Comparison of antioxidant and anti-melanogenic activity of γ-oryzanol and gallic acid by inhibiting tyrosinase activity in B16F10 mouse melanoma cells

DOI: https://doi.org/10.21203/rs.3.rs-2768343/v1

Abstract

The beneficial effects of rice bran consumption must consider the synergic interaction of all these bioactive constituents.

Rice bran is abundant in gallic acid due to the tannins which upon hydrolysis yields gallic acid. Also, hydroxycinnamic acids such as ferulic, p-coumaric and caffeic acids were also found in the extracts. γ-Oryzanol comprises a mixture of ferulic acid esters and phytosterols (sterols and triterpenic alcohols). At least 10 steryl ferulates were determined in γ-oryzanol.

To better understand the mechanisms of the two important bioactive compounds of rice bran, we compared the antioxidant and anti melanogenic activities of gallic acid and γ-oryzanol for the treatment of disorders caused by hyperpigmentation. The antioxidant activity was measured by 2, 2′-diphenyl-1-picrylhydrazyl radical (DPPH) and ferric reducing antioxidant power (FRAP) methods. Then, its effects on viability, reactive oxygen species (ROS) production, mushroom tyrosinase and melanin content as well as amount of MITF and tyrosinase proteins was investigated on B16F10 murine melanoma cell line.

The antioxidant effects of were γ-oryzanol higher than gallic acid in DPPH and FRAP tests. Concentrations of 0.001 to 100 μM γ-oryzanol and gallic acid did not show significant cytotoxic effect at all concentrations after 24 and 48 h, and also reduced the amount of ROS, but did not have a significant effect in comparison with each other. In addition, the reduction of mushroom tyrosinase activity in γ-oryzanol was more than gallic acid and the concentrations of 10, 25, 50 and 100 μM showed significant effects. A decrease in melanin content was observed in different concentrations of γ-oryzanol and gallic acid, but this decrease in two compounds was not significant in comparison to each other. Finally, western blotting results showed that the decrease in the expression of MITF and tyrosinase proteins in γ-oryzanol is higher than in gallic acid, and this decrease was significant in concentrations of 01, 5 and 10 μM. According to the obtained results and the comparison of measured parameters between γ-oryzanol and gallic acid compounds, it can be concluded that γ-oryzanol has more antioxidant effects than gallic acid. In addition, γ-oryzanol shows higher inhibitory effects in the expression pathway of proteins involved in melanin synthesis in B16F10 cells.

According to the obtained results γ-oryzanol was superior to gallic acid in case of antioxidant and anti-melanogenic activity in B16F10 cells and may has higher potential in treating disorders caused by hyperpigmentation compared to gallic acid.

Introduction

As the largest organ of the body, the skin has the duty of protecting the internal body from the external environment with an excessive effect on the beauty of the body. In general, skin diseases comprise 34% of all diseases worldwide (1). Tyrosinase is a copper-containing polyphenol involved in melanogenesis, which is present in a large amount in mushrooms, higher plants and animals (2). MITF and proteins related to tyrosinase 1 and 2 (TRP-1, 2) help in the production of melanin (3, 4). Melanin pigment is necessary to protect human skin from harmful rays. But excessive production of melanin induces hyperpigmentation disorders such as melasma, sun spots, freckles, acne scar pigments and senile lentigines (5, 6). One of the clinical applications of tyrosinase enzyme inhibitors is to prevent excessive accumulation of melanin pigment in mammals (7). A number of strong and moderate inhibitors of tyrosinase enzyme, from natural and synthetic sources such as arbutin, kojic acid and hydroquinone have been used in the last decade as bleaching or strong anti-pigmentation because they have the ability to inhibit the production of melanin in the skin (8).

γ-Oryzanol is one the non-soap phytosterol derived from rice bran oil and consists of a mixture of plant sterols esterified to phenol and ferulic acid. γ-Oryzanol was first isolated in Japan in the 1950s and used as a medication to treat anxiety, menopause symptoms, peptic ulcer and gastritis (9–10). Rice bran oil is the richest source of γ-oryzanol but this substance is also found in corn, barley, rye, wheat bran and other edible oils. Phytosterols are potent antioxidants and control the growth, development and maintenance of membrane fluidity in plants. Rice bran oil with a high concentration of γ-oryzanol contains about 1% to 10 mg/g and unripe rice bran oil contains 1.5% γ-oryzanol. γ-Oryzanol has anti-inflammatory, antioxidant and lipid-lowering activity (10). Gallic acid (3, 4, 5 trihydroxybenzoic acid) is one of the most important polyphenols in fruits, foods and various plants such as oak, tea (green and black), sumac, grape seeds, apples and rice bran (11, 12). Gallic acid prevents cellular damage by reducing oxidative stress (13). By inhibiting the activity of tyrosine, gallic acid shows antioxidant, antibacterial, antiviral, antifungal, anticancer, anti-inflammatory and detoxification activities (14). Gallic acid can reduce the effects of oxidative stress in cells by increasing total thiol and the activity of GPx enzyme and reducing the amount of MDA (14). In addition, it has been approved as a food additive and is used widely in skin care products (15). Recent researches show that γ-oryzanol may inhibit the tyrosinase with potential anti- melanogenesis activity. It was shown that out of eight substances isolated from rice bran, γ-oryzanol had significant inhibitory effects on melanin synthesis, and treatment of melanoma cells for 72 h with γ-oryzanol decreased the synthesis of melanin and mushroom tyrosinase. Also, γ-oryzanol reduces the expression of MITF and TRP1 genes through regulating the protein kinase a pathway and reducing the phosphorylation of cAMP-response element binding protein (p-CREB) (16). In addition, it has been shown that gallic acid, has both antioxidant and anti-melanogenic properties through the regulation of GSH/GSSG levels (17). Moreover, gallic acid has the ability to phosphorylate the MITF protein through the activation of the MEK-ERK kinase cascade also activate the phosphorylation of glycogen synthase kinase 3β (GSk3β) and the accumulation of β-catenin by inactivating the Wnt/β-catenin signaling pathway and finally, it reduces the expression of MITF (18). In this research we have attempted to compare the antioxidant and anti-melanogenic effects of γ-oryzanol and gallic acid and investigate the underling molecular mechanisms involved in melanin synthesis in B16F10 mouse melanoma cell line.

Materials And Methods

Determination of antioxidant activity

2, 2′-diphenyl-1-picrylhydrazyl radical (DPPH) assay

DPPH radical scavenging activity was performed in a 96-well microplate according to protocol of Bajalan, Mohammadi  with some modifications (19). 150 μL of various concentrations of γ-oryzanol and gallic acid were added to DPPH radical solution (150 μL, 0.1 mM, Merck, Germany) in methanol and incubated for 30 min in the dark at room temperature. The absorbance of the samples was measured at 517 nm. Vitamin C was used as a positive control. Antioxidant capacity of the sample was expressed as DPPH radical scavenging activity (%):

DPPH radical scavenging activity (%) = (Abs control – Abs sample /Abs control) × 100    

Where Abs control is the absorbance of DPPH radical in methanol and Abs sample is absorbance DPPH radical in sample/standard.

Ferric Reducing Antioxidant Power (FRAP) assay

Ferric reducing antioxidant potential was conducted according to Nemes, Szőllősi  (20). FRAP reagent was prepared freshly before analysis. Then, 2, 4, 6-tri (2-pirydyl)-s-triazine (TPTZ) (5 mL, 10 mM) in HCl (40 mM) were mixed with FeCl3 (5 mL, 20 mM) and with acetate buffer (50 mL, 0.3 M, pH = 3.6). The 96-well plates were then incubated at 37˚C for 30 minutes before absorbance was recorded at 593 nm. Analysis of FRAP was performed by adding 20 µL of γ-oryzanol and gallic acid to 180 µL of FRAP reagent. Results obtained for the samples were expressed as µmol Fe2+/L of extract.

Cell culture and treatment

B16F10 melanoma cells (Pasteur Institute, Iran), were cultured in Roswell Park Memorial Institute Medium (RPMI 1640; Sigma, Germany) with 10% fetal bovine serum (FBS; Gibco, -USA) and penicillin and streptomycin (PS; Gibco, USA). Then incubated at 37°C with 5% CO2 and 90% humidity. γ-Oryzanol and gallic acid (4 mM) was dissolved in methanol to provide a stock solution. 

Analysis of cell viability

First, 100 μl of suspension containing 104 B16F10 cells was transferred to each 96-well plate well. Then, cells were treated with concentrations of 0.001 to 100 μM of γ-oryzanol and gallic acid for 24 and 48 h. After a day, 20 μl of resazurin dye (sigma, Germany) was added to each well. It was shaken for 10 min and incubated for 6 h until the blue color of the wells turned pink. Finally, absorbance of samples at 600 and 570 nm was read in Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, USA). Doxorubicin (1 mM) was as positive control (21).

ROS generation 

To determinate the level of ROS generation, 104 cells were treated with concentrations of 0.001 to 10 μM of γ-oryzanol and gallic acid for 48 h. Then 100 μl of hydrogen peroxide (H2O2; Sigma, Germany) with a concentration of 24 mM was added to each well and samples incubated for 30 min. Thereafter, 2', 7'-dichlorofluorescein diacetate (DCFH-DA; Sigma, Germany) (10 μl) was added to each well and samples incubated for 20 min. Finally, the fluorescent intensity of DCF was recorded with Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, USA) at excitation wavelength of 485 nm and emission wavelength of 538 nm (21). Kojic acid (4 mM) was as positive control (22).  

Mushroom tyrosinase activity assay

104 Cells were treated with concentrations of 0.001 to 100 μM of γ-Oryzanol and gallic acid for 48 h. Then, 160 µl L-DOPA (5 mM) (Sigma, Germany) and 20 µl of mushroom tyrosinase (Sigma, Germany) was added to the cells and incubated for 30 min. Finally, the absorbance was compared at 475 nm by Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, USA) (23). 

Melanin content assay

106 cells were treated with concentrations of 0.01 to 25 μM of γ-oryzanol and gallic acid. After 48 h, cells were dissolved in 100 μl of NaOH (1 N). The final amount of melanin was read at 405 nm (22, 24).

Western blotting

106 B16F10 melanoma cells were treated with concentrations of 0.1, 5 and 10 μM of γ-oryzanol and gallic acid for 48 h. Western blot analysis of the extracted cell proteins were performed based on previously printed protocols (25). The membrane exposed to rabbit monoclonal MITF, polyclonal tyrosinase and β-actin as primary antibodies and anti-rabbit IgG (1:2000) (Cell Signaling Technology, USA) as secondary antibody. Finally, the density of each band recorded by the Gel Doc UV Alliance and all bands normalized to the related β-actin bands.

Statistical analysis

Data values and results were expressed as mean ± SD of three independent expiments in triplicates and Prism 8 software was used for statistical analysis of data and graphs. Comparisons between groups were performed using Two-way ANOVA statistical test and Sidak's multiple comparisons test and p<0.05 was considered as a significant difference.

Results

Antioxidant activity 

2, 2′-diphenyl-1-picrylhydrazyl radical (DPPH) 

The scavenging effect of γ-oryzanol and gallic acid (0.001-100 μM) was measured and compared to vitamin C (0.01-100 μM). DPPH Radical scavenging activity reduced in the order of vitamin C > γ –oryzanol> gallic acid (Table 1) (p<0.05).

Ferric Reducing Antioxidant Power (FRAP)

Antioxidant activity of γ-oryzanol and gallic acid (0.001-100 μM) was assayed and compared to vitamin C (0.01-100 μM). Antioxidant activity decreased in the order of vitamin C > γ-oryzanol > gallic acid (Table 2) (p<0.001).

Comparison of effects of γ-oryzanol and gallic acid on cell viability of B16F10 cells 

γ-Oryzanol and gallic acid (0.001 to 100 μM) did not show cytotoxic effect compared to the control group after 24 and 48 h on B16F10 cells.  Doxorubicin (1 mM) as a positive control reduced cell viability. For the following experiments, the cells were pretreated with different concentrations of γ-oryzanol and gallic acid for 48 h as the optimal time.

Comparison of effects γ-oryzanol and gallic acid on B16F10 cellular ROS level

As seen in Fig. 2, treatment with H2O2 elevated the ROS content and γ-oryzanol and gallic acid reduced the ROS content in B16F10 cells induced by H2O2. and there was not significant difference between γ-oryzanol and gallic acid. Kojic acid (4 mM) as a positive control also also reduced the ROS content (Fig. 2).

Comparison of effects of γ-oryzanol and gallic acid on mushroom tyrosinase activity 

Treatment of cells with γ-oryzanol and gallic acid reduced the activity of mushroom tyrosinase. In comparison between γ-oryzanol and gallic acid using tow way ANOVA analysis the reduction was significant in concentrations of 10, 25, 50 and 100 µM of (p<0.001) and γ-Oryzanol was more effective than gallic acid in reducing the mushroom tyrosinase activity. Kojic acid (4 mM) as a positive control also had a reducing effect on the activity of mushroom tyrosinase (p<0.001) (Fig. 3).

Comparison of effect of γ-oryzanol and gallic acid on melanin production

Both γ-oryzanol and gallic acid decreased melanin content with no significant difference between the two active compounds. Also, kojic acid (4 mM) as a positive control induced a decrease in melanin content (Fig. 4). 

Comparison of the expression of MITF and tyrosinase proteins in γ-oryzanol and gallic acid treated cells

Western blot analysis of cells treated with γ oryzanol and gallic acid done to compare the change in the amount of MITF and tyrosinase proteins. It was seen that treatment with γ- oryzanol and gallic acid reduced the amount of MITF protein with significant difference between concentrations of 0.1, 5 and 10 µM of γ-oryzanol and gallic acid (p<0.001. p<0.5 and p<0.001, respectively) (Fig. 5a). Also, treatment with γ-oryzanol and gallic acid decreased the level of tyrosinase protein with significant difference between concentrations of 5 and 10 µM γ-oryzanol and gallic acid (p<0.001 and p<0. 001) (Fig. 5b).

Discussion

In the current study, we compared the antioxidant and anti-melanogenic activity of γ-oryzanol and gallic acid in B16F10 mouse melanoma cells. Our finding indicated that γ-oryzanol has higher antioxidant and antimelanogenic effects than gallic acid on B16F10 cells. Therefore, it probably has a greater ability to deal with and treat damage caused by excessive melanin production compared to gallic acid.

Phenolic acids are phenolic compounds having one carboxylic acid group. Phenolic acids are divided into two sub-groups: hydroxybenzoic and hydroxycinnamic acid. Hydroxybenzoic acids possess a common structure of C6-C1 and derived from benzoic acid like 3, 4, 5-trihydroxybenzoic acid (Gallic acid). Hydroxycinnamic acid having a C6–C3 skeleton. The four most common hydroxycinnamic acids are ferulic, caffeic, p-coumaric, and sinapic acids. Typically, they are present in bound from such as amides, esters, or glycosides and rarely in free form. γ-Oryzanol comprises a mixture of ferulic acid esters and phytosterols (sterols and triterpenic alcohols. γ-Oryzanol was thought to be a single compound, but as mentioned earlier it is a mixture of ferulic acid esters and phytosterols (sterols and triterpenic alcohols). Unsaponifiable matter of crude rice bran oil contains high levels of components with antioxidant properties including tocopherols/tocotrienols and γ-oryzanol. Both gallic acid and γ-Oryzanol which are existing in rice bran can be used as natural antioxidants for pharmaceutical purposes (9–12).

In previous studies, Xu et al. (2001) attempted to purify, identify, and evaluate the antioxidant chemicals in rice bran. The antioxidants present in rice bran are tocopherols, tocotrienols, and γ-oryzanol, which have shown remarkable antioxidant effects in the oxidation models of cholesterol and linoleic acid. Interestingly, γ-oryzanol has more antioxidant activity in preventing cholesterol oxidation than tocopherols and tocotrienols (26). Saenjum et al. (2012) investigated the antioxidant and anti-inflammatory effects of five varieties of Thai purple rice contained 1.23 to 9.14 w/w γ-oryzanol. All extracts had acceptable antioxidant and anti-inflammatory activity on murine macrophage cells (27). Ham et al. (2014) investigated the protective effect of rice bran unsaponifiable matters on oxidative damage of HepG2 cells induced by tert-butyl hydroperoxide. The results showed that treatment with terbutyl hydroperoxide increases the activity of superoxide dismutase (SOD), catalase (CA), glutathione peroxidase (GPx), glutathione reductase (GSH), while pretreatment with unsaponifiable matters of rice bran compounds effectively reduces oxidative damage (28). Juliano et al. (2005) compared the antioxidant activity of γ-oryzanol and the synthetic antioxidants butylated hydroxyanisole (BHA) and reported relatively similar antioxidant effects.

Therefore, it can be said that γ-oryzanol prevents lipid per oxidation induced by 2, 2′-azobis (2, 4-dimethylvaleronitrile) (AMVN).

It has been shown that gallic acid in pistachio green hull has the potential to protect triglycerides in soybean oil against peroxidation due to having an electron-donating carboxylate anion (30).

To begin the study, the antioxidant activity of different concentrations of γ-oryzanol and gallic acid was determined by free radical scavenging of DPPH and FRAP assay based on the electron transfer mechanism (31). The free radical scavenging ability of antioxidants can be determined by using stable free radicals like DPPH. The purple colour of DPPH radical disappears by abstracting a hydrogen atom from the antioxidant. As shown in Table 1, the DPPH radical scavenging effects of γ-oryzanol is higher than gallic acid, but still weaker than ascorbic acid. The radical-scavenging activity of phenolic acids depends on the number of electron donor hydroxy and methoxy substitutions which increase the stability of the phenoxy radicals. FRAP determines the reduction of TPTZ to a blue sample. The compounds with antioxidant activity can reduce Fe3+ into Fe2+. As shown in Table 1, the reducing capacities of the γ-oryzanol and gallic acid increased with the increase in concentration. The results of DPPH and FRAP methods confirmed that γ-oryzanol has a higher antioxidant activity than gallic acid and a good performance in free radicals scavenging. The antioxidant activity of these compounds was mostly due to their redox properties like reducing agents, singlet oxygen scavengers and hydrogen atom donors.

Our results also showed that treatment with different concentrations of γ-oryzanol and gallic acid did not induce a significant reduction in cell viability at 24 and 48 h in melanoma B16F10 cells

As mentioned, melanin plays an important role in the absorption of free radicals produced by the cell cytoplasm and UV rays of sunlight in the skin. So, the substances with antioxidant properties can reduce the production of ROS and melanin (32). Kojic acid is a natural compound derived from the fungi Aspergillus, Acetobacter and Penicillium and is used as lightening and anti-stain agent at cosmetics formulation (33). Its antioxidant activity is through the chelating of iron ions (Fe). Also, the inhibitory activity on melanin production is attributed to copper ion (Cu) chelation which is presents in the active site of the tyrosinase enzyme (34, 35). According to the study on the effect of γ-oryzanol on human kidney stem cells in 2018 γ-oryzanol significantly reduces the production of H2O2-induced free radicals (36). Also, pretreatment of human lymphoblast cells with ascorbic acid and gallic acid 30 min before the exposure to H2O2 induces a slight inhibition on DNA damage (37). In addition, pretreatment with fucoidan-gallic acid increase cell viability and reduce ROS in pre-osteoblast-like cells induced by H2O2 toxicity (38). According to the results γ-oryzanol and gallic acid both reduced the levels of oxygen free radicals, significantly and similarly. In this study, the activity of mushroom tyrosinase as an enzyme responsible for the conversion of L-DOPA to dopamine was investigated. The concentrations of 10, 25, 50 and 100 µM γ-oryzanol and gallic acid significantly reduced the activity of mushroom tyrosinase and this decrease was more in cells treated with γ-oryzanol than those treated with gallic acid. Considering that the basic structure of γ-oryzanol is the aromatic core of ferulic acid, in a study was investigated the inhibitory effect of ferulic acid on mushroom tyrosinase and it was shown that ferulic acid is an inhibitor of this enzyme (39). In addition, the structure of the phenol ring is important in showing the activity of anti-tyrosinase and the reason for the anti-tyrosinase effect of gallic acid can be due to the presence of phenolic groups in its structure (40, 41). Furthermore, γ-oryzanol and gallic acid decrease the content of cellular melanin, significantly in a similar manner. At the end, we investigated the mechanisms involved in the anti-melanogenic effects of γ-oryzanol and gallic acid and compared the expression of two proteins involved in melanin synthesis in γ-oryzanol and gallic acid treatments. Evidence showed that γ-oryzanol and gallic acid at all concentrations was able to significantly reduce tyrosinase and MITF protein and γ-oryzanol more effective than gallic acid. By comparing the two compounds in rice bran, it can be concluded that both the antioxidant and anti-melanogenic effects of γ-oryzanol are higher than gallic acid. It was previously investigated that the anti-melanogenic effects of γ-oryzanol and gallic acid are through the modulation of PKA pathway and MEK-ERK kinase cascade, respectively and the GSk3β phosphorylation causes a decrease in MITF protein expression.

Conclusion

According to the results of this study and by comparing the antioxidant and anti-melanogenic effects between γ-oryzanol and gallic acid, γ-oryzanol showed higher reducing and inhibitory effects on activity of mushroom tyrosinase and the expression of tyrosinase and MITF proteins than gallic acid. It seems the presence of aromatic compounds called ferulic acid gives a higher effectiveness to γ-oryzanol.

Abbreviations

2, 2′-diphenyl-1-picrylhydrazyl radical, DPPH; ferric reducing antioxidant power, FRAP; reactive oxygen species, ROS; 5-hydroxy-2-hydroxymethyl-4-pyrone, Kojic acid; microphthalmia-associated transcription factor, MITF; polyphenol oxide, PPO; levodopa, L-DOPA; 2,4,6-tri(2-pirydyl)-s-triazine, TPTZ; Roswell park memorial institute medium, RPMI; fetal bovine serum, FBS; streptomycin, PS; dimethyl sulfoxide, DMSO; 2', 7'-dichlorofluorescein diacetate, DCFH-DA; superoxide dismutase, SOD; catalase, CA; glutathione peroxidase, GPx; glutathione reductase,  GSH; butylated hydroxyanisole, BHA; butylated hydroxytoluene, BHT; hydrogen peroxide, H2O2; tumor necrosis factor alpha, TNF-a; interleukin-1β, (IL-1β); interleukin-6, (IL-6); cyclooxygenase-2, (COX-2); nuclear factor kappa B, (NF-Κb); heme oxygenase-1, (HO-1); extracellular signal-regulated kinase 1 and 2, (ERK1/2); inducible nitric oxide synthase, iNOS; 2, 2′-azobis(2,4-dimethylvaleronitrile, AMVN);  malondialdehyde, MDA; glutathione/oxidized glutathione, GSH/GSSG; mitogen-activated protein kinase/ERK kinase -extracellular-signal-regulated kinase, MEK-ERK; glycogen synthase kinase 3β, GSk3β; cAMP-response element binding protein, p-CREB; tyrosinase 1 and 2,  TRP-1, 2; ultraviolet radiation,  UV;

Declarations

*Human/animals subjects were not used in the study.

Funding: This work has been supported by a grant (No. 981633) from Research Affairs of Mashhad University of Medical Sciences, Mashhad, Iran.

Conflict of interest: There is no conflict of interest in this study.

Ethics approval: This work is carried out on B16F10 murine melanoma cells and there is no need for ethical clearance.

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Tables

Table 1. DPPH radical scavenging assay (%)

Extracts (μM)

0.01

1

10

20

50

100

Gallic acid

10.16±0.24b

41.69±3.17b

58.19±6.19a

73.15±2.64b

83.15±4.2b

87.1±6.14b

γ-Oryzanol

4.1±0.03c

13.17±1.82c

21.0±3.64b

28.2±1.56c

32.09±5.01c

42.16±0.1c

Vitamin C

21.04±2.16a

35.15±3.1a

62.94±3.74a

79.3±2.89a

97.64±6.91a

98.7±7.47a

 Each value represents as mean±SD of triplicate experiments. Different letters in the column indicate significant differences (p<0.05)

Table 2. FRAP (µmol Fe2+/L)

Extracts (μM)

0.01

1

10

20

50

100

Gallic acid

19.75±0.4b

41.02±2.18b

58.13±6.1b

71.9±3.18b

136.04±10.69b

295.16±3.1b

γ-Oryzanol

15.14±2.1c

34.16±2.1c

42.1±3.18c

53.5±4.6c

87.16±4.9c

97.4±6.92c

Vitamin C

55.16±2.18a

91.15±3.15a

153.12±2.8a

201.15±21.6a

280.1±14.3a

315.05±7.6a

 Each value represents as mean±SD of triplicate experiments. Different letters in the column indicate significant differences (p<0.05)