Two New Geranylated Biphenyl Esters From Artocarpus altilis

Abstract

Two new geranylated biphenyl esters, 2,3′,4,4′-tetrahydroxy-3-geranyl-5-carboxyethyl-biphenyl methyl ester (1), 2,3′,4,4′-tetrahydroxy-3-geranyl-5-carboxymethyl-biphenyl methyl ester (2), together with 4 known compounds were isolated from the methanol extract of the leaves of Artocarpus altilis. Their structures were established by spectroscopic means and by comparison with literature data. Compound 1 possessed moderate cytotoxicity toward MDA-MB-231 (a breast carcinoma cell line) with a half-maximal inhibitory concentration value of 30 µM and inhibited the growth of 8 bacteria, including gram-negative and gram-positive organisms, but was inactive against fungi.

Keywords

geranylated biphenyl cytotoxicity antimicrobial activity

Moraceae is a large family comprising 40 genera, with over 1200 species widely distributed in the tropical and subtropical regions and to a lesser extent in temperate regions worldwide.¹ The genus Artocarpus Moraceae is an exceptionally rich source of prenylated flavonoids. Artocarpus altilis (Parkinson) Fosberg, also known as breadfruit, is native to Southeast Asia including Indonesia where its leaves have been used traditionally for treatment of liver cirrhosis, hypertension, and diabetes.² Various triterpenes and flavonoids have previously been reported from A. altilis.^3
-5 In our continuing search for bioactive compounds from plants, the leaves of A. altilis were further investigated. We report herein the isolation and structure elucidation of 2 new geranylated biphenyl esters (1, 2), along with 4 known compounds (3, 4, 5, and 6), and the structure modification of compounds 1 and 2. In addition, the 2 unreported compounds (1 and 2) were evaluated for in vitro cytotoxicity against a small panel of human cancer cell lines and for antimicrobial activity.

Results and Discussion

The butanol-soluble fraction of the methanol extract of the leaves of A. altilis was subjected to column chromatography (CC) on a macroporous resin followed by repeated high-performance liquid chromatography (HPLC) to yield compounds 1‐6. Compounds 3, 4, 5, and 6 were identified as 4-hydroxy-4,8-dimethylnona-2,7-dienoic acid (3)⁶，1-(2,4-dihydroxyphenyl)-3-[(2R)-8-hydroxy-2-methyl-2-(4-methyl-3-penten-1-yl)−2H-1-benzopyran-5-yl]−1-propanone (4)⁷，cyclocommunol (5)⁸，and 2-geranyl-2′,3,4,4′-tetrahydroxydihydrochalcone (6)⁹ by comparison with published data. The structures of the isolated compounds are shown in Figure 1. Copies of spectra for all of the isolated compounds have been provided in the Supplemental Materials file accompanying this paper.

Figure 1

Compounds isolated from Artocarpus altilis.

Compound 1 was isolated as a yellow powder. Its molecular formula was determined as C₂₆H₃₂O₆, from the high-resolution electrospray ionization mass spectrometry (HRESIMS) pseudomolecular ion (m/z 439.2126 [M-H]⁻), as well as its ¹³C nuclear magnetic resonance (NMR) spectrum. Infrared (IR) absorptions at 3439, 2925, 1339, and 1730 cm⁻¹ supported the presence of hydroxyl, methylene, and carbonyl groups. The NMR assignments of compounds 1 and 2 (Table 1) were established by a comprehensive analysis of ¹H and ¹³C-NMR, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple bond correlation (HMBC), and total correlated spectroscopy spectra. Typical signals of ABX-type aromatic spin system (δ 6.71 [1H, d, J = 8.5 Hz, H-5′], 6.31 [1H, d, J = 2 Hz, H-2′], and 6.23 [1H, dd, J = 8.5, 2.0 Hz , H-6′]) and geranyl group (δ 1.53 [3H, s, Me-8′′], 1.60 [3H, s, Me-10′′], 1.70 [3H, s, Me-9′′], 1.94 [2H, m, H-4′′], 2.01 [2H, m, H-5′′], 3.32 [2H, d, J = 6.6 Hz, H-1′′], 5.05 [1H, t, J = 6.6 Hz, H-6′′], 5.11 [1H, t, J = 6.6 Hz, H-2′′]) were observed. In the HMBC spectrum, correlations between H-1′′ (δ_H 3.32) and C-2, C-3, and C-4 (δ_C 142.3, 126.1, and 142.1, respectively) as well as C-2″ and C-3′′ (δ_C 124.0, 132.6) indicate that the geranyl group is linked to C-3. HMBC correlations between H-1′′′ (δ_H 2.18) and C-5, C-2′′′, and C-3′′′ (δ_C 128.5, 24.8, and 172.9, respectively) indicate that the carboxyethyl sidechain is attached at C-5. Significantly, HMBC correlations between H-6 (δ_H 6.32) and C-1, C-2, C-4, and C-5 (δ_C 120.0, 142.3, 142.1, and 128.5, respectively) but not C-3 (δ_C 126.1) establish that the lone proton on the A ring is situated para to the site of attachment of the geranyl group. Finally, an HMBC correlation between the protons of the O-methyl group (δ_H 3.49) and the ester carbonyl (δ_C 172.9) establish the presence of the methyl ester. Therefore, the structure of compound 1 was identified by NMR as 2,3′,4,4′-tetrahydroxy-3-geranyl-5-carboxyethyl-biphenyl methyl ester.

Table 1

¹H (600 MHz) and ¹³C (150 MHz) NMR Data of Compounds 1 and 2 in DMSO-D₆.

CompoundC	1			2
CompoundC	δ_C	DEPT	δ_H mult. (J in Hz)	δ_C	DEPT	δ_H mult. (J in Hz)
1	120.0	C	-	120.1	C	-
2	142.3	C	-	142.2	C	-
3	126.1	C	-	126.1	C	-
4	142.1	C	-	142.1	C	-
5	128.5	C	-	129.7	C	-
6	102.4	CH	6.32 s	102.4	CH	6.31 s
1′	129.8	C	-	128.8	C	-
2′	115.6	CH	6.31 d (2.0)	115.5	CH	6.33 d (2.0)
3′	155.1	C	-	155.1	C	-
4′	157.0	C	-	156.9	C	-
5′	131.3	CH	6.71 d (8.5)	131.3	CH	6.69 d (8.5)
6′	106.0	CH	6.23 dd (8.5, 2.0)	105.8	CH	6.21 dd (8.5, 2.0)
1′′	25.2	CH₂	3.32 d (6.6)	25.2	CH₂	3.30 d (6.5)
2′′	123.9	CH	5.11 t (6.6)	124.1	CH	5.09 t (6.6)
3′′	133.2	C	-	133.1	C	-
4′′	39.2	CH₂	1.94 m	39.2	CH₂	1.94 m
5′′	26.1	CH₂	2.01 m	26.1	CH₂	2.02 m
6′′	124.0	CH	5.05 t (6.6)	124.0	CH	5.05 t (6.6)
7′′	130.7	C	-	130.6	C	-
8′′	17.3	CH₃	1.53 s	17.5	CH₃	1.51 s
9′′	15.7	CH₃	1.70 s	15.8	CH₃	1.66 s
10′′	25.4	CH₃	1.60 s	25.4	CH₃	1.59 s
1′′′	34.2	CH₂	2.18 m	34.6	CH₂	2.18 m
2′′′	24.8	CH₂	2.56 m	174.1	C	-
3′′′	172.9	C	-
OCH₃	50.9	CH₃	3.49 s	48.5	CH₃	3.54 s

Abbreviations: DEPT, distortionless enhancement by polarization transfer; DMSO, dimethyl sulfoxide; NMR, nuclear magnetic resonance.

Acetylation of 1 with acetic anhydride and pyridine afforded the tetraacetate, 1-a. Its molecular formula was determined to be C₃₄H₄₀O₁₀, from the HRESIMS pseudomolecular ion (m/z 626.2958 [M+NH₄] ⁺, and m/z 609.2692 [M + H]⁺).

Compound 2 was isolated as a yellow powder. Its molecular formula was determined as C₂₅H₃₀O₆, deduced from the HRESIMS pseudomolecular ion (m/z 425.1974 [M-H]⁻). IR absorptions at 3439, 2925, 1339, and 1730 cm⁻¹ supported the presence of hydroxyl, methylene, and carbonyl groups. The ¹H and ¹³C-NMR data for 2 are essentially superimposable with similar data for 1, except for the resonances of the ester sidechain, which has lost 1 methylene group. As in 1, the presence of the methyl ester was supported by HMBC correlations between the 3-proton singlet at δ_H 3.54 and the carbonyl carbon at δ_C 174.1.

Acetylation of 2 with acetic anhydride and pyridine afforded the tetraacetate, 2-a, with a molecular formula of C₃₃H₃₈O₁₀, deduced from the HRESIMS pseudomolecular ions (m/z 612.2802 [M+NH₄] ⁺ and m/z 595.2536 [M + H]⁺).

Biological Activity

The 2 new geranylated biphenyl esters were tested for cytotoxicity against 3 human cancer cell lines (breast carcinoma MDA-MB-231, lung cancer A549, cervical carcinoma HeLa) using the standard 3-[4,5-dimethylthiazol-2-yl]−2,5-diphenyltetrazolium bromide (MTT) method with doxorubicin as a positive control. The results are summarized in Table 2. Compound 1 moderately inhibited MDA-MB-231 and weakly inhibited HeLa with half-maximal inhibitory concentration (IC₅₀) values of 30.53 and 101 µM, respectively (Table 2). Compound 2 weakly inhibited HeLa cells with IC₅₀ value of 110 µM but was inactive against the other cell lines at the highest concentration tested.

Table 2

Cytotoxicity of Compounds 1 and 2 Against Human Cancer Cell Lines.

Compound	IC₅₀ (μM)
Compound	MDA-MB-231	A549	HeLa
1	30.53	>120	101
2	>120	>120	110
Doxorubicin	2.8 ± 0.3	3.9 ± 0.3	4.5 ± 0.4

Abbreviation: IC₅₀, half-maximal inhibitory concentration.

Compound 1 inhibited the growth of Staphylococcus epidermidis (ATCC 14990), Staphylococcus aureus (ATCC 12600), and Moraxella (Branhamella) catarrhalis (ATCC 25238) with minimum inhibitory concentrations (MICs) of 136, 68, and 272 µM, respectively. Compound 2, however, exhibited little effect on the growth of the tested microorganisms. Neither 1 nor 2 had any inhibitory effect on the fungi tested. The antimicrobial activity of compounds 1 and 2 is summarized in Table 3.

Table 3

Antimicrobial Activity of Compounds 1 and 2.

ATCC no.	Organism	MIC (μM)
ATCC no.	Organism	1	2
12600	Staphylococcocus aureus	68	n/a
14990	Staphylococcus epidermidis	136	n/a
25175	Streptococcus mutans	n/a	n/a
13525	Pseudomonas fluorescens	n/a	n/a
15692	Pseudomonas aeruginosa	n/a	n/a
8043	Enterococcus hirae	n/a	n/a
25238	Moraxella (Branhamella) catarrhalis	272	n/a
6633	Bacillus subtilis subsp. spizizenii	n/a	n/a
76615	Candida albicans	n/a	n/a
22019	Candida parapsilosis	n/a	n/a
10571	Candida rugosa	n/a	n/a
750	Candida tropicalis	n/a	n/a
16888	Aspergillus niger	n/a	n/a
2601	Saccharomyces kudriavzevii	n/a	n/a
10106	Penicillium chrysogenum	n/a	n/a
4103	Rhizopus stolonifera	n/a	n/a

Abbreviations: MIC, minimum inhibitory concentration; n/a, not active.

Experimental

General

All solvents used throughout these studies were HPLC grade (Concord Technologies, Tianjin, China). CC was performed on Diaion HP-20 (Mitsubishi Chemical Corp., Tokyo, Japan). Analytical and semipreparative HPLC separations were performed on an Agilent 1260VL quad gradient system (G1311C pump, G1329B autosampler, G1316A thermostatted column compartment and G1315D photodiode array detector, Agilent Technologies, Santa Clara CA, USA), using a Hypersil GOLD C18 analytical column (3 µm, 2.1 × 150 mm, ThermoFisher Scientific, Waltham MA, USA) or a Hypersil GOLD C18 semipreparative column (5 µm, 10 × 250 mm). Preparative separations were performed on an Agilent 1260VL quad gradient system (G1311C pump, and G1315D photodiode array detector) equipped with a Rheodyne 7725i manual injection valve (IDEX Health & Science, Middleboro, MA, USA) and Shimadzu CTO-20A column oven (Shimadzu Scientific Instruments, Kyoto, Japan). Preparative separations were conducted using a Hypersil GOLD C18 column (5 µm, 21.2 × 250 mm). Flash chromatography was performed on an ISCO Combiflash Rf+ instrument (Teledyne ISCO, Lincoln, NE, USA) using prepacked columns of spherical C18 bonded silica 20‐45 µm (SepaFlash, Santai Technologies, Changzhou, China). Mass spectra were determined on a Q-Exactive HF Orbitrap LCMS with electrospray ionization coupled to an UltiMate 3000 RSLC nano HPLC system (Thermo Fisher Scientific, Waltham, MA, USA). Ultraviolet (UV) spectra were acquired using a HITACHI U3900 spectrophotometer (HITACHI, Kyoto, Japan). NMR spectra were recorded on Bruker Avance III spectrometers (Bruker Biospin Corp., Billerica, MA, USA) operating at 400 or 600 MHz (¹H) and 100 or 150 MHz (¹³C). Residual solvent resonances were used as internal reference, and chemical shifts are reported in PPM (δ). Optical densities on 96-well plates were measured with a Spectramax M2 plate reader (BioTek Instruments, Winooski, VT, USA). MTT was obtained from Solarbio Science Technology Co. Ltd (Beijing, China). 2,3,5-Triphenyltetrazolium chloride (TTC) was obtained from Biotopped Science Technology Co. Ltd. (Beijing, China).

Plant Material

Samples comprising the leaves of Artocarpus altilis (Parkinson) Fosberg were collected from a cultivated population on the grounds of the University of Hawaii at Hilo Experimental Agricultural Farm, in Panaewa, HI (19^o 30.198′ N Latitude, 155^o 2.828 W Longitude) at an elevation of approximately 100 m in May 2009. Plant material was collected and identified by Professor Norman Q. Arancon of the College of Agriculture, Forestry and Natural Resource Management, University of Hawaii at Hilo.

Extraction and Isolation

Dried and powdered leaves of Artocarpus altilis (Parkinson) Fosberg (10 kg) were extracted with MeOH (4 × 20 L) at ambient temperature and the combined methanol extracts were concentrated in vacuo to yield a tarry residue. The methanol extract was dissolved in 90% (aqueous) methanol, and extracted with n-hexane. The residual hydroalcoholic phase was freed of solvent in vacuo, suspended in water, and then sequentially extracted with dichloromethane and n-butanol (n-BuOH) to obtain a gross separation into hexane-, dichloromethane-, butanol-, and water-soluble fractions.

The n-BuOH fraction (176 g) was chromatographed on a 2-L column of Diaion HP20, eluted with a step gradient of MeOH–water (6 L each: 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH) to yield 5 fractions. The recovered weights of the 5 fractions were 69.01, 39.97, 27.18, 20.56, and 7.47 g, respectively.

Fraction 5 (7.47 g), eluted from Diaion HP-20 with 100% MeOH, was analyzed by HPLC (Hypersil GOLD C18, 10%-100% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃), which showed this fraction to be a very complicated mixture of components. Fraction 5 was rechromatographed on a 220-g column of Diaion HP20, eluting with a step gradient of MeOH–water (60% MeOH, 80% MeOH, 100% MeOH, 700 mL each). Recovered weights of 3 subfractions were 2.47 g (fraction 5‐1), 2.03 g (fraction 5‐2), and 2.95 g (Fraction 5‐3), respectively.

Fraction 5‐3 was analyzed by HPLC (Hypersil GOLD C18, 47% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃) and then purified by preparative HPLC (40 ℃, Hypersil GOLD C18, 47% acetonitrile, 15 mL/min) to obtain compound 6 as a brown solid. The retention time of compound 6 on analytical HPLC was 21.8 minutes (Hypersil GOLD C18, 47% acetonitrile, 0.1% formic acid, 0.2 mL/min, 40 ℃). Compound 6 was also detected in several other fractions and isolated from them. The total yield of 6 isolated was 61.6 mg.

Fraction 5‐2 was analyzed by HPLC (Hypersil GOLD C18, 37% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃) and then purified by preparative HPLC (Hypersil GOLD C18, 47% acetonitrile, 15 mL/min, 40 ℃) to obtain compound 5 as a brown solid (4.8 mg). Retention time of compound 5 was 21.1 minutes (Hypersil GOLD C18, 37% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃). Recovered side cuts were pooled, concentrated, and then analyzed by HPLC (Hypersil GOLD C18, 50% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃). Increasing the solvent concentration to 50% aqueous acetonitrile allowed further separation of a yellow solid, compound 4 (6.5 mg) by semi-preparative HPLC. The retention time of compound 4 was 18.1 minutes (Hypersil GOLD C18, 50% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40℃).

Fraction 4 (20.56 g), eluted from Diaion HP-20 with 80% MeOH, was analyzed by HPLC (Hypersil GOLD C18, 10%-100% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃). Fraction 4 was separated by repeated flash chromatography on a C18-bonded silica column (330 g) eluting with a step gradient consisting of 60% MeOH (720 mL) 80% MeOH (360 mL) and 100% MeOH (540 mL) at 18 mL/min to give 7 subfractions (each 180 mL).

Fraction 4‐4 (1.39 g), eluting from HP-20 with 60% MeOH, was analyzed by HPLC (Hypersil GOLD C18, 47% acetonitrile containing 0.1% formic acid at 0.2 mL/min, 40 ℃) and then fractionated by preparative HPLC (Hypersil GOLD C18, 47% acetonitrile at 15 mL/min, 40 ℃) to yield compound 1 as a brown oil (80.9 mg). The retention time of compound 1 was 16.9 minutes (Hypersil GOLD C18, 45% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃). Recovered side cuts were pooled, concentrated, and then analyzed by HPLC (Hypersil GOLD C18, 38% acetonitrile containing 0.1% formic acid, 0.2 mL/min, 40 ℃). Decreasing the solvent concentration to 38% acetonitrile allowed further separation of compound 2 as a brown oil (17.8 mg). The retention time of compound 2 is 13.7 minutes under these conditions.

Subfraction 4‐5 was analyzed by HPLC (Hypersil GOLD C18, 24% acetonitrile containing 0.1% formic acid at 0.2 mL/min, 40 ℃) then purified by semipreparative HPLC (Hypersil GOLD C18, 24% acetonitrile at 3 mL/min, 40℃) to yield compound 3 (3.9 mg). The retention time of compound 3 is 16.8 minutes under these conditions.

2,3′,4,4′-tetrahydroxy-3-geranyl-5-carboxyethyl-biphenyl methyl ester (1)

Brown oil. ¹H and ¹³C NMR, see Table 1. UV (MeOH) λ _max 204, 282 nm. Fourier transform infrared (FTIR) (KBr) 3439, 2925, 1339, and 1730 cm⁻¹. HRESIMS m/z 439.2126 [M-H]⁻ (calcd 439.2121 for C₂₆H₃₁O₆).

2,3′,4,4′-tetrahydroxy-3-geranyl-5-carboxymethyl-biphenyl methyl ester (2)

Yellowish oil. ¹H and ¹³C NMR, see Table 1. UV (MeOH) λ _max 204, 282 nm. FTIR (KBr) 3439, 2925, 1339, and 1730 cm⁻¹. HRESIMS [M-H]^- m/z 425.1974 (calculated 425.1964 for C₂₅H₂₉O₆)

Acetylation of 1 and 2

Five milligrams of compound 1 and 2 were individually dissolved in 800 µL anhydrous pyridine (stored over molecular sieves). Two milliliters of acetic anhydride was added and the mixture was stirred for 24 hours at room temperature. After evaporation, the reaction mixture was partitioned between water and ethyl acetate. Finally, the organic extract was analyzed by liquid chromatography mass spectrometry.

2,3′,4,4′-tetraacetoxy-3-geranyl-5-carboxyethyl-biphenyl methyl ester (1-a)

HRESIMS [M+NH₄]⁺ m/z 626.2958 (calculated 626.2965 for C₃₄H₄₄NO₁₀), [M + H]⁺ m/z 609.2692 (calcd 609.2700 for C₃₄H₄₁O₁₀).

2,3′,4,4′-tetraacetoxy-3-geranyl-5-carboxymethyl-biphenyl methyl ester (2-a)

HRESIMS [M+NH₄]⁺ m/z 612.2802 (calculated 612.2809 for C₃₃H₄₂NO₁₀), [M + H]⁺ m/z 595.2536 (calcd 595.2543 for C₃₃H₃₉O₁₀).

Cytotoxicity Assay

Three human cancer cell lines including MDA-MB-231 (breast carcinoma cell line), A549 (lung cancer cell line), and HeLa (cervical carcinoma cell line) were used for cytotoxicity evaluation. Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere (95% air and 5% CO₂). Exponentially growing cells were used throughout the experiments. The inhibitory effects of compounds on the growth of human cancer cell lines were determined by measuring metabolic activity using a MTT assay as previously described.¹⁰

Briefly, human cancer cell lines (4 × 10³ cells/mL) were treated for 2 days with a series of concentrations (0, 10, 20, 40, 60, and 80 µM) of the compounds dissolved in dimethyl sulfoxide (DMSO; 10 µL). After incubation, 0.5 mg/mL MTT solution (100 µL) was added to each well, and the cells were then incubated at 37 °C for 4 hours. The medium was then carefully aspirated. DMSO (100 µL) was then added to each well to dissolve the formazan crystals. The plates were read immediately at 490 nm on a microplate reader. All experiments were performed 3 times, and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the untreated controls. A dose–response curve was generated, and the IC₅₀ was determined for each compound against each cell line.

Antimicrobial Assay

Well diffusion antimicrobial assay

Crude extracts, fractions, and purified compounds were all evaluated for antimicrobial activity against a panel of bacteria and fungi using a standard well diffusion assay.¹¹ The microorganisms used and the culture conditions are shown in Table 4. Nutrient agar, brain–heart infusion agar, yeast malt agar, and potato dextrose agar media were prepared according to the manufacturer’s instructions, sterilized at 120 °C for 15 minutes, allowed to cool to room temperature, and then incubated at 37 °C for 24 hours to ensure sterility prior to use.

Table 4

Microbial Culture Conditions.

ATCC no.	Organism	Media	Temperature (°C)	Antibiotic standard
12600	Staphylococcocus aureus	NA	37	Penicillin/ampicillin
14990	Staphylococcus epidermidis	NA	37	Penicillin/ampicillin
25175	Streptococcus mutans	BHI	37	Penicillin/ampicillin
13525	Pseudomonas fluorescens	NA	26	Ciprofloxacin/gentamicin
15692	Pseudomonas aeruginosa	NA	37	Ciprofloxacin
8043	Enterococcus hirae	BHI	37	Amoxicillin/cephalexin
25238	Moraxella (Branhamella) catarrhalis	BHI	37	Amoxicillin/cephalexin
6633	Bacillus subtilis subsp spizizenii	BHI	30	Vancomycin
76615	Candida albicans	YM	24 °C-26 °C	Clotrimazole/fluconazole
22019	Candida parapsilosis	YM	24 °C-26 °C	Clotrimazole/fluconazole
10571	Candida rugosa	YM	24 °C-26 °C	Clotrimazole/fluconazole
750	Candida tropicalis	YM	24 °C-26 °C	Clotrimazole/fluconazole
16888	Aspergillus niger	PDA	30 °C	Amphotericin B
2601	Saccharomyces kudriavzevii	YM	25 °C-30 °C	Clotrimazole/fluconazole
10106	Penicillium chrysogenum	PDA	25 °C	Amphotericin B
4103	Rhizopus stolonifer	PDA	24 °C	Amphotericin B

Abbreviations: BHI, brain-–heart infusion agar; NA, nutrient agar; PDA, potato dextrose agar; YM, yeast malt agar.

Hexane-, dichloromethane-, butanol-, and water-soluble fractions of Artocarpus altilis and subfractions of the butanol fraction were dissolved in DMSO to obtain sample solutions with the concentration of 10 mg/mL. Pure compounds were dissolved in DMSO at a concentration of 10 mM. In addition, positive and negative controls were applied on each plate. Control antibiotics were prepared at 1 mg/mL (Table 4). DMSO was used as the negative control.

Minimum inhibitory concentrations

MICs were determined for the purified compounds which showed activity in the well diffusion assays using a broth dilution method with TTC as the chromogenic agent as previously described.^12,13 Pure compounds were dissolved in DMSO to obtain sample solutions at a concentration of 1000 µg/mL. Positive and negative controls were also applied on each 96-well plate. Control antibiotics for each organism (Table 3) were prepared at 1 mg/mL, and DMSO was used as negative control.

The chromogenic reagent, TTC, was used in this study as a redox indicator measuring microbial respiration. In the presence of living microorganisms, the colorless TTC is converted to the red 1,3,5-triphenylformazan. TTC dissolved in sterilized water was added to liquid media to a final concentration of 12.5 mg/L.

Microbial cultures were suspended in sterile distilled water and adjusted to a concentration equivalent to McFarland standard No. 0.5 and then diluted 100-fold with culture medium to afford a microorganism suspension (5 × 10⁵ CFU/mL).

Compounds were tested using 2-fold serial dilutions starting at 100 µg/mL performed in 96-well microtiter plates. After 20‐24 hour incubation at the preferred temperature for each organism, microbial growth/viability was assessed by measuring the optical density at 570 nm using a microplate reader. The results were expressed as % reduction of microorganism growth/viability compared with control, and MIC values were obtained using GraphPad Prism software.

Supplemental Material

Supplementary Material 1 - Supplemental material for Two New Geranylated Biphenyl Esters From Artocarpus altilis

Supplemental material, Supplementary Material 1, for Two New Geranylated Biphenyl Esters From Artocarpus altilis by Yitong Zhang, Xinghua Jin, Norman Q. Arancon and Robert P. Borris 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 financially supported by the National Basic Research Program (973) of China (2015CB856500), and by the Hawaii EPSCoR program under NSF (USA) award EPS-0903833.

ORCID iD

Robert P. Borris

Supplemental Material

Supplemental material for this article is available online.

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