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Original Research Article

DTT 2022; 1(1): 12-18

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.001

Copyright © The Pharmaceutical Society of Korea.

Anti-Adipogenic Effect of Terminalin Isolated from the Seeds of Wild Mango (Irvingia gabonensis) on 3T3-L1 Preadipocytes

Bum Soo Lee1, Heesun Kang1, Min Jeong Yoo1, Se Yun Jeong1, Yoon-Joo Ko2, Ki Hyun Kim1

1School of Pharmacy, Sungkyunkwan University, Suwon, Korea
2Laboratory of Nuclear Magnetic Resonance, National Center for Inter-University Research Facilities (NCIRF), Seoul National University, Seoul, Korea

Correspondence to:Ki Hyun Kim, khkim83@skku.edu

Received: April 11, 2022; Revised: May 10, 2022; Accepted: June 2, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Irvingia gabonensis(Irvingiaceae), commonly known as “wild mango,” is a herbaceous and multifunctional fruit tree native to tropical Africa. Wild mango fruits have been primarily used as a food source and in traditional medicinal applications. As part of ongoing research to explore biologically active natural products, chemical analysis of the aqueous extract of I. gabonensis seeds was performed using a liquid chromatography-mass spectrometry-ultraviolet-based analysis, which led to the isolation of one major compound. The structure of the compound was identified as terminalin using nuclear magnetic resonance spectroscopic analysis and electrospray ionization-mass spectrometry. Terminalin was evaluated for its effects on lipid metabolism and adipogenesis during adipocyte maturation. It substantially inhibited lipid accumulation compared to the control. Consistently, the mRNA expression of mature adipocyte marker genes, Adipoq and Fabp4, was reduced upon incubation with terminalin. We evaluated the effects of terminalin on lipid metabolism by measuring the transcription of lipolytic and lipogenic genes. Exposure to terminalin significantly elevated the expression of the lipolytic gene, ATGL, during adipogenesis. In contrast, exposure to terminalin significantly reduced the expression of lipogenic genes, FASN and SREBP1. Our findings provide insights into the therapeutic potential of terminalin as an anti-obesity agent.

KeywordsIrvingia gabonensis, wild mango, terminalin, 3T3-L1 preadipocytes

Obesity is a serious health problem caused due to complex genetic, dietary, lifestyle, and environmental factors (Spiegelman and Flier 2001). It is associated with various pathological disorders, including hypertension, atherosclerosis, diabetes, and cancer (Kopelman 2000). Obesity is aggravated by excess accumulation of lipids and triglycerides in the adipose tissue (Trujillo and Scherer 2006), which plays a crucial role in the regulation of energy homeostasis via the control of food intake and energy expenditure (Trujillo and Scherer 2006). Obesity is associated with the expansion of adipose tissue, accompanied by the differentiation of preadipocytes into mature adipocytes, along with the accumulation of lipid droplets in adipocytes (Rosen and Spiegelman 2006; Yang et al. 2008). Hence, the identification of compounds that prevent adipogenesis and lipogenesis is an essential strategy to alleviate obesity and develop therapeutic avenues for metabolic diseases.

Irvingia gabonensis, commonly known as “African mango” or “wild mango,” is a herbaceous and multifunctional fruit tree belonging to the family Irvingiaceae. It is native to tropical Africa. Its fruits are similar to mango and have been primarily used as a food source and in traditional medicinal applications (Okoronkwo et al. 2014; Fungo et al. 2016; Ofundem et al. 2017). Recently, the fruit showed a significant effect on weight management (Mateus-Reguengo et al. 2020), rendering it popular in the nutraceuticals. Traditionally, the seeds of I. gabonensis fruit are an important part of the diet in West and Central Africa, as they contain carbohydrates and proteins (Mateus-Reguengo et al. 2020). The seeds of I. gabonensis fruit also contain high-soluble fibers, which aid in delaying gastric emptying and act as a laxative (Mateus-Reguengo et al. 2020). Pharmacological studies of the seeds have demonstrated various biological activities, such as improvement of blood sugar levels in diabetes (Oben et al. 2008) and a positive effect on obesity via reduction of total cholesterol, triglyceride, low-density lipoprotein (LDL), and blood glucose levels and elevation of high-density lipoprotein (HDL)-cholesterol levels (Ngondi et al. 2005). Previous phytochemical studies of I. gabonensis seed extract revealed diverse chemical constituents, including steroids, flavonoids, alkaloids, cardiac glycosides, volatile oils, terpenoids, tannins, saponins (Giami et al. 1994), and gallotannins, demonstrating a potent antioxidant effect (Arogba et al. 2012).

As part of our ongoing studies for the identification of biologically active natural products from diverse natural sources (Lee et al. 2020a; Yu et al. 2020; Lee et al. 2021a; Lee et al. 2021b), phytochemical investigation of an aqueous extract of I. gabonensis seeds was performed using liquid chromatography/mass spectrometry (LC/MS)-guided isolation, which led to the isolation of one major compound. Based on the comparison of 1H and 13C nuclear magnetic resonance (NMR) spectroscopic and physical data with the previously reported values and LC/MS analyses, the compound was identified as terminalin. Herein, the isolation, elucidation of the structure of terminalin, and evaluation of its effects on adipogenesis and lipid metabolism in adipocytes are described.

General experimental procedures

The ultraviolet (UV) spectrum data was evaluated using Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were acquired using Varian UNITY INOVA 800 NMR spectrometer (Varian, Palo Alto, CA, USA) operating at 800 MHz (1H) with chemical shifts reported in ppm (δ). Preparative high-performance liquid chromatography (HPLC) was performed using Waters 1525 Binary HPLC pump equipped with Waters 996 Photodiode Array Detector (Waters Corporation, Milford, CT, USA). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan) was used for open-column chromatography. Semi-preparative HPLC was performed using Shimadzu Prominence HPLC System equipped with SPD-20A/20AV Series Prominence HPLC UV-Vis Detector (Shimadzu, Tokyo, Japan). LC/MS analyses were performed using Agilent 1200 Series HPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 Series electrospray ionization (ESI) mass spectrometer with an analytical Kinetex HPLC column (4.6 × 100 mm, 3.5 μm). Merck pre-coated silica gel F254 and RP-18 F254s plates were used for thin-layer chromatography (TLC). Spots were detected on TLC plates under UV light or upon heating after spraying with anisaldehyde sulfuric acid.

Plant materials

I. gabonensis seeds were provided by the Korean health functional food company FromBIO Co., Ltd. in June 2019. The material was authenticated by one of the authors (K. H. K.). A voucher specimen of the material (WM-FB-2019-06) was deposited at the R&D Center, FromBIO Co., Ltd.

Extraction and isolation

I. gabonensis seeds (500 g) were dried, crushed, and then extracted twice with distilled water at 30℃ for 24 h. The extract was filtered using a centrifuge, and the filtrate was concentrated under a vacuum using a rotary evaporator at 25-30℃. The resultant extract was lyophilized to obtain the crude aqueous extract powder (35 g). Using an in-house-built UV library as a reference, LC/MS analysis of the extract revealed the presence of one main peak with m/z 601.0 [M-H] and another major component with m/z 301.0 [M-H], which was identified as ellagic acid. To isolate the compound corresponding to the unidentified peak, the extract (5.0 g) was subjected to analysis using Diaion HP-20 column in 100% H2O to eliminate the sugar portion, and fraction M was obtained by elution with 100% MeOH. The fraction M (2.5 g) was fractionated by preparative reverse-phase HPLC using a gradient solvent system of MeOH/H2O (10-100% MeOH in 50 min, flow rate of 5 mL/min) to obtain four fractions (M1-M4). LC/MS analysis of the fractions derived via HPLC separation revealed the presence of the target peak with m/z 601.0 [M-H] in the fraction M3. Finally, the fraction M3 (90 mg) was purified via semi-preparative reverse-phase HPLC with 40% MeOH/H2O (flow rate of 2 mL/min) to isolate the compound corresponding to the target peak (tR = 18.5 min, 50 mg), which was identified as terminalin.

Terminalin: bright-yellow powder; UV (MeOH) λmax (log ε) 382 (1.5), 258 (4.3) nm; IR (KBr) νmax 3350, 2942, 1715, 1583, 1502, and 1025 cm−1; 1H and 13C NMR (800 and 200 MHz, respectively) (Table 1); negative ESIMS m/z 601.1 [M-H]; positive HR-ESIMS m/z 603.0045 [M+H]+ (calculated for C28H11O16, 603.0047).

Table 1 1H (800 MHz) and 13C NMR (200 MHz) data of terminalin in DMSO-d6 (δ ppm)a

PositionTerminalin
δH (J in Hz)δC
1, 23139.0 s
2, 24139.6 s
3, 25148.3 s
4, 267.52 s110.7 d
5, 27108.7 s
6, 28159.5 s
7, 20136.3 s
8, 21123.3 s
9, 22112.7 s
10, 17107.2 s
11, 16112.8 s
12, 15136.6 s
13, 19146.1 s
14, 18158.3 s

aJ values are represented in Hz and shown in parentheses; 13C NMR assignments are based on HMBC experiments.


Cell culture and differentiation

3T3-L1 pre-adipocytes obtained from the American Type Culture Collection (ATCC® CL-173TM) were grown on Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% penicillin/streptomycin (P/S) and 10% bovine calf serum in a humidified incubator containing 5% CO2. For adipogenic differentiation, 3T3-L1 cells were incubated for two days in an MDI induction medium. The MDI induction medium comprised DMEM with 10% fetal bovine serum (FBS), 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg/mL of insulin. Subsequently, the medium was replaced with DMEM containing 10% FBS, 1% P/S, and 10 μg/mL insulin on alternate days until day 10. To assess the effects of terminalin on adipogenesis, 3T3-L1 cells were treated with terminalin at concentrations of 0, 5, 10, and 50 μM during the process of adipogenesis. The same volume of DMSO was used as a negative control. On day 8, we used Oil Red-O staining to visualize the lipid droplets and harvested the cells for quantitative real-time polymerase chain reaction (RT-qPCR).

Oil Red O staining

Oil Red O staining was performed to visually detect the lipid droplets in differentiated adipocytes. Oil Red O powder (300 mg) was dissolved in 100 mL of 99% isopropyl alcohol to prepare the Oil Red O stock solution. Then, 30 mL of the prepared stock solution was diluted with 20 mL of distilled water to prepare the Oil Red O working solution before use. After adipogenesis (day 8), mature adipocytes were fixed with 10% formaldehyde for 15 min and then washed with 60% isopropyl alcohol. The fixed cells were stained with the Oil Red O working solution for 1 h at room temperature, and the cells were washed with phosphate-buffered saline (PBS). The stained lipids were visualized using a Leica DMi1 inverted microscope (Leica Microsystems Korea).

Reverse transcription and quantitative real-time polymerase chain reaction (RT- qPCR)

The Easy-Blue reagent (Intron Biotechnology, Seongnam, Korea) was utilized to extract total RNA from the adipocytes. For reverse transcription, 1 μg of total RNA extracted with Maxim RT-PreMix Kit (Intron Biotechnology) was reverse transcribed into cDNA using the ImProm-II Reverse Transcription System (Promega, Fitchburg, WI, USA). The cDNA was mixed with KAPATM SYBR FAST qPCR (Kapa Biosystems, Wilmington, MA, USA); the primers used for RT-qPCR are indicated in Table 2. The qPCR reaction data were analyzed using a CFX96TM or Chromo4 real-time PCR detector (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was quantified and normalized to that of β-actin for each reaction.

Table 2 Sequences of primers used for RT-qPCR

GeneForwardReverse
β-Actin5′-ACGGCCAGGTCATCACTATTG-3’5′-TGGATGCCACAGGATTCCA-3′
Adipoq5′-CAGGCCGTGATGGCAGAGATG-3’5′-GGTTTCACCGATGTCTCCCTTAG-3′
Fabp45′-AAGGTGAAGAGCATCATAACCCT-3’5′-TCACGCCTTTCATAACACATTCC-3′
ATGL5′-TTCACCATCCGCTTGTTGGAG-3’5′-AGATGGTCACCCAATTTCCTC-3′
FASN5'-TTGACGGCTCACACACCTAC-35'-CGATCTTCCAGGCTCTTCAG-3'
SREBP15′-AACGTCACTTCCAGCTAGAC-3’5′-CCACTAAGGTGCCTACAGAGC-3′

Statistical analysis

The averages and error bars are expressed as the standard error of the mean (SEM; n = 3 samples). The statistical significance was analyzed using a two-tailed Student’s t-test performed using Excel, and p-values were evaluated. **p < 0.01, and ***p < 0.001 vs. the control group.

Isolation and identification of the compounds

Dried I. gabonensis seeds were crushed and extracted with water at 30℃ to obtain the crude aqueous extract via rotary evaporation and freeze-drying. The crude extract was analyzed using LC/MS with reference to an in-house UV library database. The results demonstrated the presence of one main peak with m/z 601.0 [M-H] and a unique UV spectrum of the extended conjugated system (λmax 382 and 258 nm) and another major component with m/z 301.0 [M-H], which was clearly identified without isolation through the reference to our in-house UV library. To isolate the compound corresponding to the unidentified main peak, the crude extract was subjected to Diaion HP-20 column using 100% H2O to eliminate the sugar portion, and the fraction M was obtained by elution with 100% MeOH. The LC/MS/UV-guided fractionation of fraction M using the preparative reverse-phase HPLC and semi-preparative HPLC resulted in the isolation of the compound corresponding to the peak with m/z 601.0 [M-H] (Fig. 1). Based on the comparison of 1H and 13C NMR spectroscopic and physical data with the previously reported values and LC/MS analyses (Yoon et al. 2022), the compound was identified as terminalin (Fig. 2).

Figure 1.Separation of terminalin.

Figure 2.Chemical structure of terminalin.

Evaluation of the effects of terminalin on adipogenesis and lipid metabolism

To investigate the effects of terminalin on adipogenesis, 3T3-L1 pre-adipocytes were treated with terminalin at concentrations of 5, 10, and 50 μM during the process of adipogenesis for eight days (Fig. 3). The cells were differentiated for eight days, and lipid droplets within the mature adipocytes were stained using Oil Red O staining solution (Yi et al. 2020). The images of Oil Red O solution-stained adipocytes showed that terminalin substantially prevented the de novo generation of adipocytes and lipid accumulation within adipocytes compared to those observed in the control, especially at a high concentration of 50 μM (Fig. 3). Thus, we performed RT-qPCR to assess the expression of adipogenic markers. Terminalin reduced the mRNA expression of mature adipocyte marker genes, Adipoq and Fabp4, in a dose-dependent manner during adipocyte maturation, although the transcription level of Adipoq showed a minor increase upon treatment with terminalin at a concentration of 10 μM (Fig. 4). These data demonstrate that terminalin prevents adipogenesis of 3T3-L1 preadipocytes.

Figure 3.Inhibitory effect of terminalin on adipogenesis. 3T3-L1 cells were treated with terminalin at various concentrations. Images of adipocytes stained with Oil Red O after incubation with 5, 10, and 50 μM of terminalin during adipogenesis.

Figure 4.Relative mRNA expression of Adipoq, Fabp4, ATGL, FASN, and SREBP1 during adipogenesis in 3T3-L1 adipocytes incubated with terminalin. The data represent the mean ± SEM for n = 3. **p < 0.01, ***p < 0.001.

Next, we assessed the capacity of terminalin to regulate lipid metabolism via the expression of the lipolytic gene, ATGL, and lipogenic genes, FASN and SREBP1 (Fig. 4). The mRNA expression of the lipolytic gene (ATGL) was significantly upregulated upon exposure to terminalin at concentrations of 5 and 10 μM during adipogenesis, whereas the expression of lipogenic genes (FASN and SREBP1) was significantly downregulated at all treatment concentrations (5, 10, and 50 μM) of terminalin (Fig. 4). These data indicated that terminalin inhibited adipogenesis and facilitated lipid metabolism via the promotion of lipolysis and inhibition of lipogenesis.

The growth of adipose tissues occurs with the differentiation of preadipocytes in the adipose tissues into adipocytes and the synthesis and accumulation of lipid droplets in adipocytes (Smith and Kahn 2016). Therefore, the identification of active compounds that prevent adipogenesis and lipogenesis has been considered a potential therapeutic strategy for the management of obesity and other related metabolic diseases. We evaluated the effects of terminalin on regulating lipid metabolism during adipogenesis. Adipocyte differentiation involves multiple processes with changes in hormone sensitivity and morphology. It is regulated by transcription factors and signaling pathways (Cho et al. 2004). 3T3-L1 cells are a well-established in vitro assay system to assess adipogenesis and adipocyte differentiation (Kong et al. 2009).

Examination of the anti-adipogenic activity of terminalin in 3T3-L1 cells revealed that terminalin inhibited adipogenesis and suppressed the enlargement of lipid droplets. Additionally, the mRNA expression of adipocyte markers, Adipoq and Fabp4, decreased noticeably following treatment with terminalin. Terminalin also promoted lipid metabolism by upregulating the expression of the lipolytic gene, ATGL, and downregulating the expression of lipogenic genes, FASN and SREBP1.

As part of our continued search for natural products with biological properties, our group has been investigating anti-adipogenic natural products derived from natural sources. We demonstrated that withasilolides G–I, identified as novel withanolides derived from the roots of Indian ginseng (Withania somnifera), inhibited adipogenesis and enlargement of lipid droplets, indicated by reduced mRNA expression levels of Fabp4 and Adipsin. Active withasilolides G–I also promoted lipid metabolism by upregulating the expression of lipolytic genes, HSL and ATGL, and downregulating the expression of the lipogenic gene, SREBP1 (Lee et al. 2022). According to our previous study, two new C10-polyacetylene glycosides, (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside and (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside, were isolated from the florets of safflower (Carthamus tinctorius). (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside inhibited adipogenesis in 3T3-L1 preadipocytes, whereas (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside promoted adipogenesis (Baek et al. 2021). (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside also prevented lipid accumulation by suppressing the expression of lipogenic genes and increasing that of lipolytic genes (Baek et al. 2021). In addition, our group found an anti-adipogenic pregnane steroid isolated from Hydractinia-associated fungus, Cladosporium sphaerospermum SW67 (Lee et al. 2020b). In the study, we showed that 3α-hydroxy-pregn-7-ene-6,20-dione inhibited lipid accumulation along with the expression of the adipocyte marker gene (Adipsin). The expression of the lipolytic gene, ATGL, was elevated and that of lipogenic genes, FASN and SREBP1, were inhibited by 3α-hydroxy-pregn-7-ene-6,20-dione (Lee et al. 2020b). In contrast, 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid and α-dimorphecolic acid isolated from the aerial parts of Lespedeza cuneata induced adipocyte differentiation, as evidenced by the upregulated mRNA expression of Fabp4 in 3T3-L1 pre-adipocytes (Kang et al. 2021). Furthermore, compounds 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid and α-dimorphecolic acid regulated lipid metabolism by inducing lipolytic and lipogenic gene expression (Kang et al. 2021).

In conclusion, terminalin was isolated from I. gabonensis seeds using LC/MS-guided process. Terminalin was evaluated for its anti-adipogenesis activity and lipid metabolism throughout different stages of adipocyte differentiation in 3T3-L1 cells. Terminalin efficiently inhibited the differentiation of 3T3-L1 preadipocytes into adipocytes by downregulating the mRNA expression of Adipoq and Fabp4. Furthermore, it upregulated the expression of the lipolytic gene, ATGL, and downregulated the expression of lipogenic genes, FASN and SREBP1. Our findings support the metabolic role of terminalin in the prevention of excessive lipid accumulation in obesity.

No potential conflict of interest relevant to this article was reported.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (2019R1A5A2027340 and 2021R1A2C2007937).

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Article

Original Research Article

DTT 2022; 1(1): 12-18

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.001

Copyright © The Pharmaceutical Society of Korea.

Anti-Adipogenic Effect of Terminalin Isolated from the Seeds of Wild Mango (Irvingia gabonensis) on 3T3-L1 Preadipocytes

Bum Soo Lee1, Heesun Kang1, Min Jeong Yoo1, Se Yun Jeong1, Yoon-Joo Ko2, Ki Hyun Kim1

1School of Pharmacy, Sungkyunkwan University, Suwon, Korea
2Laboratory of Nuclear Magnetic Resonance, National Center for Inter-University Research Facilities (NCIRF), Seoul National University, Seoul, Korea

Correspondence to:Ki Hyun Kim, khkim83@skku.edu

Received: April 11, 2022; Revised: May 10, 2022; Accepted: June 2, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Irvingia gabonensis(Irvingiaceae), commonly known as “wild mango,” is a herbaceous and multifunctional fruit tree native to tropical Africa. Wild mango fruits have been primarily used as a food source and in traditional medicinal applications. As part of ongoing research to explore biologically active natural products, chemical analysis of the aqueous extract of I. gabonensis seeds was performed using a liquid chromatography-mass spectrometry-ultraviolet-based analysis, which led to the isolation of one major compound. The structure of the compound was identified as terminalin using nuclear magnetic resonance spectroscopic analysis and electrospray ionization-mass spectrometry. Terminalin was evaluated for its effects on lipid metabolism and adipogenesis during adipocyte maturation. It substantially inhibited lipid accumulation compared to the control. Consistently, the mRNA expression of mature adipocyte marker genes, Adipoq and Fabp4, was reduced upon incubation with terminalin. We evaluated the effects of terminalin on lipid metabolism by measuring the transcription of lipolytic and lipogenic genes. Exposure to terminalin significantly elevated the expression of the lipolytic gene, ATGL, during adipogenesis. In contrast, exposure to terminalin significantly reduced the expression of lipogenic genes, FASN and SREBP1. Our findings provide insights into the therapeutic potential of terminalin as an anti-obesity agent.

Keywords: Irvingia gabonensis, wild mango, terminalin, 3T3-L1 preadipocytes

Introduction

Obesity is a serious health problem caused due to complex genetic, dietary, lifestyle, and environmental factors (Spiegelman and Flier 2001). It is associated with various pathological disorders, including hypertension, atherosclerosis, diabetes, and cancer (Kopelman 2000). Obesity is aggravated by excess accumulation of lipids and triglycerides in the adipose tissue (Trujillo and Scherer 2006), which plays a crucial role in the regulation of energy homeostasis via the control of food intake and energy expenditure (Trujillo and Scherer 2006). Obesity is associated with the expansion of adipose tissue, accompanied by the differentiation of preadipocytes into mature adipocytes, along with the accumulation of lipid droplets in adipocytes (Rosen and Spiegelman 2006; Yang et al. 2008). Hence, the identification of compounds that prevent adipogenesis and lipogenesis is an essential strategy to alleviate obesity and develop therapeutic avenues for metabolic diseases.

Irvingia gabonensis, commonly known as “African mango” or “wild mango,” is a herbaceous and multifunctional fruit tree belonging to the family Irvingiaceae. It is native to tropical Africa. Its fruits are similar to mango and have been primarily used as a food source and in traditional medicinal applications (Okoronkwo et al. 2014; Fungo et al. 2016; Ofundem et al. 2017). Recently, the fruit showed a significant effect on weight management (Mateus-Reguengo et al. 2020), rendering it popular in the nutraceuticals. Traditionally, the seeds of I. gabonensis fruit are an important part of the diet in West and Central Africa, as they contain carbohydrates and proteins (Mateus-Reguengo et al. 2020). The seeds of I. gabonensis fruit also contain high-soluble fibers, which aid in delaying gastric emptying and act as a laxative (Mateus-Reguengo et al. 2020). Pharmacological studies of the seeds have demonstrated various biological activities, such as improvement of blood sugar levels in diabetes (Oben et al. 2008) and a positive effect on obesity via reduction of total cholesterol, triglyceride, low-density lipoprotein (LDL), and blood glucose levels and elevation of high-density lipoprotein (HDL)-cholesterol levels (Ngondi et al. 2005). Previous phytochemical studies of I. gabonensis seed extract revealed diverse chemical constituents, including steroids, flavonoids, alkaloids, cardiac glycosides, volatile oils, terpenoids, tannins, saponins (Giami et al. 1994), and gallotannins, demonstrating a potent antioxidant effect (Arogba et al. 2012).

As part of our ongoing studies for the identification of biologically active natural products from diverse natural sources (Lee et al. 2020a; Yu et al. 2020; Lee et al. 2021a; Lee et al. 2021b), phytochemical investigation of an aqueous extract of I. gabonensis seeds was performed using liquid chromatography/mass spectrometry (LC/MS)-guided isolation, which led to the isolation of one major compound. Based on the comparison of 1H and 13C nuclear magnetic resonance (NMR) spectroscopic and physical data with the previously reported values and LC/MS analyses, the compound was identified as terminalin. Herein, the isolation, elucidation of the structure of terminalin, and evaluation of its effects on adipogenesis and lipid metabolism in adipocytes are described.

Materials and Methods

General experimental procedures

The ultraviolet (UV) spectrum data was evaluated using Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). NMR spectra were acquired using Varian UNITY INOVA 800 NMR spectrometer (Varian, Palo Alto, CA, USA) operating at 800 MHz (1H) with chemical shifts reported in ppm (δ). Preparative high-performance liquid chromatography (HPLC) was performed using Waters 1525 Binary HPLC pump equipped with Waters 996 Photodiode Array Detector (Waters Corporation, Milford, CT, USA). Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan) was used for open-column chromatography. Semi-preparative HPLC was performed using Shimadzu Prominence HPLC System equipped with SPD-20A/20AV Series Prominence HPLC UV-Vis Detector (Shimadzu, Tokyo, Japan). LC/MS analyses were performed using Agilent 1200 Series HPLC System (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector and a 6130 Series electrospray ionization (ESI) mass spectrometer with an analytical Kinetex HPLC column (4.6 × 100 mm, 3.5 μm). Merck pre-coated silica gel F254 and RP-18 F254s plates were used for thin-layer chromatography (TLC). Spots were detected on TLC plates under UV light or upon heating after spraying with anisaldehyde sulfuric acid.

Plant materials

I. gabonensis seeds were provided by the Korean health functional food company FromBIO Co., Ltd. in June 2019. The material was authenticated by one of the authors (K. H. K.). A voucher specimen of the material (WM-FB-2019-06) was deposited at the R&D Center, FromBIO Co., Ltd.

Extraction and isolation

I. gabonensis seeds (500 g) were dried, crushed, and then extracted twice with distilled water at 30℃ for 24 h. The extract was filtered using a centrifuge, and the filtrate was concentrated under a vacuum using a rotary evaporator at 25-30℃. The resultant extract was lyophilized to obtain the crude aqueous extract powder (35 g). Using an in-house-built UV library as a reference, LC/MS analysis of the extract revealed the presence of one main peak with m/z 601.0 [M-H] and another major component with m/z 301.0 [M-H], which was identified as ellagic acid. To isolate the compound corresponding to the unidentified peak, the extract (5.0 g) was subjected to analysis using Diaion HP-20 column in 100% H2O to eliminate the sugar portion, and fraction M was obtained by elution with 100% MeOH. The fraction M (2.5 g) was fractionated by preparative reverse-phase HPLC using a gradient solvent system of MeOH/H2O (10-100% MeOH in 50 min, flow rate of 5 mL/min) to obtain four fractions (M1-M4). LC/MS analysis of the fractions derived via HPLC separation revealed the presence of the target peak with m/z 601.0 [M-H] in the fraction M3. Finally, the fraction M3 (90 mg) was purified via semi-preparative reverse-phase HPLC with 40% MeOH/H2O (flow rate of 2 mL/min) to isolate the compound corresponding to the target peak (tR = 18.5 min, 50 mg), which was identified as terminalin.

Terminalin: bright-yellow powder; UV (MeOH) λmax (log ε) 382 (1.5), 258 (4.3) nm; IR (KBr) νmax 3350, 2942, 1715, 1583, 1502, and 1025 cm−1; 1H and 13C NMR (800 and 200 MHz, respectively) (Table 1); negative ESIMS m/z 601.1 [M-H]; positive HR-ESIMS m/z 603.0045 [M+H]+ (calculated for C28H11O16, 603.0047).

Table 1 . 1H (800 MHz) and 13C NMR (200 MHz) data of terminalin in DMSO-d6 (δ ppm)a.

PositionTerminalin
δH (J in Hz)δC
1, 23139.0 s
2, 24139.6 s
3, 25148.3 s
4, 267.52 s110.7 d
5, 27108.7 s
6, 28159.5 s
7, 20136.3 s
8, 21123.3 s
9, 22112.7 s
10, 17107.2 s
11, 16112.8 s
12, 15136.6 s
13, 19146.1 s
14, 18158.3 s

aJ values are represented in Hz and shown in parentheses; 13C NMR assignments are based on HMBC experiments..



Cell culture and differentiation

3T3-L1 pre-adipocytes obtained from the American Type Culture Collection (ATCC® CL-173TM) were grown on Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% penicillin/streptomycin (P/S) and 10% bovine calf serum in a humidified incubator containing 5% CO2. For adipogenic differentiation, 3T3-L1 cells were incubated for two days in an MDI induction medium. The MDI induction medium comprised DMEM with 10% fetal bovine serum (FBS), 1% P/S, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 10 μg/mL of insulin. Subsequently, the medium was replaced with DMEM containing 10% FBS, 1% P/S, and 10 μg/mL insulin on alternate days until day 10. To assess the effects of terminalin on adipogenesis, 3T3-L1 cells were treated with terminalin at concentrations of 0, 5, 10, and 50 μM during the process of adipogenesis. The same volume of DMSO was used as a negative control. On day 8, we used Oil Red-O staining to visualize the lipid droplets and harvested the cells for quantitative real-time polymerase chain reaction (RT-qPCR).

Oil Red O staining

Oil Red O staining was performed to visually detect the lipid droplets in differentiated adipocytes. Oil Red O powder (300 mg) was dissolved in 100 mL of 99% isopropyl alcohol to prepare the Oil Red O stock solution. Then, 30 mL of the prepared stock solution was diluted with 20 mL of distilled water to prepare the Oil Red O working solution before use. After adipogenesis (day 8), mature adipocytes were fixed with 10% formaldehyde for 15 min and then washed with 60% isopropyl alcohol. The fixed cells were stained with the Oil Red O working solution for 1 h at room temperature, and the cells were washed with phosphate-buffered saline (PBS). The stained lipids were visualized using a Leica DMi1 inverted microscope (Leica Microsystems Korea).

Reverse transcription and quantitative real-time polymerase chain reaction (RT- qPCR)

The Easy-Blue reagent (Intron Biotechnology, Seongnam, Korea) was utilized to extract total RNA from the adipocytes. For reverse transcription, 1 μg of total RNA extracted with Maxim RT-PreMix Kit (Intron Biotechnology) was reverse transcribed into cDNA using the ImProm-II Reverse Transcription System (Promega, Fitchburg, WI, USA). The cDNA was mixed with KAPATM SYBR FAST qPCR (Kapa Biosystems, Wilmington, MA, USA); the primers used for RT-qPCR are indicated in Table 2. The qPCR reaction data were analyzed using a CFX96TM or Chromo4 real-time PCR detector (Bio-Rad, Hercules, CA, USA). Relative mRNA expression was quantified and normalized to that of β-actin for each reaction.

Table 2 . Sequences of primers used for RT-qPCR.

GeneForwardReverse
β-Actin5′-ACGGCCAGGTCATCACTATTG-3’5′-TGGATGCCACAGGATTCCA-3′
Adipoq5′-CAGGCCGTGATGGCAGAGATG-3’5′-GGTTTCACCGATGTCTCCCTTAG-3′
Fabp45′-AAGGTGAAGAGCATCATAACCCT-3’5′-TCACGCCTTTCATAACACATTCC-3′
ATGL5′-TTCACCATCCGCTTGTTGGAG-3’5′-AGATGGTCACCCAATTTCCTC-3′
FASN5'-TTGACGGCTCACACACCTAC-35'-CGATCTTCCAGGCTCTTCAG-3'
SREBP15′-AACGTCACTTCCAGCTAGAC-3’5′-CCACTAAGGTGCCTACAGAGC-3′


Statistical analysis

The averages and error bars are expressed as the standard error of the mean (SEM; n = 3 samples). The statistical significance was analyzed using a two-tailed Student’s t-test performed using Excel, and p-values were evaluated. **p < 0.01, and ***p < 0.001 vs. the control group.

Results

Isolation and identification of the compounds

Dried I. gabonensis seeds were crushed and extracted with water at 30℃ to obtain the crude aqueous extract via rotary evaporation and freeze-drying. The crude extract was analyzed using LC/MS with reference to an in-house UV library database. The results demonstrated the presence of one main peak with m/z 601.0 [M-H] and a unique UV spectrum of the extended conjugated system (λmax 382 and 258 nm) and another major component with m/z 301.0 [M-H], which was clearly identified without isolation through the reference to our in-house UV library. To isolate the compound corresponding to the unidentified main peak, the crude extract was subjected to Diaion HP-20 column using 100% H2O to eliminate the sugar portion, and the fraction M was obtained by elution with 100% MeOH. The LC/MS/UV-guided fractionation of fraction M using the preparative reverse-phase HPLC and semi-preparative HPLC resulted in the isolation of the compound corresponding to the peak with m/z 601.0 [M-H] (Fig. 1). Based on the comparison of 1H and 13C NMR spectroscopic and physical data with the previously reported values and LC/MS analyses (Yoon et al. 2022), the compound was identified as terminalin (Fig. 2).

Figure 1. Separation of terminalin.

Figure 2. Chemical structure of terminalin.

Evaluation of the effects of terminalin on adipogenesis and lipid metabolism

To investigate the effects of terminalin on adipogenesis, 3T3-L1 pre-adipocytes were treated with terminalin at concentrations of 5, 10, and 50 μM during the process of adipogenesis for eight days (Fig. 3). The cells were differentiated for eight days, and lipid droplets within the mature adipocytes were stained using Oil Red O staining solution (Yi et al. 2020). The images of Oil Red O solution-stained adipocytes showed that terminalin substantially prevented the de novo generation of adipocytes and lipid accumulation within adipocytes compared to those observed in the control, especially at a high concentration of 50 μM (Fig. 3). Thus, we performed RT-qPCR to assess the expression of adipogenic markers. Terminalin reduced the mRNA expression of mature adipocyte marker genes, Adipoq and Fabp4, in a dose-dependent manner during adipocyte maturation, although the transcription level of Adipoq showed a minor increase upon treatment with terminalin at a concentration of 10 μM (Fig. 4). These data demonstrate that terminalin prevents adipogenesis of 3T3-L1 preadipocytes.

Figure 3. Inhibitory effect of terminalin on adipogenesis. 3T3-L1 cells were treated with terminalin at various concentrations. Images of adipocytes stained with Oil Red O after incubation with 5, 10, and 50 μM of terminalin during adipogenesis.

Figure 4. Relative mRNA expression of Adipoq, Fabp4, ATGL, FASN, and SREBP1 during adipogenesis in 3T3-L1 adipocytes incubated with terminalin. The data represent the mean ± SEM for n = 3. **p < 0.01, ***p < 0.001.

Next, we assessed the capacity of terminalin to regulate lipid metabolism via the expression of the lipolytic gene, ATGL, and lipogenic genes, FASN and SREBP1 (Fig. 4). The mRNA expression of the lipolytic gene (ATGL) was significantly upregulated upon exposure to terminalin at concentrations of 5 and 10 μM during adipogenesis, whereas the expression of lipogenic genes (FASN and SREBP1) was significantly downregulated at all treatment concentrations (5, 10, and 50 μM) of terminalin (Fig. 4). These data indicated that terminalin inhibited adipogenesis and facilitated lipid metabolism via the promotion of lipolysis and inhibition of lipogenesis.

Discussion

The growth of adipose tissues occurs with the differentiation of preadipocytes in the adipose tissues into adipocytes and the synthesis and accumulation of lipid droplets in adipocytes (Smith and Kahn 2016). Therefore, the identification of active compounds that prevent adipogenesis and lipogenesis has been considered a potential therapeutic strategy for the management of obesity and other related metabolic diseases. We evaluated the effects of terminalin on regulating lipid metabolism during adipogenesis. Adipocyte differentiation involves multiple processes with changes in hormone sensitivity and morphology. It is regulated by transcription factors and signaling pathways (Cho et al. 2004). 3T3-L1 cells are a well-established in vitro assay system to assess adipogenesis and adipocyte differentiation (Kong et al. 2009).

Examination of the anti-adipogenic activity of terminalin in 3T3-L1 cells revealed that terminalin inhibited adipogenesis and suppressed the enlargement of lipid droplets. Additionally, the mRNA expression of adipocyte markers, Adipoq and Fabp4, decreased noticeably following treatment with terminalin. Terminalin also promoted lipid metabolism by upregulating the expression of the lipolytic gene, ATGL, and downregulating the expression of lipogenic genes, FASN and SREBP1.

As part of our continued search for natural products with biological properties, our group has been investigating anti-adipogenic natural products derived from natural sources. We demonstrated that withasilolides G–I, identified as novel withanolides derived from the roots of Indian ginseng (Withania somnifera), inhibited adipogenesis and enlargement of lipid droplets, indicated by reduced mRNA expression levels of Fabp4 and Adipsin. Active withasilolides G–I also promoted lipid metabolism by upregulating the expression of lipolytic genes, HSL and ATGL, and downregulating the expression of the lipogenic gene, SREBP1 (Lee et al. 2022). According to our previous study, two new C10-polyacetylene glycosides, (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside and (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside, were isolated from the florets of safflower (Carthamus tinctorius). (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside inhibited adipogenesis in 3T3-L1 preadipocytes, whereas (8Z)-decaene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside promoted adipogenesis (Baek et al. 2021). (8S)-deca-4,6-diyne-1,8-diol-1-O-β-D-glucopyranoside also prevented lipid accumulation by suppressing the expression of lipogenic genes and increasing that of lipolytic genes (Baek et al. 2021). In addition, our group found an anti-adipogenic pregnane steroid isolated from Hydractinia-associated fungus, Cladosporium sphaerospermum SW67 (Lee et al. 2020b). In the study, we showed that 3α-hydroxy-pregn-7-ene-6,20-dione inhibited lipid accumulation along with the expression of the adipocyte marker gene (Adipsin). The expression of the lipolytic gene, ATGL, was elevated and that of lipogenic genes, FASN and SREBP1, were inhibited by 3α-hydroxy-pregn-7-ene-6,20-dione (Lee et al. 2020b). In contrast, 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid and α-dimorphecolic acid isolated from the aerial parts of Lespedeza cuneata induced adipocyte differentiation, as evidenced by the upregulated mRNA expression of Fabp4 in 3T3-L1 pre-adipocytes (Kang et al. 2021). Furthermore, compounds 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid and α-dimorphecolic acid regulated lipid metabolism by inducing lipolytic and lipogenic gene expression (Kang et al. 2021).

In conclusion, terminalin was isolated from I. gabonensis seeds using LC/MS-guided process. Terminalin was evaluated for its anti-adipogenesis activity and lipid metabolism throughout different stages of adipocyte differentiation in 3T3-L1 cells. Terminalin efficiently inhibited the differentiation of 3T3-L1 preadipocytes into adipocytes by downregulating the mRNA expression of Adipoq and Fabp4. Furthermore, it upregulated the expression of the lipolytic gene, ATGL, and downregulated the expression of lipogenic genes, FASN and SREBP1. Our findings support the metabolic role of terminalin in the prevention of excessive lipid accumulation in obesity.

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (2019R1A5A2027340 and 2021R1A2C2007937).

Fig 1.

Figure 1.Separation of terminalin.
Drug Targets and Therapeutics 2022; 1: 12-18https://doi.org/10.58502/DTT.22.001

Fig 2.

Figure 2.Chemical structure of terminalin.
Drug Targets and Therapeutics 2022; 1: 12-18https://doi.org/10.58502/DTT.22.001

Fig 3.

Figure 3.Inhibitory effect of terminalin on adipogenesis. 3T3-L1 cells were treated with terminalin at various concentrations. Images of adipocytes stained with Oil Red O after incubation with 5, 10, and 50 μM of terminalin during adipogenesis.
Drug Targets and Therapeutics 2022; 1: 12-18https://doi.org/10.58502/DTT.22.001

Fig 4.

Figure 4.Relative mRNA expression of Adipoq, Fabp4, ATGL, FASN, and SREBP1 during adipogenesis in 3T3-L1 adipocytes incubated with terminalin. The data represent the mean ± SEM for n = 3. **p < 0.01, ***p < 0.001.
Drug Targets and Therapeutics 2022; 1: 12-18https://doi.org/10.58502/DTT.22.001

Table 1 1H (800 MHz) and 13C NMR (200 MHz) data of terminalin in DMSO-d6 (δ ppm)a

PositionTerminalin
δH (J in Hz)δC
1, 23139.0 s
2, 24139.6 s
3, 25148.3 s
4, 267.52 s110.7 d
5, 27108.7 s
6, 28159.5 s
7, 20136.3 s
8, 21123.3 s
9, 22112.7 s
10, 17107.2 s
11, 16112.8 s
12, 15136.6 s
13, 19146.1 s
14, 18158.3 s

aJ values are represented in Hz and shown in parentheses; 13C NMR assignments are based on HMBC experiments.


Table 2 Sequences of primers used for RT-qPCR

GeneForwardReverse
β-Actin5′-ACGGCCAGGTCATCACTATTG-3’5′-TGGATGCCACAGGATTCCA-3′
Adipoq5′-CAGGCCGTGATGGCAGAGATG-3’5′-GGTTTCACCGATGTCTCCCTTAG-3′
Fabp45′-AAGGTGAAGAGCATCATAACCCT-3’5′-TCACGCCTTTCATAACACATTCC-3′
ATGL5′-TTCACCATCCGCTTGTTGGAG-3’5′-AGATGGTCACCCAATTTCCTC-3′
FASN5'-TTGACGGCTCACACACCTAC-35'-CGATCTTCCAGGCTCTTCAG-3'
SREBP15′-AACGTCACTTCCAGCTAGAC-3’5′-CCACTAAGGTGCCTACAGAGC-3′

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