Cytotoxic Phenolic Compounds from Fruit Glandular Trichomes of Macaranga tanarius

A new flavonoid, macatanarin D (1), together with five known stilbenes (2-6), was isolated from fruit glandular trichomes of Macaranga tanarius. Their structures were elucidated on the basis of spectroscopic methods and through comparison with data reported in the literature. All isolated compounds were evaluated for their cytotoxic activities against KB and MCF-7 cell lines. Compounds 3, 4, and 5 showed the strongest activities against both cell lines with IC50 values in the range of 0.03–0.12 μM, and compound 2 only showed a significant cytotoxicity against KB cell line (IC50 = 0.26 μM) and a moderate cytotoxicity against MCF-7 (IC50 = 10.4 μM). Compounds 1 and 6 showed weak cytotoxic activities against KB cell line with IC50 values of 29.3 and 24.7 μM, respectively.


Introduction
Macaranga (Euphorbiaceae) is a large genus of about 300 species mainly distributed in Southern Asia, of which 13 species are native to Vietnam [1,2]. In Vietnam, several species of this genus known as "Ba soi" have been used in traditional medicine for the treatment of swellings, wounds, and diarrhoea [2,3]. Phytochemical studies of Macaranga species have led to the discovery of various compounds such as flavonoids [4][5][6] and stilbenes [7,8], which are regarded as the main constituents [9]. ey are responsible for the cytotoxic and antioxidant activities generally found in plants of this genus [9]. Macaranga tanarius is known as "Bach dan nam" in Vietnam. e dried roots are used as an emetic agent, whereas fresh leaves are used as an anti-inflammatory drug to heal wounds [10].
A previous chemical investigation of Macaranga tanarius fruits led to the isolation of seven new and six known prenylated stilbenes [11]. In another study, it was also demonstrated that vedelianin, one of the most potent cytotoxic metabolites of this chemical series, was located in the glandular trichomes present on the surface of fruits of this species [12]. Plant glandular trichomes are considered to be natural cell factories of high biotechnological interest [13].
is result, combined with the highly cytotoxic activity of an AcOEt extract of glandular trichomes of Macaranga tanarius fruits, led us to further investigate chemically these epidermal outgrowths. Herein, we report the isolation of five known prenylated stilbenes (2-6) and the structure elucidation of the new flavonoid macatanarin D (1) and their cytotoxic activities against KB and MCF-7 cancer cell lines. e glandular trichomes were then separated and collected for further investigations by gently hand-rubbing dried fruits on a sieve of stainless-steel mesh.

Extraction and Isolation.
Dry glandular trichomes (200 g) were successively extracted with EtOH (5 × 0.5 L). e extracts were combined and concentrated under diminished pressure. e residue (24 g) was suspended in water (70 mL) and extracted successively with n-hexane and EtOAc. e n-hexane and EtOAc solutions were concentrated under reduced pressure to afford 4.9 g and 10.5 g, respectively.
e water solution was concentrated under vacuum to give 7.1 g of dry extract.

Cytotoxic Assay.
e cytotoxicity assays were carried out in triplicate in 96-well microtiter plates against KB cell line (mouth epidermal carcinoma cells) and MCF-7 cell line (breast cancer cells). Cells were maintained in Dulbecco's DMEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100 μg/mL), and gentamicin (10 μg/mL). Stock solutions of compounds were prepared in DMSO/ H 2 O (1/9), and the cytotoxicity assays were carried out in 96-well microtiter plates against cancer or normal cells (3 × 10 3 cells/mL) using a modification of the published method [14]. After 72 h incubation at 37°C in air/CO 2 (95 : 5) with or without test compounds, cell growth was estimated by colorimetric measurement of living cells stained by neutral red. Optical density was determined at 540 nm with a Titertek Multiskan photometer. e IC 50 value was defined as the concentration of the sample necessary to inhibit the cell growth to 50% of the control. Ellipticine was used as a reference compound. e UV absorption maximum at 366, 327, and 271 nm was typical for a flavonol-type compound [15]. e presence of a substituted flavonol skeleton was suggested by the analysis of 1 H and 13 C-NMR spectroscopic data (Table 1). e NMR spectroscopic data of 1 were similar to those of macakurzin B, which has been previously isolated from M. kurzii, except for the presence of a prenyl, acetyl, and OH groups [6]. In the 1 H-NMR spectrum, the presence of an ABX system at δ H 6.92 (d, e analysis of 13 C-NMR data and 2D HSQC spectrum of 1 revealed the presence of 27 carbons, corresponding to a flavonol derivative with one acetyl group and two isoprene moieties (Table 1). e HMBC correlations of H-1‴ (δ H 3.28) with C-2′ (δ C 128.2), C-3′ (δ C 127.5), C-4′ (δ C 156.1), C-2‴ (δ C 122.6), C-3‴ (δ C 131.6) and OH (δ H 7.29) with C-4′ (δ C 156.1), C-5′ (δ C 114.9), and C-3′ (δ C 127.5) determined the linkage of the isoprenyl chain with C-3′ and position of OH group at C-4′ on ring B (Figure 1). Furthermore, the COSY correlations of CH 2 -4″/H-5″ and the HMBC cross peaks of H-4″ (δ H 2.40 and 2.77) with C-5″/C-6″/C-6/C-5/C-7 and OH (δ H 3.09) with C-4″ (δ C 26.0), C-5″ (δ C 67.1), and C-6″ (δ C 77.5) were confirmed that the position of OH group was at C-5″ of pyrano ring and 2,2-dimethyldihydropyranol moiety was attached to ring A. From the 1 H-NMR spectrum, absence of resonance for a hydrogen-bonded hydroxy proton, led to the assumption that a free hydroxy group was not present at C-5. Two possible isomers could be considered at this stage. In the first one, the dihydropyranol moiety is fused to ring A via C-5 and C-6 and the O-acetyl group is located at C-7, while in the second possible isomer, the pyranol moiety is fused to ring A via C-6 and C-7 and the O-acetyl group is attached in position C-5. Since only the correlations of the methyl protons of acetyl group at δ H 1.88 with methylene protons of CH 2 -4″ at δ H 2.40 and 2.77 and no NOE interaction between the aromatic singlet H-8 (δ H 6.45) and the methyl protons of acetyl group were observed on the NOESY spectrum, the pyranol moiety is supposed to be attached at C-6 and C-7 and the acetyl group in C-5 on ring A. is suggestion agreed with NMR data found for structurally close prenylated flavonoids. In particular, when a pyrane moiety is fused via C6 and C-7 on ring A, the resonance of C-7 is around δ C 161.0-164.0 (δ C 161.2 for dinklagin B [16] and 163.7 for tanariflavanone B [17], δ C 161.1 for compound 1), whereas a pyrane moiety fused via C5 and C-6 leads to a upfield-shifted carbon C-5 signal at about 155.0 (δ C 155.5 and 155.7 for vogelins I and J, respectively [18]). e relative configuration of C-5″ was established by proton coupling constant analysis and NOESY spectrum. e pseudoaxial orientation of H-5″ can be deduced from its proton coupling constants with a gauche (J � 5.5 Hz) and an anti (7.5 Hz). is observation was confirmed by the NOESY data analysis, which showed NOE correlations between H-5″ (δ H 3.67) and CH 3 -7″ (δ H 1.34) and proton H b -4″ (δ H 2.77) of the 4″-CH 2 group. Based on these observations, the structure of the newly isolated compound 1 was determined as 5-O-acetyl-6,7-(2,2-dimethyl-3-hydroxydihydropyrano)-3′-prenyl kaempferol ( Figure 2) and named "macatanarin D." e structures of the known stilbenes: schweinfurthin H (2) [19], vedelianin (3) [20], schweinfurthin F (4), schweinfurthin E (5) [19], and 4′deprenyl-mappain (6) [21] were determined by analysis of spectroscopic data and comparison with reported data. So far, about 90% of the isolated compounds come from the leaves of Macaranga genus while 10% were isolated from other plant parts such stem and root barks, fruits, seeds, and flowers. No phytochemical studies had been conducted to date on glandular trichomes of Macaranga fruits. It is important to note that collecting time clearly influences the harvesting yield of glandular trichomes. While the young fruits do not have glandular trichomes and overripe fruits contain low yield of glandular trichomes, the adult/mature fruits, with clearly visible trichome glands, give the best results. In Vietnam, it is best to harvest mature fruits in June.

Results and Discussion
Since prenylated stilbenes and flavonoids of Macaranga genus are reported to have potent cytotoxic activities [9,22], compounds 1-6 were evaluated for their cytotoxic activity against KB and MCF-7 human cancer cell lines. Ellipticine was used as a reference compound. e results are shown in Table 2. Compounds 1 and 6 showed moderate cytotoxic activities against KB cell line with IC 50 values of 29.3 and  24.7 μM, respectively. Compounds 3, 4, and 5 showed the strongest activities against both KB and MCF-7 cell lines with IC 50 values in the range of 0.03-0.12 μM, which is evenly stronger than ellipticine. It was worth noting that three aforementioned compounds possessed the same hexahydroxanthene moiety but a variable number of hydroxy groups, which may explain the difference in their cytotoxic potencies. Compound 2 also showed a significant cytotoxicity against KB cell line (IC 50 � 0.26) but compared to compounds 3, 4, and 5, cytotoxicity appears to be much less active against the MCF-7 cell line with an IC 50 value of 10.4.

Conclusion
An undescribed flavonoid, macatanarin D (1), together with five known prenylated stilbenes (2-6) were isolated from glandular trichomes of fruits of Macaranga tanarius. Most of the compounds isolated have shown potent cytotoxic activities against the two cancer cell lines KB and MCF-7. It is postulated that these specialized metabolites are an important first line of defense against herbivorous insects and/or pathogens.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.