# Piper Essential Oils Inhibit Rhizopus oryzae Growth, Biofilm Formation, and Rhizopuspepsin Activity

Authors: Catia A. Almeida; Mariana M. B. Azevedo; Francisco C. M. Chaves; Marcelo Roseo de Oliveira; Igor A. Rodrigues; Humberto R. Bizzo; Paola E. Gama; Daniela S. Alviano; Celuta S. Alviano
Journal: Canadian Journal of Infectious Diseases and Medical Microbiology
 (2018)

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## Abstract

Piper is the largest genus of the Piperaceae family. The species of this genus have diverse biological activities and are used in pharmacopeia throughout the world. They are also used in folk medicine for treatment of many diseases in several countries including Brazil, China, India, Jamaica, and Mexico. In Brazil, Piper species are distributed throughout the national territory, making this genus a good candidate for biological activity screening. During our studies with Piper essential oils, we evaluated its activity against Rhizopus oryzae, the main agent of mucormycosis. The main compounds of seven Piper essential oils analyzed were Piper callosum—safrole (53.8%), P. aduncum—dillapiole (76.0%), P. hispidinervum—safrole (91.4%), P. marginatum—propiopiperone (13.2%), P. hispidum—γ-terpinene (30.9%), P. tuberculatum—(E)-caryophyllene (30.1%), and Piper sp.—linalool (14.6%). The minimum inhibitory concentration of Piper essential oils against R. oryzae ranged from 78.12 to >1250 μg/mL. The best result of total inhibition of biofilm formation was obtained with Piper sp. starting from 4.88 μg/mL. Considering the bioactive potential of EOs against planktonic cells and biofilm formation of R. oryzae could be of great interest for development of antimicrobials for therapeutic use in treatment of fungal infection.


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## Body

## 1. Introduction

Piper is the largest genus of the Piperaceae family. The species of this genus have diverse biological activities and are used in pharmacopeia throughout the world. They are also used in folk medicine for treatment of many diseases in several countries including Brazil, China, India, Jamaica, and Mexico. In Brazil, Piper species are distributed throughout the national territory. Among the aromatic flora of the Amazon region, there are more than a dozen species that provide essential oils that are used by the population for therapeutic purposes. The tea of the decoction of Piper hispidum leaves is useful for the treatment of malaria. Piper marginatum is used as a tonic, carminative, stimulant, diuretic, and sudorific agent against stomach, liver and gallbladder pain, toothaches, and snake and insect bites [1]. Regasini et al. [2] related trypanocidal activity of the Piper tuberculatum extract.Zygomycosis, also referred to as phycomycosis or mucormycosis, is an aggressive and rapidly progressive infection that primarily occurs in immunocompromised patients. Members of the generaRhizopus, Mucor, and Absidia are the organisms most commonly isolated from patients with zygomycosis. Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea are other zygomycetes that have been implicated in human diseases. Amphotericin B, as well as its lipid formulation, has been essential for treatment for several decades [3].The purpose of the present work was to evaluate the anti-Rhizopus oryzae activity of Piper aduncum, P. hispidinervum, P. callosum, P. hispidum, P. tuberculatum, P. marginatum, and Piper sp. essential oil of leaves.


## 2. Materials and Methods

### 2.1. Plant Material and Essential Oil Extraction

Plant material was obtained from EMBRAPA Experimental Farm, Amazonas, Brazil. A voucher of each specimen was deposited at Federal Agrotechnical School of Machado Herbarium (Table1). Leaves of Piper species were collected between 8 and 9 a.m., dried at room temperature, and coarsely ground into powder just before distillation. The oil was obtained by hydrodistillation in a modified Clevenger apparatus for 5 h [4].Table 1
Deposit number and deposit location of plant material.
Plant material
Deposit number
Deposit location
Name of herbarium
Piper aduncum
10,480
INPA1
INPA herbarium
Piper tuberculatum
6,797
IFAM2
EAFM herbarium
Piper hispidum
6,796
IFAM
EAFM herbarium
Piper marginatum
6,798
IFAM
EAFM herbarium
Piper callosum
6,794
IFAM
EAFM herbarium
Piper hispidinervum
IFAM
EAFM herbarium
Piper sp.
IFAM
EAFM herbarium
1National Institute of Amazonas Research; 2Federal Institute of Amazonas.


### 2.2. Essential Oil Analyses

Sample of eachPiper essential oil was analyzed in an Agilent 6890 N gas chromatograph fitted with a 5% diphenyl-95% dimethylpolysiloxane capillary column (DB-5MS, 30 m × 0.25 mm × 0.25 μm). The results were compared to data from the literature [5].


### 2.3. Antifungal Activity Assay

The antifungal activity ofPiper essential oils was evaluated against R. oryzae (UCP1506). The strain used belongs to the culture collection of the “Universidade Católica de Pernambuco,” located in the Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Brazil, NPCIAMB/UNICAP. The culture collection is registered in the WFCC.The microdilution broth method was used according to CLSI reference document M38-A [6] for filamentous fungi. Briefly, the cells were grown in RPMI-MOPS (pH 7.2) for 18 h at 30°C in the presence of different concentrations (1.22 to 1250 μg/mL) of each essential oil. Positive and negative growth controls were performed. Amphotericin B (Sigma) was used as a reference drug, and stock solution was made at 20 mg/mL in sterile distilled water. All experiments were performed in duplicate and repeated twice.In order to evaluate the fungicide/fungistatic properties ofPiper essential oils, a 10 μL aliquot was collected from the inhibited cultures and dropped on the surface of potato dextrose agar. The absence or presence of growth in the solid medium was evaluated after 48 h incubation period at 30°C.


### 2.4. Biofilm Formation

The influence ofPiper essential oils on biofilm formation was determined as described by Singh et al. [7]. Briefly, spores of R. oryzae were put in 96-well microtiter plate at 5 × 104 cells per mL in RPMI and treated with twofold serial dilution of each Piper essential oil. After incubation for 18 h at 30°C, the culture media was removed and the wells were washed twice with PBS 0.01 M and pH 7.2. Biofilms were stained with 200 µL of 0.1% safranin for 5 min. Then, the supernatants were removed, and the wells were washed twice with PBS. Finally, 200 µL of 30% glacial acetic acid was added to the microplates in order to elute safranine from the matrix. Biofilm formation was estimated by spectrophotometry (SpectraMax M5) at 490 nm.


### 2.5. Red Blood Cell Lysis Assay

The hemolytic activity was evaluated by Franca Rodrigues et al. [8] by mixing 80 μL of a 5% suspension of fresh human red blood cells (O+) in PBS with 20 μL of different concentrations of Piper sp. essential oil and incubating at 37°C for 1 h. The reaction was slowed by adding 200 μL of PBS, and the suspension was centrifuged (1000 g for 10 min). The supernatant was transferred to a 96-well plate, and cell lysis was quantified by spectrophotometrical measurement of absorbance at 540 nm, as previously described. The maximal lysis and blank control were obtained by replacing the extract sample with an equal volume of PBS or distilled water, respectively.


### 2.6. Rhizopuspepsin Inhibition

In order to evaluate a possible mode of action of thePiper essential oils, the inhibition of rhizopuspepsin (Sigma) activity was determined as previously described by Buroker-Kilgore and Wang with some modifications [9]. First, 59 μL of the rhizopuspepsin solution was mixed with 1 μL inhibitor, 20 μL BSA (1 mg/mL), and 20 μL buffer (pH 3.0). After 1 h incubation at 37°C, 100 μL of Bradford solution (0.025% Coomassie Blue G-250, 11.75% ethanol, and 21.25% phosphoric acid) previously diluted (1 : 1) was added. Negative control was performed by adding the substrate immediately after the incubation period. Finally, the plate was read on a spectrophotometer (SpectraMax M5) at 595 nm. One unit of enzyme activity was defined as the total enzyme that causes an increase of 0.001 in unit of absorbance under the conditions of the standard assay. The inhibitors tested were Piper essential oils (48 μg/mL) and 10 mM Pepstatin A (standard inhibitor).


### 2.7. Antioxidant Activity ofPiper spp. Essential Oils

The antioxidant activity was evaluated qualitatively [10, 11] by application of 0.5 μL of each essential oil and 7-hydroxycalamenene (as standard) on a plate of silica gel 60 F254 and eluted with hexane-ethyl acetate (9 : 1). The plates were treated with a 0.2% methanolic solution of DPPH and read just after spraying and after 45 min.


## 2.1. Plant Material and Essential Oil Extraction

Plant material was obtained from EMBRAPA Experimental Farm, Amazonas, Brazil. A voucher of each specimen was deposited at Federal Agrotechnical School of Machado Herbarium (Table1). Leaves of Piper species were collected between 8 and 9 a.m., dried at room temperature, and coarsely ground into powder just before distillation. The oil was obtained by hydrodistillation in a modified Clevenger apparatus for 5 h [4].Table 1
Deposit number and deposit location of plant material.
Plant material
Deposit number
Deposit location
Name of herbarium
Piper aduncum
10,480
INPA1
INPA herbarium
Piper tuberculatum
6,797
IFAM2
EAFM herbarium
Piper hispidum
6,796
IFAM
EAFM herbarium
Piper marginatum
6,798
IFAM
EAFM herbarium
Piper callosum
6,794
IFAM
EAFM herbarium
Piper hispidinervum
IFAM
EAFM herbarium
Piper sp.
IFAM
EAFM herbarium
1National Institute of Amazonas Research; 2Federal Institute of Amazonas.


## 2.2. Essential Oil Analyses

Sample of eachPiper essential oil was analyzed in an Agilent 6890 N gas chromatograph fitted with a 5% diphenyl-95% dimethylpolysiloxane capillary column (DB-5MS, 30 m × 0.25 mm × 0.25 μm). The results were compared to data from the literature [5].


## 2.3. Antifungal Activity Assay

The antifungal activity ofPiper essential oils was evaluated against R. oryzae (UCP1506). The strain used belongs to the culture collection of the “Universidade Católica de Pernambuco,” located in the Nucleus of Research in Environmental Sciences, Catholic University of Pernambuco, Brazil, NPCIAMB/UNICAP. The culture collection is registered in the WFCC.The microdilution broth method was used according to CLSI reference document M38-A [6] for filamentous fungi. Briefly, the cells were grown in RPMI-MOPS (pH 7.2) for 18 h at 30°C in the presence of different concentrations (1.22 to 1250 μg/mL) of each essential oil. Positive and negative growth controls were performed. Amphotericin B (Sigma) was used as a reference drug, and stock solution was made at 20 mg/mL in sterile distilled water. All experiments were performed in duplicate and repeated twice.In order to evaluate the fungicide/fungistatic properties ofPiper essential oils, a 10 μL aliquot was collected from the inhibited cultures and dropped on the surface of potato dextrose agar. The absence or presence of growth in the solid medium was evaluated after 48 h incubation period at 30°C.


## 2.4. Biofilm Formation

The influence ofPiper essential oils on biofilm formation was determined as described by Singh et al. [7]. Briefly, spores of R. oryzae were put in 96-well microtiter plate at 5 × 104 cells per mL in RPMI and treated with twofold serial dilution of each Piper essential oil. After incubation for 18 h at 30°C, the culture media was removed and the wells were washed twice with PBS 0.01 M and pH 7.2. Biofilms were stained with 200 µL of 0.1% safranin for 5 min. Then, the supernatants were removed, and the wells were washed twice with PBS. Finally, 200 µL of 30% glacial acetic acid was added to the microplates in order to elute safranine from the matrix. Biofilm formation was estimated by spectrophotometry (SpectraMax M5) at 490 nm.


## 2.5. Red Blood Cell Lysis Assay

The hemolytic activity was evaluated by Franca Rodrigues et al. [8] by mixing 80 μL of a 5% suspension of fresh human red blood cells (O+) in PBS with 20 μL of different concentrations of Piper sp. essential oil and incubating at 37°C for 1 h. The reaction was slowed by adding 200 μL of PBS, and the suspension was centrifuged (1000 g for 10 min). The supernatant was transferred to a 96-well plate, and cell lysis was quantified by spectrophotometrical measurement of absorbance at 540 nm, as previously described. The maximal lysis and blank control were obtained by replacing the extract sample with an equal volume of PBS or distilled water, respectively.


## 2.6. Rhizopuspepsin Inhibition

In order to evaluate a possible mode of action of thePiper essential oils, the inhibition of rhizopuspepsin (Sigma) activity was determined as previously described by Buroker-Kilgore and Wang with some modifications [9]. First, 59 μL of the rhizopuspepsin solution was mixed with 1 μL inhibitor, 20 μL BSA (1 mg/mL), and 20 μL buffer (pH 3.0). After 1 h incubation at 37°C, 100 μL of Bradford solution (0.025% Coomassie Blue G-250, 11.75% ethanol, and 21.25% phosphoric acid) previously diluted (1 : 1) was added. Negative control was performed by adding the substrate immediately after the incubation period. Finally, the plate was read on a spectrophotometer (SpectraMax M5) at 595 nm. One unit of enzyme activity was defined as the total enzyme that causes an increase of 0.001 in unit of absorbance under the conditions of the standard assay. The inhibitors tested were Piper essential oils (48 μg/mL) and 10 mM Pepstatin A (standard inhibitor).


## 2.7. Antioxidant Activity ofPiper spp. Essential Oils

The antioxidant activity was evaluated qualitatively [10, 11] by application of 0.5 μL of each essential oil and 7-hydroxycalamenene (as standard) on a plate of silica gel 60 F254 and eluted with hexane-ethyl acetate (9 : 1). The plates were treated with a 0.2% methanolic solution of DPPH and read just after spraying and after 45 min.


## 3. Results and Discussion

The average oil yield obtained was 0.65% (dry wt.). The compounds present in the essential oils fromPiper species used are shown in Table 2. Quantitative and/or qualitative variations were observed among samples of Piper.Table 2
Main components fromPiper spp. essential oils.
Area (%)
Peak
LRI calc
LRI lit
Identification
P. callosum
P. aduncum
P. hispidinervum
P. marginatum
P. hispidum
P. tuberculatum
Piper sp.
1
924
924
α-Thujene
0.1
0.1
0.1
2
931
932
α-Pinene
12.2
1.5
0.1
2.0
1.3
9.4
3
946
946
Canphene
0.4
0.1
0.1
4
971
969
Sabinene
3.0
0.1
5
975
974
β-Pinene
7.7
3.5
1.5
1.1
15.0
6
985
981
6-Methyl-5-hepten-2-one
0.6
7
989
990
Myrcene
0.6
0.1
1.3
1.2
8
1004
1002
α-Phellandrene
0.1
1.5
0.5
9
1009
1008
δ-3-Carene
0.2
11.3
0.4
10
1014
1014
α-Terpinene
0.7
0.2
14.4
11
1022
1022
p-Cimene
0.3
0.3
0.3
0.3
12.1
12
1025
1024
Limonene
0.7
0.4
0.2
1.6
13
1026
1025
β-Phellandrene
1.0
1.4
14
1028
1026
1,8-Cineole
3.7
15
1034
1032
(Z)-β-Ocimene
0.4
0.3
6.0
16
1044
1044
(E)-β-Ocimene
0.8
0.7
8.3
17
1055
1054
γ-Terpinene
1.8
0.3
30.9
18
1085
1086
Terpinolene
0.5
1.2
0.3
7.3
19
1098
1098
Linalool
0.3
0.2
1.1
14.6
20
1134
1135
trans-Pinocarveol
0.2
21
1152
1155
Isoborneol
0.1
22
1173
1174
Terpinen-4-ol
0.7
1.0
23
1182
1179
p-8-Cymenol
1.0
24
1187
1186
α-Terpineol
0.5
0.1
25
1193
1194
Myrtenol
0.1
26
1314
1285
Safrole
53.8
91.4
4.6
27
1332
1335
δ-Elemene
0.3
28
1370
1374
α-Copaene
0.5
0.5
4.8
0.5
1.3
29
1379
1387
β-Bourbonene
0.9
30
1385
1387
β-Cubebene
0.3
31
1387
1389
β-Elemene
0.6
3.0
32
1402
1403
Methyl eugenol
7.6
5.4
33
1413
1417
(E)-Caryophyllene
0.7
6.0
0.3
6.3
5.3
30.1
14.4
34
1423
1430
β-Copaene
0.3
2.8
35
1438
1439
Aromadendrene
1.4
36
1447
1452
α-Humulene
0.1
0.9
0.7
0.4
7.1
37
1450
—
n.i.
0.3
38
1456
1457
Croweacin
0.9
39
1468
1471
4,5-Di-epi-aristolochene
0.3
40
1475
1476
β-Chamigrene
1.6
41
1480
1489
β-Selinene
1.7
8.1
2.6
5.5
42
1489
1498
α-Selinene
9.0
1.7
5.0
43
1499
1500
Epizonarene
0.1
44
1471
1478
γ-Muurolene
0.4
1.6
45
1474
1484
Germacrene D
1.0
0.6
2.9
46
1488
1493
Epi-cubebol
0.4
47
1490
1494
Bicyclogermacrene
0.5
1.0
3.9
48
1491
1494
Sarisan
0.3
49
1495
1500
Pentadecane
0.3
0.2
50
1498
1505
Germacrene A
0.2
51
1500
1500
α-Muurolene
0.2
52
1500
1506
β-Bisabolene
9.1
53
1509
1514
Cubebol
0.8
54
1510
—
n.i.
1.6
55
1518
1517
Myristicin
2.4
2.0
56
1513
1513
γ-Cadinene
0.4
3.5
57
1516
1520
7-epi-α-Selinene
0.2
58
1501
1511
δ-Amorphene
0.3
59
1518
1522
δ-Cadinene
0.4
0.8
1.1
1.2
60
1530
1545
Propiopiperone
13.2
1.2
61
1544
1548
Elemol
1.1
62
1554
1555
Elemicin
1.4
2.7
63
1559
1561
(E)-Nerolidol
0.5
1.0
6.5
13.8
64
1571
1577
Spathulenol
0.5
0.7
4.1
2.2
2.5
65
1576
1582
Caryophyllene oxide
1.5
1.8
13.3
10.1
66
1580
1601
α-Cedrol
3.1
67
1582
1590
Globulol
1.2
68
1584
1592
Viridiflorol
1.2
69
1593
1624
Selina-6-en-4-ol
7.3
70
1606
—
n.i.
0.6
71
1618
—
n.i.
0.1
72
1625
1620
Dillapiole
76.0
73
1625
1627
1-epi-Cubenol
0.9
74
1631
1642
2-Hydroxy-3,4-methylenedioxypropiophenone
1.0
75
1637
1638
epi-α-Cadinol
0.5
76
1640
1644
α-Muurolol
0.2
77
1648
1649
β-Eudesmol
0.2
0.9
78
1651
1658
Selin-11-en-4α-ol
2.0
79
1652
1652
α-cadinol
1.2
1.5
3.0
80
1655
1658
neo-Intermedeol
0.5The essential oils ofP. aduncum, P. hispidinervum, P. callosum, P. marginatum, P. hispidum, P. tuberculatum, and Piper sp. were analyzed by GC and GC-MS, and the percentage of identified components is given in Table 2.The major compounds ofP. aduncum and P. hispidinervum were identified as dillapiole (76%) and safrole (91.4%), respectively. In the oil of P. callosum, the main components were safrole (53.8%) and α-pinene (12.2%). Major components of P. marginatum were propiopiperone (13.2%) and δ-3-carene (11.3%). P. hispidum presented the terpinene isoforms γ-terpinene (30.9%) and α-terpinene (14.4%) as main compounds. β-Pinene (15%) and caryophyllene oxide (13.3%) were the major constituents of P. tuberculatum, while the sesquiterpenes linalool (14.6%) and nerolidol (13.8%) were identified in the Piper sp. oil.Dillapiole has been described as acaricidal (Rhipicephalus (Boophilus) microplus), larvicidal and insecticidal (Anopheles marajoara, Aedes aegypti, and Solenopsis saevissima), and antifungal (Aspergillus fumigatus) agent. Safrole demonstrated antileishmanial (L. major, L. mexicana, L. braziliensis, and L. donovani) activity. Propiopiperone exhibited antifungal activity against Cladosporium cladosporioides and C. sphareospermum. Oyedemi et al. [12] showed the activity of γ-terpinene against Proteus vulgaris and Escherichia coli. Our group previously described [13] the activity of (+)-β-pinene against Cryptococcus neoformans and Candida albicans. Other promising activity described by our group [14] was linalool-rich essential oil of Lippia alba against two dermatophytes Trichophyton rubrum and Epidermophyton floccosum [12–21].The results of the MIC assay ofPiper essential oils and amphotericin B against R. oryzae are shown in Table 3.Table 3
MIC values (μg/ml) of Piper essential oils and amphotericin B against R. oryzae.
Essential oil
MIC
MFC
Piper aduncum
>1250
ND
Piper hispidinervum
1250
ND
Piper callosum
>1250
ND
Piper marginatum
156.25
>1250
Piper hispidum
312.5
>1250
Piper tuberculatum
625
>1250
Piper sp.
78.12
>156.5
Amphotericin B
0.98
1.95
Posaconazole
1.56
1.56
MIC: minimal inhibition concentration; MFC: minimal fungicide concentration; ND: not determined.Sartoratto et al. [22] considered MIC values between 50 and 500 µg/mL as strong activity, MIC values between 600 and 1500 µg/mL as moderate activity, and above 1500 µg/mL as weak activity [21]. According to this classification, it could be stated that Piper sp., P. marginatum, and P. hispidum essential oils present high activity, P. tuberculatum and P. hispidinervum present moderate activity, and P. aduncum and P. callosum against R. oryzae planktonic cells present weak activity.Based on previous MIC results, the essential oils tested on biofilm formation wereP. hispidum, P. marginatum, and P. tuberculatum; Piper sp. Rhizopus oryzae biofilm formation in the presence of each Piper essential oil was inhibited in lower concentration than MIC for all species tested (Figure 1).Figure 1
Effect ofPiper essential oils against R. oryzae biofilm formation. The plates were incubated at 30°C for 18 h.In their natural environments, most of bacteria and fungi change from a planktonic to a sessile state forming the so-called biofilms. Biofilms are sessile microbial and fungal communities that are strongly attached to surfaces and to each other; in such phase, they are protected by a polymeric extracellular matrix (ECM), constituted primarily of polysaccharides. According to Singh et al. [7], the major compounds of biofilm matrix are GlcN and GlcNAc. The cell wall of zygomicetes is also mainly formed by GlcN and GlcNAc polymer constituents of chitosan and chitin, respectively. Then, our results on MIC and inhibition of biofilm formation could be associated with each other. The essential oil of Piper sp. showed the most active agent against the two cell forms, planktonic and biofilm [7, 23].Piper sp. essential oil was the most active agent against planktonic cells and biofilm formation (78.12 and 4.88 μg/mL, resp.). However, this essential oil displayed hemolytic activity (Figure 2) at higher concentration (2500 μg/mL), making it a promising antifungal candidate.Figure 2
Hemolytic assay after treatment with various concentrations ofPiper sp. essential oil.Other important mechanism of action is the inhibition of rhizopuspepsin and/or saps, a class of enzymes secreted forR. oryzae and other Rhizopus species [24]. The results in Figure 3 showed inhibition of proteolytic activity of rhizopuspepsin when Piper essential oils were used, mainly P. hispidum and P. tuberculatum which inhibited 11.8% and 12.05% of enzymatic activity, respectively.Figure 3
Proteolytic activity of rhizopuspepsin after overnight treatment with 48µg/ml of Piper essential oils. The plates were incubated at 37°C.The antioxidant activity was evaluated after TLC ofPiper essential oils. It was not possible to identify regions containing substances with activity even after 45 min of application of DPPH. Terpenes are the most significant class of compounds present in essential oils. Among them, several monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpenes are often reported as weak antioxidant agents [25]. However, due to the complexity of essential oils’ composition, some antioxidant activity was expected. Thus, further investigation will be necessary in order to evaluate other antioxidant methods.


## 4. Conclusion

This study showed the promising anti-Rhizopus oryzae activity of Piper tuberculatum, P. hispidum, and Piper sp. against planktonic cells, biofilm formation, and rhizopuspepsin which makes these essential oils useful in formulating strategies to limit the growth of R. oryzae.



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*Source: 5295619-2018-07-05.xml*