Int J Pharm Pharm Sci, Vol 12, Issue 2, 15-19Original Article
ASSESSING COSMECEUTICALS PROPERTIES OF SOME MACROFUNGI FOR IMPROVED SKINCARE
AKASH SARASWAT1, PURVI MATHUR1, DOYELI SANYAL1*
1TERI-Deakin Nano Biotechnology Centre, Sustainable Agriculture Division, The Energy and Resources Institute, New Delhi 110003, India
Email: doyeli.sanyal@teri.res.in
Received: 14 Oct 2019, Revised and Accepted: 10 Dec 2019
ABSTRAC
Objective: This study has investigated cosmeceutical properties namely, antibacterial, sun protection factor and total phenolics contents of some selected macrofungi. The studies were conducted on five reference cultures collected from Indian type culture collection center (IMTEC, Chandigarh, India) and five isolates collected from TERI-Deakin Nano Biotechnology Centre facility at TERI Gram, Gurugram, India.
Methods: The cosmeceutical properties of the crude extracts from selected macrofungi were analyzed using standard bioassay techniques. Antibacterial activity was analyzed against Staphylococcus epidermidis, Escherichia coli, Micrococcus luteus, Bacillus megaterium, Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii using Agar well diffusion method. The sun protection factor was estimated and calculated using the Mansur equation. Free radical scavenging activity using DPPH was performed to assess the antioxidant activity of the extracts.
Results: Ethyl acetate extracts of the broth from P. florida and TERI-G1 cultures showed a broad-spectrum antibacterial activity against S. epidermidis, E. coli, M. luteus and B. megaterium. Ethyl acetate extracts of the broth from TERI-G3 showed the highest SPF activity of 34.02 at 200 µg. ml-1 concentration. Ethyl acetate extract of the broth from F. velutipes, P. florida, P. ostreatus, and TERI-G1 showed comparable antioxidant activity of 66.86%, 79.51%, 82.02%, and 69.58% respectively when compared to ascorbic acid (85.83%) and quercetin (83.09%) taken as positive control in the study and their total phenolic contents were found to be 6.93, 43.68, 20.88 and 13.77 Gallic acid equivalent (GAE) per gram. The minimal inhibitory concentration of F. velutipes, P. florida, P. ostreatus, and TERI-G1 was found to be 3552.89 µg. ml-1, 1250 µg. ml-1, 2418.9 µg. ml-1 and 3219 µg. ml-1 respectively.
Conclusion: The work is in progress to identify and characterize TERI-G1and TERI G3 cultures. Further studies on the anti-inflammatory, anti-tyrosinase, elastase inhibition properties of the cultures will be assessed to identify potential cosmeceutical active ingredients with promising applications in cosmeceutical products.
Keywords: Chemical analysis, Microbiology, Spectroscopy, Macrofungi, bioassay studies
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2020v12i2.36074. Journal homepage: https://innovareacademics.in/journals/index.php/ijpps
INTRODUCTION
Presently, there is increasing consumer demand for cosmetics that incorporate organic and natural ingredients as a result of which the cosmeceutical division of the personal care industry is rapidly thriving [1]. Cosmeceuticals are products that comprise creams, lotions, and ointments and have both cosmetic and therapeutic effects due to the presence of bioactive ingredients which enhances skin health [2]. Recently macrofungi have attracted a lot of attention as an interesting source of unique bioactive. These contain substances like carotenoids, resveratrol, omega fatty acids, ceramides, etc [3] which makes them a potent candidate for use in the cosmetic industry. They are easy to grow and maintain and their extracted secondary metabolites can be used as enhanced analogs with minimum side-effects. These macrofungi have been well documented as to the source of polyphenolic, phenolics, polysaccharides, terpenoids, selenium, vitamins and volatile organic compounds. Many of these compounds have been associated with excellent antioxidant, anti-inflammatory, anti-bacterial, anti-viral, anti-aging, anti-wrinkle, skin renewal, skin whitening and moisturizing effects [4-6]. Macrofungi like Reishi (Ganoderma lucidum), Shiitake (Lentinula edodes), Maitake (Grifola frondosa), cauliflower mushroom (Sparassis latifolia), Fu Ling (Wolfiporia extensa), etc are popularly used in facial creams and serums in East Asian countries including Korea, Japan and China [7-9]. Tinder fungus (Fomes fomentarius), oyster mushroom (Pleurotus ostreatus), elm oyster mushroom (Hypsizygus ulmarius), Portobello mushroom (Agaricus bisporus) prevail more in the Western region [10-12].
These macrofungi also show interesting anti-microbial activities. Lentinula edodes is very efficient against both gram-positive and gram-negative bacteria. Ganoderma, Lepista and Boletus species also show similar properties [13]. These macrofungi contain phenolic compounds that safeguard against viruses, bacteria, insects, and UV light [14].
Many well-known cosmetic brands nowadays use extracts of macrofungi in their products. For instance, the popular product range Aveeno Positively Ageless by Johnson and Johnson contains extract of Ganoderma lucidum and Lentinula edodes. Another brand, Vitamega cosmetics uses the extract of Agaricus subrufescens in their shampoo and moisturizing cream range. Earthherbs L. L. C. uses the extract of Siberian Chaga mushroom (Inonotus obliquus) in their antioxidant rejuvenating cream [15].
The present work is focused on exploring cosmeceutical properties namely; antibacterial, SPF, antioxidant activities and phenolics content of few selected identified (Pleurotus species, Flammulina species), and uncharacterized mushrooms collected from the TDNBC (TERI-Deakin Nano Biotechnology Centre) facility at TERI Gram, Gurugram, India.
MATERIALS AND METHODS
Reagents
All the reagents and chemicals used in the experiments were of analytical grade. Culture media (Nutrient Agar, Nutrient Broth, Potato Dextrose broth, Potato Dextrose Agar, Mueller Hinton agar no. 2) were collected from HiMedia Laboratories. Solvents (methanol, ethyl acetate, ethanol) and chemicals (Ascorbic acid, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Quercetin, Folin-Ciocalteu reagent, gallic acid) were purchased from Sigma Chemical Co., USA. Milli-Q water used in the studies has been obtained from Milli-Q® Integral by Merck.
Apparatus
1000 ml Erlenmeyer flask, Buchner funnel assembly, rotary vacuum evaporator, UV visible spectrophotometer (SHIMADZU, UV 2450) were used in the study.
Microbial culture
Mushroom cultures namely, Flammulina velutipes (MTCC-543), Pleurotus florida (MTCC-9194), Pleurotus ostreatus (MTCC-1801, 1802) and Pleurotus sajor caju (MTCC-141) and bacterial cultures for antimicrobial studies namely, Staphylococcus epidermidis (MTCC-3615), Escherichia coli (MTCC-443), Micrococcus luteus (MTCC-106), Bacillus megaterium (MTCC-453), Pseudomonas aeruginosa (MTCC-424), Staphylococcus aureus (MTCC-1144) and Acinetobacter baumannii (MTCC-1425) were collected from Microbial Type Culture Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India. The fungal cultures were maintained in Potato Dextrose Agar plates at 28 °C. The bacterial cultures were maintained in Nutrient Agar plates at 28 °C except for E. coli which was maintained at 37 °C.
Preparation of extracts
Three 6 mm discs of the mushroom cultures from their respective agar plates were used to inoculate 1000 ml Erlenmeyer flask containing 300 ml of Potato Dextrose broth in triplicate and incubated under shaking at 28 °C at 180 rpm for 21 d. After the completion of the incubation period, the mycelia were separated from the broth aseptically using a sieve (BSS 500 micron) and air-dried. The mycelia were extracted with methanol (50 ml) in a shaker for 24 h at 200 rpm. The extracts were filtered through Buckner funnel assembly and were washed twice with 25 ml methanol each. The extracts were collected and concentrated using a rotary vacuum evaporator at 45 °C. The broth samples were extracted with 100 ml ethyl acetate using liquid-liquid partitioning in a 2 liter separating funnel. The samples were further extracted two more times with 50 ml of ethyl acetate each. All the extracts were collected and concentrated using a rotary vacuum evaporator set at 45 °C. The concentrated crude extracts were then used for bioassay studies.
Table 1: Macrofungi and corresponding codes for the extracts
| Microorganism | MTCC Number | Solvent | Extract | Fungal Code |
| Flammulina velutipes (FV) | 543 | Ethyl acetate | Broth | V1B |
| Flammulina velutipes (FV) | Methanol | Mycelia | V1M | |
| Pleurotus florida (PF) | 9194 | Ethyl acetate | Broth | F1B |
| Pleurotus florida (PF) | Methanol | Mycelia | F1M | |
| Pleurotus ostreatus (PO-S1) | 1801 | Ethyl acetate | Broth | O1B |
| Pleurotus ostreatus (PO-S1) | Methanol | Mycelia | O1M | |
| Pleurotus ostreatus (PO-S2) | 1802 | Ethyl acetate | Broth | O2B |
| Pleurotus ostreatus (PO-S2) | Methanol | Mycelia | O2M | |
| Pleurotus sajor caju (PSC) | 141 | Ethyl acetate | Broth | C1B |
| Pleurotus sajor caju (PSC) | Methanol | Mycelia | C1M | |
| TERI-G1 (TG-1) | Ethyl acetate | Broth | T1B | |
| TERI-G1 (TG-1) | Methanol | Mycelia | T1M | |
| TERI-G2 (TG-2) | Ethyl acetate | Broth | T2B | |
| TERI-G2 (TG-2) | Methanol | Mycelia | T2M | |
| TERI-G3 (TG-3) | Ethyl acetate | Broth | T3B | |
| TERI-G3 (TG-3) | Methanol | Mycelia | T3M | |
| TERI-G4 (TG-4) | Ethyl acetate | Broth | T4B | |
| TERI-G4 (TG-4) | Methanol | Mycelia | T4M | |
| TERI-G5 (TG-5) | Ethyl acetate | Broth | T5B |
In vitro antimicrobial study
Agar well-diffusion method described by Perez et al. [16] was used to determine the antimicrobial activity. Mueller Hinton agar no. 2 was prepared and cooled to 40-45 °C and the bacterial inoculum (1.5×108 CFU. ml-1, 0.5 McFarland) prepared above was then added aseptically to the molten agar and poured into sterile petri dishes to give a solid plate. Wells (6 mm diameter) were made in each of these plates using a sterile cork borer. The wells were filled with 50 μl of test extract (5 µg. ml-1). The plates were first incubated at 4 °C for 15 min and then at 37 °C for 18 h under aerobic conditions. The antimicrobial activity of the extract was determined by calculating the zone of inhibition. The diameters of zones of inhibition (ZOI) produced by the test were recorded and compared with those produced by positive control (Tetracycline at 5 µg. ml-1, Sigma-Aldrich). For negative control, pure solvent (autoclaved Milli-Q water) was used in place of the extract. The experiment was performed two times in triplicates to minimize error and to check the reproducibility of results. The average values were recorded. The % antimicrobial activity was calculated with equation 1.
% Inhibition = 100-{ *100}Equation 1
Sun protection factor
SPF determination was performed as described in Mansur et al. 1986 [17]. Absorbance readings of the crude extracts (200 µg. ml-1 concentration) dissolved in ethanol were taken in the wavelength range of 290 to 320 nm at 5 nm interval. A dilution to 100 µg. ml-1 was done with crude extracts showing exceeding spectroscopic readings. Mansur equation as mentioned in equation 2 was used to determine the SPF values of the crude extracts.
SPF= CF… Equation 2
where CF is 10 (correction factor), EE (λ) is erythemogenic effect of radiation at wavelength λ, I (λ) is the intensity of solar light at wavelength λ, and abs (λ) is the absorbance of wavelength λ by a solution of the preparation. The obtained absorbance values were multiplied by the EE (λ) values; their summation was taken and multiplied by the correction factor 10.
Antioxidant assay
Antioxidant activity of the crude extracts was determined as described by Clarke et al. 2013 [18] with slight modification. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity was assessed with 20 ul of crude extract of 5000 µg. ml-1 concentration dissolved in methanol, mixed with 0.1 mmol of DPPH followed by incubation in the dark for 30 min. Absorption was taken at 517 nm with the help of a UV-Visible Spectrophotometer. Appropriate blank (methanol) and positive control (Ascorbic acid and Quercetin) were used. The inhibition of the DPPH free radical in percentage (I %) was calculated following equation 3.
% Inhibition = { * 100}…. Equation 3
Where Acontrol is the absorbance of the control reaction (full reaction, without the tested extract or Quercetin) and Asample was the absorbance in the presence of the sample. A fresh solution of DPPH was prepared before each experiment. Decreased absorbance of the reaction mixture indicates stronger DPPH free radical-scavenging activity. The minimum inhibitory concentration (MIC) values of the extracts showing good antioxidant properties were evaluated using the two-fold serial dilution method [19]. The samples were dissolved in methanol to obtain 5 mg. ml-1 stock solutions. With the help of serial dilution concentrations 500 µg. ml-1, 250 µg. ml-1 and 125 µg. ml-1 were achieved and readings were taken at 517 nm wavelength.
Total phenolics content
The total phenolic content (TPC) was determined calorimetrically using a Folin-Ciocalteu 96-well microplate assay as described by Zhang et al. (2006) [20]. The total phenolic content was calculated from the linear equation of a standard calibration curve prepared with gallic acid and expressed as Gallic Acid Equivalent (GAE) per gram of extract.
Statistical analysis
All data are presented as mean±standard error of the means (SEM). Data for antimicrobial studies were analyzed using a 2way analysis of variance (ANOVA) using GraphPad Prism version 8.0.2. Differences were accepted as statistically significant when p-value<0.05.
RESULTS AND DISCUSSION
Antimicrobial studies
Continued search for antibacterial activity assessment of new potential active ingredients is crucial as gram-positive (S. epidermidis, M. luteus, B. megaterium, S. aureus) and gram-negative (E. coli, P. aeruginosa, A. baumannii) bacteria considered for this study are associated with systemic infections namely, urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and has been reported to have developed resistance against several commercial antibiotics [21]. As enumerated in table 2, extracts displayed different levels of antibacterial activities against the tested pathogenic bacteria. Broth culture of P. florida and TERI-G1 showed a broad-spectrum antibacterial activity against S. epidermidis, E. coli, M. luteus, B. megaterium cultures, with P. florida showing specific activity against P. aeruginosa and TERI-G1 against A. baumannii and S. aureus. Antibacterial activity as observed in remaining extracts was negligible or less than 50% relative to tetracycline, the positive control taken in the study. The antimicrobial activity of fungal extracts against different bacteria was found to be significant during statistical analysis (p-value<0.05).
Table 2: Antibacterial activities of the crude fungal extracts
| Fungal code | Antibacterial activity (%)* | ||||||
| S. epidermidis | E. coli | M. luteus | B. megaterium | P. aeruginosa | A. baumannii | S. aureus | |
| V1B | 0.00±0.00 | 0.00±0.00 | 28.41±1.14 | 40.00±3.64 | 41.18±1.96 | 0.00±0.00 | 0.00±0.00 |
| V1M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 39.22±3.92 | 0.00±0.00 | 0.00±0.00 |
| F1B | 21.51±0.00 | 26.67±0.00 | 22.73±0.0 | 45.45±1.82 | 43.14±3.92 | 0.00±0.00 | 0.00±0.00 |
| F1M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 43.14±3.92 | 0.00±0.00 | 0.00±0.00 |
| O1B | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 34.55±1.82 | 43.14±3.92 | 0.00±0.00 | 0.00±0.00 |
| O1M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 60.00±5.45 | 39.22±0.00 | 0.00±0.00 | 0.00±0.00 |
| O2B | 0.00±0.00 | 0.00±0.00 | 22.73±0.00 | 36.36±0.00 | 35.29±3.92 | 0.00±0.00 | 0.00±0.00 |
| O2M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 32.73±3.64 | 41.18±1.96 | 0.00±0.00 | 0.00±0.00 |
| C1B | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 49.09±5.45 | 47.06±3.92 | 0.00±0.00 | 0.00±0.00 |
| C1M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 39.22±0.00 | 0.00±0.00 | 0.00±0.00 |
| T1B | 50±0.00 | 51.28±0.00 | 44.44±0.00 | 56.86±9.8 | 0.00±0.00 | 33.85±3.08 | 64.79±5.63 |
| T1M | 21.43±0.00 | 0.00±0.00 | 25.56±1.11 | 56.86±9.8 | 0.00±0.00 | 0.00±0.00 | 50.7±0.00 |
| T2B | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| T2M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| T3B | 0.00±0.00 | 51.28±0.00 | 27.78±1.11 | 35.29±3.92 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| T3M | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| T4B | 0.00±0.00 | 69.23±7.69 | 28.89±2.22 | 35.29±3.92 | 0.00±0.00 | 0.00±0.00 | 26.76±1.41 |
| T4M | 0.00±0.00 | 51.28±0.00 | 27.78±1.11 | 35.29±3.92 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| T5B | 0.00±0.00 | 61.54±0.00 | 35.56±2.22 | 35.29±3.92 | 0.00±0.00 | 0.00±0.00 | 25.35±2.82 |
The % antimicrobial activity was calculated using equation 1 and expressed as a mean±standard deviation to the mean. (n=3) *(Data was found to be significant, p<0.05).
Fig. 1: Sun protection factor of the fungal extracts, SPF values were calculated using Mansur equation (equation 2) and expressed in fig. 1. (n=1)
Sun protection factor
Calculated values of SPF of the crude extracts isolated from the selected macrofungi were found to be in the range of 3.67-34.02. Dutra et al. (2004) [22] analyzed several commercial products containing a combination of active ingredients viz., benzophenone-3, octyl methoxycinnamate and octyl salicylate (in varying proportions) for their SPF values and compared them with the declared values in their product label and found to be in the range of 15-20. In the present study, crude extracts of TERI-G3 (T3B) showed promising SPF activity of 34.02 at 200 µg. ml-1. P. florida (F1B), TERI-G1 (T1B), TERI-G2 (T2B), TERI-G4 (T4B) and TERI-G5 (T5B) also showed comparable SPF of 19.63, 25.50, 22.44, 22.31 and 22.87 respectively.
Antioxidant activity
Ascorbic acid and Quercetin were used as a positive control in this study which showed as 85.83% and 83.09% DPPH free radical scavenging activity respectively. Broth of P. florida, F. velutipes, P. ostreatus, TERI-G1 showed comparable scavenging activity as positive control with values of 79.51, 66.8, 82.02 and 69.58% respectively. Broth of P. florida, F. velutipes, P. ostreatus and TERI-G1-B showed MIC at 1250, 3552.89, 2418.9 and 3219 µg. ml-1. Mycelia of P. florida showed MIC at 2903.76 µg. ml-1.
Fig. 2: DPPH free radical scavenging activity of the fungal extracts, the % antioxidant activity was calculated using equation 3 and expressed as mean±standard error to the mean (n=2)
Fig. 3: Minimum inhibitory concentration of the selected fungal extracts, the MIC values are expressed as mean±standard error to the mean (n=2)
Total phenolic content
Phenolic compounds have been well documented for their contribution to antioxidant activity. These compounds are good electron donors, which directly contribute to antioxidant action [23]. Some phenolic compounds also stimulate the synthesis of endogenous antioxidant molecules in the cell [24]. Therefore, knowledge of total phenolic content is necessary for our study. Broth of P. florida, F. velutipes, P. ostreatus and TERI-G1-B showed total phenolics content of 43.68, 6.93, 20.88 and 13.77 gallic acid equivalent (GAE) per gram. Mycelia of P. florida showed a total phenolics content of 5.96 gallic acid equivalent (GAE) per gram.
Table 3: Total phenolic contents of the selected crude fungal extracts
| Extract code | Total phenolics content (Gallic acid equivalent (GAE) per gram) |
| T1B | 13.77± |
| F1B | 43.68± |
| F1M | 05.96± |
| V1B | 06.93± |
| O2B | 20.88± |
Total phenolic content was plotted from a standard curve of Gallic acid equivalent. (n=2)
CONCLUSION
The most promising activity was shown by TERI G1, TERI G3, and P. florida extracts. The work is in progress to identify and characterize TERI-G1and TERI G3 cultures. Further studies on the anti-inflammatory, anti-tyrosinase, elastase inhibition properties of the cultures will be assessed to identify potential cosmeceutical active ingredients with promising applications in cosmeceutical products.
ACKNOWLEDGMENT
We thank The Director, Sustainable Agriculture Division, TD-NBC, TERI, India for providing necessary facilities to carry out research work.
AUTHORS CONTRIBUTIONS
All authors contributed extensively to the work presented in this paper.
CONFLICT OF INTERESTS
The authors declared no conflict of interest.
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