Chemical Constituents from the Stem Bark of Macaranga capensis (Baill.) Benth. ex Sim

Abstract

Background and objectives

Various parts of Macaranga capensis are widely used in traditional folk medicine due to their health-promoting effects. This study aims to investigate the phytochemical constituents in the stem bark of M. capensis.

Methodology

The stem bark of M. capensis was extracted using a methanol/dichloromethane (1:1) mixture, and the crude extract was subjected to chromatographic techniques to isolate the phytochemical constituents.

Results

Chemical investigation of the stem bark of M. capenesis led to isolation of eight previously reported metabolites: chrysoeriol (1), isorhamnetin (2), kaempferol (3), 8-hydroxy-6-methoxy-3-pentylisocoumarin (4), scopoletin (5), 3,3′-di-O-methylellagic acid-4′-O-α-L-rhamnopyranoside (6), 3,3ʹ,4′-trimethoxyellagic acid (7), and betulin (8). The structures of these compounds were elucidated using spectroscopic analysis and by comparison with those reported in the literature. It is worth noting that all the compounds identified in this study are reported from Macaranga capensis for the first time. While isocoumarins are a class of lactonic natural products commonly found in microbes and higher plants, the identification of compound 4 represents the first report of its occurrence within the Euphorbiaceae family.

Conclusion

The isolation of these compounds contributes to a deeper understanding of the chemical diversity within the Macaranga genus and underscores its potential therapeutic applications.

Introduction

Macaranga capensis (Baill.) Benth. ex Sim (Euphorbiaceae) is a deciduous tree characterized by pale gray bark, spirally arranged, broadly ovate leaves, and short thorns on its young stems. It bears densely packed, yellowish-green, dehiscent fruits and purplish brown to blackish seeds. M. capensis, an inhabitant of evergreen forests (ranging from 305-2133 m), is considered an indicator of forest invasion. It is found along lake and stream banks of East Africa (Kenya and Ethiopia) to South Africa and can grow up to 30 meters in height.^1–3

Traditionally, M. capensis stem bark has been used in KwaZulu-Natal, South Africa, to treat various skin conditions, such as sunburn.^1,4 The bark decoction is used to manage diarrhea in local communities in Kenya ⁵ and South Africa. ⁶ The Washambaa people of Tanzania use M. capensis leaves to manage allergies. ⁷ In South Africa, an aqueous decoction of the leaves is used to treat chest complications, coughs, and bloody sputum. ⁸ In Zimbabwe and Ethiopia, root-based formulations of M. capensis are used to manage male impotence.^9–11 Additionally, traditional healers in Vietnam and Thailand utilize M. capensis to treat various diseases, including diabetes. ¹²

Phytochemical studies of various Macaranga species have identified the genus as a source of prenylated stilbenes and flavonoids, coumarin, ellagic acids, and terpenoids, which exhibit intriguing biological properties.^13–18 Additionally, a previous study by Majeed ¹² reported the presence of phytochemicals, such as 3α-hydroxyaleuritolic acid 2-p-hydroxybenzoate and 3-acetoxyoleanolic acid, in the crude methanolic extract of M. capensis stem bark. Our recent investigation examined the antibacterial activities of crude extracts from different parts of M. conglomerata, M. kilimandscharica, and M. capensis against 13 bacterial strains expressing multi-drug resistance (MDR) phenotypes. ¹⁹ Among these, the stem bark of M. capensis exhibited significant antibacterial activity, with minimum inhibitory concentration (MIC) values ranging from 4 to 32 μg/mL and bactericidal effects (with minimum bactericidal concentration (MBC)/MIC ratio ≤ 4) against Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Providencia stuartii, Pseudomonas aeruginosa, and Staphylococcus aureus. ¹⁹ Despite the traditional uses of various parts of M. capensis and the promising pharmacological potential of their crude extracts, limited information is available on the phytochemical characterization of this species. Therefore, this study aims to fill this gap through the isolation and identification of chemical constituents from the stem bark of M. capensis. As a result, phytochemical investigation of the stem bark of M. capensis led to the isolation of different classes of secondary metabolites, and their structure elucidation is reported herein.

Methodology

Experimental Procedures

Silica gel (Kieselgel 60; mesh sizes 60-120, 70-230, and 230-400; Merck, Darmstadt, Germany) and Sephadex LH–20 (25-100 μm; Sigma Aldrich, USA) were used as stationary phases for column chromatography (CC) and gel filtration, respectively. Thin Layer Chromatography (TLC) was performed on pre-coated silica gel 60 F₂₅₄ plates (0.25 mm; Merck, Darmstadt, Germany). Various solvent systems were tested, with optimal separation achieved using hexane-ethyl acetate or dichloromethane-methanol (v/v), depending on the polarity of the compound. TLC Plates were visualized under UV light at 254 nm and 365 nm, and further treated by spraying with a 5% (v/v) H₂SO₄–H₂O solution followed by gentle heating. All solvents, including methanol, dichloromethane, and n-hexane, were of analytical grade and purchased from either Fischer Scientific or Sigma Aldrich (USA). NMR spectra were recorded on Bruker Advance Neo spectrometers operating at 400, 500, or 600 MHz for ¹H NMR and at 100, 125, or 150 MHz for ¹³C NMR, respectively (Bruker BioSpin GmbH, Rheinstetten, Germany). Samples were dissolved in deuterated solvents, and all the spectra were recorded at room temperature using standard pulse sequences. Spectral data were processed using MestReNova 14.1 software and referenced to the residual solvent peaks.

Plant Collection

The stem bark of Macaranga capensis (Baill.) Benth. ex Sim was harvested in March 2019 from mature trees growing in Ngangao Forest, Taita-Taveta County, Kenya (3°25ʹ S, 38°20ʹ E). Collection was performed using a sterile spud to gently remove sections of bark without damaging the main trunk or compromising the health of the tree, ensuring a sustainable harvesting approach. Botanical identification of the plant was confirmed by Mr Patrick C. Mutiso, a taxonomist from the Faculty of Science and Technology (FST), University of Nairobi, where a voucher specimen (HIUON 2019/003) was deposited at the University of Nairobi Herbarium for future reference.

Extraction and Isolation

The stem bark of M. capensis was cut into pieces, air-dried under shade, ground into fine powder (600 g), and extracted three times at room temperature using a 1:1 mixture of methanol and dichloromethane for 24 h each. The combined extracts were filtered and concentrated under reduced pressure using a rotary evaporator, yielding a dark brown crude extract (65.9 g), corresponding to an extraction yield of 11.0%, calculated using the formula: yield (%) = (weight of dried extract/weight of dried plant material) × 100. Approximately 60.0 g of stem bark crude extract was subjected to column chromatography (CC) on silica gel (60-120 mesh), eluted with EtOAc/n-hexane (0:10 to 10:0), yielding 350 fractions of 100 mL each. These fractions were combined based on their TLC profiles into six fractions (F_A−F). Fraction F_C (580.5 mg) was then subjected to another CC on silica gel (230-400 mesh), eluted with an isocratic system of EtOAc/n-hexane (3:7) to give compound 8 (18.7 mg) and three sub-fractions (Fr_C1−3)_. Sub-fraction Fr_C2 (83.2 mg) was further purified by chromatotron using an EtOAc/n-hexane (1:9 to 10.0) system to yield compounds 4 (4.0 mg), 5 (1.5 mg), and 6 (2.5 mg). Repeated chromatotron of sub-fraction Fr_C3 (43.2 mg) with a ternary system of CH₃OH/EtOAc/n-hexane (0.5:2.5:7) led to the isolation of compound 1 (2.3 mg) and a mixture (1.9 mg) of compounds 2 and 3. Size exclusion chromatography on fraction F_D (270.0 mg) with CH₂Cl₂/CH₃OH (1:1) as mobile phase was done to afford four subfractions (Fr_D1−4). Sub-fraction F_D3 (90.6 mg) was loaded onto a silica gel column (230-400 mesh) and eluted with a binary system of CH₂Cl₂/n-hexane (2:8), affording compound 7 (3.5 mg). The overall isolation and purification workflow is illustrated in Figure 1.

Figure 1.

Schematic Representation of the Isolation and Purification Workflow of the Compounds from the Stem Bark Extract of M. capensis.

Results

Identification of Compounds

Systematic fractionation of the stem bark extract, followed by chromatographic purification of subfractions, led to the isolation of eight secondary metabolites, including flavonoids (1-3), coumarins (4 and 5), ellagic acid derivatives (6 and 7), and terpenoid (8). The isolated compounds were identified by comparing their NMR spectroscopic data (NMR spectra, Supplemental material) with those reported in the literature. These compounds were identified as chrysoeriol (1), ²⁰ isorhamnetin (2), ²¹ kaempferol (3), ²² 8-hydroxy-6-methoxy-3-pentylisocoumarin (4), ²³ scopoletin (5), ²⁴ 3,3′-di-O-methylellagic acid-4′-O-α-L-rhamnopyranoside (6), ²⁵ 3,3ʹ,4′-trimethoxyellagic acid (7) ²⁵ and betulin (8) ²⁶ (Figure 2).

Figure 2.

Chemical Structure of Compounds 1–8 from the Stem Bark of M. capensis.

Chrysoeriol (1): A yellowish solid; ¹H-NMR (600 MHz, CDCl₃): δ_H 7.53 (1H, s, H-2′), 7.52 (1H, d, J = 1.6, H-6′), 6.90 (1H, d, J = 8.8, H-5′), 6.86 (1H, s, H-3), 6.48 (1H, d, J = 2.1, H-8), 6.16 (1H, d, J = 2.1, H-6), 3.86 (3H, s, 3′-OCH₃); ¹³C-NMR (150 MHz, CDCl₃): δ δ_C 181.8 (C-4), 164.2 (C-7), 163.7 (C-2), 161.5 (C-5), 157.4 (C-8a), 150.8 (C-4′), 148.1 (C-3′), 121.5 (C-1′), 120.4 (C-6′), 115.8 (C-5′), 110.2 (C-2′), 103.7 (C-4a), 103.2 (C-3), 98.9 (C-6), 94.1 (C-8), 56.0 (C-3′-OCH₃).

Isorhamnetin (2): A yellowish solid; ¹H-NMR (600 MHz, CDCl₃): δ_H7.72 (1H, d, J = 2.2, H-2′), 7.65 (1H, dd, J = 9.0, 2.2, H-6′), 6.89 (1H, d, J = 9.0, H-5′), 6.39 (1H, d, J = 2.1, H-8), 6.14 (1H, d, J = 2.1, H-6), 3.81 (3H, s, 3′-OCH₃); ¹³C-NMR (150 MHz, CDCl₃): δ_C 175.9 (C-4), 164.1 (C-7), 160.7 (C-5), 156.2 (C8a), 148.8 (C-4′), 147.4 (C-3′), 146.8 (C-2), 135.7 (C-3), 122.0 (C-1′), 121.7 (C-6′), 115.5 (C-5′), 111.7 (C-2′), 103.0 (C-4a), 98.3 (C-6), 93.5 (C-8), 55.8 (C-3′-OCH₃).

Kaempferol (3): A yellowish solid; ¹H-NMR (600 MHz, CDCl₃): δ_H 8.00 (2H, d, J = 9.0, H-2′/6′), 6.89 (2H, d, J = 9.0, H-3′/5′), 6.39 (1H, d, J = 2.1, H-8), 6.14 (1H, d, J = 2.1, H-6); ¹³C-NMR (150 MHz, CDCl₃): δ_C 175.9 (C-4), 164.1 (C-7), 160.7 (C-5), 159.2 (C-4′), 156.2 (C8a), 146.8 (C-2), 135.7 (C-3), 129.5 (C-2′/6′), 122.0 (C-1′), 115.5 (C-3′/5′), 103.0 (C-4a), 98.3 (C-6), 93.5 (C-8).

8-Hydroxy-6-methoxy-3-pentyl-1H-isochromen-1-one (4): White solid; ¹H-NMR (600 MHz, CDCl₃): δ_H 6.46 (1H, d, J = 2.3 Hz, H-7), 6.31 (1H, d, J = 2.3 Hz, H-5), 6.17 (1H, s, H-4), 2.49 (2H, m, H-9), 1.69 (2H, m, H-10), 1.34 (2H, m, H-11), 1.35 (2H, m, H-12), 0.91 (3H, m, H-13), 3.87 (3H, s, 6-OCH₃); ¹³C-NMR (150 MHz, CDCl₃): δ_C 166.9 (C-6), 166.7 (C-1), 163.8 (C-8), 158.2 (C-3), 139.6 (C-4a), 104.1 (C-4), 101.2 (C-5), 100.3 (C-7), 100.1 (C-8a), 55.8 (C-6-OCH₃), 33.4 (C-9), 31.3 (C-11), 26.7 (C-10), 22.5 (C-12), 14.1 (C-13).

Scopoletin (5): White solid; ¹H-NMR (600 MHz, CDCl₃): δ_H 7.60 (1H, d, J = 9.5, H-2), 6.92 (1H, s, H-8), 6.85 (1H, s, H-5), 6.27 (1H, d, J = 9.5, H-4), 3.95 (3H, s, 6-OCH₃); ¹³C-NMR (150 MHz, CDCl₃): δ_C 161.6 (C-2), 150.4 (C-7), 149.8 (C-10), 144.1 (C-6), 143.4 (C-3), 113.6 (C-4), 111.7 (C-9), 107.6 (C-5), 103.4 (C-8), 56.6 (C-6-OCH₃).

3,3′-di-O-methylellagiciacid-4′-O-α-L-rhamnopyranoside (6): White amorphous solid; ¹H-NMR (600 MHz, CDCl₃): δ_H 7.78 (1H, s, H-5), 7.50 (1H, s, H-5′), 5.55 (1H, d, J = 1.8, H-1′′), 4.03 (3H, s, 3-OCH₃), 4.02 (3H, s, 3′-OCH₃), 3.93 (1H, d, J = 2.7, H-3′′), 3.68 (1H, dd, J = 9.2, 6.2, H-4′′), 3.49 (1H, dd, J = 9.4, 6.2, H-5′′), 3.14 (1H, d, J = 5.87, H-2′′), 1.11 (3H, d, J = 6.2, H-6′′); ¹³C-NMR (150 MHz, CDCl₃): δ_C 158.6 (C-7), 158.4 (C-7′), 150.2 (C-4), 141.9 (C-3), 141.7 (C-4′), 141.1 (C-2), 141.1 (C-2′), 140.3 (C-3′), 114.2 (C-6), 114.2 (C-6′), 112.8 (C-1′), 112.0 (C-1), 111.8 (C-5′), 111.7 (C-5), 99.8 (C-1′′), 71.6 (C-2′′), 70.5 (C-4′′), 70.3 (C-5′′), 70.1 (C-3′′), 61.7(C-3-OCH₃), 61.0 (C-3′-OCH₃), 17.9 (C-6′′).

3,3ʹ,4′-trimethoxyellagiciacid (7): White amorphous solid; ¹H-NMR (500 MHz, CDCl₃): δ_H 8.21 (1H, s, H-5′), 7.63 (1H, s, H-5), 4.10 (3H, s, 3-OCH₃), 4.03 (3H, s, 3′-OCH₃), 3.99 (3H, s, 4-OCH₃); ¹³C-NMR (125 MHz, CDCl₃): δ_C 158.5 (C-7), 158.4 (C-7′), 154.4 (C-4), 147.6 (C-4′), 143.3 (C-3′), 141.4 (C-6′), 140.9 (C-3), 117.6 (C-5′), 114.2 (C-1′), 113.0 (C-6), 112.9 (C-1), 111.6 (C-2), 111.6 (C-2′), 107.5 (C-5), 61.5 (C-3-OCH₃), 61.4 (C-3′-OCH₃), 56.8 (C-4-OCH₃).

Betulin (8): White crystal; ¹H-NMR (400 MHz, CDCl₃): δ_H 4.68 (1H, d, J = 2.3, H-29a), 4.57 (1H, m, H-29b), 3.79 (1H, dd, J = 10.9, 2.0, H-28a), 3.33 (1H, d, J = 10.8, H-28b), 3.18 (1H, dd, J = 11.2, 4.9, H-3), 2.38 (1H, td, J = 10.8, 5.8, H-19), 1.93 (1H, m, H-21a), 1.86 (1H, m, H-22a), 1.67 (2H, m, H-1), 1.67 (3H, s, H-30), 1.63 (2H, m, H-2), 1.63 (1H, m, H-13), 1.62 (2H, m, H-12), 1.58 (1H, m, H-18), 1.52 (2H, m, H-6), 1.49 (1H, m, H-21b), 1.38 (2H, m, H-7), 1.38 (2H, m, H-11), 1.25 (1H, m, H-9), 1.22 (2H, m, H-16), 1.02 (2H, m, H-15), 1.02 (3H, s, H-26), 1.01 (1H, m, H-22b), 0.97 (3H, s, H-27), 0.96 (3H, s, H-23), 0.82 (3H, s, H-25), 0.75 (3H, s, H-24); ¹³C-NMR (100 MHz, CDCl₃): δ_C 150.6 (C-20), 109.8 (C-29), 79.1 (C-3), 60.7 (C-28), 56.4 (C-17), 55.4 (C-5), 50.5 (C-9), 48.9 (C-18), 47.9 (C-19), 42.8 (C-14), 41.0 (C-8), 39.0 (C-4), 38.8 (C-1), 37.4 (C-10), 37.3 (C-13), 34.4 (C-7), 34.1 (C-22), 29.9 (C-21), 29.3 (C-16), 28.1 (C-23), 27.5 (C-2), 27.2 (C-15), 25.3 (C-12), 21.1 (C-11), 19.2 (C-30), 18.4 (C-6), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.9 (C-27).

Discussion

This study presents the chemical investigation of M. capensis, a plant endemic to Kenya. All the compounds reported here were isolated from this plant for the first time. However, some of these compounds have been previously reported from other Macaranga species, supporting M. capensis's taxonomic position within the genus. Notably, this is evident for kaempferol (3), reported from M. indica, ¹⁶ scopoletin (5), isolated from M. triloba ²⁷ and M. sampsonii, ²⁸ and 3,3ʹ,4′-trimethoxyellagiciacid (7), previously found in M. conglomerata and M. occidentalis.^29,30 Although chrysoeriol (1), isorhamnetin (2), 3,3′-di-O-methylellagiciacid-4′-O-α-L-rhamnopyranoside (6), and betulin (8) have been obtained from other plants, they were isolated from the Macaranga genus for the first time. Ellagic acid glycosides, including compound 6, have been previously isolated from Aphananthe aspera (Ulmaceae) ³¹ and Prosopis juliflora (Leguminosae). ³² Betulin (8), previously unknown in the Macaranga genus, is a common, naturally occurring triterpene. This compound has been reported from other species belonging to the family Euphorbiaceae, including Euphorbia micractina, ³³ Euphorbia spinidens, ³⁴ and Bridelia ferruginae. ³⁵ While coumarins and ellagic acid derivatives have been previously reported from the Macaranga genus,^{27,29,30,36–38} the identification of 8-hydroxy-6-methoxy-3-pentylisocoumarin (4) is reported here for the first time in the family Euphorbiaceae. This compound was previously reported from the stem bark of Xylosma longifolia (Flacourtiaceae) ³⁹ and Tessmannia densiflora (Caesalpiniaceae). ²³ The discovery of this compound within the family Euphorbiaceae helps to increase the chemical diversity of secondary metabolites in the genus Macaranga. The biosynthesis of this isocoumarin (4) derivative may be influenced by environmental factors such as geography (altitude, aspect), climate (temperature, precipitation, seasonality), and soil conditions, which were reported to significantly impact the growth, physiology, and secondary metabolite biosynthesis in plants. ⁴⁰ These factors may account for variations in the presence and concentration of bioactive compounds within M. capensis and other related species, underscoring the importance of considering environmental context in phytochemical and pharmacological studies.

The distinct chemical components isolated in this study may provide an explanation for some of the ethnomedicinal uses of M. capensis in regions where it is found. Additionally, these metabolites may contribute to the bactericidal activity observed in the stem bark extract, as demonstrated in our recent investigation against bacterial strains exhibiting MDR phenotypes. ¹⁹ It is important to note that several of these compounds have already been studied for various biological activities, including antibacterial. For instance, Nascimento et al ⁴¹ investigated the antibacterial activity of chrysoeriol (1) against Enterococcus faecalis, Bacillus subtillis, S. aureus, P. aeruginosa, K. pneumoniae, and E. coli. The authors found significant activity with MIC values of 0.06 µg/mL for E. coli, 0.12 µg/mL for P. aeruginosa, 0.25 µg/mL for K. pneumonia, 1 µg/mL for E. faecalis, 1 µg/mL for B. subtillis, and 0.25 µg/mL for S. aureus. Furthermore, isorhamnetin (2) has been reported to exhibit antimycobacterial effects on Mycobacterium tuberculosis H37Rv and multidrug-resistant clinical isolates with MICs of 158 and 316 μM, respectively. ⁴² The presence of this compound in M. capensis validates its traditional use for treating chest complications, coughs, and bloody sputum. Scopoletin (5) has been reported to exert anti-allergic activity by inhibiting the production of cytokines (interleukin (IL)-4, IL-5, IL-10, and interferon (IFN)-γ) and suppressing nuclear factor and GATA3 expression in activated T cells and phorbol myristate acetate (PMA)/ionomycin-induced EL-4 T cells at a concentration of 50 μμ. ⁴³ Several studies have also reported that betulin (8) has protective effects against diabetes, including restoring insulin resistance by improving glucose tolerance, inhibiting lipid peroxidation in the hippocampus, modifying basal learning performance, reducing inflammatory cytokines (IL-6, IL-1β, tumor necrosis factor (TNF)α), and inhibiting nuclear factor kappa B (NFκB) signaling axis in diabetic rats.^44–46 A key limitation of this study is the limited quantity of material available from the natural source. This constraint hindered the comprehensive evaluation of the biological activity of the identified compounds. To address these limitations, future research should prioritize bioassay-guided fractionation to isolate active compounds in sufficient quantities for in-depth biological testing.

Conclusion

Eight (08) secondary metabolites, including flavonoids, isocoumarin, and its chemical cousin, coumarin, ellagic acid, and terpenoids, are reported from the stem bark of Macaranga capensis. The occurrence of these compounds supports the plant's traditional medicinal use and underscores its rich phytochemical profile. Notably, the identification of certain compounds for the first time within the Macaranga genus may help chemotaxonomic studies. These findings contribute to a deeper understanding of the chemical diversity of secondary metabolites found in the genus Macaranga and their potential therapeutic applications. Future studies should focus on a comprehensive pharmacological evaluation of these compounds.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251372423 - Supplemental material for Chemical Constituents from the Stem Bark of Macaranga capensis (Baill.) Benth. ex Sim

Supplemental material, sj-docx-1-npx-10.1177_1934578X251372423 for Chemical Constituents from the Stem Bark of Macaranga capensis (Baill.) Benth. ex Sim by Hashim Ibrahim, Justus Mukavi, John Mmari Onyari, Leonidah Kerubo Omosa, Vaderament-A. Nchiozem-Ngnitedem and Inna Popova in Natural Product Communications