Isolation of Six Isoprenylated Biflavonoids from the Leaves of Garcinia subelliptica

Abstract

The acetone-soluble parts of Garcinia subelliptica leaves were analyzed and six new biflavonoids were isolated, i.e., garciniaflavones A–F (1–6), as well as the five known biflavonoids amentoflavone (7), podocarpusflavone A (8), (+)-morelloflavone (9), (+)-morelloflavone-7″-O-β-glucopyranoside (10), and (+)-4‴-O-methylmorelloflavone (11) and the three triterpenoids oleanan-3-one, β-amyrin, and cycloartenol. The structures of the isolates were established based on spectroscopic analyses, including a detailed NMR spectroscopic investigation. The new biflavonoids are rare mono-isoprenylated derivatives that have a flavone-(3′–8″)-flavone core (1–4: amentoflavone type) and a flavanone-(3–8″)-flavone core (5, 6: morelloflavone type). The absolute configurations of the morelloflavone-type biflavonoids (5, 6) were confirmed by circular dichroism to be 2R,3S. The biflavonoids with an isoprenyloxy group (1) and a 2-hydroxy-3-methyl-3-butenyl group (2), and the morelloflavone-type biflavonoids with a C₅ unit are the first examples in nature. We found that 7, one of the major biflavonoids, strongly inhibited hypoxia-inducible factor-1 in human embryonic kidney 293 cells under hypoxic conditions.

Plants belonging to the genus Garcinia are well known for their abundant sources of polyphenols such as biflavonoids, xanthones, and benzophenones.^1–6) Knowledge of the chemical characteristics of biflavonoids from the genus has accumulated since the 1970s, and research into many species has shown that the majority of these biflavonoids are simply 3′–8″ and 3–8″ linked types, which are represented by amentoflavone and morelloflavone.

Xanthones and benzophenones were the main subjects of our previous studies on the chemical composition of Garcinia species. Their chemical diversity and various biological activities were demonstrated based on a chemical library that contained isoprenylated and simple oxygenated xanthones, isoprenylated benzophenones, and flavonoids.^7–14) During the last decade, the bioactivities of biflavonoids from various families have been investigated by many researchers, which has led to the identification of the different bioactivities of amentoflavone.^15–17) Our previous study also demonstrated that Garcinia leaves contain high quantities of biflavonoids, although the systematic preparation of a biflavonoid library and an analysis of their biological activities were not conducted. The leaves of G. subelliptica are not used as medicines, but their high biflavonoid content means they could be an important source of bioactive components.

During our examination of several Garcinia extracts and their fractions, we found that an acetone extract of the leaves of G. subelliptica contained various biflavonoids with a C₅ unit according to electrospray ionization mass spectrometry (LC-ESI-MS) analysis, which lead us to initiate the current phytochemical study focused on biflavonoids. A comprehensive investigation of the chemical constituents in the extract led to the isolation and characterization of six new biflavonoids, garciniaflavones A–F (1–6) that had a C₅ moiety derived from an isoprenyl unit, as well as five biflavonoids (7–11) and three triterpenoids. The structures of the new compounds (1–6) were elucidated using 2 dimensional (2D)-NMR techniques such as ¹H–¹H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence (HMQC), and heteronuclear multiple-bond correlation (HMBC), as well as high-resolution (HR) FAB-MS.

Results and Discussion

An acetone extract of the leaves of G. subelliptica was subjected to open column chromatography (CC) on silica gel and further purification by gel filtration using a Sephadex LH-20 CC. Subsequent reverse-phase CC on octadecyl silica (ODS) and preparative HPLC using two stationary phases [(ODS and polyethylene glycol (PEG)] allowed the isolation of 11 biflavonoids (1–11). The known biflavonoids were identified based on their spectral data as amentoflavone (7),¹⁸⁾ podocarpusflavone A (8),¹⁹⁾ (+)-morelloflavone⁴⁾ (syn. fukugetin) (9), (+)-morelloflavone-7″-O-β-glucopyranoside (syn. fukugiside)²⁰⁾ (10), and (+)-4‴-O-methylmorelloflavone (11).²¹⁾ Oleanan-3-one,²²⁾ β-amyrin,²³⁾ and cycloartenol²⁴⁾ were also isolated. The new biflavonoids (1–6) were obtained as pale yellow solids and exhibited positive reactions to FeCl₃ and H₂SO₄ reagents.

Garciniaflavone A (1) is the first example of a biflavonoid with an isoprenyl ether group from nature. The detailed structural elucidation was carried out as follows: The molecular formula (C₃₅H₂₆O₁₀) was deduced based on the pseudo-molecular ion ([M+Na]⁺) at m/z 629.1450 in the positive-ion HR-FAB-MS. The ¹H- and ¹³C-NMR signals of 1 (Tables 1, 2) was analyzed using double-quantum filtered correlation spectroscopy (DQF-COSY), the HMQC spectrum, and the HMBC spectrum (Fig. 1), which confirmed the existence of two apigenin units (rings A–C and D–F) and an isoprenyl group. The ¹H-NMR spectrum of 1 had signals attributable to rings A–F, as follows: δ_H 6.20 (1H, d, J=1.6 Hz, H-6) and δ_H 6.46 (1H, d, J=1.6 Hz, H-8): ring A; δ_H 8.00 (1H, br s, H-2′), δ_H 7.16 (1H, d, J=8.4 Hz, H-5′), and δ_H 8.01 (1H, br d, J=8.4 Hz, H-6′): ring B; δ_H 6.81 (1H, s, H-3): ring C; δ_H 6.66 (1H, s, H-6″): ring D; δ_H 7.60 [2H, d, J=7.6 Hz, H-2‴(6‴)] and δ_H 6.74 [2H, d, J=7.6 Hz, H-3‴(5‴)]: ring E; and δ_H 6.83 (1H, s, H-3″): ring F. The ¹H-NMR spectrum exhibited signals that were attributed to five phenolic OH groups [δ_H 13.00 (chelated OH-5), 10.82 (OH-7), 10.34 (OH-4′), 13.24 (chelated OH-5″), and 10.30 (OH-4‴)], which disappeared after the addition of D₂O. The presence of an isoprenyl group was also verified by the ¹H-NMR signals [δ_H 4.67 (2H, m, H-1″″), δ_H 5.33 (1H, m, H-2″″), and δ_H 1.68 (6H, s, H-4″″, H-5″″)].

Fig. 1. CH Long-Range Correlations for 1–6 Based on Their HMBC Spectra

Table 1. ¹H-NMR Data of Garciniaflavones A–F (1–6)^a)

Position	1	2	3	4	5a	5b	6a	6b
2					5.74 (d 11.8)	5.54 (d 12.6)	5.78 (d 12.2)	5.61 (d 12.2)
3	6.81 (s)	6.82 (d 1.2)	6.89 (s)	6.87 (s)	4.92 (d 11.8)	4.99 (d 12.6)	4.90 (d 12.2)	5.00 (d 12.2)
6	6.20 (d, 1.6)	6.20 (br s)	6.21 (d 2.4)	6.20 (br s)			5.93 (s)	5.98 (s)
8	6.46 (d, 1.6)	6.47 (br s)	6.49 (d 2.0)	6.48 (br s)	5.95 (s)	6.08 (s)
2′	8.00 (br s)	7.99 (br s)	7.97 (d 2.4)	7.91 (br s)	7.15 (d 7.8)	7.10 (d 7.8)	7.17 (d 8.4)	7.12 (d 8.6)
3′					6.41 (d 7.8)	6.46 (d 7.8)	6.41 (d 8.4)	6.62 (d 8.6)
5′	7.16 (d, 8.4)	7.16 (d 8.8)			6.41 (d 7.8)	6.46 (d 7.8)	6.41 (d 8.4)	6.62 (d 8.6)
6′	8.01 (br d, 8.4)	8.00 (d 8.8)	7.93 (d 2.4)	7.95 (br s)	7.15 (d 7.8)	7.10 (d 7.8)	7.17 (d 8.4)	7.12 (d 8.6)
3″	6.83 (s)	6.85 (s)	6.82 (s)	6.82 (s)	6.59 (s)	6.62 (s)	6.57 (s)	6.61 (s)
6″	6.66 (s)	6.68 (s), 6.69 (s)b)	6.42 (s)	6.40 (s)	6.25 (s)	6.15 (s)	6.23 (s)	6.07 (s)
2‴	7.60 (d, 7.6)	7.61 (d 8.8)	7.58 (d 8.8)	7.57 (d 8.4)	7.43 (br s)	7.19 (br s)	7.41 (br s)	7.23 (br s)
3‴	6.74 (d, 7.6)	6.75 (d 8.8)	6.70 (d 8.8)	6.73 (d 8.4)
5‴	6.74 (d, 7.6)	6.75 (d 8.8)	6.70 (d 8.8)	6.73 (d 8.4)	6.92 (d 8.0)	6.95 (d 8.1)	6.91 (d 8.0)	6.96 (d 8.6)
6‴	7.60 (d, 7.6)	7.61 (d 8.8)	7.58 (d 8.8)	7.57 (d 8.4)	7.44 (br d 8.0)	6.62 (br d 8.1)	7.43 (br d 8.0)	6.50 (br d 8.6)
1″″	4.67 (m)	4.04 (m)	6.62 (d 10.0)	2.94 (t)	2.55 (br s)	2.55 (br s)	2.50 (m)	2.50 (br s)
		4.11 (m)
2″″	5.33 (m)	4.11 (m)	5.86 (d 10.0)	1.74 (m)	1.80 (br s)	1.80 (br s)	1.75 (m)	1.75 (m)
				1.83 (m)
3″″
4″″	1.68 (s)	4.65 (s), 4.71 (s)b)	1.15 (s)	1.18 (s)	1.33 (s)	1.32 (s)	1.31 (s)	1.31 (s)
		4.80 (s), 4.85 (s)b)
5″″	1.68 (s)	1.61 (s), 1.62 (s)b)	1.31 (s)	1.02 (s)	1.33 (s)	1.32 (s)	1.32 (s)	1.32 (s)
OH-5	13.00 (s)	13.00 (s)	12.94 (s)	12.98 (s)	12.66 (s)	12.55 (s)	12.03 (s)	11.98 (s)
OH-7	10.82 (s)
OH-4′	10.34 (s)
OH-5″	13.24 (s)	13.27 (s)	13.12 (s)	13.12 (s)	13.09 (s)	13.00 (s)	13.09 (s)	12.99 (s)
OH-4‴	10.30 (s)

a) In acetone-d₆; at 400 MHz, δ in ppm, J in Hz. b) Signals for a pair of conformers. a Series, e.g., 5a, represents major conformer; b series, e.g., 5b, represents minor conformer.

Table 2. ¹³C-NMR Data of Garciniaflavones A–F (1–6)^a)

Position	1	2	3	4	5a	5b	6a	6b
2	163.8	163.9	163.4	163.7	80.9	81.9	81.0	81.1
3	102.6	102.9	103.5  f)	103.2	48.5	47.6	48.2	47.4
4	181.7	181.7	181.7	181.7	196.8	197.1	196.7	196.8
5	161.4	161.8	161.4	161.4	160.9	161.0	160.5	160.7
6	98.8	98.9	98.9	98.9	101.9 g)	101.8 i)	96.6	96.7
7	164.1	164.3	163.7	164.2	162.0 h)	162.7	162.0   j)	162.1k)
8	94.0	94.0	94.1	94.1	95.8	95.9	100.7	100.7
9	153.7	157.4	157.4	157.4	160.1	160.1	159.8	160.0
10	103.7	103.6	103.7	103.7	101.9 g)	101.8 i)	101.8	101.9
1′	120.9	120.9	122.3	120.9	128.2	127.7	128.4	128.7 l)
2′	131.4	131.8	130.8	128.9	128.8	128.8	128.3	128.7l)
3′	119.6	119.3	120.6	121.1	114.4	114.6	114.4	114.6
4′	159.4	159.4	154.2	155.4	157.3	157.6	157.3	157.4
5′	116.1	116.0	121.3	121.8	114.4	114.6	114.4	114.6
6′	127.9	127.7	124.3	127.7	128.8	128.8	128.3	128.7 l)
2″	164.0	164.1	163.6	163.5	163.4	163.4	163.4	163.4
3″	102.6	102.6	102.5	102.3	102.2	103.2	102.2	102.3
4″	182.3	182.3	182.1	182.1	181.6	181.7	181.6	181.7
5″	161.1b)	161.2	161.0	160.4	160.5	160.6	160.9	161.1
6″	96.5	96.3	98.5	98.6	98.7	98.0	98.7	98.8
7″	161.9	161.9	161.8	161.8	162.0 h)	162.4	162.0   j)	162.1k)
8″	105.2	105.2	103.1	103.8	100.6	100.2	100.6	100.6
9″	157.4	153.7	154.2	155.4	155.2	154.6	155.3	155.4
10″	104.1	104.2	103.5  f)	103.5	103.0	103.6	103.0	103.1
1‴	121.3	121.3	121.3	121.2	121.0	121.6	121.1	121.1
2‴	128.3	128.4	128.2	128.2	119.3	118.2	119.3	119.3
3‴	115.8	115.8	115.7	115.7	145.7	146.0	145.6	145.9
4‴	161.2 b)	161.4	160.6	161.1	149.7	149.7	149.6	149.8
5‴	115.8	115.8	115.7	115.7	116.1	115.1	116.1	116.2
6‴	128.3	128.4	128.2	128.2	113.3	113.8	113.3	113.4
1″″	65.7	71.9	121.3	22.1	15.5	15.5	16.0	15.0
2″″	119.2	71.4, 71.6 e)	31.7	31.6	31.2	32.1	31.2	31.2
3″″	137.8	145.0	77.7	75.7	76.3	76.5	76.2	76.3
4″″	18.1	111.6, 111.8 e)	28.2	25.6	26.3c)	26.6d)	26.1	26.1
5″″	25.4	18.0, 18.2 e)	28.0	27.7	26.4c)	26.7d)	26.8	26.8

a) In acetone-d₆; at 100 MHz, δ in ppm, J in Hz. a Series, e.g., 5a, represents major conformer; b series, e.g., 5b, represents minor conformer. b–d) Interchangeable. e) Signals for a pair of conformers. f–l) Overlapping.

The HMQC and HMBC spectra facilitated the complete assignment of all the protonated and quaternary carbons of the two apigenin units and the C₅ unit, as shown in Table 2. The interflavonoid linkage corresponding to the amentoflavone series (3′–8″ linked) was confirmed by the HMBC correlation between H-2′ and C-8″. These data were similar to those for 7, except for the isoprenyl group. In the ¹³C-NMR spectrum, the signal due to C-1″″ appeared at δ_C 65.7, indicating that the carbon was an oxymethylene group. The position of the isoprenyloxy group was confirmed to be C-7″ by HMBC, which detected a ³J correlation between H-1″″ and C-7″. Therefore, garciniaflavone A (1) was 8-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-hydroxyphenyl)-5-hydroxy-2-(4-hydroxyphenyl)-7-((3-methylbut-2-en-1-yl)oxy)-4H-chromen-4-one.

The compositions of garciniaflavones B (2) to D (4) were determined to be C₃₅H₂₆O₁₁ (2), C₃₅H₂₄O₁₀ (3), and C₃₅H₂₆O₁₀ (4) based on the MS spectra, which detected the corresponding pseudo-molecular ions: 2, ([M−H]⁻, m/z 621.1392 in FAB-MS); 3, ([M−H]⁻, m/z 603.1284 in FAB-MS); and 4, ([M−H]⁻, m/z 605.1453 in FAB-MS). Their structures had the same core structure of a 3′–8″-linked biflavonoid [amentoflavone (7)]. The difference was because the C₅ units differed with respect to their substitution position, oxidative degree, and the formation of a further heterocyclic ring (dimethylchromene or dimethylchromane). The presence of a different C₅ moiety in 2–4 was supported by NMR spectral data (Tables 1, 2). For example, 2 had the typical NMR signals of a 2-hydroxy-3-methylbut-3-en-1-yloxy group based on carbon signals (C-1″″–C-5″″), an oxymethylene proton (H-1″″), an oxymethine proton (H-2″″), two olefinic protons (H-4″″), and a methyl protons (H-5″″) instead of the isoprenyloxy group in 1.

In differential NOE experiments, irradiation of H-1″″ and H-2″″ enhanced the signal for H-6″, supporting the location of the 2-hydroxy-3-methylbut-3-en-1-yloxy group at C-7″. Based on the NMR data, the C₅ units of 3 and 4 were found to compose the dimethylpyran ring and dimethyldihydropyran ring, respectively, which condensed to C-4′ and C-5′ of the B-ring. The ¹H- and ¹³C-NMR data for 2–4 were similar to those of 1 and/or amentoflavone (7), except for the C₅ unit. The 2D-NMR spectra (Fig. 1) confirmed the structures of garciniaflavones B (2) to D (4) [i.e., 8-(5-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2-hydroxyphenyl)-5-hydroxy-7-((2-hydroxy-3-methylbut-3-en-1-yl)oxy)-2-(4-hydroxyphenyl)-4H-chromen-4-one (2), 5,5″,7,7″-tetrahydroxy-2″-(4-hydroxyphenyl)-2′,2′-dimethyl-2′H,4H,4″H-[2,6′:8′,8″-terchromene]-4,4″-dione (3), and 8-(6-(5,7-dihydroxy-4-oxo-4H-chromen-2-yl)-2,2-dimethylchroman-8-yl)-5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (4)]. Garciniaflavone B (2) was identified as a racemic mixture, because 2 had no optical rotation or Cotton effects in the circular dichroism (CD) spectrum. The multiple NMR signals of 2 (H-6″, H-4″″, H-5″″, C-2″″, C-4″″, C-5″″) showed the presence of a pair of conformers (1 : 1), which would be explained by an atropisomerism due to the restricted rotation about the interflavonyl 3′–8″ linkage.

Garciniaflavones E (5) and F (6) were obtained as optically active, pale yellow amorphous solids ([α]_D²² +139.6 (5), +59.4 (6)). The same molecular formula (C₃₅H₂₈O₁₁) was deduced from the pseudo-molecular ions in the FAB-MS spectra (5: [M−H]⁻, m/z 623.1557; 6: [M−H]⁻, m/z 623.1545). Compounds 5 and 6 had an exchangeable atropisomeric feature in the NMR spectra (Tables 1, 2, major conformers: 5a, 6a; minor conformers: 5b, 6b). The presence of a dimethylchromane ring in these compounds was supported by their NMR spectra. The ¹H- and ¹³C-NMR data of 5 and 6 were very similar to those of the flavanone-(3–8″)-flavone-type biflavonoid [morelloflavone (9)], except for the dimethylchromane ring. The HMBC spectra (Fig. 1) confirmed the structures and regioisomerism of their dimethylchromane ring, which fused linearly in 5 and angularly in 6. The configuration of their two asymmetric carbons (C-2, C-3) were determined as follows. The ¹H-NMR spectral data detected large coupling constants (J=11.8 Hz for 5; J=12.2 Hz for 6), which suggested the trans orientation of H-2 and H-3. As demonstrated by Gaffield, the Cotton effect due to the π→π* transition near 290 nm is more reliable for the determination of the C-2 stereochemistry than the n→π* transition near 340 nm, which was demonstrated unequivocally because (+)-(2R,3R)-taxifolin and (+)-(2R,3S)-epitaxifolin exhibited a stronger negative Cotton effect, whereas (−)-(2S,3S)-taxifolin and (−)-(2S,3R)-epitaxifolin exhibited a stronger positive Cotton effect.²⁵⁾ On the basis of Gaffield’s results, Li et al. elucidated the absolute structure of (+)-morelloflavone based on the inspection of its chiroptical property due to the π→π* transition, which resulted in the determination of the 2R,3S-configurations of a γ-pyrone ring.²⁶⁾ However, Ding et al. suggested that the empirical ECD rules derived from monomeric flavonoids might not be applicable to the configurational assignment of complex 3,8″-biflavonoids based on the theoretical calculations of the electronic CD of (+)-morelloflavone using the time-dependent density functional theory.²⁷⁾ They also achieved the unequivocal assignment of its 2R,3S absolute configuration by determining the contribution of the acetophenone π→π* transition of the ABC-flavanone moiety and the electronic transition within the DEF-flavone moiety to the Cotton effect near 290 nm. The CD spectra of 5, 6, 9, and 10 displayed Cotton effects near 340 and 290 nm, which were attributed to the n→π* and π→π* transitions, respectively [5: 351 nm (Δε +4.0), 291 nm (Δε +13.8); 6: 346 nm (Δε +2.8), 290 nm (Δε +7.9); 9: 350 nm (Δε +3.7), 289 nm (Δε +11.7); 10: 350 nm (Δε +5.3), 290 nm (Δε +12.6)] (Fig. 2). The high-amplitude positive Cotton effects near 290 nm indicated the 2R absolute configuration for all four compounds based on the data accumulated by Li et al. and Ding et al. Thus garciniaflavones E (5) and F (6) were elucidated to be (2R,3S)-3-(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-8-yl)-5-hydroxy-2-(4-hydroxyphenyl)-8,8-dimethyl-2,3,7,8-tetrahydropyrano[3,2-g]chromen-4(6H)-one and (2R,3S)-3-(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-8-yl)-5-hydroxy-2-(4-hydroxyphenyl)-8,8-dimethyl-2,3,9,10-tetrahydropyrano[2,3-f]chromen-4(8H)-one, respectively.

Fig. 2. Steric View of Conformers 5a and 5b

The molecule was minimized based on the MMFF94 calculation using the PCMODEL 9.3 molecular modeling program.

The CD spectra also exhibited the 2R,3S-configurations of the aglycone of 10. The 2R,3S-configurations of 11 were confirmed based on the optical rotation ([α]_D²⁵ +61.8). Li et al.²⁶⁾ also performed a conformational study of the two atropisomeric conformers of 9 based on computational calculations, which was further supported by the NMR spectral evidence. Their study concluded that 9 had two conformers bearing an extended structure (major conformer) and a compact structure (minor conformer).²⁶⁾ The atropisomeric ratio of the major and minor conformers of 5 (1 : 0.3), 6 (1 : 0.21), and 11 (1 : 0.21) were similar to that of 9 (1 : 0.27) and the ¹H-NMR signals of the morelloflavone core displayed no reversed ratio. Thus, it was also concluded that the major and minor conformers of each corresponded to the extended and compact forms, respectively. Supporting evidence for the nonreversal ratio of the extended and compressed rotamers was obtained based on the similarity of their CD curve, because Ding et al. also demonstrated that the two rotamers of 9 had different chiroptical properties, which indicated that the conformational exchange (ratio reversal) significantly affected the ECD of 3,8″-biflavonoids.²⁷⁾ The optimized 3D structures of 5a and 5b are shown in Fig 3. Interestingly, the ratio of the conformers of 10 based on the ¹H-NMR spectrum was 1 : 1 in acetone solution, while the Cotton effects near 230 nm (negative), 290 nm (positive), and 350 nm (positive) had a stronger intensity than those of 9, which may have been due to its compressed conformer.

Fig. 3. CD Spectra of Biflavonoids (5, 6, 9, 10)

Biflavonoids are major constituents in the leaves of G. subelliptica, and the current study revealed several new aspects of their structural diversity. The most recent findings are as follows: A flavone-(3′–8″)-flavone core (1–4, 7, 8: amentoflavone type) and a flavanone-(3–8″)-flavone core (5, 6, 9–11: morelloflavone type) were the major cores of a biflavonoid constituent. Isolation of the 3′–8″-coupled biflavonoids from this plant are the first example, although the 3–8″-coupled biflavonoids are representative constituents of the woody parts and fruit of the genus Garcinia.^4,6,28) Biflavonoids with a C₅ unit are very rare in nature and the seven known examples were isolated from the leaves of two Calophyllum species (Clusiaceae: Calophyllum venulosum^29,30) and Calophyllum inophylloide³¹⁾). All of these previously known biflavonoids with a C₅ unit belong to the amentoflavone type and they have a C-isoprenylated substituent on C-6″, which is representative of a pyranoamentoflavone. Compounds 1 and 2 are the first biflavonoids to be isolated that possess an isoprenyloxy substituent, while the presence of a C₅ unit that substitutes for the C-5′ of amentoflavone is also the first example, which form a dimethylchromene ring (3) and a dimethylchromane ring (4), respectively. Compounds 5 and 6 are the first examples of morelloflavone-type biflavonoids with a C₅ unit. The leaves of G. subelliptica are considered to have a high expression system for the biosynthetic pathway of two major biflavonoids (7, 9), which is followed by their structural modification to produce a biflavonoid scaffold. Monomeric flavonoids, i.e., apigenin, naringenin, luteolin, and their isoprenylated derivatives, have not been isolated from this material, which indicates that amentoflavone is the biosynthetic precursor of 1–4 whereas (+)-morelloflavone is the precursor of 5, 6, 10, and 11.

We evaluated plant-derived organic extracts to determine the hypoxia-inducible factor (HIF)-1 inhibitors using a stable transformant of human embryonic kidney (HEK) 293 cells in a previously established HIF-1-dependent promoter assay.³²⁾ We observed that the acetone-soluble part of the leaves of G. subelliptica inhibited HIF-1-dependent luciferase activity under hypoxic conditions. The effects of one major biflavonoid (7) on HIF-1 activity were examined further under hypoxic conditions, which we compared with aerobic exposure in the HEK293 clone (Fig. 4). The treatment of cells with 7 significantly reduced the luciferase activity at a concentration of 100 µm, whereas the other major biflavonoid 9 had no statistically significant effect on HIF-1 activity at a concentration of 100 µm (data not shown). The cytotoxicity of 7 was evaluated in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays. At the maximum concentration that dissolved in the medium (100 µm), 7 had no cytotoxic effect under hypoxic and aerobic conditions. Fang et al. demonstrated that apigenin, which is a monomeric flavonoid found in various foods, has an inhibitory effect on tumor-specific angiogenesis via the downregulation of vascular endothelial growth factor (VEGF) expression at the transcriptional level by inhibiting HIF-1α expression in human ovarian cancer cells (OVCAR-3 and A2780/CP70).³³⁾ It is interesting that 7, which is a biflavonoid with a dimeric apigenin structure, also had an inhibitory effect on tumor specific-angiogenesis by inhibiting the production of various endogenous factors such as VEGF, although only a correlative effect on HIF-1α has been reported.³⁴⁾ Further studies on the mechanism of action of 7 and a structure–activity relationship study using amentoflavone derivatives may provide new insights into the possible pharmacologic targeting of HIF-1 for therapeutic purposes, which will be described in future studies.

Fig. 4. Dose–Response Relationship of Amentoflavone (7) in the HIF-1-Dependent Reporter Assay

Data shown are means from three independent experiments performed in triplicate; the bars represent standard deviations. Asterisks indicate a statistically significant difference compared with the hypoxic control. ** p<0.01.

Recently, the multifunctional bioactivity of 7 and 9 has been documented for the treatment and prevention of disease.^15–17) The present study and our knowledge of polyphenols suggest that the leaves of the family Clusiaceae are a major source of useful polyphenols and terpenoids, which could be applied for medicinal purposes and/or for the prevention of disease.

Experimental

General Experimental Procedures

The following instruments were used: optical rotations, JASCO P-1020 polarimeter; UV spectra, Shimadzu UV-3100 spectrophotometer (in methanol solution); CD spectra, JASCO J-820 spectrometer (in MeOH solution); IR spectrum: PerkinElmer Spemtrum 100 FT-IR spectrometer; ¹H- and ¹³C-NMR spectra, JEOL JNM ECA-500 and JEOL JNM AL-400 [chemical shift values in ¹H-NMR spectra are presented as δ values with tetramethylsilane (TMS) as internal standard]; ESI-MS, Shimadzu LCMS-IT-TOF instrument; and FABMS, JEOL JMS-DX-300 instrument.

The following adsorbents were used for purification: analytical TLC, Merck Kieselgel 60 F₂₅₄ (0.25 mm); preparative TLC, Merck Kieselgel 60 F₂₅₄ (0.5 mm); CC, Merck Kieselgel 60, Pharmacia Fine Chemicals AB Sephadex LH-20, and Fuji Silysia Chemical Chromatorex DMS; and vacuum-liquid chromatography (VLC), Merck Kieselgel 60. A Waters Sep-Pak C₁₈ cartridge was used for small-scale reversed-phase (RP) open CC. The following system was used for preparative HPLC: LC-6AD pump, SIL-10AXL auto injector, SCL-10AVP system controller, and SPD-10AV UV/Vis absorbance detector, equipped with CLASS-VP software. The separation was performed on a Capcell Pak C18 UG120 S-5 column (5 µm, 250×10.0 mm; Shiseido, Japan) and Discovery HS PEG (5 µm, 250×10.0 mm; Supelco, U.S.A.) at 40°C. The flow rate of the mobile phase was 5 mL/min, and detection was performed at 280 nm.

Energy-minimized stereostructures were obtained using PCMODEL 9.3 (Serena Software, U.S.A.).

Plant Material

The leaves of G. subelliptica were collected on Okinawa Island, Japan, during September 2007 and identified by one of the co-authors (M. Iinuma). A voucher specimen (number GSL_0709) was deposited in the herbarium of Gifu Pharmaceutical University.

Extraction and Isolation

The dried and ground leaves (5.0 kg) were subsequently extracted at room temperature with acetone (18 L×3 times) and 70% methanol (18 L×2 times). The extracts were then concentrated in vacuo, which produced a dry solid mass of 530 g and 820 g, respectively. Part of the acetone extract (497 g) was subjected to CC (120×12 cm) on silica gel (2.2 kg), eluted with a mixture of CHCl₃–MeOH with increasing polarity; eight fractions (frs. 1–8) were obtained. Oleanan-3-one (1.4 g), β-amyrin (41.5 mg), and cycloartenol (6.3 mg) were obtained from fr. 1 (CHCl₃, 87 g) after repeated purification on CC over silica gel (n-hexane–EtOAc: open system and VLC) and Sep-Pak cartridges (H₂O–acetone) and recrystallization (n-hexane). Purification of fr. 2 (CHCl₃–MeOH 20 : 1, 30 g) by recrystallization (n-hexane–EtOAc) allowed the isolation of 9 (25 g). Fraction 3 (CHCl₃–MeOH 10 : 1, 185 g) was further fractionated by reversed-phase CC (10×100 cm) on DMS (1.5 kg) eluted with a mixture of MeOH–H₂O. Then the MeOH ratio was increased stepwise, and the eluates were combined in TLC analysis to produce 13 subfractions (frs. 3_A–3_M). The seventh subfraction (fr. 3_G) was further purified on Sephadex LH-20 CC (MeOH), DMS CC (H₂O–MeCN), and Sep-Pak C₁₈ CC (H₂O–MeCN), followed by purification on HPLC using a Capcell Pak C18 UG120 column (H₂O–MeOH) to yield 7 (196 mg), 8 (900 mg), and 11 (6 mg). Purification of fr. 3_H on CC over silica gel (CHCl₃–MeOH), Sephadex LH-20 CC (MeOH), and Sep-Pak C₁₈ CC (H₂O–MeCN), followed by HPLC using a Discovery HS PEG column (H₂O–MeOH) allowed the isolation of 1 (12 mg), 2 (10 mg), 3 (12 mg), 4 (9 mg), 5 (17 mg), and 6 (9 mg). Compound 10 (2.5 g) was obtained from fr. 5 (CHCl₃–MeOH 5 : 1; 35 g) after purification using the same procedure on CC with DMS (MeCN–H₂O), Sephadex LH-20 (MeOH), and a Sep-Pak C₁₈ cartridge (H₂O–MeCN).

Garciniaflavone A (1): A pale yellow solid; [α]_D²⁵ −4.5° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 332 (4.45), 269 (4.49) nm; IR ν (KBr disk): 3391, 1653, 1609, 1583, 1497, 1443, 1374, 1349, 1289, 1241, 1219, 1160, 1116, 1030, 834 cm⁻¹; HR-FAB-MS m/z: 629.1450 ([M+Na]⁺, Calcd for C₃₅H₂₆O₁₀Na: 629.1418); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

Garciniaflavone B (2): A pale yellow solid; [α]_D²⁵ −4.4° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 335 (4.47), 269 (4.53) nm; IR ν (KBr disk): 3403, 1654, 1605, 1583, 1497, 1445, 1354, 1373, 1352, 1287, 1165, 1117, 836 cm⁻¹; HR-FAB-MS m/z: 621.1392 ([M−H]⁻, Calcd for C₃₅H₂₅O₁₁: 621.1397); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

Garciniaflavone C (3): A pale yellow solid; [α]_D²³ −0.6° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 341 (4.45), 269 (4.67) nm; IR ν (KBr disk): 3428, 1652, 1612, 1575, 1507, 1426, 1363, 1287, 1259, 1240, 1163, 1115, 838 cm⁻¹; HR-FAB-MS m/z: 603.1284 ([M−H]⁻, Calcd for C₃₅H₂₃O₁₀: 603.1291); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

Garciniaflavone D (4): A pale yellow solid; [α]_D²⁶ +14.8° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 337 (4.59), 270 (4.61) nm; IR ν (KBr disk): 3431, 1653, 1610, 1576, 1506, 1425, 1363,, 1283, 1238, 1168, 1111, 836 cm^–1; HR-FAB-MS m/z: 605.1453 ([M−H]^–, Calcd for C₃₅H₂₅O₁₀: 605.1447); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

Garciniaflavone E (5): A pale yellow solid; [α]_D²² +139.6° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 349 (3.53), 293 (3.64) nm; CD (c=16.0 µm, MeOH) nm (Δε): 351 (+4.0), 291 (+13.8), 229 (−14.3) nm; IR ν (KBr disk): 3410, 1642, 1614, 1580, 1516, 1445, 1371, 1259, 1156, 1116, 1091, 833 cm⁻¹; HR-FAB-MS m/z: 623.1557 ([M−H]⁻, Calcd for C₃₅H₂₇O₁₀: 623.1554); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

Garciniaflavone F (6): A pale yellow solid; [α]_D²² +59.4° (c=0.1, MeOH); UV (c=0.001, MeOH) λ_max (log ε): 349 (3.48), 287 (3.54) nm; CD (c=16.0 µm, MeOH) nm (Δε): 346 (+2.8), 290 (+7.9), 228 (−6.2) nm; IR ν (KBr disk): 3410, 1642, 1612, 1585, 1517, 1484, 1446, 1369, 1261, 1157, 1116, 1093, 835 cm⁻¹; HR-FAB-MS m/z: 623.1545 ([M−H]⁻, Calcd for C₃₅H₂₇O₁₀: 623.1554); ¹H-NMR (acetone-d₆, 400 MHz) and ¹³C-NMR (acetone-d₆, 100 MHz), see Tables 1 and 2, respectively.

HIF-1-Dependent Luciferase Assay

Compounds were prepared as stock solutions in dimethyl sulfoxide (DMSO) and stored as aliquots at −20°C. The final concentration of DMSO was 0.1% (v/v) in the biological assays. HEK293 clone cells were stably transfected with p2.1 HIF-1-dependent luciferase vector and used for the assay. The cell culture, hypoxia treatment, and cell-based luciferase reporter assay were performed according to protocols described in a previous study.³²⁾

MTT Assay

HEK293 cells were plated at a density of 6.0×10³ cells/well (96-well plate, TPP Techno Plastic Products AG) in 100 µL of McCoy’s 5A medium with 10% FBS and antibiotics, incubated for 24 h, and exposed to the test compounds under aerobic or hypoxic conditions for 24 h. The MTT reagent (0.5 mg/mL, Sigma-Aldrich, Japan) was added to the media. After 4-h incubation at 37°C, the media were removed, and the cells were lysed with DMSO. Absorbance at 570 nm was measured on a Multiskan JX plate reader (Thermo Fisher Scientific).

Clonogenic Assay

HEK293 cells were plated at a density of 3.0×10² cells in a 6-well plate (TPP Techno Plastic Products AG) and treated with the test compounds for 24 h under normoxic or hypoxic conditions. On day 7, after colonies formed, the cells were washed with PBS, fixed in methanol for 30 min at room temperature, stained with Giemsa’s solution over 3 h, and washed with water. These colonies were then counted.

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