Total phenol content:

The total phenol content of the fungal crude extract was done using a Folin-Ciocalteau reagent-based assay. 100 µL of the fungal crude extract (1 mg/ml) and 500 µL of 1N Folin- Ciocalteau reagent was taken in a test tube. To this mixture, 1.5 ml of 20% sodium carbonate was taken and distilled water was added to make it into 5ml of the final volume. The content was kept for incubation at room temperature for 30 min and absorbance was measured at 765 nm. The calibration curve was used to calculate the total phenol content and it is expressed in mg of Gallic acid equivalent per g dry weight.¹⁷

 

Free radical scavenging assay:

The changes in optical density of the free radicals were used to study antioxidant activity. 1 mg/mL concentration of the fungal crude extract was used. DPPH generates violet color in an alcohol solution that is stable and nitrogen-centered free radical. When a substrate is added to DPPH solution (prepared in 95% methanol) it will be reduced to a yellow color product, diphenylpicryl hydrazine by the donation of the hydrogen atom. The fungal crude extract (1 mg/ml) was diluted in methanol and at different concentrations, it was added in 2ml of DPPH solution. The content was incubated at room temperature for 30 min in darkness and absorbance was measured at 517 nm. The DPPH free radical scavenging activity was calculated using the following equation¹⁸

 

                                            Control absorbance-sample absorbance

Inhibition of DPPH radical (%) = –––––––––––––––––––––––– × 100

                                                               Control absorbance

 

RESULT:

Isolation and fungi identification:

Endophytic fungi were isolated from the healthy stem of Boerhaavia diffusa and its identification was done based on colony morphology and microscopic examinations. The results were shown in Table 1 and Figure 1a and 1b.

 

Table 1: colony morphology of isolated fungus

 

Figure 1a: Single morphology colony of mycelia sterilia

 

Figure 1b: Microscopic observation of mycelia sterilia

 

Qualitative phytochemical analysis:

Table 2: Preliminary qualitative phytochemical analysis of various fungal crude extracts of mycelia sterilia.

+++: Highly present, ++: Moderately present, +: Low,-: Absent

 

The preliminary phytochemical screening of mycelia sterilia of various extracts showed the presence of phytoconstituents. In the present study butanol, methanol mat and DCM extracts showed the presence of phenol, flavonoids, tannin, anthraquinones and alkaloids whereas saponins were present only in butanol and terpenoids were present only in methanol mat extract as depicted in Table 2. The presence of different secondary metabolites indicates that fungal extracts have a wide range of pharmacological applications.

 

Total phenol content:

Table 3: Total phenol content of fungal crude extracts of butanol, methanol mat and DCM.

 

 

 

Based on the Folin-Ciocalteu assay the total phenol content of the fungal extracts was determined. This method is based on the redox potential of sample extracts which is reported as total phenol content.¹⁹ The result revealed that methanolic extract showed maximum phenol content as compared to butanol and DCM extracts which are about 10.38mg GAE/g for butanol extract, 17.36mg GAE/g for methanol extract and 9.26mg GAE/g for DCM extract. (Table 3) Higher content of phenol in the fungal extract is responsible for biological activity; as a result of this, the fungal extracts are anticipated to show good results in antioxidant activity. The results were acquired from a calibration curve (y=0.027x+0.026) and stated in gallic acid equivalents (GAE) per gram dry weight of extracts.

 

Antioxidant activity:

Radical scavenging effects of the fungal extracts were determined by DPPH assay with absorption at 570nm. Absorption decreases as the anti-oxidant give proton to these free radicals. At various concentrations, the fungal extracts were examined against free radical and as the absorbance value decreases, it specifies the level of radical scavenging effect. The phenol compound is considered to have an important role in the stabilization of lipid oxidation that is in interrelation with free radical scavenging assay. The scavenging activity of the crude extract was assessed along with ascorbic acid as the standard. All three fungal extracts have shown radical scavenging effects up to different extents ranging from 12% to 59% (Fig 2). Among these extracts, the methanol mat has shown high antioxidant capacity compared to the other two extracts.

 

Fig 2: Free radical scavenging assay of different extracts of mycelia sterilia

 

CONCLUSION:

The current study concludes that the endophytic fungi isolated from this plant have shown phytochemical presence and antioxidant activity because of the presence of bioactive compounds. These active substances have possibilities of implementing in several medicinal products. Thus, a natural antioxidant and phenol compound in mycelia sterilia having the ability to be employed in medicine and also in food systems to maintain the quality of food. Still, furthermore extended clinical biochemistry and molecular investigations are to be done to find out the active substances responsible for numerous biological activities by the fungal extracts. Further research will focus on the structural interpretation of biologically active compounds and their application in various sectors. Though several studies have been done on a different part of this plant, it has not yet been formulated as a drug by pharmaceutical companies. Therefore a brief study should be done on this plant for documentation of indigenous knowledge for further use.

 

ACKNOWLEDGMENT:

The authors thank the Vellore Institute of Technology, Vellore for providing lab facilities to carry out this research.

 

CONFLICT OF INTEREST:

The author declares no conflict of interest.

 

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Accepted on 22.08.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1109-1112.