Abstract: Background and Objective: Study on medicinal plant extract is gradually interested and distributed, especially their biological activities. The present study aimed to determine the enzyme inhibition and antimicrobial activities of the fractionated extracts of wild grape (Ampelocissus martinii Planch.) seeds. Materials and Methods: Wild grape seeds in different growth stages were extracted with methanol before fractionation by silica gel chromatography. The anti-glucosidase and anti-tyrosinase enzyme activities of the extracts were then tested by using UV-Vis spectrophotometry and antimicrobial activities were observed from MIC, MBC values and time killing assay. Results: The sub-fraction of immature stage eluted by ethyl acetate/methanol at 75/25 (%v/v) has the highest enzyme inhibition activity and the most potent efficiency for time kills profiles. The MIC values of the potent immature, mature and ripe fractioned extracts were ranging from 1.25-50.00, 1.25-50.00 and 1.56-25.00 mg mL–1, respectively, while the MBC values ranged from 3.12-6.25, 3.12-25.00 and 3.12-25.00 mg mL–1, respectively. Conclusion: The wild grape seed composed of α-glucosidase and tyrosinase inhibition and antibacterial activities compounds. The wild grape seed extracts may be used as active ingredients sources of health-supporting products or cosmetics.
INTRODUCTION
The medicinal plants or herbs are popularly used for health promotion, personalized treatment and disease prevention worldwide1. The active ingredients in plants are known as phytochemicals. They have been proved for using in medicinal and nutritional applications1,2. The substances obtained from plants are huge groups including; phenolics, flavonoids, quinines, tannins, alkaloids, terpenoids, saponins, phytosterols and plant essentials oils3-5. These substances showed various biological activities such as; antioxidant, antibacterial, anti-inflammatory, anti-diabetic and anti-aging6-11.
During the past decade, different types of health supplement products from grape, especially seed extracts have been produced and commercially advertised4,12,13. Many studies of phytochemicals extracted from grapes have been reported14-18, confirming that all parts of grapes have high phytochemical contents4. The authors are interested in phytochemicals found in a native herb, namely “wild grape (Ampelocissus martinii Planch.)”. This plant has a long history of use as an herb in the north and northeast of Thailand. Its stem, fruit and leaf were similar with the planted grape. The previous study indicated that the fruit extracts of wild grapes have a high content of phytochemicals and biological activities such as; antioxidant and antibacterial19-21. However, a widely detailing study on the biological activity of the wild grape is necessary in order to increase its efficiency. Therefore, this study the crude extract of wild grape seed was firstly fractionated by using silica gel column chromatography. The obtained fractions were then analyzed for antioxidant, anti-glucosidase and anti-tyrosinase inhibition and antibacterial activities.
MATERIALS AND METHODS
This study done for six months from 1st September, 2018 to 29 February, 2019. The experiment performed at the Department of Chemistry, Faculty of Science, Mahasarakham University, Thailand.
Materials: The wild grape (Ampelocissus martinii Planch.) fruits at different growth stages (immature, mature and ripe) were collected from September-October, 2018, from Roi-Et province, Thailand. They were separated seeds and pulps and only seeds were dried and ground by using a mortar and then stored at room temperature in desiccators until further analysis.
Methods
Crude extract and fractionation: The extraction process was performed by following the previous report4 with slight modification. The mixture solution of methanol:HCl (99:1 v/v) was used as a solvent. A rotary vacuum evaporator was used for concentration of the solvent. The obtained crude extract was dissolved in methanol and loaded on a 60×4.5 cm i.d., glass column packed with silica gel (60-200 mesh). The column was then eluted with the different polarity of solvent mixtures of ethyl acetate/methanol in different ratios. The absorbance at 280 nm was performed to identify each fraction. Sub-fractions were stored at -4°C until analysis.
Tyrosinase inhibition activity assay: The tyrosinase inhibition of the extracts was performed by following the previous report22 with some modifications. A tyrosinase substrate, DOPA was mixed with the enzyme and extracts. The reaction was measured absorbance at 475 nm. All samples were analyzed in triplicate. Kojic acid was used as positive control and expressed the tyrosinase inhibition activity as an IC50 value.
Alpha-glucosidase inhibition activity assay: The α-glucosidase inhibition activity of the samples were determined by following the previous report23. The p-NPG was used as an enzyme substrate. The reaction was stopped by adding sodium carbonate solution. The α-glucosidase activity was determined by measuring the p-nitrophenol release from p-NPG by using absorbance measurements made at 405 nm. Acarbose was used as positive control. The α-glucosidase inhibitory activity was expressed as an IC50 value.
Antibacterial activity assay
Test bacteria and fungi: The eight reference strains of bacteria including; Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Salmonella Typhi DMST 22842, Shigella dysenteriae DMST 15110, S. flexneri DMST 4423, methicillin susceptible Staphylococcus aureus (MSSA) DMST 2933, methicillin resistant Staphylococcus aureus (MRSA) DMST 20651 and Vibrio cholerae (O1) DMST 9700 were used in this study. One strain of pathogenic fungus Candida albicans NCYC854 was also included in this study.
Preliminary screening for antibacterial activity: The extracts were screened for activity against eight pathogenic bacteria and one pathogenic fungus using an agar well diffusion method by following the previous report24. The inhibition zones in each plate were measured compared with the reference standards antibiotic tetracycline at concentrations of 250 μg mL–1 (for bacteria) and amphotericin B at concentrations of 25 μg mL–1 (for fungi). Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) were determined by using the microdilution method as previously described24,25.
Time-kill assay: The best extract was selected to investigate for the antibacterial or fungal activity using time-kill assay by a method modified from White et al.26 and Perim et al.27. The effects of the most potent extract on the cell morphology of pathogens were also investigated by using a Scanning Electron Microscope (SEM).
Statistical analysis: The mean±Standard Deviation (SD) and Duncan’s new multiple range tests were used to evaluate the significant differences with p<0.05. Pearson’s correlation coefficient (r) used to indicate data correlation.
RESULTS
Enzyme inhibition activity: As shown in Table 1, the crude extracts of all stages showed higher α-glucosidase inhibition activity than standard acarbose, which has the highest activity in the crude extract of the mature stage. Among all fractionated extracts, the extract of immature stage eluted by methanol (G-Fr5) has the highest α-glucosidase inhibition activity. Comparison between the growth stages, the α-glucosidase inhibition activity was followed by immature >mature>ripe extracts. The mixture of ethyl acetate and methanol with an equal ratio (50:50 %v/v) could be extracted the substances containing higher α-glucosidase inhibition activity than other systems. The fractionated extracts in all stages have averaged lower tyrosinase inhibition activity than kojic acid. The modulate tyrosinase inhibition activity found in immature and mature stages eluting by ethyl acetate/methanol at 75/25 and 50/50 (v/v) with IC50 values in the range of about 440-824 μg mL–1.
Preliminary screening for antimicrobial activity: As shown in Table 2, some of the fractionated extracts exhibited antibacterial activity against two strains of Gram-negative bacteria (V. cholerae O1 DMST 9700 and S. dysenteriae DMST 15110) and three strains of Gram-positive bacteria (S. aureus (MSSA) DMST 2933, S. aureus (MRSA) DMST 20651 and B. cereus ATCC 11778). In contrast, no antifungal activity against C. albicans NCYC 854 was observed. Among the immature fractioned extracts, the fraction eluted by ethyl acetate:methanol of 75:25 (%v/v, G-Fr2) showed more significant potent activity against 4 of the bacterial strains, V. cholera O1 DMST 9700, B. cereus ATCC 11778, S. aureus (MSSA) DMST 2933 and S. aureus (MRSA) DMST 20651. For mature (R) and ripe (B) fractioned extracts, the fraction eluted by ethyl acetate:methanol of 75:25 (%v/v, R-Fr2) and ethyl acetate (B-Fr1) extracts had more excellent antibacterial activity than those of the mature and ripe fractioned. Interestingly, the test bacteria, S. aureus (MRSA) DMST 20651 were more sensitive to the mention fractionated extracts above (G-Fr2, R-Fr2 and B-Fr1) than tetracycline.
| Table 1: | Fractionation yields and α-glucosidase and tyrosinase inhibition activities of fractionated wild grape seed extracts |
Results are expressed as mean±SD of triplicate measurements. Means with different letters in the same column represent significant differences at p<0.05, G (green): Immature stage, R (red): Mature stage, B (black): Ripe stage, F: Sub-fraction | |
| Table 2: | Screening of antibacterial and antifungal activity of the extract against pathogenic bacteria and fungal strains |
| ND: Not determined | |
| Table 3: | MIC and MBC values of selected extracts against 5 strains of Gram-positive and Gram-negative pathogenic bacteria |
| MIC: Minimum inhibition concentration, MBC: Minimum bactericidal concentration | |
Table 3 showed the MIC and MBC values of the selected fractions on bacterial. The MIC value of the potent immature fractioned extracts were ranging from 1.25-50 mg mL–1, whereas, the MIC values of the potent mature and ripe extracts were 1.25-50 and 1.56-25 mg mL–1, respectively. The MIC value of immature fraction eluted by ethyl acetate:methanol of 75:25 (%v/v, G-Fr2) had higher potent activity against all tested bacterial strains, but it showed quite a low MIC value (3.12 mg mL–1).
| Fig. 1(a-c): | In vitro antibacterial activity of the extract against (a) Staphylococcus aureus (MSSA) DMST 2933, (b) S. aureus (MRSA) DMST 20651 and (c) B. cereus ATCC 11778 at concentrations of 1×MIC of the extract |
Moreover, the fractions eluted by ethyl acetate:methanol of 75:25 (%v/v, R-Fr2) and ethyl acetate (B-Fr1) had lower MICs value against the tested bacterial strains than those of the mature and ripe fractioned. A similar pattern of results was observed with the MBC values. The greatest potential efficiency for fractionated extracts of each growth state (G-Fr2, R-Fr2 and B-Fr1) gave the lowest MBC values. The MBC values of the G-Fr2, R-Fr2 and B-Fr1 fractioned extracts ranged from 3.12-6.25, 3.12-25.00 and 3.12-25.00 mg mL–1, respectively.
| Fig. 2(a-c): | In vitro bacteriostatic activity of the extract G-Fr2 against (a) Staphylococcus aureus (MSSA) DMST 2933, (b) S. aureus (MRSA) DMST 20651 and (c) B. cereus ATCC 11778 at various concentrations |
Time-kill assay: The time-kill assay of the best antibacterial activity of the fractionated extracts was shown in Fig. 1. The G-Fr2 fractioned at 1x MIC of the extract had the bacteriostatic activity against only bacterial pathogen S. aureus (MSSA) DMST 2933, whereas, no activity against S. aureus (MRSA) DMST 20651 and B. cereus ATCC 11778 (Fig. 1a-c). The 1x MIC of R-Fr2 and B-Fr1 fractioned extracts did not show antibacterial activity against S. aureus (MSSA) DMST 2933 (Fig. 1a), S. aureus (MRSA) DMST 20651 (Fig. 1b) and B. cereus ATCC 11778 (Fig. 1c). Therefore, the G-Fr2 fractioned extract was chosen for further investigations.
| Fig. 3(a-j): | Scanning electron micrographs of (a-e) Staphylococcus aureus (MSSA) DMST 2933 and (f-j) S. aureus (MRSA) DMST 20651 after treatment with the extract at 2×MIC of (c and h) extract B-Fr1, (d and i) G-Fr2, (e and j) R-Fr2 and (b and g) Antibiotic tetracycline at 250 μg mL–1 compared with the (a and f) Untreated control |
| Fig. 4(a-e): | Scanning electron micrographs of B. cereus ATCC 11778 after treatment with (a) Untreated control, (b) Antibiotic tetracycline at 250 μg mL–1, (c) Extract B-Fr1, (d) G-Fr2 and (e) R-Fr2 at 2×MIC |
Figure 2a-c showed the bacteriostatic or bactericidal of the various concentrations (1×, 2× and 4×MIC) of the G-Fr2 fractioned extract was investigated. The results showed that the extract at concentration of 2× and 4× MIC had the bacteriostatic activity against both S. aureus strains and B. cereus ATCC 11778. The result suggested that the G-Fr2 fractioned extract has time and a concentration-dependent bacteriostatic activity against S. aureus (MSSA) DMST 2933, S. aureus (MRSA) DMST 20651 and B. cereus ATCC 11778.
Effects of the extracts on morphology of bacteria: Figure 3a-j showed effects of the fractionated extracts on cell morphology of bacteria. Untreated control cells appeared undamaged (Fig. 3a and f), whereas, the treatment of both strains of S. aureus with 2×MIC levels of all extracts induced several alterations, some bacterial cells increased or decreased in size (Fig. 3c-e and h-j). Similar morphological alterations occurred when these bacterial pathogens were treated with the antibiotic tetracycline (Fig. 3b and g).
Figure 4 showed the morphology of B. cereus ATCC 11778 after treatment with 2× MIC level of the G-Fr2, R-Fr2 and B-Fr1 fractioned extracts. Untreated control cells appeared undamaged (Fig. 4a), while the B. cereus ATCC 11778 cells treated with antibiotic tetracycline, cell lysis or bacterial cell cavities was observed (Fig. 4b). The B. cereus ATCC 11778 cell shape was abnormal when compared with their original size (Fig. 4c-e). Moreover, some other cells appearing collapsed were observed in the G-Fr2 fractioned extract treatment (Fig. 4d).
DISCUSSION
Attractive in biological activities of plant extracts have focused according to their safety for using on human health. From the past until now, natural products, mainly plants have been used in traditional medicine28. The plants composed of several phytochemicals that possess various biological effects29. In this study, the methanolic extracts of different growth stage wild grape seeds were fractionated via silica gel column chromatography. The variable yields of the fractionated extracts were obtained. This may be affected from polarity of the mobile phase using for fractionation of the extract30. Plants are rich in phytochemicals, which widely used as medicinal remedies31. Besides biological activities, anti-α-glucosidase activity was chosen as like as anti-tyrosinase. The reason for choosing these two enzymes are searching for plants materials could be helped to protect diabetes and as cosmetics ingredient. The results indicated that the wild grape seed extracts in all growth stages have a high potential for α-glucosidase inhibition activity. The obtained results were in agreement with previous reports about grape seed32 and skin33 extracts that showed higher α-glucosidase inhibition activity than acarbose. On the other hand, similar potential activity to peel extract of Citrus mitis Blanco (IC50 = 870 μg mL–1)34 and Hawthorn berry seed extract (IC50 = 870 μg mL–1)35 on the inhibition of tyrosinase activity. Many reports indicated that natural products could be inhibited various types of micro-organisms. These alterations and antibacterial activity against bacterial pathogens may be due to the phytochemical constituent involving total phenolic and flavonoid compounds that present in wild grape fruit36. Phenolic compounds play an essential role in inhibiting bacterial growth by disrupting the bacterial cytoplasmic membrane, then causing a change in membrane permeability and finally causing leakage of constituents such as; proteins, nucleic acids and inorganic ions37. Flavonoids could inhibit bacterial pathogens by many actions such as; inhibit of DNA gyrase, inhibit cytoplasmic membrane function and inhibit of energy metabolism38.
In the future study, characterization and quantification of phenolic components in each sub-fraction and in vivo assay for biological activity were performed to obtain more information on the active compounds containing in the fractionated extracts of wild grape seeds.
CONCLUSION
This study indicated that the fractionated extracts of the immature (G-Fr2) wild grape seeds have significant potent α-glucosidase inhibition activity and has modulate tyrosinase inhibition activity. The G-Fr2 sub-fraction showed bacteriostatic activity and caused affection on abnormal shape and size of bacterial cells.
SIGNIFICANCE STATEMENT
This study discovers the enzyme inhibition and antimicrobial activities of the fractionated extracts of wild grape seed that can be beneficial for use as active ingredients in health supplement products or cosmetics. This study will help the researcher to uncover the critical areas of natural products from local wisdom that many researchers were not able to explore. Thus, a new information on biological activities of wild grape seed fractionated extracts may be arrived at.
ACKNOWLEDGMENT
This research was financially supported by Mahasarakham University (Grant year 2019). The authors would like to thank the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Thailand for partial financial support.