A New Phenolic Constituent From Carica papaya Flowers and Its Tyrosinase Inhibitory Activity

Abstract

A new phenolic (caricapapayol, 1) and 8 known compounds (2-9) were isolated from the flowers of Carica papaya. Their structures were determined by analysis of HR-ESI-MS, NMR spectral data, and comparison with the literature. Among known compounds, compound 2 has not been reported from natural source. Compounds 1, 2, and 4 exhibited tyrosinase inhibitory activity with IC₅₀ values of 14.3 ± 2.7, 25.5 ± 1.9, and 19.8 ± 3.0 µM, respectively, in comparison with positive control kojic acid IC₅₀ 11.3 ± 1.6 µM.

Carica papaya is a well-known tropical plant producing edible fruit called papaya, papaw, or pawpaw. Papaya and its products are listed as a significant nutritional source for provitamin A (carotenoids), folate, potassium, and ascorbic acid. ¹ The content of carotenoids and ascorbic acid depend on the ripeness of the fruit, growing location, and cultivar. ² Different parts of C. papaya such as barks, flowers, fruits, latex, leaves, roots, and seeds have been used in traditional medicinal remedies. ^3,4 Literature surveys indicate that C. papaya contains broad diversity of phytochemicals such as carotenoids, phenolics, alkaloids, glucosinolates, ascorbic acid, amino acids, and enzymes (papain and caricain). ¹ In this report, the flowers of C. papaya were subjected for chemical study and lead to the isolation of 9 metabolites comprising a new phenolic compound (1, Figure 1).

Compound 1 was isolated as a yellowish amorphous powder. Its molecular formula was determined to be C₂₉H₃₄O₄ by a quasi-molecular ion peak at m/z 447.2526 [M + H]⁺ (calcd for C₂₉H₃₅O₄, 447.2535) in the HR-ESI-MS and in conjunction with ¹³C-NMR data. The ¹H-NMR spectrum of 1 contained signals characteristic for a symmetric 1,3,4,5-tetrasubstituted phenyl group [δ_H 6.59 (2H, br s)], an asymmetric 1,3,4,5-tetrasubstituted phenyl group [δ_H 6.88, 6.69 (each 1 H, d, J = 2.0 Hz)], a cis-vinylene group [δ_H 6.35, 5.69 (each 1 H, d, J = 10.0 Hz)], a trans-vinylene group [δ_H 6.84, 6.76 (each 1 H, d, J = 16.0 Hz)], 2 olefinic protons [δ_H 5.27, 5.09 (each 1 H t, J = 7.0 Hz)], and 5 methyl groups [δ_H 1.78, 1.64, 1.58, 1.44, 1.44 (each 3 H, s)]. The ¹³C-NMR and DEPT spectral data of 1 showed 29 carbons comprising 4 sp² oxygenated tertiary (δ_C 157.2, 157.2, 146.4, 141.2), 6 sp² quaternary, 1 sp³ oxygenated tertiary (δ_C 77.8), 10 sp² methine, 3 sp³ methylene, and 5 methyl carbons. The HMBC correlations between H-1′ (δ_H 6.84) and C-3 (δ_C 114.3)/ C-5 (δ_C 117.1)/ C-3′ (δ_C 137.4), H-2′ (δ_H 6.76) and C-4′ (δ_C 105.8)/ C-8′ (δ_C 105.8)/ C-4 (δ_C 132.0) indicated that 1,3,4,5-tetrasubstituted phenyl groups connected each other via a trans-vinylene group (Figure 1). The presence of a geranyl group was deduced by HMBC correlations between H₃-9″ (δ_H 1.64)/H₃-10″ (δ_H 1.58) and C-7″ (δ_C 125.5)/ C-8″ (δ_C 131.9), H₂-6″ (δ_H 2.07) and C-7″/ C-8″/ C-5″ (δ_C 40.9)/ C-3″ (δ_C 134.9), H₃-4″ (δ_H1.78) and C-5″/ C-3″/ C-2″ (δ_C 124.5), H₂-1″ (δ_H 3.33) and C-3″/ C-2″. Additionally, HMBC correlations between H₂-1″ and C-5′ (δ_C 157.2)/ C-7′ (δ_C 157.2)/ C-6′ (δ_C 116.1) in conjunction with the downfield shifted values of δ_{C-5′,C-7′} 157.2 indicated the presence of hydroxy groups at C-5′, C-7′ and geranyl group at C-6′ to form a symmetric 1,3,4,5-tetrasubstituted phenyl moiety. The HMBC correlations between H-3 (δ_H 6.88) and C-1 (δ_C 141.2)/ C-2 (δ_C 146.4) suggested the assignment of2 oxygenated sp² carbons C-1 and C-2. The presence of a 3-methylbut-1-enyl side chain and its binding location at C-6 were confirmed by HMBC correlations between H₃-10 (δ_H 1.44)/ H₃-11 and C-9 (δ_C 77.8)/ C-8 (δ_C 132.3), H-7 (δ_H 6.35) and C-1 (δ_C 141.2)/ C-6 (δ_C 123.2)/ C-5 (δ_C 117.1). An ether bridge between C-1 and C-7 was deduced by a signal of sp³ oxygenated tertiary carbon at δ_C 77.8 (C-9). Consequently, the structure of compound 1 was established to be a new phenolic compound and named as caricapapayol.

Figure 1

Chemical structure and important HMBC correlations of 1.

Other compounds were determined to be 1-benzyl-5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (2), ⁵ benzyl O-β-d-glucopyranoside (3), ⁶ lariciresinol (4), ⁷ dehydrodiconiferyl alcohol (5), ⁸ vitexsoid (6), ⁹ 6-hydroxy-2,6-dimethyl-2,7-octadienoic acid (7), ¹⁰ 2,6-dimethylocta-2,7-diene-1,6-diol (8), ¹¹ 6-hydroxy-2,6-dimethyloct-7-enoic acid (9) ¹² by comparison their NMR spectral data with those reported in the literature (Supplemental Material). To the best of our knowledge, this is the first report on the isolation of compound 2 from natural source. Because of tyrosinase inhibitory effect of the methanol extract (62.7% at 200 µg/mL), compounds 1 to 9 were then evaluated their effects on the activity of mushroom tyrosinase using l-tyrosine as a substrate. Tyrosinase inhibitors can be prevented the formation of o-quinone from phenolic substrates, suppressing the browning processes. ¹³ Compounds 1, 2, and 4 showed potent inhibitory effect with their IC₅₀ values of 14.3 ± 2.7, 25.5 ± 1.9, and 19.8 ± 3.0 µM, respectively. Compounds 3, 5 to 9 displayed weak inhibitory effect in comparison with kojic acid as a positive control (Table 1).

Table 1

Tyrosinase Inhibitory Activity of Compounds 1 to 9.

Comp	IC50 (µM)	Comp	IC50 (µM)
1	14.3 ± 2.7	6	>100
2	25.5 ± 1.9	7	36.8 ± 2.5
3	>100	8	82.1 ± 3.6
4	19.8 ± 3.0	9	47.5 ± 2.9
5	76.4 ± 3.3	Pos a	11.3 ± 1.6

^aKojic acid was used as a positive control.

Experimental

General

NMR spectra, Bruker AM500 FT-NMR spectrometer; HR-ESI-MS, Agilent 6530 Accurate Mass Q-TOF LC/MS system.

Plant Material

The flowers of Carica papaya Linn. were collected at Da Nang, Vietnam in December 2016. Its scientific name was identified by botanist Dr Ngo Van Trai, Vietnam National Institute of Medicinal Materials. A voucher specimen No. DD001 was deposited at the Herbarium of the Institute of Chemistry, Vietnam Academy of Science and Technology.

Extraction and Isolation

The dried powdered C. papaya flowers (5 kg) were ultrasonically extracted with MeOH for 3 times (each 8 L, 50°C in 30 minutes). After filtration, the filtrate was evaporated in vacuo to give methanolic extract (300 g). This extract was suspended in water and successively partitioned with n-hexane, methylene chloride (MC), ethyl acetate, and n-butanol to obtain corresponding n-hexane (54 g), MC (52 g), ethyl acetate (20 g), and n-butanol (70 g) residues. The MC residue was roughly separated on a silica gel column, eluting with MC/methanol (0-100% volume of methanol) to give5 fractions CPD1-CPD5. Fraction CPD2 was further chromatographed on a silica gel column, eluting with MC/methanol (20/1, v/v) to give 4 smaller fractions CPD2A-CPD2D. Compounds 2 (5 mg, yield 1.0 × 10⁻⁴% w/w dried material) and 6 (7 mg, yield 1.4 × 10⁻⁴% w/w) were isolated from fraction CPD2A (1.2 g) using reverse phase C-18 (RP-18) column and acetone/water (2/1, v/v) as an eluent. Fraction CPD2B (0.8 g) was loaded on a silica gel column and eluted with MC/ethyl acetate (2/1, v/v) to give compounds 4 (5 mg, yield 1.0 × 10⁻⁴% w/w) and 5 (2.5 mg, yield 5.0 × 10⁻⁵% w/w). Fraction CPD2C was purified on a RP-18 column, eluting with methanol/water (2/1, v/v) to give compound 1 (9 mg, yield 1.8 × 10⁻⁴% w/w). Fraction CPD2D (0.5 g) was first chromatographed on a RP-18 column eluting with methanol/water (2/1, v/v) and further purified on a silica gel column eluting with MC/acetone (5/1, v/v) to give compound 3 (3 mg, yield 6.0 × 10⁻⁵% w/w). Fraction CPD4 (3.5 g) was separated on a silica gel column, eluting with MC/methanol (20/1, v/v) to give 3 fractions CPD4A-CPD4C. Fraction CPD4A was repeatedly chromatographed on a silica gel column, eluting with MC/ethyl acetate (4/1, v/v) to give 4 fractions CPD4A1-CPD4A4. Compounds 7 (10 mg, yield 2.0 × 10⁻⁴% w/w) and 9 (8 mg, yield 1.6 × 10⁻⁴% w/w) were isolated from fraction CPD4A4 using silica gel column and MC/methanol (10/1, v/v) as eluent. Fraction CPD4C was separated on RP-18 column eluting with methanol/water (2/1, v/v) and then purified on a silica gel column eluting with MC/acetone (3/1, v/v) to give compound 8 (7 mg, yield 1.4 × 10⁻⁴% w/w).

Caricapapayol (1)

Yellowish amorphous powder.

¹H-NMR and ¹³C-NMR (CD₃OD): Table 2.

Table 2

¹H (500 MHz) and ¹³C (125 MHz) NMR Data for Compound 1 in CD₃OD.

No.	δC	δH (mult., J in Hz)	No.	δC	δH (mult., J in Hz)
1	141.2	-	5′	157.2	-
2	146.4	-	6′	116.1	-
3	114.3	6.88 (d, 2.0)	7′	157.2	-
4	132.0	-	8′	105.8	6.49 (s)
5	117.1	6.69 (d, 2.0)	1″	23.2	3.33 (d, 7.0)
6	123.2	-	2″	124.5	5.27 (t, 7.0)
7	123.4	6.35 (d, 10.0)	3″	134.9	-
8	132.3	5.69 (d, 10.0)	4″	16.3	1.78 (s)
9	77.8	-	5″	40.9	1.97 (t, 7.0)
10	28.0	1.44 (s)	6″	27.8	2.07 (m)
11	28.0	1.44 (s)	7″	125.5	5.09 (t, 7.0)
1′	128.2	6.84 (d, 16.0)	8″	131.9	-
2′	128.0	6.76 (d, 16.0)	9″	25.8	1.64 (s)
3′	137.4	-	10″	17.7	1.58 (s)
4′	105.8	6.49 (s)

Assignment were done by HSQC and HMBC experiments.

HR-ESI-MS: m/z 447.2526 [M + H]⁺ calcd for C₂₉H₃₅O₄, 447.2535.

Supplemental Material

Supplementary Material - Supplemental material for A New Phenolic Constituent From Carica papaya Flowers and Its Tyrosinase Inhibitory Activity

Supplemental material, Supplementary Material, for A New Phenolic Constituent From Carica papaya Flowers and Its Tyrosinase Inhibitory Activity by Giang Thi Kim Lien, Do Thi Thuy Van, Dao Hung Cuong, Pham Hai Yen, Bui Huu Tai, and Phan Van Kiem in Natural Product Communications