In vitro Antioxidant activity, FTIR analysis and Total Phenolic content of Endophytic Fungus Penicillium sp. isolated from Boerhaavia diffusa L.
Saranya Shankar, Uma Anitha K.P.G, Mythili Sathiavelu*
School of Biosciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu.
*Corresponding Author E-mail: smythili@vit.ac.in
ABSTRACT:
Endophytes from medicinal plants are a significant source of various novel biologically active secondary metabolites. Endophytic fungi are a group of fungi that colonize the internal part of the plants without causing any immediate harmful effects. Our study mainly focuses on the isolation and identification of endophytic fungi from the medicinal plant Boerhaavia diffusa Linn belonging to the family Nyctaginaceae. Penicillium sp., a fungal endophyte isolated from Boerhaavia diffusa L. leaves for their bioactive potential. The fungal culture was further grown and extracted by various solvents such as dichloromethane (DCM), butanol and methanol. Different crude extracts were used to study their phytochemical composition, total phenolic content, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and fourier transform infrared spectroscopy (FTIR) analysis. The present study reveals that phytochemical screening of different fungal crude extracts indicates the presence of phenol, flavonoids, saponins, tannin, and terpenoids. Dichloromethane crude extract exhibited the highest total phenolic content of 14.4±0.018mg of GAE/g of extract and DPPH scavenging activity of 96% when compared to other solvent extracts. FTIR analysis revealed various functional groups of the compounds present in the extracts. The results indicated that the endophytic fungi Penicillium sp. isolated from medicinal plant Boerhaavia diffusa L. could be a significant source of bioactive compounds.
KEYWORDS: Endophytic fungi, Penicillium sp., Boerhaavia diffusa L., Phytochemicals, Total phenolic content, Antioxidant activity.
INTRODUCTION:
Various reports revealed that the use of fungal bioactive compounds in the field of pharmaceutical and agricultural industries6. According to recent studies, endophytic fungi have the potential to produce bioactive metabolites production like pestaloside, taxol, torreyanic acid and a few enzymes such as asparaginase, xylanase7. Modern clinical drugs (50%) are from the source of natural products. In the pharmaceutical industry natural product play a crucial role in drug development programs8. To enhance the quality and quantity of plant resources various methods, strategies, new modern techniques and plant associations have been put into use9. Any chemical substance called secondary metabolites in plants is responsible for medicinal properties. The exact function is produced when it enters the human body10. In the present study, the leaves of Boerhaavia diffusa L. an important medicinal plant was used for the isolation of endophytic fungi and to evaluate their potential bioactive metabolites.
The medicinal plant Boerhaavia diffusa L. belonging to the family Nyctaginaceae was found in India, Nigeria and many other countries11. In Tamil, the plant B.diffusa is known as Mukkarattai and Punarnava in Sanskrit. The leaves investigation gave 1.3g/100g of minerals, 27mg/100g of vitamin C and 61Kcal/100g of energy12. The whole plant as well as its plant extracts were used to cure different disorders in the medicine of traditional and folklore systems13. A well-known Dutch physician Hermann Boerhaave named the plant Boerhaavia diffusa L. in the18th century14. It is a rasayana herb in Ayurveda and it is reported to maintain various properties such as re-establishing youth, strengthening life, anti-aging, disease prevention, all of which increases the protection of the body against any ailments and this plant contains some of the secondary metabolites such as steroids, flavonoid glycosides, isoflavonoids (rotenoids) and phenolic and lignin glycosides15. It is also used in the night blindness, elephantiasis and corneal ulcers treatment16. The Boerhaavia diffusa plant contains a huge number of compounds such as punarnavoside17, lirodendrin18, ursolicacid19 and hypoxanthine 9-Larabinofuranoside20. Punarnava also contains β-Ecdysone, hexacosonoic, β-Sitosterol, α-2-sitosterol, arachidic acid, urosilic acid, tetracosanoic and triacontanol etc21.
MATERIALS AND METHODS:
Isolation and identification of endophytic fungi:
The fresh and healthy plant Boerhaavia diffusa L. was collected from Brahmapuram, Vellore, Tamil Nadu, India using polythene bags and brought to the laboratory and was used for the isolation of endophytic fungi. The whole plant sample was rinsed with tap water to remove dust and debris. The plant parts were surface-sterilized and cut into small segments using a sterile blade. The fragments were placed on potato dextrose agar medium and incubated for 1 week at 27ºC22. The fungal endophytes isolated were identified based on their aerial mycelium, colony or hyphal morphology, surface texture, pigmentation and characteristics of the spores using various standard manuals. Microscopic studies were done by lactophenol cotton blue staining23.
Fermentation and extraction of endophytic fungal metabolites:
Endophytic fungal isolates were inoculated in 100ml potato dextrose broth and incubated for 21 days at room temperature. To separate the filtrate and mycelia,the broth culture was filtered. Then the mycelia mat was allowed to soak in methanol and kept in a shaker for 2 days. Different solvents like dichloromethane (DCM) and butanol were used for the solvent extraction procedure and the crude extract was obtained. After 2 days, the methanolic extract of the mat was collected and the crude is extracted using a solvent extraction procedure24.
Phytochemical screening:
The different endophytic fungal crude extracts were subjected to phytochemical tests for the presence of phenolics, tannins, flavonoids, alkaloids, saponins, sterol and terpenoids using standard methodology25,26.
Determination of total phenol content:
Foli–Ciocalteau Photometric method was used to identify the total phenol content of crude extracts and gallic acid was used as a standard. About 1mg/ml of extracts were dissolved in methanol.Then 500µl of 50% Foli –Ciocalteau reagent and 1.5ml of 20% Na2Co3 were added to the mixture. The final volume was made to 5ml by adding distilled water, and the mixture was shaken well and incubated for 30mins in dark conditions. Absorbance was measured at 725nm. The concentration of polyphenols in samples was derived from a standard curve of gallic acid ranging from 10-50µg/ml. The TPC was obtained as mg of gallic acid equivalents in crude extracts by using the standard curve27.
1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity:
The free radical scavenging effect of fungal crude extracts was determined by the standard DPPH method28. Aliquots of fungal extracts at different concentration (50, 100, 150µg/ml) was added to 2ml of DPPH methanolic solution (0.1mM) and by adding dimethyl sulphoxide, the final volume was made to 3ml.The mixture was incubated for 10 mins at room temperature. After the incubation period, the absorbance of the reaction mixture was read at 517nm using a UV-Visible spectrophotometer against a blank. Ascorbic acid was used as a control. The radical scavenging activity on DPPH radicals was calculated using the following equation:
Scavenging ability (%) = [(ΔA517 of control – ΔA517 of sample) / ΔA517 of control] × 100.
Fourier Transform Infrared Spectroscopy (FTIR) Analysis:
The different endophytic fungal crude extracts were subjected to FTIR analysis to identify the functional groups present in the phytochemicals29.
RESULTS:
Isolation and identification of endophytic fungi:
The endophytic fungi were isolated and purified from the leaf segments of the plant Boerhaavia diffusa L. The purified culture (Code: BDL3) was mass multiplied for further studies. Fungi were identified through macroscopic and microscopic techniques which are shown in Figures 1A and 1B. The colony strain of BDL3 was identified as white to pale brown, spore-forming with irregular mycelium, thick edge growth on the PDA media. Microscopic characteristic of the fungal strain was observed by lactophenol cotton blue staining method to identify the conidiophores, hyphae. Conidia are round and hyaline. Based on the macroscopic and microscopic analysis, the isolated fungus was identified as Penicillium sp.
Figure 1: Macroscopic and microscopic colony morphologies of the endophytic fungal strain isolated from Boerhavia diffusa L
A. BDL3 (Penicillium sp.), B. BDL3 (Microscopic view)
Fermentation and extraction of endophytic fungal metabolites:
The Penicillium sp. was cultured in potato dextrose broth (PDB) and incubated for 21 days. During the incubation period, the color of the medium turns pale brown. Solvents with different polarities such as DCM, methanol and butanol were used to extract the metabolites from fungal broth and used for further analysis.
Phytochemical Screening:
The phytochemical that occurs naturally in the plants were consists of chemical compounds. Phytochemical screening exhibits the presence of secondary metabolites30. Phytochemical screening of various fungal crude extracts demonstrated the presence of flavonoids, tannins, saponins, phenol, and triterpenes. Among the test results, flavonoids were present in all the extracts. Phenols were observed in dichloromethane (DCM), butanol and methanol extracts. Tannins were observed in all the extracts except methanol extract. Saponins were present in dichloromethane and methanol extracts. Alkaloids and steroids were absent in all the extracts. Terpenoids were present in dichloromethane, butanol and methanol extracts. Table 1 shows the results for the qualitative phytochemical screening of fungal crude extracts.
Table 1: Phytochemical screening of Boerhaavia diffusa L.fungal crude extracts (+: present; -: absent)
|
Phytochemical test |
Dichloromethane |
Butanol |
Methanol |
|
Phenol |
+ |
+ |
+ |
|
Flavonoids |
+ |
+ |
+ |
|
Alkaloids |
- |
- |
- |
|
Saponins |
+ |
- |
+ |
|
Steroids |
- |
- |
- |
|
Tannins |
+ |
+ |
- |
|
Terpenoids |
+ |
+ |
+ |
Determination of total phenolic content:
The method Folin-Ciocalteu was used to determine the total phenolic content of various crude extracts of the plant Boerhaavia diffusa L. All three extracts were tested for total phenol content. Gallic acid was used as a standard compound and the total phenols were expressed as mg of GAE/g of extract. Among these three extracts, dichloromethane extract was demonstrating a higher concentration of total phenolic content (14.4±0.018mg of GAE/g of extract) than that of the other solvent extracts.The results are given in Table 2.
Table 2: Total phenolic content of Boerhaavia diffusa L. fungal crude extracts
|
SI. No |
Solvent used to prepare fungal crude extracts |
Total phenolic content (mg of GAE/g of extract) |
|
1 |
DCM |
14.4±0.018 |
|
2 |
Butanol |
14.1±0.008 |
|
3 |
Methanol |
14.2±0.010 |
DPPH radical scavenging activity:
Antioxidants may consider as a defense against free radical-mediated toxicity by securing the harm which produces by free radicals31. Antioxidant activity was measured for all the three crude extracts at various concentrations (50, 100, 150µg/ml). In the present study, dichloromethane crude extract showed the highest scavenging activity of 96% in 150µg/ml concentration than that of the methanol and butanol. Ascorbic acid was used as a standard showing 97.91% radical scavenging activity. The percentage of DPPH radical scavenging activity of different crude extracts and ascorbic acid is shown in Figure 2.
Figure 2: DPPH free radical scavenging activity of different crude extracts of Penicillium sp.
FTIR analysis:
The FTIR spectral analysis of the dichloromethane crude extract of Penicillium sp. showed the major peaks with an intensity of 3336.85cm-1 indicates the presence of an alcohol (O-H stretch) group. The second and third major peaks at 2943.37cm-1 and 2833.43cm-1 showed the presence of the alkanes group (C-H stretch) group. The fourth major peak at 1373.32cm-1 indicates the alkane group (C-H stretch) and the fifth peak at 1026.13cm-1 revealed the ethers (C-O stretch) as in figure 3A. The butanol crude extract of Penicillium sp. showed the peak at 3336.85cm-1 revealed the presence of an alcohol (O-H stretch). The peak at 2958.80cm-1, 2933.73cm-1 and 2872.01cm-1 refers to the presence of alkanes (C-H stretch). The peak at 1734.01cm-1 correspondsto the carboxylic acid group (C=O stretch). A peak at 1462.04cm-1 denotesthe alkane group (C-H stretch). A peak of 1070.49cm-1 showed the presence of esters (C-O stretch). A peak of 846.75cm-1 revealed the alkenes (C-H stretch). The results are shown in figure 3B.
FTIR spectroscopic analysis of methanol crude extract (Boerhaavia diffusa L.) as shown in figure 3C. The peaks at 3332.99cm-1, 2945.30cm-1 and 2833.43cm-1, 1708.93cm-1 , 1446.61cm-1, 1018.41cm-1 and 536.21cm-1 refers to the presence of functional group such as alcohol (O-H stretch), alkanes (C-H stretch), carboxylic acid (C=O stretch), alkene (C-H stretch), esters (C–O stretch) and halide (C-H stretch).
Figure 3: FTIR spectrum for different crude extracts (A- DCM, B- Butanol and C- methanol) from Penicillium sp.
DISCUSSION:
Endophytic fungi residing in medicinal plants are well-known sources of secondary metabolites32. In Arab countries, B.diffusa was used for the prevention of stress, abdominal pain, jaundice, inflammation etc33. In this study, endophytic fungi were isolated from Boerhaavia diffusa L. leaves and examined for the presence of various phytochemicals, total phenolic content and antioxidant activity.The qualitative phytochemical analysis of ethyl acetate extract of the endophytic fungus Penicillium sp. isolated from the stems of Phragmanthera capitata (Loranthaceae) indicates the presence of flavonoids, anthraquinones, tannins, phenols, steroids, coumarins and terpenoids34. These biologically active secondary metabolites are noted to have many biological and therapeutic applications35,36,37. Among different antioxidant assays, DPPH free radical scavenging assay was considered as a sensitive and most accurate screening method used for the antioxidant activity within the short time but the use of stable DPPH in the assay has taken the maximum attention owing to its convenience. DPPH is also not overwhelmed by metals or inhibition of enzyme38,39. The increase in radical scavenging ability may have been due to an increase in the concentration of total phenol compounds40. Ankush Garg et al., 2010 reported that the free hydroxyl group present in the aryl moiety is one of the factors in deciding the DPPH scavenging activity of the compound. DPPH scavenging activity was intensely decreased by free hydroxyl group protection41.
Natural antioxidants from plant sources are very powerful and possess free radical scavenging capacities leads to the improvement of new natural antioxidant drugs by the presence of phenolic compounds like phenolic acids, ascorbic acid, flavonoids and carotenoids42. It is familiar that free radicals play a vital role in various diseases. Free radicals contribute to more than a hundred human disorders including central nervous system injury, cancer, diabetes mellitus, arthritis, ischemia, gastritis, AIDS, reperfusion injury of many tissues and atherosclerosis43.In this present study, all fungal extracts had a broad range of scavenging abilities. Among that, dichloromethane crude extract showed the highest scavenging activity of 96% than that of methanol and butanol. Ascorbic acid was used as standard showing 97.91% antioxidant activity with a significant total phenolic concentration of 14.4±0.018mg of GAE/g of extract. Compounds like oxidation inhibitors or antioxidants prevent the oxidation and in common lengthen the life of the oxidizable matter44. Seemadhankhar et al., 2012 reported that the acetonic extract of the endophytic fungus Penicillium chrysogenum from Salvadora oleoides DECNE showed high antioxidant activity. The antioxidant capacity of the host plant is due to the presence of phenolic compounds45. Akanksha Bhardwajet al., 2015 isolated fungal endophytes Penicillium frequentans from spikes of the Pinus roxburghi. The ethyl acetate crude extract of this fungus showed maximum total phenolic content of 288.34 (mg/g) and also antibacterial activity of Penicillium frequentans showed 20.20 mm, 24.50 mm, 17 mm, 10 mm zone of inhibition against S. aureus, E. coli, S. Typhimuriu and C. Albicans respectively46. FTIR analysis of the different extracts of endophytic fungi Penicillium sp. showed the presence of various chemical constituents. FTIR spectral analysis of Aerva lanata leaf extract stated that the strong absorption band was noticed around 3373-3422 cm-1 may be due to the presence of bond N-H/C-H/O-H stretching of amines and amides compounds which is responsible for potential medicinal properties47.
CONCLUSION:
Plants provide shelter and nutrition to endophytes and in return, endophytic fungi increase the resistance to different stresses and also secrete functional products. The present study reveals that Penicillium sp., a fungal endophyte isolated from the medicinal plant Boerhaavia diffusa L. leaves showed potent antioxidant activity due to the presence of biologically active compounds. The bioactive natural compounds have a potential application as antioxidant agents in many pharmaceutical products. Hence, we conclude that the endophytic fungi isolated from the medicinal plant Boerhaavia diffusa L. play an important role in discovering and developing new drugs which will be more effective with no side effects.
ACKNOWLEDGEMENT:
The authors thank the Vellore Institute of Technology,Vellore for providing ‘VIT SEED GRANT’ for carrying out this research work.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 08.12.2020 Modified on 21.06.2021
Accepted on 09.10.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2022; 15(7):2951-2956.
DOI: 10.52711/0974-360X.2022.00492