Total Phenolic and Flavonoid Content and Antibacterial Properties of Polygonatum orientale Desf and Tilia dasystyla

AUTHORS

Parichehr Hanachi ORCID 1 , * , Roshanak Zarringhalami ORCID 1 , Ertugrul Kaya ORCID 2

1 Department of Biotechnology, Faculty of Biological Science, Alzahra University, Tehran, Iran

2 Medical Pharmacology, Faculty of Medicine, Duzce University, Düzce, Turkey

How to Cite: Hanachi P, Zarringhalami R, Kaya E. Total Phenolic and Flavonoid Content and Antibacterial Properties of Polygonatum orientale Desf and Tilia dasystyla, Hormozgan Med J. Online ahead of Print ; In Press(In Press):e102953. doi: 10.5812/hmj.102953.

ARTICLE INFORMATION

Hormozgan Medical Journal: In Press (In Press); e102953
Published Online: December 2, 2020
Article Type: Research Article
Received: March 25, 2020
Revised: September 5, 2020
Accepted: October 6, 2020
Uncorrected Proof scheduled for 25 (1)
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Abstract

Background: Secondary metabolites of plants such as phenol and flavonoids can neutralize free radicals. The antioxidant and cytotoxic activities of plants are related to phenolic or flavonoids compounds. The occurrence of drug resistance to antimicrobial drugs has led to the use of medicinal herbs in the treatment of infections. Antibiotic resistant of Staphylococcus aureus has become a major problem in the treatment of diseases.

Objectives: The present study aimed to determine total phenolic content (TPC) and total flavonoids content (TFC) of Polygonatum orientale Desf and Tilia dasystyla and evaluate their antibacterial effects on Staphylococcus aureus bacterium. Total phenolic content of Polygonatum orientale Desf and Tilia dasystyla has not been previously investigated.

Methods: Total phenolic and flavonoid content of P. orientale Desf and T. dasystyla extracts were determined using colorimetric methods of Folin-Ciocalteu and aluminum chloride. Antimicrobial activities of the extracts were evaluated by microdilution broth and disc diffusion methods to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values.

Results: The results showed that total flavonoids content of Polygonatum orientale Desf with the value of 7.9 ± 0.040 mg/g DW extracted with diluted water solvent and boiling method and total phenolic contents of Tilia dasystyla with the value of 62.13 ± 0.073 mg/g DW extracted with methanol solvent and boiling method were the highest amount. Methanol extract of Polygonatum orientale Desf had more antibacterial activities with the MBC and MIC values of 0.140 mg/ mL and 8 ± 0.4 mm zone of inhibition.

Conclusions: T. dasystyla and P. orientale Desf contain bioactive compounds such as phenolic and flavonoids that can be used as a promising option in pharmacognostical studies for the treatment of S. aureus infections.

1. Background

Secondary metabolites in plants such as phenolic and flavonoid compounds have antioxidant properties. Since plants are one of the significant sources of antioxidants, they can protect cells against oxidative damage. Natural antioxidants increase the potency of plasma antioxidants and reduce the risk of certain diseases such as cancer, heart disease, and stroke (1-3).

The precise mechanism of the antibacterial activity of the plant extracts has not yet been fully investigated. It appears that the phenolic compounds of the plants exert their antibacterial activity by altering the structure and function of the cell membrane. Studies show that phenolic compounds increase the permeability of the cell membrane, so the cell swells and dies. In gram-positive bacteria such as Staphylococcus aureus, antimicrobial agents as well as phenolic compounds can easily destroy the cell wall of the bacterium and its membrane, causing the bacterial material to leak out into the environment. Other antimicrobial activities of phenolic compounds include forming soluble compounds with proteins, disrupting bacterial surface receptors, and ultimately disrupting protein synthesis in bacteria (4, 5).

Medicinal plants are more preferred than prescription medicine due to unwanted side effects of prescription medicine (6, 7). Plants are one of the important sources of antioxidants. Antioxidant compounds from plants protect cells from oxidative damage (8). Secondary metabolites such as phenol and flavonoid compounds, show antioxidant effects and are derived from whole parts of the plant, like leaves, fruits, roots, and skin (9, 10). Because of the high prevalence of chronic diseases, it is logical to use plants to provide the antioxidants needed by the body, particularly the plants with high levels of phenol and flavonoid. Antioxidants are substances that are able to prevent oxidation caused by active oxygen species (11).

Although Staphylococcus aureus bacteria threatens public health through infectious diseases and food poisoning, it resists multiple antibiotics simultaneously. Thus, new methods and therapies have emerged for its treatment. The uncontrolled use of these antimicrobials can increase the drug resistance to antibiotics in most bacteria, which is one of the reasons for the growing use of plants as low-risk, accessible, and cost-effective natural substances for synthetic antibiotics in the treatment of bacterial infections caused by the increasing global studies and the introduction of antibacterial effects of different plants in recent years. Among all the identified substances contained in effective compounds of plants, phenolic compounds or secondary compounds without nitrogen have many biological effects, including antibacterial activity (12-14).

Polygonatum orientale Desf comes from the asparagus family in the north of Iran. Its rhizome has some medical properties such as wounds healing, anti-diabetic and antibacterial effects, gynecological disorders, anti-gout, and rheumatism, aphrodisiac (15).

Tilia is a genus from the family of Tiliaceae with about thirty species of trees that are mostly native to the northern hemisphere. Flowers and leaves of Tilia species (linden) are commonly used for cold symptoms. Tilia is also a traditional medicine as mosaic and diuretic, anti-inflammatory, anti-diarrhea, anti-spasmodic, anti-hair loss, anti-anxiety, and sedative (16, 17).

2. Objectives

The present study aimed to investigate the total phenol and flavonoid content of P. orientale Desf and T. dasystyla using three different solvents and two methods of extraction and determination of antimicrobial effects of two plants on Staphylococcus aureus species.

3. Methods

3.1. Chemicals and Reagents

We purchased methanol, ethanol, Folin-Ciocalteu reagent, sodium carbonate, gallic acid, aluminum chloride, potassium acetate, quercetin, Mueller–Hinton broth, Mueller–Hinton agar, and other chemicals from Merck.

3.2. Plant Material

Polygonatum orientale Desf and Tilia dasystyla species were provided from Bagh Firuze, Tehran, Iran and were identified at Alzahra University herbarium. In this study, we used the leaf of Tilia dasystyla and rhizome of Polygonatum orientale Desf.

3.3. Sample Preparation for Total Phenolic and Flavonoid Assay

A 0.1 g of dried samples was added to each solvent, including 10 mL of 80% aqueous methanol, 10 mL of 80% aqueous ethanol, and 10 mL distilled water. An ultrasonic bath (ultrasonic cleaner set, Model: WUC-A0H) was used for extraction. Samples remained 20 min in an ultrasonic bath, then the extracts were centrifuged for 15 min at 3000 rpm (18).

A 0.1 g of dried sample was added to 10 mL 80% aqueous methanol, and 10 mL 80% aqueous ethanol and 10 mL distilled water, this time water bath method was used for extraction. Samples were heated in a water bath for 60 min at a temperature of 70°C, then the extracts were centrifuged for 20 min at 2000 rpm (4).

3.4. Determination of the Total Phenolic Compounds

Phenolic contents of samples were determined by the Folin–Ciocalteu method. A volume of 200 µL extract samples were added to 1 mL of 1:10 diluted Folin–Ciocalteu reagent. Then, 800 µL of saturated sodium carbonate (75 g/L) was added after 4 min. The absorbance at 765 nm was measured after 2 h of incubation at room temperature. The results were expressed as gallic acid equivalent (GAE) mg/g dry weight of crude extract (2).

3.5. Determination of the Total Flavonoid Compounds

Extracts of samples (0.5 mL) were mixed with 1.5 mL of 95% ethanol, 0.1 mL of 10% aluminum chloride, 2.8 mL of distilled water, and 0.1 mL of 1 M potassium acetate. The absorbance was measured at 415 nm with a spectrophotometer after incubation at room temperature for 30 min. The stock solution of quercetin was used to make a serial dilution of concentrations (10 – 100 μg/mL) in methanol to make the calibration curve (19).

3.6. Antibacterial Activity

3.6.1. Sample Preparation for Antibacterial Assay

Samples (100 mg) were extracted with 80% methanol and ethanol and diluted water (5 mL) at 70°C temperature for 2 h. The extracts were centrifuged at 3000 g for 15 min and used to determine antibacterial properties (20).

3.6.2. The Preparation of Microorganism

ATCC 25923 stock cultures were kept at 4°C on tubes of nutrient agar. Active cultures were prepared by transferring a loop full of bacteria from the stock cultures to test tubes of Mueller–Hinton broth (MHB) and incubated for 24 h at 37°C. The cultures were diluted with fresh MHB to get densities corresponding to 2.0 × 106 colony-forming units.

3.6.3. Antimicrobial Susceptibility Test

The disc diffusion method was used to investigate the antimicrobial activity. Fifteen mL of molten media was poured into sterile petri plates to prepare the MHA plates. After 5 min, the plates were solidified. Then, 0.1% microorganism suspension was spread and dried after 5 min. Thirty microliters of extracts were added on 8 mm sterile disc. The disc was located on the surface of the medium. After 5 min, the compounds was diffused, and the plates were kept at 37°C for 24 h for incubation. Gentamicin discs were positive control. Inhibition zones were formed at the end of incubation around the disc and were measured with a ruler in millimeters (21).

3.6.4. Minimum Inhibitory Concentration (MIC) Determination

1 mL of TSB medium was added to nine autoclaved tubes. One mL sanitizing agent was added to the first and the second tubes of the series; tube 2 was stirred and 1 mL of solvent from tube 2 was transferred to tube 3. The transfer of solvents was repeated until tube 8. Then, 0.1 mL of microorganism was added to all flasks, except for flask number 8. Then all tubes were incubated for 24 h. Tubes 8 and 9 were positive and negative controls, concentration of extracts were 9 mg/mL, 4.5 mg/mL, 2.25 mg/mL, 1.125 mg/mL, 0.562 mg/mL, 0.281 mg/mL, and 0.140 mg/mL, respectively (22).

3.7. Statistical Analysis

All data are the average of three times analyses. ANOVA test by SPSS version 24 program was used to perform statistical analysis, and P-value < 0.05 was regarded as significant. Data were shown as means ± standard deviation.

4. Results

The content of phenolic compounds (mg/g DW) in ethanol, methanol, and diluted water extracts, determined from regression.

The content of flavonoids compounds (mg/g DW) in ethanol, methanol, and diluted water extracts, determined from regression.

Figures 1 and 2 indicate that TPC values are higher in water bath (boiling) method and methanol solvent. Methanol solvent had a higher amount of TPC. Phenolic compounds of P. orientale Desf methanol extract was 11.17 ± 0.45 mg/g DW, and T. dasystyla was 62.13 ± 3.53 mg/g DW. According to the results, T. dasystyla was richer source of phenolic compound than P. orientale Desf . Statistic results indicate that the two methods of extraction have significant difference (P < 0.05) in total phenolic content of P. orientale Desf, which the most TFC value in water bath method was about 11.17 ± 0.054 mg/g DW, however; this amount was about 9.97 ± 0.54 mg/g DW in sonication method. Also, there is a significant difference between solvents and total phenolic content of T. dasystyla. The highest TPC value is methanol solvent with 62.13 ± 3.53 mg/g DW, and the lowest is ethanol solvent with 28.97 ± 2.94 mg/g DW.

Figure 1. Total phenolic contents (mg/g DW) of P. orientale Desf and T. dasystyla by sonication method and three solvents (W: water, M: methanol, E: ethanol); values in each column with different letter are significantly different (P < 0.05).
Figure 2. Total phenolic contents (mg/g DW) of P. orientale Desf and T. dasystyla by water bath method and three solvents (W: water, M: methanol, E: ethanol); values in each column with different letters are significantly different (P < 0.05).

According to Figures 3 and 4, diluted water solvent and water bath method are more efficient in extracting flavonoid compounds of P. orientale Desf with the value of 7.9±0.049 mg/g DW. Ethanol solvent and water bath (boiling) method are more efficient in extracting total flavonoid compounds of T. dasystyla with the value of 4.1 ± 0.056 mg/g DW, it shows that P. orientale Desf has a richer source of flavonoid compounds than T. dasystyla.

Figure 3. Total Flavonoid contents (mg/g DW) of P. orientale Desf and T. dasystyla by sonication method and three solvents (W: water, M: methanol, E: ethanol); values in each column with different letter are significantly different (P < 0.05).
Figure 4. Total Flavonoid contents (mg/g DW) of P. orientale Desf and T. dasystyla by water bath method and three solvents (W: water, M: methanol, E: ethanol); values in each column with different letter are significantly different (P < 0.05).

Table 1 shows methanol solvent as the lowest concentration of MIC and MBC in both plants with an amount of 0.140 (mg/ mL). Results of Table 2 showed that the widest ZOI in P. orientale Desf with the size of 8 ± 0.4 is 20 (mg/ mL, dry wt) concentration of methanol solvent.

Table 1. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of P. orientale Desf and T. dasystyla Extracts Against S.aureus
Plant ExtractsMBC ( mg/mL)MIC (mg/mL)
P. orientaleDesf
Water0.2810.140
Ethanol0.5620.281
Methanol0.2810.140
T. dasystyla
Water1.1250.562
Ethanol0.2810.140
Methanol0.2810.140
Table 2. Zone of Inhibition (mm) of P. orientale Desf And T. dasystyla Extracts against S.aureus
plant Extracts20 (mg/mL, dry wt)10 (mg/mL, dry wt)5 (mg/mL, dry wt)
P. orientaleDesf
Water___
Ethanol6 ± 0.2__
Methanol8 ± 0.4__
T. dasystyla
Water___
Ethanol___
Methanol5 ± 0.3__

5. Discussion

Statistical results showed that the two methods of extraction are different (P < 0.05) in total flavonoid content of T. dasystyla and P. orientale Desf. In T. dasystyla, the highest amount of TFC is 4.1 ± 0.056 mg/g DW with water bath method; however, this amount is 1.6 ± 0.22 mg/g DW with the sonication method. Also, in P. orientale Desf, the highest amount of TFC is 7.9 ± 0. 53 mg/g DW with water bath method, and this amount is 1.7 ± 0.21 mg/g DW with sonication method.

The results showed that methanol was the best solvent for the isolation of polyphenols, as the previous reports showed that aqueous methanol was the best solvent for polyphenols explained by the polarity of phenolic compound (23). The water bath method was more effective due to the high temperature, which would reduce the solubility of any compounds. The water bath method indicated the highest amount of phenolic and flavonoid compounds in both plant species (24).

According to Hanachi et al. (2018), first methanol extracts showed more antioxidant properties than water extracts of T. dasytyla and P. orientale Desf (25). Thus, there is a direct correlation between phenolic compounds and antioxidant properties in these two plants. Methanol solvent can extract more phenolic compounds and show more antioxidant properties in both plant species (25).

According to the results, methanol extracts of these two plants showed the most antibacterial activities due to the high amount of TPC and TFC in methanol solvent. Also, P. orientale Desf had more antibacterial activities than T. dasystyla, and there was a direct relationship between the amount of phenol and antibacterial properties. Therefore, a higher amount of phenol content leads to more antibacterial activity. Also, the hydroxyl group in phenol compounds can lead to bacterial inhibition and damage to bacterial DNA (26).

5.1. Conclusion

Considering the results and increasing the resistance of bacteria to chemical antibiotics, it is suggested that with further studies on these plants, antibacterial compounds of them can be used to treat infectious diseases. We can conclude that the highest amount of total phenolic compound belonged to T. dasystyla methanol extract by water bath method. However, the purification of polyphenol extracts and in vivo evaluation should be further studied.

Footnotes

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