β-Pinene Derived Products With Enhanced In Vitro Antimicrobial Activity

Abstract

The development of new antimicrobials has always been a research hotspot. In this study, β-pinene-based derivatives were synthesized, and their antimicrobial activity was evaluated. The purpose was to develop some novel, promising new fungicides. Three β-pinene derivatives containing bis-hydronopyl were prepared, and their antifungal and antibacterial activities were evaluated against 6 plant pathogenic fungi and 4 bacterial species; a preliminary structure-activity relationship is discussed. The results indicated that the derivatives containing the blend of alkyl group and bis-hydronopyl had potent inhibitory activities against plant fungal pathogens and bacteria. Among these molecules, bis-hydronopyl dimethyl ammonium bromide showed excellent effects on Colletotrichum acutatum with a half-maximal effective concentration of 0.538 µg/mL, which was lower than that of carbendazim. Scanning electron microscope showed that after administration of bis-hydronopyl dimethyl ammonium bromide (compound 3a), the C. acutatum mycelia were sunken and deformed in comparison with the control group. Furthermore, the inhibitory activities of the methyl derivatives against the plant pathogens were better than those of the ethyl derivatives. These results provide new insights into the inhibition of fungi and bacteria by β-pinene derivatives, which may lead them to be used as precursor molecules for novel pesticides and antimicrobials in further research.

Keywords

β-pinene hydronopyl antifungal activity phytopathogenic fungi bacteria

Plant pathogenic fungi have become a serious threat to global crop production and food security, which has resulted in huge losses to human beings.^1
-3 In addition, pathogenic bacteria are the cause of many human diseases.⁴ In the past few decades, the large-scale use of chemical fungicides and antibiotics has been the principal tool to eliminate these disasters.⁵ However, the widespread application of chemical fungicides and antibiotics often leads to drug resistance of fungi and bacteria. Drug-resistant bacterial and fungal strains are causing a serious threat to global human health, and so it is very urgent to look for new alternatives.^6,7 Therefore, the development of safe and eco-friendly alternatives can not only control the increase of microorganisms but also reduce the environmental risks.⁸

β-Pinene is a natural compound with antibacterial activity, which can be used to participate in many chemical reactions.^9

-13 A large number of β-pinene derivatives can be synthesized by chemical modification, and some of these derivatives have been proved to have increased antibacterial activities.^14

-19 For example, Gavrilov et al²⁰ synthesized 3 series of β-pinene-based derivatives, and the reaction mixes of the sulfoxides had high activity against Penicillium tardum. Recently, cationic antimicrobial agents have been shown to be very effective bacteriostatic agents and are used in many applications, such as pesticides, hospital protective clothing, medical implants, wound dressings, and food packaging materials.^21,22 Among these cationic antimicrobial agents, quaternary ammonium salts are probably the most widely developed and used.^23
-25 Jin et al reported that hydronopyl quaternary ammonium salts from β-pinene have good antifungal activity against various plant pathogenic fungi. N-hydronopyl-γ-dimethylamino pyridine ammonium bromide was shown to exert good antifungal activity against Colletotrichum glecosporioides and Pestalotiopsis actinidia .²⁶

Herein, a series of quaternary ammonium salts were synthesized from β-pinene in order to obtain derivatives with potent antimicrobial activity. This study can be used as a guide for the development of new and efficient botanical antimicrobial agents.

Materials and Methods

Synthetic Procedures and Structural Characterization of the Title Compounds

Materials and structural characterization techniques

Pure chemicals were purchased from Jiangxi Jingke Scientific Instrument Co., Ltd., China. Fourier transform infrared (FT-IR) spectroscopy was performed using a Nicolet IS10 FT-IR spectrometer (America). Melting points (m.p.) were determined with a WRS-2 melting point apparatus (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) and are uncorrected. Proton nuclear magnetic resonance (NMR) was performed on a Bruker 400 spectrometer with tetramethylsilane and deuterated chloroform (CDCl₃) used as internal control and solvent, respectively. Mass spectrometry (MS) was carried out on a Bruker mass SL spectrometer (Bruker, Germany).

The synthesis of quaternary ammonium salts

(1R,2R,5R)-Hydronopyl bromide (1), (1R,2R,5R)-hydronopyl chloride (2), and (1R,2R,5R)-hydronopyl ammonium halide (2a-c) were synthesized from β-pinene as previously reported.^27,28 Then, bis-hydronopyl quaternary ammonium salts (3a-c) were obtained by the reaction between (1R,2R,5R)-hydronopyl bromide (1) and (1R,2R,5R)-hydronopyl ammonium halide (2a-c). The synthetic routes and structures of compounds 3a-c are shown in Scheme 1.

Scheme 1

Synthetic routes of bis-hydronopyl quaternary ammonium salts (3a-c).

(1R,2R,5R)-Hydronopyl dimethyl amine (2a, 0.05 mol) and compound 1 (0.05mol) were slowly added to 30 mL acetone at 60 °C. After 48 hours of reaction, the acetone was vacuum removed. Following 3 light petroleum washes and recrystallization from ethyl acetate, a white solid bis-hydronopyl dimethyl ammonium bromide(3a) was obtained; m.p., 213.7-215.2 °C; Yield, 82%. FT-IR v (cm⁻¹): 2997.43-2867.23 (C-H), 1227.71 (C-N); ¹H NMR (CDCl₃, 400 MHz) δ _H: 3.41 (4H, t, J = 8.8 Hz, 2 ₁₁-CH₂), 3.41 (10 H, s, 2 ₁₁-CH₂, 2 _α-CH₃), 2.33 (2 H, m, 2 ₂-CH), 1.99-1.70 [16H, m, 2 (₇-CH, ₁₀-CH₂, _1-CH, ₅-CH, ₃-CH, ₄-CH₂)], 1.40 (2 H, m, 2 ₃-CH), 1.15 (6H, s, 2 ₉-CH₃), 0.97 (6 H, s, 2 ₈-CH₃), 0.86 (2 H, d, J = 10 Hz, 2 ₇-CH); ¹³C NMR (CDCl₃, 100 Hz) δ _C: 62.29 (C_-11), 51.61 (C_-α), 46.41 (C-₂), 41.03 (C-₅), 38.56 (C-₆), 38.34 (C-₁), 33.32 (C-₁₀), 30.16 (C-₇), 27.90 (C-₉), 26.02 (C-₄), 23.30 (C-₈), 22.12 (C-₃); LC-MS, C₂₄H₄₄NBr: 346.3 (M⁺ − Br), 504.0, 506.0, 508.0 (M⁺ + Br).

(1R,2R,5R)-Hydronopyl diethyl amine (2b, 0.04 mol) and compound 1 (0.05 mol) were slowly added to 40 mL ethyl acetate at 60 °C. After 72 hours of reaction, the ethyl acetate was vacuum removed. Following 3 light petroleum washes and recrystallization from ethyl acetate, a white solid bis-hydronopyl ethyl ammonium bromide (3b) was obtained; m.p., 198-201.7 °C; Yield, 80%. FT-IR v (cm⁻¹): 2985.86‐2866.27 (C-H), 1121.63 (C-N); ¹H NMR (CDCl₃, 400 MHz) δ _H: 3.47 (4H, q, J = 6.8 Hz, 2 _α-CH₂), 3.23 (4H, t, J = 8.4 Hz, 2 ₁₁-CH₂), 2.33 (2H, m, 2 ₂-CH), 2.06-1.67 [18 H, m, 2 (₇-CH, ₁₀-CH₂, _1-CH, ₅-CH, ₃-CH₂, ₄-CH₂)], 1.38 (6H, m, 2 _β-CH₃), 1.17 (6H, s, 2 ₉-CH₃), 0.99 (6H, s, 2 ₈-CH₃), 0.85 (2H, d, J = 10 Hz, 2 ₇-CH); ¹³C NMR (CDCl₃, 100 Hz); δ _C: 57.02 (C_-11), 54.08 (C_-α), 46.25 (C-₂), 41.01 (C-₅), 38.57 (C-₆), 38.49 (C-₁), 33.34 (C-₁₀), 29.53 (C-₇), 27.92 (C-₉), 26.04 (C-₄), 23.33 (C-₈), 23.31 (C-₃), 8.16 (C-_β); LC-MS, C₂₆H₄₈NBr: 374.4 (M⁺ − Br), 532.4, 534.4, 536.4 (M⁺+ Br).

(1R,2R,5R)-Hydronopyl piperidine (2 c, 0.05 mol) and compound 1 (0.06 mol) were slowly added to 40 mL ethyl acetate at 60 °C. After 72 hours of reaction, the ethyl acetate was vacuum removed. Following 3 light petroleum washes and recrystallization from ethyl acetate, a white solid bis-hydronopyl piperidine ammonium bromide (3 c) was obtained; m.p., 226.9-230.3 °C; Yield, 86.5%. FT-IR v (cm⁻¹): 2975.25‐2867.23 (C-H), 1066.65 (C-N); ¹H NMR (CDCl₃, 400 MHz) δ _H: 3.74 (4H, s, 2 _α-CH₂), 3.48-3.20 (4H, m, 2 ₁₁-CH₂), 2.32 (2H, m, 2 ₂-CH), 1.98-1.63 [22H, m, 2 ₁₀-CH₂, 2 ₇-CH, 2 ₁-CH, 2 ₅-CH, 2 _3-CH, 2 _β-CH₂, _γ-CH₂, 2 ₄-CH₂], 1.35 (2H, m, 2 ₅-CH), 1.13 (6H, s, 2 ₉-CH₃), 0.98 (6H, s, 2 ₈-CH₃), 0.86 (2H, d, J = 10 Hz, 2 ₇-CH); ¹³C NMR (CDCl₃, 100 Hz) δ _C: 58.96 (C_-11), 56.96 (C_-α), 46.48 (C-₂), 41.11 (C-₅), 38.52 (C-₆), 38.44 (C-₁), 33.30 (C-₁₀), 29.04 (C-₇), 27.88 (C-₉), 26.07 (C-₄), 23.29 (C-₈), 22.16(C-₃), 20.43(C-_γ), 19.93(C-_β); LC-MS, C₂₄H₄₄NBr: 386.4 (M⁺ − Br), 544.0, 546.0, 548.0 (M⁺ + Br).

Antimicrobial Tests

Fungal pathogens and bacteria were obtained from East China Woody Fragrance and Flavor Engineering Research Center of National Forestry and Grassland Administration. The microorganisms included 6 typical phytopathogenic fungi (Thanatephorus cucumeris, Phytophthora parasitica var. nicotianae, Colletotrichum gloeosporioides, Coriolus versicolor, Colletotrichum acutatum, and Fusarium oxysporum); Gram-negative bacteria Escherichia coli and Pseudomonas putida; and Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. Fungal pathogen mycelial morphology was evaluated under an FEI Quanta250 scanning electron microscope (SEM) (America).

Determination of mycelial growth inhibition of fungal pathogens

The antifungal activities of the samples were tested using the mycelial growth inhibition (MGI). The compounds were added to sterilized water to generate respective stock solutions (2.0 × 10⁴ mg/L), which were further diluted to 200, 100, 50, 25, and 12.5 µg/mL with potato dextrose agar, respectively. Then, a 5-mm disc containing mycelia was transferred to the center of the medicated media and incubated at 28 °C.^29,30 Each experiment was conducted in triplicate. The diameter of the fungal colony was measured using a cross method and averaged. Sterilized water was used as the no-treatment control and chlorothalonil (98%) as the positive control. MGI was calculated using Eq. (1).

M G I = (D_{a} - D_{b}) ∕ D_{a} \times 100 %

(1)

where D _a and D _b were the radial growth of the fungal colonies in the control plate and the treatment plate, respectively. The radial growth of the fungal colony was calculated by Eq. (2):

R a d i a l g r o w t h (m m) = A v e r a g e d i a m e t e r o f f u n g a l c o l o n y (m m) - 5 (m m)

(2)

Determination of effective concentration

The antimicrobial activities of 3a-c against phytopathogenic fungi were evaluated using the half-maximal effective concentration (EC₅₀) values. The regression equation was obtained by using the natural logarithm of the compound concentrations as the abscissa, and the biometric probability value calculated by MGI, as the ordinate. The EC₅₀ values were calculated using the regression equation when the MGI values were 50%.

Determination of minimum inhibitory concentration of bacteria

The antimicrobial properties of 3a-c against E. coli, P. putida, S. aureus, and B. subtilis were evaluated by the resazurin coloration method.^31,32 The concentrations of bacterial suspensions were 1.5 × 10⁸ CFU/mL (D _625nm =0.08-0.10). One hundred microliters of the bacterial suspension and 100 µL of the compound solutions with different concentrations were transferred to a 96-well plate, after which 20 µL of a 0.1% resazurin solution was added into the 96-well plate, and the contents of each well were mixed by vortex shaking. Instead of the compound solution, 0.9% saline was added as a control plate. Kanamycin sulfate (98%) was used as the positive control. Then, the mixtures were incubated at 37 °C for 24 hours.

Results and Discussion

Chemistry

The FT-IR spectrum of 3a showed an absorption band at 2997-2867 cm⁻¹ due to the new amide V C-H group and the characteristic stretching frequency of δ C-H was observed at 1450 cm⁻¹. The characteristic absorption band of the C-N group appeared at 1227 cm⁻¹. The absorption peaks at 2985-2866, 1469, and 1121 cm⁻¹ represented the infrared absorption bands of saturated V C-H, δ C-H, and C-N, respectively, which indicated that the expected structure of 3b had been formed. The FT-IR spectrum of 3c exhibited absorption wavelengths at 2975‐2867 cm⁻¹ corresponding to a V C-H group, and at 1434 cm⁻¹ belonging to the δ C-H group. The C-N group absorption was observed at 1066 cm⁻¹. In the ¹H NMR spectrum, the proton signal of the CH₂ group attached to the N group was observed at δ 3.2-3.6 ppm as a doublet of doublets; for example, in the spectrum of compound 2b, this was seen at δ 3.471 and 3.226 ppm. In the ¹³C NMR spectra, the carbon signal of the CH₂ group attached to the N group was observed at δ 51.640-62.354 ppm; for example, in the spectrum of compound 3b, this was seen at δ 57.018 and 54.060 ppm. The mass spectrum of 3a and 3b showed the expected pseudomolecular ion peaks at m/z 346.3 (M⁺ − Br) and 504.0, 506.0, 508.0 (M⁺ + Br) and 374.4 (M⁺ − Br) and 532.4, 534.4, 536.4 (M⁺ + Br), corresponding to the molecular formula. There was a difference in (CH₂)₂ (28) between 3a and 3b.

Biological Activity

Activity against phytopathogenic fungi

The antifungal activities of the β-pinene-based derivatives against six plant pathogens are shown in Tables 1 -4. For all the tested samples, the inhibitory rates improved with the increase in sample concentration. The EC₅₀ values of the β-pinene-based derivatives against the 6 typical phytopathogenic fungi were less than 30 µg/mL. Compound 3a showed the highest antifungal activity against T. cucumeris with an EC₅₀ value of 11.30 µg/mL. For P. nicotianae, the EC₅₀ values of the 3 compounds (3a-c) were below 7 µg/mL. Among them, compound 3a showed the best activity with an EC₅₀ value of 5.72 µg/mL; for C. gloeosporioides, compound 3c exhibited the best antifungal activity (EC₅₀ = 16.56 µg/mL). The 3 derivatives had certain inhibitory effects on C. versicolor, the best being compound 3a with an EC₅₀ of 19.12 µg/mL. For C. acutatum, compound 3a also exhibited an outstanding antifungal activity, with an EC₅₀ value of 0.54 µg/mL. Compound 3c showed the best effect on F. oxysporum, with an EC₅₀ value of 10.15 µg/mL. Compared with the positive control, chlorothalonil, the β-pinene-based derivatives exhibited better antifungal activity against P. nicotianae, C. versicolor, and C. acutatum.

Table 1

Antifungal Activities of 3a Against Plant Pathogenic Fungi.

Strain	MGI (%) at a concentration of (µg/mL)
Strain	200	100	50	25	12.5	EC₅₀	y = a + bx	R ²	95% CI
T. cucumeris	100	100	92.96	83.74	79.37	11.3034	y = 0.6695 + 4.1117x	0.9086	4.4754-28.5483
P. nicotianae	100	100	100	62.94	14.69	5.7174	y = 0.7298 + 5.6395x	0.9356	3.3465-9.7678
C. gloeosporioides	99.16	93.82	76.40	54.78	35.96	20.6670	y = 1.9761 + 2.2991x	0.9929	17.2874-24.7072
C. versicolor	100	95.97	83.89	60.40	49.33	19.1249	y = 0.0967 + 3.8259x	0.9006	9.0370-9.0370
C. acutatum	98.28	93.70	91.40	89.68	86.82	0.5380	y = 5.2022 + 0.7512x	0.9254	0.0627-4.6136
F. oxysporum	100	98.25	96.79	90.38	65.01	11.4824	y = 1.4668 + 3.3331x	0.9144	4.7350-27.8448

Abbreviations: EC₅₀, half-maximal effective concentration; MGI, mycelial growth inhibition.

Table 2

Antifungal Activities of 3b Against Plant Pathogenic Fungi.

Strain	MGI (%) at a concentration of (μg/mL)
Strain	200	100	50	25	12.5	EC₅₀	y = a + bx	R ²	95% CI
T. cucumeris	100	100	96.84	79.71	35.98	15.8611	y = −0.9362 + 4.9454x	0.9636	9.8771-25.4705
P. nicotianae	100	100	100	88.51	66.32	6.6406	y = 3.4287 + 1.9110x	0.9962	6.0250-7.3190
C. gloeosporioides	94.49	84.02	67.77	51.79	31.40	24.6157	y = 2.6365 + 1.6989x	0.9982	22.6831-26.7129
C. versicolor	99.18	81.97	71.04	48.91	32.51	24.6701	y = 1.9254 + 2.2085x	0.9582	16.4039- 37.1017
C. acutatum	87.70	75.39	63.35	48.17	32.72	27.5795	y = 3.1110 + 1.3113x	0.9989	25.9851-29.2718
F. oxysporum	100	98.33	90.83	66.11	56.67	16.6141	y = 0.3879 + 3.7790x	0.9243	8.3404-33.0952

Abbreviations: EC₅₀, half-maximal effective concentration; MGI, mycelial growth inhibition.

Table 3

Antifungal Activities of 3c Against Plant Pathogenic Fungi.

Strain	MGI (%) at a concentration of (μg/mL)
Strain	200	100	50	25	12.5	EC₅₀	y = a + bx	R ²	95% CI
T. cucumeris	100	100	96.89	87.33	63.78	12.2333	y = 0.2481 + 4.3694x	0.9489	6.4143-23.3311
P. nicotianae	100	100	100	85.85	67.92	6.0693	y = 3.7100 + 1.6472x	0.9957	5.4709-6.7331
C. gloeosporioides	99.89	91.13	80.11	65.86	49.46	16.5630	y = 2.1283 + 2.3555x	0.9401	9.0367-30.3576
C. versicolor	100	94.72	76.25	58.84	37.73	21.6848	y = −0.3352 + 3.9930x	0.9014	10.7659-43.6778
C. acutatum	100	96.83	79.37	62.96	46.56	19.5593	y = −0.0183 + 3.8861x	0.9055	9.5255-40.1623
F. oxysporum	100	100	98.94	90.48	74.07	10.1548	y = 0.8533 + 4.1192x	0.9573	5.3585-19.2444

Abbreviations: EC₅₀, half-maximal effective concentration; MGI, mycelial growth inhibition.

Table 4

Antifungal Activities of Chlorothalonil Against Plant Pathogenic Fungi.

Strain	MGI (%) at a concentration of (μg/mL)
Strain	200	100	50	25	12.5	EC₅₀	y = a + bx	R ²	95% CI
T. cucumeris	91.40	80.23	76.51	75.93	64.88	3.5676	y = 4.6122 + 0.7020x	0.9336	1.0504-12.1164
P. nicotianae	67.96	54.70	52.76	48.62	44.20	31.0515	y = 4.3172 + 0.4576x	0.9415	19.9710-48.2796
C. gloeosporioides	59.83	57.89	56.23	54.02	45.15	19.0705	y = 4.6428 + 0.2790x	0.9215	9.8998-36.7365
C. versicolor	49.75	56.85	44.92	24.37	15.74	111.6972	y = −3.0509 + 0.9517x	0.9119	58.5682-213.0209
C. acutatum	71.14	69.30	50.89	23.93	18.71	59.4580	y = -2.5805 + 1.3637x	0.9663	44.0478-80.2596
F. oxysporum	79.05	72.86	70.48	62.86	59.29	4.3285	y = 4.6984 + 0.4739x	0.9895	2.7994-6.6930

Abbreviations: EC₅₀, half-maximal effective concentration; MGI, mycelial growth inhibition.

Of all the compounds tested, 3a exhibited the most significant antifungal activity against C. acutatum (EC₅₀ = 0.538 µg/mL). The ultrastructural features of C. acutatum mycelium upon administration of 3a were examined by SEM, as shown in Figure 1. After being cultured for 72 hours Figure 1(A and B) show that the mycelia were smooth and full in the control experiment, indicating that the fungus was in good condition. However, the morphology of the mycelia treated with an EC₅₀ concentration of 3a was changed. Serious mycelial deformation was observed, with sunken surfaces and the mycelia were easily fractured when harvested (Figure 1(C and D)).

Figure 1

Effect of bis-hydronopyl dimethyl ammonium bromide (3a) on mycelial morphology in Colletotrichum acutatum (A, C): 2000 times, plates untreated and treated with EC₅₀ value concentration of 3a; (B, B): 6000 times, plates untreated and treated with EC₅₀ value concentration of 3a. EC₅₀, half-maximal effective concentration.

Although it was hard to give a comprehensive structure-activity relationship to these derivatives, we could find some interesting hints from the antifungal experiment. For T. cucumeris, P. nicotianae, C. versicolor, and C. acutatum, the antifungal activities of the compounds were 3b < 3c <3a. For C. gloeosporioides and F. oxysporum, the antifungal activities of the compounds were 3b < 3a < 3c. In our previous report, for quaternary ammonium salts containing a hydronopyl group, the antifungal activities of 2 ethyl groups linked on the hydrophilic group N⁺ was stronger than that of 2 methyl groups.³³ However, when there were 2 hydronopyl groups in the structure of the quaternary ammonium salts, the rule for the antifungal activity was opposite. Several reports suggest that polycations can kill various microbes by adsorption to the cell membranes and disruption of their integrity.^34
-36 Therefore, quaternary ammonium salts could effectively inhibit the growth of fungi because of their polycationic nature,^37,38 but the molecular weight of the quaternary ammonium salt and its inhibitory effect might not be positively correlated under different conditions. The molecular weight of 3b with an ethyl alkyl chain was higher than that of 3a with a methyl alkyl chain. However, the ethyl in 3b could hinder the combination of the positively charged amino groups of the quaternary ammonium salt with the negatively charged substances on the cell wall to form polyelectrolyte complexes, which cause the integrity of the hard cell membrane to be destroyed. Researchers found that the antifungal activity of amides bearing piperidine moieties against Cladosporium sphaerospermuma was comparable with that observed for the reference compounds miconazole and nystatin.³⁹ Interestingly, the introduction of piperidine to the hydrophilic group N⁺ of the derivatives (3c) could improve the antifungal activity against C. gloeosporioides and F. oxysporum.

Antibacterial activity

Screening of the activities of bis-hydronopyl quaternary ammonium salts against bacteria was performed using Gram-negative (E. coli, P. putida) and Gram-positive bacteria (S. aureus, B. subtilis). The minimum inhibitory concentration (MIC) values were determined using the resazurin coloration method. Resazurin is a redox indicator used to differentiate between metabolically active and inactive cells. The resazurin reagent is blue; however, it gives off a bright-red color when reduced, indicating the presence of live bacteria.⁴⁰ The MIC values for the bis-hydronopyl quaternary ammonium salts and kanamycin sulfate against E. coli, P. putida, S. aureus, and B. subtilis are shown in Figure 2.

Figure 2

MIC values of bis-hydronopyl quaternary ammonium salts against Escherichia coli, Pseudomonas putida, Staphylococcus aureus, and Bacillus subtilis. MIC, minimum inhibitory concentration.

Compared with the positive control kanamycin sulfate, 3a had better bacteriostatic activity against P. putida, S. aureus, and B. subtilis (MIC <3 µg/mL). Compound 3b had strong bacteriostatic activity against P. putida, and its MIC (2.5 µg/mL) was lower than that of kanamycin sulfate. Compound 3c had strong bacteriostatic activity against S. aureus, and its MIC value is equal to that of kanamycin sulfate. In addition, for E. coli and P. putida (Gram-negative bacteria), the bacteriostatic activities of the compounds were 3c < 3b <3a. The molecular weights and length of the alkyl chain were 3c > 3b >3a. Zhong et al reported that the molecular weight and chain length of the compound affect its bacteriostatic activity and the factor of chain length played a dominant role.⁴¹ However, our result showed that the bacterial activities of the ammonium salts containing bis-hydronopyl were less dependent on molecular weight. Peptidoglycan is a major part of the bacterial cell walls with open network structures. Both Gram-positive and Gram-negative bacteria have a layer of peptide polysaccharides, while the latter have an outer membrane structure in the cell wall, forming an additional barrier to foreign molecules.^23,42 This allows compounds with lower molecular weight more easily to diffuse through the cell walls of the Gram-negative bacteria to achieve an effective bacteriostatic effect.

According to the recent literature, the introduction of piperidine can make the compounds have antioxidant, bacteriostatic, and antimalarial activities.^43
-45 Subba Poojari et al found that some novel piperidine derivatives had obvious inhibitory activity against S. aureus with a MIC value of 0.4 µg/mL.⁴⁶ Similarly, this study found that compound 3c containing piperidine showed significant antibacterial activity against S. aureus in the tested bacteria with a MIC of 5 µg/mL.

Conclusions

Derivatives of β-pinene (3a-c) were produced, and their inhibitory properties against phytopathogenic fungi and bacteria were assessed. Results showed that compound 3a containing the methyl and bis-hydronopyl group had a higher activity in comparison with the positive control. Specifically, compound 3a against P. nicotianae, C. versicolor, C. acutatum, P. putida, S. aureus, and B. subtilis exhibited an EC₅₀ or MIC value lower than that of compounds 3b, 3 c and the positive control. SEM images showed that compound 3a could alter the morphological structure of the mycelium of C. acutatum. The structure-activity relationship analysis showed that the antibacterial activity of ammonium salts containing bis-hydronopyl against plant pathogens and bacteria were less dependent on molecular weight. The introduction of piperidine to the hydrophilic group N⁺ could improve the antibacterial activity against C. gloeosporioides, F. oxysporum, and S. aureus. Therefore, the β-pinene-based derivatives can be used as precursor molecules for further pesticide and bacteriostatic agent development.

Supplemental Material

Supplementary Material 1 - Supplemental material for β-Pinene Derived Products With Enhanced In Vitro Antimicrobial Activity

Supplemental material, Supplementary Material 1, for β-Pinene Derived Products With Enhanced In Vitro Antimicrobial Activity by Xuezhen Feng, Zhuanquan Xiao, Yuling Yang, Shangxing Chen, Shengliang Liao, Hai Luo, Lu He, Zongde Wang and Guorong Fan in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship,and/or publication of this article: This work was supported by the Key Projects of Key R&D Program of Jiangxi Province (20192ACB60011).

ORCID iDs

Xuezhen Feng

Shangxing Chen

Guorong Fan

Supplemental Material

Supplemental material for this article is available online.

References

Zhang

Tan

Zhang

et al. Synthesis, characterization, and the antifungal activity of chitosan derivatives containing urea groups. Int J Biol Macromol. 2018;109(4):1061-1067.doi:10.1016/j.ijbiomac.2017.11.092

29155157

Knogge

. Fungal infection of plants. Plant Cell. 1996;8(10):1711-1722.doi:10.2307/3870224

12239359

Mendgen

Hahn

. Plant infection and the establishment of fungal biotrophy. Trends Plant Sci. 2002;7(8):352-356.doi:10.1016/S1360-1385(02)02297-5

12167330

Asghar

MA.

Zahir

Shahid

et al. Iron, copper and silver nanoparticles: Green synthesis using green and black tea leaves extracts and evaluation of antibacterial, antifungal and aflatoxin B₁ adsorption activity. LWT. 2018;90(3):98-107.doi:10.1016/j.lwt.2017.12.009

Yang

Jin

Fan

Wang

Xie

. Synthesis, characterization and antifungal activity of coumarin-functionalized chitosan derivatives. Int J Biol Macromol. 2018;106(8):179-184.doi:10.1016/j.ijbiomac.2017.08.009

28797818

Lagrouh

Dakka

Bakri

. The antifungal activity of Moroccan plants and the mechanism of action of secondary metabolites from plants. J Mycol Med. 2017;27(3):303-311.doi:10.1016/j.mycmed.2017.04.008

28506565

Łukowska-Chojnacka

Mierzejewska

Milner-Krawczyk

Bondaryk

Staniszewska

. Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity. Bioorg Med Chem. 2016;24(22):6058-6065.doi:10.1016/j.bmc.2016.09.066

27745991

Xie

Wang

Huang

Zhang

. Antifungal activity of several essential oils and major components against wood-rot fungi. Ind Crops Prod. 2017;108(6):278-285.doi:10.1016/j.indcrop.2017.06.041

Rivas da Silva

AC.

Lopes

PM.

Barros de Azevedo

MM.

Costa

DCM.

Alviano

CS.

Alviano

. Biological activities of α-pinene and β-pinene enantiomers. Molecules. 2012;17(6):6305-6316.doi:10.3390/molecules17066305

22634841

10.

de Macêdo Andrade

AC.

Rosalen

PL.

Freires

et al. Antifungal activity, mode of action, docking prediction and anti-biofilm effects of (+)-β-pinene enantiomers against Candida spp. Curr Top Med Chem. 2018;18(29):2481-2490.doi:10.2174/1568026618666181115103104

30430938

11.

Wilson

CL.

Solar

JM.

El Ghaouth

Wisniewski

ME.

Wisniewski

. Rapid evaluation of plant extracts and essential oils for antifungal activity against Botrytis cinerea . Plant Dis. 1997;81(2):204-210.doi:10.1094/PDIS.1997.81.2.204

30870898

12.

Feng

XZ.

Xiao

Zhang

et al. Antifungal activity of β-pinene-based hydronopyl quaternary ammonium salts against phytopathogenic fungi. Nat Prod Commun. 2020;15(8):1-6.doi:10.1177/1934578X20948365

13.

Moreira

ACP.

Lima

EDO.

Souza

ELD.

Castellano

LR.

Castro

. Inhibitory effect of Cinnamomum zeylanicum Blume (Lauraceae) essential oil and beta-pinene on the growth of dematiaceous moulds. Digestion. 1972;7(38):33-38.

14.

Nikitina

LE.

Startseva

VA.

Vakulenko

et al. Synthesis and antifungal activity of compounds of the pinane series. Pharm Chem J. 2009;43(5):251-254.doi:10.1007/s11094-009-0282-3

15.

Liao

Shang

Shen

et al. One-pot synthesis and antimicrobial evaluation of novel 3-cyanopyridine derivatives of (-)-β-pinene. Bioorg Med Chem Lett. 2016;26(6):1512-1515.doi:10.1016/j.bmcl.2016.02.024

26898336

16.

Tian

Gao

Shang

Feng

Zhang

. A value-added use of volatile turpentine: antifungal activity and QSAR study of β-pinene derivatives against three agricultural fungi. RSC Adv. 2015;5(82):66947-66955.doi:10.1039/C5RA10660E

17.

Gao

Hao

Song

Shang

. Structural modification of turpentine with natural chiral preservation and low-risk application prospects in crop protection. ACS Omega. 2019;4(4):6392-6398.doi:10.1021/acsomega.9b00241

18.

Gao

Wang

Shang

Song

. Improved application of natural forest product terpene for discovery of potential botanical fungicide. Ind Crops Prod. 2018;126(210):103-112.doi:10.1016/j.indcrop.2018.10.008

19.

Gao

Song

Shang

Rao

. High add valued application of turpentine in crop production through structural modification and QSAR analysis. Molecules. 2018;23(2):356.doi:10.3390/molecules23020356

29419733

20.

Gavrilov

VV.

Startseva

VA.

Nikitina

et al. Synthesis and antifungal activity of sulfides, sulfoxides, and sulfones based on (1S)-(-)-β-pinene. Pharm Chem J. 2010;44(3):126-129.doi:10.1007/s11094-010-0413-x

21.

Hoque

Akkapeddi

Yadav

et al. Broad spectrum antibacterial and antifungal polymeric paint materials: synthesis, structure-activity relationship, and membrane-active mode of action. ACS Appl Mater Interfaces. 2015;7(3):1804-1815.doi:10.1021/am507482y

25541751

22.

Zhang

Tan

Luan

et al. Synthesis of quaternary ammonium salts of chitosan bearing halogenated acetate for antifungal and antibacterial activities. Polymers. 2018;10(5):530. doi:10.3390/polym10050530

30966564

23.

Muñoz-Bonilla

Fernández-García

. Polymeric materials with antimicrobial activity. Prog Polym Sci. 2012;37(2):281-339.doi:10.1016/j.progpolymsci.2011.08.005

24.

Wan

Zhang

Deng

et al. Effects of interaction between a polycation and a nonionic polymer on their cross-assembly into mixed micelles. Soft Matter. 2015;11(21):4197-4207.doi:10.1039/C5SM00380F

25882114

25.

Gilbert

Moore

. Cationic antiseptics: diversity of action under a common epithet. J Appl Microbiol. 2005;99(4):703-715.doi:10.1111/j.1365-2672.2005.02664.x

16162221

26.

Jin

LL.

Xiao

ZQ.

Fan

et al. Synthesis and antifungal activity of a series of N-hydronopol pyridine ammonium halides. Chem Ind Forest Prod. 2017;37(3):122-128.

27.

Chen

JZ.

Xiao

ZQ.

Wang

ZD.

Hang

. The synthesis and structural analysis of hydronopyl tertiary amine compounds. J Jiangxi Normal U: Nat Sci Ed. 2016;40(2):179-182.

28.

Zhao

LH.

Xiao

ZQ.

Chen

JZ.

Wang

ZD.

Fan

GR.

Chen

. Synthesis and structural analysis of hydronopol and its halides. Chem Ind Forest Prod. 2012;32(1):39-42.

29.

Shirakawa

Uehara

Tanaka

Makoto

Iwao

Megumi

. Mycorrhizosphere bacterial communities and their sensitivity to antibacterial activity of ectomycorrhizal fungi. Microbes Environ. 2019;34(2):191-198.doi:10.1264/jsme2.ME18146

31080215

30.

Abdel-Aziz

AA-M.

Asiri

YA.

Al-Agamy

MHM.

Design

A-AMHM

. Design, synthesis and antibacterial activity of fluoroquinolones containing bulky arenesulfonyl fragment: 2D-QSAR and docking study. Eur J Med Chem. 2011;46(11):5487-5497.doi:10.1016/j.ejmech.2011.09.011

21982337

31.

Lubna

Fahim

. Establishment and application of the resazurin micro-plate method for rapid diagnosis of bovine mastitis-causing staphylococci. Pak J Bio Sci. 2011;11(1):2017.

32.

Guembe

Alonso

Cruces

Sanchez-Carrillo

Perez

. Comparison of the XTT and resazurin assays for quantification of the metabolic activity of Staphylococcus aureus biofilm. Agro Food Ind Hi Tec. 2016;27(6):135-137.

33.

Feng

XZ.

Xiao

ZQ.

Fan

GR.

Wang

. Synthesis and antibacterial activity of hydronopol dimethyl alkyl ammonium halide. Chem Ind Forest Prod. 2019;39(1):35-40.

34.

Lee

SB.

Koepsel

RR.

Morley

SW.

Matyjaszewski

Sun

Russell

. Permanent, nonleaching antibacterial surfaces. 1. synthesis by atom transfer radical polymerization. Biomacromolecules. 2004;5(3):877-882.doi:10.1021/bm034352k

15132676

35.

Lin

Qiu

Lewis

Klibanov

. Mechanism of bactericidal and fungicidal activities of textiles covalently modified with alkylated polyethylenimine. Biotechnol Bioeng. 2003;83(2):168-172.doi:10.1002/bit.10651

12768622

36.

Kügler

Bouloussa

Rondelez

. Evidence of a charge-density threshold for optimum efficiency of biocidal cationic surfaces. Microbiology. 2005;151(Pt 5):1341-1348.doi:10.1099/mic.0.27526-0

15870444

37.

Guo

Xing

Liu

et al. The influence of molecular weight of quaternized chitosan on antifungal activity. Carbohydr Polym. 2008;71(4):694-697.doi:10.1016/j.carbpol.2007.06.027

38.

Badawy

MEI

. Structure and antimicrobial activity relationship of quaternary N -alkyl chitosan derivatives against some plant pathogens. J Appl Polym Sci. 2010;117(2):960-969.doi:10.1002/app.31492

39.

Navickiene

HM.

Alécio

AC.

Kato

et al. Antifungal amides from Piper hispidum and Piper tuberculatum . Phytochemistry. 2000;55(6):621-626.doi:10.1016/S0031-9422(00)00226-0

11130674

40.

O’Brien

Wilson

Orton

Pognan

. Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421-5426.doi:10.1046/j.1432-1327.2000.01606.x

10951200

41.

Zhong

Dong

Liuyang

et al. Controllable synthesis and antimicrobial activities of acrylate polymers containing quaternary ammonium salts. Reactive and Functional Polymers. 2017;121(12):110-118.doi:10.1016/j.reactfunctpolym.2017.10.010

42.

Chen

CZ.

Beck-Tan

NC.

Dhurjati

van Dyk

TK.

LaRossa

RA.

Cooper

. Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective antimicrobials: structure-activity studies. Biomacromolecules. 2000;1(3):473-480.doi:10.1021/bm0055495

11710139

43.

Kim

JH.

Shyam

PK.

Kim

MJ.

Lee

H-J.

Lee

JT.

Jang

H-Y

. Enantioselective synthesis and antioxidant activity of 3,4,5-substituted piperidine derivatives. Bioorg Med Chem Lett. 2016;26(13):3119-3121.doi:10.1016/j.bmcl.2016.04.092

27177825

44.

Murata

Miyamoto

Ueda

. Antimicrobial and anti-plaque activity of N’-alkyl-N-(2-aminoethyl)piperidine against dental plaque bacteria. J Pharm Sci. 1991;80(1):26-28.doi:10.1002/jps.2600800107

2013844

45.

Rokhyatou

Abdoulaye

Sandrine

Christian

. Synthesis and antimalarial activity of 1,4-disubstituted piperidine derivatives. Molecules. 2020;25(2):299-315.

46.

Subba

Parameshwar Naik

Krishnamurthy

Jithendra Kumara

KS.

Sunil Kumar

Sathish

. Anti-inflammatory, antibacterial and molecular docking studies of novel spiro-piperidine quinazolinone derivatives. J Taibah Univ Sci. 2017;11(3):497-511.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

29.99 MB