In our previous study, we have found that persimmon, guava, and sweetsop owned considerably high antioxidant activity and contained high total phenolic contents as well. In order to further supply information on the antibacterial and antioxidant activity of these three tropic fruits, they were extracted by 80% methanol. We then examined the extractions about their phenolic compounds and also studied the extractions and phenolic contents about their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against twelve targeted pathogens including 8 standard strains (
The discovery of antimicrobial drugs was a monumental event in human medicine history. In recent years, however, with the widespread use of antibiotics, more and more adverse factors, such as antimicrobial resistance, have attracted the attention of researchers, pharmaceutical company, and even common people. In addition, the increasing worries about negative effects of synthetic food antioxidants and growing good wishes for pursuit of better healthy life prompt scientists to research to replace synthetic medicines and antioxidants.
It is pleasing for scientists to be equipped with a new option from studying of natural plant in this field. Actually, in many countries, such as China, India, and Japan, plants have been used to enhance health and even as medicinal cure for thousands of years.
In our previous study, we tested 62 fresh fruits that were sold in Guangzhou market and found that persimmon, guava, and sweetsop had very high antioxidant activity and high total phenolic contents [
Persimmon is a native fruit of China. The persimmon tested grows in Conghua, Guangzhou. It is cylindrical in shape and looks like chicken heart. It was reported that persimmon could be used for treatment of diarrhea, dry coughs, and hypertension [
Guava is a tropic and subtropic fruit. The most prevailing guava in Guangzhou has green peel and white pulp, whose shape is sphere and oval. It was reported that guava could benefit diabetes, caries, wounds, diarrhea, inflammation, and hypertension. The extracts from guava showed antispasmodic, anti-inflammatory, anticancer, antioxidant, and hypoglycemic effect [
Sweetsop was native to tropic America and introduced to China about 400 years ago. It has much medicinal use such as against oxidative and hepatic damage, tumors, diabetes, epilepsy, cardiac problems, constipation, and ulcers [
Fruits and vegetables had many kinds of health-promoting compounds such as high concentrations of phenolic compounds, vitamins, fiber, and minerals, among which phenolic compounds might protect people from some chronic diseases, although they were not essential for life sustaining [
Phenolic compounds standards, including gallic acid, (+)-catechin, (−)-epicatechin, chlorogenic acid, caffeic acid, ferulic acid, quercetin, luteolin, kaempferol, TPTZ (2,4,6-tri(2-pyridyl)-s-triazine/tripyridyltriazine), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), and ABTS (2,2-azino-di-(3-ethylbenzothiazoline-sulfonic acid)), were purchased from Sigma-Aldrich. All the chemicals were of analytical grade or better. Mueller-Hinton Broth and nutrient agar were purchased from Guangdong Huankai Microbial SCI. & TECH. Co. Ltd. (China).
Fresh and mature fruits were selected and bought in Guangzhou market. The samples for antioxidant study could be fresh or dry, but those for antibacterial study and HPLC analysis should only be dried. The fruits were washed with deionized water before being peeled and cut into slices. Then they were put into the oven to be dried at 60°C for 48 hours and ground before being passed through a 40-mesh sieve to produce dry samples. Around 2.0 g dry samples or slurry of edible portion from fresh samples was ultrasonic extracted with 20 mL methanol-water (80 : 20, v/v) at room temperature for 30 min, then refluxed, and collected into 20 mL. The collection was centrifuged at 12000/min, 4°C for 10 min. The supernatant from dried samples was used for the evaluation of HPLC analysis, and those from dried and fresh samples were used for antioxidant capacity study. In order to get samples with high concentration for antibacterial study, about 50.0 g dried samples were ultrasonic extracted with methanol-water (80 : 20, v/v) at room temperature for 60 min. The extraction was filtered and the collected fluid was concentrated to 5.0 g/mL for sweetsop and 2.5 g/mL for persimmon and guava. All the samples were stored at 4°C until being used for experiment. To the phenolic compounds detected in the fruit, 5 mg/mL solution of each standard substance was used for antibacterial study.
Separation was carried out using Diamosil (R) C18 column (
Eight standard strains included four Gram-positive bacteria, that is,
Twofold microdilution broth method [
200
The FRAP assay was carried out according to the literature with slight modifications [
The TEAC assay was determined according to the method with minor modifications [
All the experiments were performed in triplicate, and the results were expressed as mean ± SD (standard deviation). Statistical analysis was performed using Excel 2007 and SPSS 13.0.
There were 8 phenolic compounds detected in this study, only caffeic acid excluded. The contents of different phenolic compounds in persimmon, guava, and sweetsop were showed in Table
Content of phenolic compounds in the three fruits (mg/Kg).
Number | Phenolic compounds | Persimmon | Guava | Sweetsop |
---|---|---|---|---|
1 | Gallic acid | 377.11 ± 18.47 | 99.15 ± 1.62 | 256.52 ± 14.33 |
2 | (+)-Catechin | 125.29 ± 9.61 | 391.93 ± 15.08 | 144.06 ± 7.90 |
3 | (−)-Epicatechin | 58.43 ± 4.70 | ||
4 | Chlorogenic acid | 43.56 ± 3.72 | ||
5 | Caffeic acid | |||
6 | Ferulic acid | 186.05 ± 17.02 | ||
7 | Quercetin | 102.65 ± 4.96 | 122.23 ± 10.14 | 73.64 ± 2.43 |
8 | Luteolin | 48.97 ± 0.86 | 51.39 ± 3.44 | 73.12 ± 3.36 |
9 | Kaempferol | 29.16 ± 1.16 | 38.06 ± 2.00 |
Previous study about persimmon showed that gallic, protocatechuic, caffeic, p-coumaric, and ferulic acids and myricetin were found, among which gallic acid was the most abundant phenolic compound (287.5 ± 5.31
For the 8 standard strains, the three fruits showed different antimicrobial activities. As seen from Table
MIC, MBC, and MBC/MIC results of the fruits and phenolic compounds to 8 standard strains.
Antibacterial properties | Phenolic compounds | Gram-positive bacteria | Gram-negative bacteria | ||||||
---|---|---|---|---|---|---|---|---|---|
| | | | | | | | ||
MIC (mg/mL) | Persimmon | 312.50 | 625.00 | 625.00 | 1250.00 | 1250.00 | 1250.00 | 1250.00 | 625.00 |
Guava | 1250.00 | 312.50 | 78.13 | 1250.00 | 625.00 | 625.00 | 625.00 | 312.50 | |
Sweetsop | 1250.00 | 1250.00 | 312.50 | 2500.00 | 2500.00 | 2500.00 | 2500.00 | 2500.00 | |
Gallic acid | 0.63 | 2.50 | 0.63 | 5.00 | 2.50 | 2.50 | 2.50 | 2.50 | |
(+)-Catechin | 1.25 | 1.25 | 1.25 | 2.50 | 5.00 | 2.50 | 2.50 | 2.50 | |
(−)-Epicatechin | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 1.25 | |
Chlorogenic acid | 5.00 | 2.50 | 0.63 | 5.00 | 5.00 | 2.50 | 2.50 | 2.50 | |
Ferulic acid | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | |
Quercetin | 2.50 | 2.50 | 1.25 | 2.50 | 2.50 | 2.50 | 2.50 | 2.50 | |
Luteolin | 0.63 | 5.00 | 0.63 | 5.00 | 2.50 | 2.50 | 2.50 | 5.00 | |
Kaempferol | 2.50 | 2.50 | 1.25 | 2.50 | 2.50 | 2.50 | 1.25 | 1.25 | |
| |||||||||
MBC (mg/mL) | Persimmon | 312.50 | 625.00 | 625.00 | 2500.00 | 1250.00 | 1250.00 | 1250.00 | 625.00 |
Guava | 2500.00 | 312.50 | 156.25 | 2500.00 | 1250.00 | 625.00 | 625.00 | 312.50 | |
Sweetsop | 2500.00 | 1250.00 | 2500.00 | >5000.00 | 5000.00 | >5000.00 | 5000.00 | 2500.00 | |
Gallic acid | 0.63 | 2.50 | 0.63 | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 | |
(+)-Catechin | 1.25 | 1.25 | 1.25 | >5.00 | 5.00 | 2.50 | 2.50 | 2.50 | |
(−)-Epicatechin | 2.50 | >5.00 | 5.00 | 5.00 | 2.50 | 2.50 | 2.50 | >5.00 | |
Chlorogenic acid | 5.00 | 2.50 | >5.00 | >5 | 5.00 | 5.00 | 5.00 | 2.50 | |
Ferulic acid | 5.00 | 2.50 | 5.00 | 5.00 | >5.00 | 5.00 | 5.00 | 5.00 | |
Quercetin | 5.00 | 2.50 | 2.50 | 5.00 | 5.00 | 2.50 | 5.00 | 5.00 | |
Luteolin | 2.50 | 5.00 | 1.25 | 5.00 | >5.00 | 5.00 | 5.00 | 5.00 | |
Kaempferol | 5.00 | 2.50 | 1.25 | 5.00 | 5.00 | 5.00 | 5.00 | 5.00 | |
| |||||||||
MBC/MIC | Persimmon | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 1 |
Guava | 2 | 1 | 2 | 2 | 2 | 1 | 1 | 1 | |
Sweetsop | 2 | 1 | 8 | / | 2 | / | 2 | 1 | |
Gallic acid | 1 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | |
(+)-Catechin | 1 | 1 | 1 | / | 1 | 1 | 1 | 1 | |
(−)-Epicatechin | 1 | / | 2 | 2 | 1 | 1 | 1 | / | |
Chlorogenic acid | 1 | 1 | / | / | 1 | 2 | 2 | 1 | |
Ferulic acid | 2 | 1 | 2 | 2 | / | 2 | 2 | 2 | |
Quercetin | 2 | 1 | 2 | 2 | 2 | 1 | 2 | 2 | |
Luteolin | 4 | 1 | 2 | 1 | / | 2 | 2 | 1 | |
Kaempferol | 2 | 1 | 1 | 2 | 2 | 2 | 4 | 4 |
Note: “/” means the MBC/MIC result cannot be provided because MBC was greater than the original concentration.
However, it is more interesting to get the data related to the 4 multidrug-resistant strains (Table
MIC, MBC, and MBC/MIC results of the fruits and phenolic compounds to 4 multidrug-resistant bacteria (mg/mL).
Samples | MRAS | ESBL | Carbapenems-resistant | Multidrug-resistant | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
MIC | MBC | MBC/MIC | MIC | MBC | MBC/MIC | MIC | MBC | MBC/MIC | MIC | MBC | MBC/MIC | |
Persimmon | / | / | / | / | / | / | / | / | / | / | / | / |
Guava | 312.50 | 312.50 | 1 | 625.00 | 1250.00 | 2 | 312.50 | 312.50 | 1 | 312.50 | 312.50 | 1 |
Sweetsop | 5000.00 | 5000.00 | 1 | / | / | / | / | / | / | / | / | / |
Gallic acid | 2.50 | 2.50 | 1 | 2.50 | 5.00 | 2 | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 |
(+)-Catechin | 1.25 | 2.50 | 2 | 2.50 | 2.50 | 1 | 2.50 | 5.00 | 2 | 2.50 | 2.50 | 1 |
(−)-Epicatechin | 2.50 | 2.50 | 1 | 2.50 | 5.00 | 2 | 2.50 | 5.00 | 2 | 2.50 | 2.50 | 1 |
Chlorogenic acid | 2.50 | 5.00 | 2 | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 |
Ferulic acid | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 |
Quercetin | 2.50 | 2.50 | 1 | 5.00 | 5.00 | 1 | 1.25 | 2.50 | 2 | 1.25 | 2.50 | 2 |
Luteolin | 2.50 | 2.50 | 1 | 2.50 | 2.50 | 1 | 1.25 | 2.50 | 2 | 2.50 | 2.50 | 1 |
Kaempferol | 2.50 | 5.00 | 2 | 2.50 | 2.50 | 1 | 5.00 | 5.00 | 1 | 2.50 | 2.50 | 1 |
Note: “/” means the MIC, MBC, and MBC/MIC result cannot be provided.
Other studies found that persimmon leaves extract showed antibacterial activity and inhibition against
As far as the ration of MBC to MIC was concerned, bactericidal effect was defined as MBC/MIC ratio ≤4, while bacteriostatic effect was determined as MBC/MIC >4 [
We also studied the antibacterial activity of phenolic compounds detected in the three fruits. In Table
Another study suggested that antibacterial activity was in a positive correlation with the amount of phenolic compounds in the plant [
In our study, all the phenolic compounds possessed bactericidal activity against the test strains, when MBC was no less than 5 mg/mL. The antibacterial activity of these phenolic contents confirmed by our study should involve many mechanisms, including alteration of the physicochemical properties of the plasma membrane, pore formation, inhibition of DNA gyrase and nucleic acid synthesis, and toxicity through the generation of hydrogen peroxide [
FRAP assay is a widely used method for evaluating antioxidant capacity. However, most known natural antioxidants are not single-functional, and so it is required to introduce different antioxidant activity assessments, which can consider various mechanisms of antioxidant action [
The regression equation of FeSO4 standard curve in FRAP assay was
As seen from the data in Table
FRAP and TEAC results of the persimmon, guava, and sweetsop.
Fruit | FRAP ( | TEAC ( | |
---|---|---|---|
Fresh | Persimmon | 12.59 ± 0.55 | 5.10 ± 0.08 |
Guava | 26.07 ± 0.32 | 9.13 ± 0.42 | |
Sweetsop | 34.37 ± 0.49 | 21.30 ± 0.92 | |
| |||
Dried | Persimmon | 51.50 ± 1.51 | 20.14 ± 0.97 |
Guava | 95.25 ± 3.99 | 31.91 ± 2.54 | |
Sweetsop | 71.54 ± 2.69 | 37.32 ± 0.75 |
It should be pointed out that the results from FRAP for fresh samples this time (12.59 ± 0.55, 26.07 ± 0.32, 34.37 ± 0.49
FRAP values and TEAC values of phenolic compounds detected in the study were shown in Table
FRAP and TEAC results of phenolic compounds.
Phenolic compounds | FRAP (mmol Fe2+/g) | TEAC (mmol Trolox/g) |
---|---|---|
Gallic acid | 35.45 ± 1.46 | 23.56 ± 1.17 |
(+)-Catechin | 13.41 ± 0.26 | 12.64 ± 0.18 |
(−)-Epicatechin | 13.91 ± 0.12 | 13.00 ± 0.06 |
Chlorogenic acid | 11.33 ± 0.22 | 4.10 ± 0.04 |
Ferulic acid | 15.56 ± 0.36 | 13.19 ± 0.07 |
Quercetin | 25.96 ± 0.06 | 13.32 ± 0.57 |
Luteolin | 14.87 ± 0.60 | 5.22 ± 0.60 |
Kaempferol | 15.90 ± 0.14 | 5.32 ± 0.02 |
Note: the result of FRAP and TEAC about ascorbic acid tested in this study is 12.66 ± 0.13 mmol
Phenolics owned perfect chemistry for antioxidant activity because they had high reactivity to donate hydrogen or electron and were capable of chelating metal ions [
In addition, a highly positive correlation (
The three kinds of tropical fruits, including persimmon, guava, and sweetsop, possessed antioxidant and antibacterial activity that supported the possibility of developing the fruits into new natural resource food and functional food as well as new natural antimicrobial agent and food preservatives. Moreover, phenolic compounds detected in the fruits could be used as a potential natural antibacterial agent and antioxidant. Further studies should be carried out to evaluate in vivo activities so that potential clinical drug and health products could be developed.
The authors declare that they have no competing interests.
This research was supported by Science and Technology Scheme of Liwan District, Guangzhou (no. 20141215057).