2014 Volume 37 Issue 7 Pages 1119-1123
Persimmon, a deciduous tree of the family Ebenaceae, is found throughout East Asia and contains high levels of tannins. This class of natural compounds exhibit favorable toxicity profiles along with bactericidal activity without the emergence of resistant bacteria, suggesting potential medical applications. Consistent with these observations, persimmon leaves show antibacterial activity. However, the mechanism of persimmon antibacterial activity remains unknown. In the present work, we demonstrate that the antibacterial activity of persimmon reflects the generation of reactive oxygen from tannins. The identification and quantification of reactive oxygen generated from persimmon and the level of antibacterial activity were determined.
Persimmon (Diospyros kaki THUNB.) is a deciduous tree of the family Ebenaceae that is endemic to East Asia. In Japan, persimmon fruit is eaten raw or in processed form. Estimates suggest that there are more than 1000 varieties of persimmon, and cultivars can be divided into either of two classes, sweet and astringent. Both kinds are known to contain high levels of tannins. Amounts of these compounds can be further enhanced by fermentation and aging of juice obtained from immature persimmons. This persimmon juice is widely used for waterproofing; for reinforcing of other materials such as wood, paper, cloth, and fishnet; for food preservation; and as a colorant or dye. Persimmon fruit and leaves also contain high levels of vitamin C, vitamin A, and potassium, and often are used in health teas purported to provide vascular and hemostatic benefits.
In recent years, persimmon has been the focus of attention for potential medicinal applications for prevention of cancer,1) viral and bacterial infectious diseases,2,3) and dental caries.4–6) Based on their antimicrobial properties, persimmon leaf-based products have been incorporated into athlete’s foot socks and soap, and persimmon leaves have been used as a sushi ingredient. At present, since natural ingredients show no emergence of resistant bacteria and favorable safety profiles, further medical applications are expected. However, the mechanism of antimicrobial activity for persimmon leaves remains poorly characterized. In this paper, we demonstrate that the antibacterial activity of persimmon reflects the generation of reactive oxygen from tannin. The identity and quantity of reactive oxygen species generated by persimmon were determined.
Reagents were obtained from the following suppliers: hydrogen peroxide 30% and iron(II) sulfate heptahydrate from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); bis(2,4,6-trichlorophenyl) oxalate (TCPO) from Tokyo Chemical Industries Inc. (Tokyo, Japan); 8-anilinonaphthalene sulphonate ammonium salt (ANS) from MERCK (Germany); diethyrentriamine penta acetic acid (DTPA) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) from Dojindo Laboratories (Kumamoto, Japan); catalase (from Bovine liver) from Roche (Tokyo, Japan); bovine serum albumin (BSA) from Sigma. All other chemicals were obtained as reagent-grade.
The breed of persimmon used as reagents are shown in Table 1.
The bacteria used for antimicrobial test were as follows: Escherichia coli the American Type Culture Collection (ATCC) 10798 (E. coli), Staphylococcus aureus ATCC6538 (S. aureus), Bacteroides thetaiotaomicron ATCC29148 (B. thetaiotaomicron), Campylobacter sputorum biovar. sputorum Centers for Disease Control and Prevention (CDC) 43563 (C. sputorum), Enterococcus faecalis Japan Collection of Microorganisms (JCM) 5803 (E. faecalis), Streptococcus salivarius ssp. thermophilus JCM20026 (S. thermophilus), Lactobacillus casei JCM1134 (L. casei), Bifidobacterium longum ssp. infantis JCM1222 (B. longum).
Reactive oxygen generated from persimmon was determined by the peroxalate chemiluminescent method.
Standard Sample: Hydrogen peroxide (H2O2) was diluted to 1.0×10−3–10−6 mol/L with 0.1 mol/L carbonate buffer (pH 8.0). The resulting peroxide solutions were used to generate a standard curve for sample measurement.
I) Fresh persimmon (fruit, peel, stem), dried persimmon (fruit, stem), and persimmon leaves were finely cut into little pieces (about 2 mm2) or grind to mush using a mortar with pestle prior to extraction.
II) Twenty milligram of sample was suspended in 1 mL distilled water, heated for 10 min at 100°C, and cooled. Then, the sample was filtered on a chromatdisk 13A (0.45 µm).
III) One hundred microliters of filtrate (or hydrogen peroxide standard) were mixed with 900 µL of 0.1 mol/L carbonate buffer (pH 8.0), and incubated for 30 min at 37°C to generate reactive oxygen. For effect of catalase, 10 µL of 2 mg/mL catalase was added to persimmon extract filtrate, which was then incubated under the same conditions. A blank solution was prepared in a similar manner without persimmon.
Prior to the assay, an ANS solution was prepared by dissolving ANS (20 mg) and BSA (0.1 g) in 100 mL of 0.2 mol/L barbital buffer (pH 9.0). This ANS solution was stored at 4°C before use.
Thus, 100 µL each of sample solution, 100 µL ANS solution, and 200 µL of 5 mmol/L TCPO in ethyl acetate solution were mixed, after 15s the luminescent intensity was then measured for 10 s using Luminescence Reader BLR-301 (ALOKA, Tokyo, Japan).
The measurements were performed in triplicate.
Hydrogen peroxide used as a standard was analyzed according to ESR method using the Fenton reaction; after hydroxyl radicals generated with the Fenton reaction were captured with DMPO spin trap agents (5,5-dimethyl-1-pyppoline-N-oxide), its spectrum was measured by ESR spectrometer FR30EX (JEOL, Tokyo, Japan).
1.0×10−3 mol/L of hydrogen peroxide was used to obtain standard spectrum of OH-DMPO aduct.
Preparation of Bacterial Suspension: Cell cultures were harvested by centrifugation (3000×g, 15 min). The final concentration of cells was 1.0×106 colony-forming units (CFU)/mL by using phosphate buffer (pH 7.0) if there is not a mention.
Sample solution (155 µL) and bacterial suspension (845 µL) were mixed and incubated for 18 h at 37°C. For the controls, the sample solution was replaced by an equivalent volume of buffer solution (negative control); catalase pre-treated sample solution (catalase effect test); or catalase pre-treatment buffer solution (control for catalase effect test).
Following incubation, the sample-bacteria mixtures were subjected to 10-fold serial dilutions and then spread or spotted to agar medium to determine growth, as described below.
Antimicrobial activity was measured using the agar dilution method and agar spot tests, as described below.
The number of viable cells of E. coli and S. aureus was forming units. Samples were subjected to 10-fold serial dilution to 1.0×10−9. Aliquots (100 µL/dilution) were spread to agar plates and cultured under appropriate conditions. Cultured by three plates per each dilution, number of viable cells was calculated from the dilution factor and the average number of colonies.
For other bacterial strains, the growth of colonies was observed with the agar spot method. Samples were subjected to 10-fold serial dilution to 1.0×10−4. Aliquots (5 µL) were spotted to agar plates and then cultured under appropriate conditions.
Colony density on the plates was scored as follows: +++, dense growth; ++, density reduced compared to control, but too numerous to count; +, number of colonies can be counted: −, no colonies.
We have found that the catechin in green tea shows the antibacterial activity, the effect is caused by hydrogen peroxide generated from catechin.9) In this report, we have studied the mechanism and antimicrobial activity of persimmon in the same way.
The validations of this method were studied in advance, the results was as follows; standard curve for hydrogen peroxide was linear line between from 1×10−6 to 1×10−3 mol/L, CV% (n=5) was from 0.8% to 2.3%, mean=1.5%, detection limit was 1×10−6 mol/L (100 pmol/assay) as blank+3SD value. Accuracy of this method was confirmed by dilution curve experiment and recovery test of persimmon sample prepared with addition of hydrogen peroxide. Persimmon sample containing high concentration of hydrogen peroxide was diluted by H2O, such as ×1, ×10, ×100, ×1000 fold and hydrogen peroxide was measured using standard curve. The regression curve between concentration of hydrogen peroxide and dilutions showed straight line of correlation coefficient value r2=0.999. In addition, recovery of hydrogen peroxide (1.0×10−4 and 1.0×10−5 mol/L as final concentration at 20 mg/mL) added to fruit of persimmon was measured using standard curve of hydrogen peroxide. The recovery % showed that addition of 1.0×10−4 and 1.0×10−5 mol/L were 97.2% (CV=4.5%) and 107.0% (CV=5.1%), respectively. These results show that hydrogen peroxide in persimmon can be measured by peroxalate chemiluminescence method.
The chemiluminescent intensity of persimmon samples (leaves, fruit, stem at 20 mg/mL) were measured according to the method described in “Determination of Reactive Oxygen Generated from Persimmon.”
Persimmon was collected in June 2009, its degree of maturation was a blue-green before ripening. The results are shown in Table 2. S/N ratio was calculated using the signal (S) of chemiluminescence obtained from the sample extract and the noise (N) using distilled water. Strong chemiluminescent intensity was observed in leaf, with lower intensity seen in stem of persimmon. On the other hand, little chemiluminescence was detected from the fruit. When catalase which would decompose hydrogen peroxide was added to these samples, the chemiluminescent intensity was suppressed. These results indicated that the chemiluminescent intensity from the persimmon is generated from the hydrogen peroxide.
For each sample, 20 mg tissue was extracted with 1 mL of extraction solution.
In order to confirm whether the chemiluminescence generated from persimmon leaf extract is unique for the persimmon, we compared the intensity to that of azalea leaf found in some roadside trees. Leaves of persimmon and azalea were gathered from trees in Tokyo during June of 2009.
The results showed that persimmon leaves and azalea leaves are 6.2×10−5 mol/mL and 7.2×10−6 mol/mL as hydrogen peroxide, respectively. Persimmon leaf extract showed higher chemiluminescent intensity than azalea leaf extract. It is thought that this difference of chemiluminescent intensity depended on the tannin content in the leaves of azalea and persimmon.
Reactive oxygen was analyzed in detail by ESR method. Figure 1 shows the results of ESR measurements that were carried out based on a combination of methods using the Fenton reaction and DMPO spin trap reagent. When the hydrogen peroxide was assayed by the spin trap method, a typical ESR spectrum (with peak height ratios of 1 : 2 : 2 : 1) was obtained, as shown in Fig. 1. When an extract of persimmon leaf was assayed instead of hydrogen peroxide, a similarly peak pattern was observed; these peaks disappeared upon addition of catalase. Therefore, we hypothesize that reactive oxygen generated from persimmon takes the form of hydrogen peroxide.
The amount of hydrogen peroxide in the sample extracted from persimmon was determined from standard curve using a hydrogen peroxide as standard.
For experiments assaying the generation of hydrogen peroxide and luminescence measurement, 0.1 mol/L carbonate buffer (pH 8.0), which corresponds to the pH in the intestine, was used. Next, we measured the amount of hydrogen peroxide for 100 mg/mL persimmon (pericarp, fruit, stem) picked from different sources (localities) which were commercially-supplied.
At the site of the persimmon, there are differences depending on the variety, and the stem showed 2.8×10−6–4.8×10−5 mol/L of hydrogen peroxide.
On the other hand, hydrogen peroxide for the fruit and pericarp was as low as 1.1×10−6–1.1×10−5 mol/L. Even for samples derived from the same breed, persimmon from different origins (difference sources) showed different concentrations of hydrogen peroxide. These results are shown in Table 3.
The dry persimmon yielded hydrogen peroxide concentrations as low as 7.8×10−4–1.3×10−5 mol/L, values that were reduced compared with fresh persimmon. This difference between fresh and dried persimmon presumably reflects differences in water content, which would affect the concentration of other components, such as vitamin C.
The above studies revealed that persimmon extract generates hydrogen peroxide, with highest levels obtained from persimmon leaves. Therefore, the antibacterial effect of hydrogen peroxide generated from persimmon leaf was tested using intestinal bacteria.
In the agar dilution assay, persimmon leaf extract, which hydrogen peroxide is corresponding to 4.8–5.7×10−4 mol/L hydrogen peroxide, was examined for antibacterial activity and its inhibition by the addition of catalase. We observed 96.6% growth inhibition against E. coli at 8.7×105 CFU/mL and 98.2% growth inhibition against S. aureus at 2.2×106 CFU/mL following treatment with the extract of persimmon leaf. When persimmon leaf extract was treated with catalase, the number of E. coli was restored to 3.4×105 CFU/mL and S. aureus was 4.1×105 CFU/mL. This result suggested that the growth inhibitory effects of persimmon leaf extract against E. coli and S. aureus are due to hydrogen peroxide.
We examined the antibacterial activity of the persimmon leaf extract against B. thetaiotaomicron, C. sputorum, E. faecalis, B. longum, L. casei and S. thermophilus. The results are shown in Table 4. When various bacteria were exposed to persimmon leaf extract (harboring hydrogen peroxide at >5.7×10−4 mol/L), the growth of C. sputorum, S. mutans, and B. thetaiotaomicron were inhibited. This growth inhibition was not observed when catalase was added to the persimmon leaf extract. Therefore, we propose that hydrogen peroxide generated from the leaves of persimmon is primarily responsible for antimicrobial activity against these bacteria.
+++: High antibacterial activity++: Middle antibacterial activity+: Low antibacterial activity
In contrast, growth inhibition by persimmon leaf extract was not observed for E. faecalis, L. casei, or B. longum. This result is consistent with the known low susceptibility of E. faecalis, L. casei, and B. longum to hydrogen peroxide. For S. thermophilus, antibacterial activity was observed at lower hydrogen peroxide concentrations extracted from persimmon leaf than the concentration obtained in the experiment directly testing the antimicrobial activity of hydrogen peroxide. Based on this observation, we hypothesize that an additional component of the persimmon leaf extract is involved in the antibacterial activity against S. thermophilus.
Subsequently, when the extract of persimmon stem (corresponding to hydrogen peroxide 6.8–6.9×10−4 mol/L) was tested on four kinds of bacteria (C. sputorum, B. thetaiotaomicron, B. longum, L. casei), the growth of C. sputorum and B. thetaiotaomicron were inhibited. This growth inhibition was not observed when catalase was added to the persimmon stem extract. Therefore, we propose that hydrogen peroxide generated from the stem of persimmon is primarily responsible for antimicrobial activity against C. sputorum and B. thetaiotaomicron. In contrast, growth inhibition of B. longum and L. casei was not observed with persimmon stem extract.
This result is consistent with the known low susceptibility of B. longum and L. casei to hydrogen peroxide.
In this study, we speculated that the known deodorant, antiseptic, and antibacterial activities of persimmon are all the result of reactive oxygen species. Therefore the identification and quantification of reactive oxygen were examined by means of ESR and peroxalate chemiluminescent method. As a result, it was found that hydrogen peroxide is generated from the persimmon.
The observed concentration of hydrogen peroxide in persimmon extract corresponded to approximately 1.0×10−4 mol/L, a level equivalent to the reactive oxygen species generated from herbal medicines known to have effects in intestinal regulators and anti-diarrheals.
Therefore, the effects of persimmon on eight intestinal bacteria were examined. A control experiment was carried out in a similar manner using a standard hydrogen peroxide solution. As a result, it was found that hydrogen peroxide is generated from persimmon tissues, and the bactericidal activity of persimmon extracts correlated with bacterial sensitivity to hydrogen peroxide.
L. casei, E. faecalis, S. thermophilus, and B. longum, which are known to contribute to the maintenance of homeostasis in the gut, exhibited low susceptibility to hydrogen peroxide. E. coli (associated with diarrhea), S. aureus and C. sputorum (associated with food poisoning), and B. thetaiotaomicron (associated with opportunistic infections) exhibited greater susceptibility to hydrogen peroxide. Notably, cultures of E. coli and S. aureus were sterilized by exposure to persimmon leaf extract. E. faecalis, L. casei, and B. longum, which have lower susceptibility to hydrogen peroxide, were less susceptible to the bactericidal activity of the extracts of persimmon stem and leaf.
On the other hand, the extract of persimmon leaf exhibited more potent antibacterial activity against S. thermophilus than attributable solely to hydrogen peroxide, suggesting that another component of persimmon leaf extract contributes to antibacterial activity against this species.
Direct medical efficacy of persimmon itself is difficult to explain. However, we propose that hydrogen peroxide generated from the persimmon affects the balance of bacteria in the intestine, in turn altering intestinal regulation. Given that persimmon is grown all over the country, the use of a tissue (leaf) that is not itself edible is expected to provide a useful resource.