The anti-obesity effect of mulberry leaf (Mori Folium) extracts was increased by bioconversion with Pectinex

Mulberry leaf (Mori Folium) extract (MLE) is known to have anti-obesity effects. In this study, the enhanced effects of MLE after bioconversion treatment using Pectinex (BMLE) on obesity

were explored, and the underlying mechanisms were investigated using the active components, neochlorogenic acid (5-CQA) and cryptochlorogenic acid (4-CQA), whose amounts were increased by

bioconversion of MLE. Both MLE and BMLE inhibited lipid accumulation in 3T3-L1 adipocytes without cytotoxicity and suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBPα).

In addition, MLE and BMLE decreased high-fat diet-induced adipose tissue mass expansion. Notably, BMLE significantly increased antiadipogenic and anti-obesity effects compared to MLE in

vitro and in vivo. The active ingredients increased by bioconversion, 5-CQA and 4-CQA, inhibited the protein levels of C/EBPα and the mRNA levels of stearoyl-CoA desaturase 1 (Scd1). These

findings provide new insights into the therapeutic possibility of using bioconversion of MLE, by which upregulation of 5-CQA and 4-CQA potently inhibits adipogenesis.

Obesity is a disease in which surplus energy caused by an increase in food intake is transformed into triglycerides and stored in adipose tissue, leading to weight gain1. Maintaining a

healthy body weight is important because obesity can cause complications such as type 2 diabetes, atherosclerosis, and heart attack2,3,4. Since the incidence of obesity has been increasing

recently, it is indispensable to discover effective pharmacological drugs that potently inhibit fat accumulation for treating obesity and metabolic disease.

Transcription factors and adipogenic genes function in the molecular mechanism of adipogenesis. Adipogenic transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) family

members and peroxisome proliferator-activated receptor gamma (PPARγ), are essential in the adipogenesis process5. After Cebpb and Cebpd genes are induced to be expressed quickly in the early

stages of adipocyte differentiation, these proteins increase the expression of PPARγ and C/EBPα, which are important for terminal adipocyte differentiation6,7. In addition, C/EBPα-mediated

adipogenic transcription activation, such as stearoyl-CoA desaturase 1 (SCD1), has been revealed8,9,10.

SCD is an endoplasmic reticulum (ER) enzyme that catalyzes biosynthesis with monounsaturated fatty acids (MUFAs), which contribute greatly to lipid synthesis11. SCD has isoforms of SCD1, 2,

3, 4, 5, of which SCD1 and SCD5 are known to be expressed in humans. Furthermore, SCD1 is expressed at higher levels in adult white adipose tissue than SCD512,13,14. Several studies have

shown a link between SCD1 expression and obesity based on the finding that Scd1 gene deficiency inhibits lipid accumulation15,16. Therefore, the regulation of SCD1 via C/EBPα can be a

treatment strategy for obesity.

Mulberry is a plant that originated from Asia, such as Korea, China, and Japan. Traditionally, all parts of plants, such as leaves, roots, and fruits, have been used for the purpose of

treating obesity or diabetes and for the purpose of eating17,18. Recent studies have reported the effects of mulberry leaves, such as anti-obesity19,20,21, cholesterol reduction22,23,

anti-inflammatory24, antioxidant25, blood pressure improvement26, thrombosis27, and anti-diabetes effects28,29.

Bioconversion is a technology that induces the production of physiologically active ingredients by modifying the chemical structure of natural products using biological methods such as

microorganisms and enzyme-mediated fermentation30. Several studies have reported the effect of potent therapeutic effects through bioconversion. For example, bioconverted Jeju hallabong

tangor (Citrus kiyomi × ponkan) by cytolase showed antioxidant and anti-inflammatory effects31, and barley fermented with Lactobacillus plantarum dy-1 showed effects in weight loss, lipid

and inflammation improvement compared with natural products32. Moreover, the bioconversion of Citrus unshiu peel extract with cytolase showed an increased inhibitory effect on adipogenic

activity compared with Citrus unshiu peel extract33, and fermentation of Panax notoginseng by lactic acid bacteria showed an increased anti-obesity effect34.

Pectinex used commercially in the food industry is a fungal enzyme complex derived from Aspergillus aculeatus35. Recent studies showed that enzymatic hydrolysis of tea seed extract using

Pectinex newly synthesized leucoside36, extracts treated with Pectinex showed a higher antioxidant effect37,38, and pectins extracted from rapeseed cake using Pectinex showed the effect of

inhibiting cancer cell growth39. Therefore, it is expected that mulberry leaves extracted by grafting bioconversion technology using Pectinex will increase therapeutic activity.

We previously reported that mulberry leaf extract (MLE) after bioconversion treatment using Viscozyme L was superior to unaltered MLE in controlling diabetes both at the cellular and

diabetic animal model levels40. In this study, we will investigate whether MLE after bioconversion treatment using Pectinex (BMLE) are more effective against obesity than MLE in 3T3-L1

adipocytes and high-fat diet-induced mouse models.

To investigate the effect of the bioconversion of MLE on adipogenesis, we investigated the effect of lipid droplet reduction in 3T3-L1 adipocytes (8-day differentiated cells) using Oil red O

staining. Compared to differentiated adipocytes, 400 and 800 μg/mL MLE dose-dependently reduced lipid droplet formation by 11.73% and 15.83%, respectively (Fig. 1a). (−)-epigallocatechin

gallate (50 μM, EGCG) was used as a positive control for antiadipogenesis41,42,43. Interestingly, 400 or 800 μg/mL BMLE reduced lipid droplets by 19.00 and 25.19%, respectively, indicating

that bioconversion using Pectinex significantly enhanced the inhibitory effect of lipid accumulation.

Effects of mulberry leaf extract (MLE) and bioconverted mulberry leaf extract with Pectinex (BMLE) on lipid accumulation, cytotoxicity, and adipogenic factors in 3T3-L1 cells. (a) Effects of

MLE and BMLE on lipid accumulation in 3T3-L1 cells. MLE (400 and 800 μg/mL), BMLE (400 and 800 μg/mL) or (−)-epigallocatechin gallate (50 μM, EGCG) were treated 3T3-L1 adipocytes during

differentiation periods. Oil red O staining was performed as described in the Materials and methods section. Pre: preadipocytes, Diff: 8-day differentiated cells. (b) Effects of MLE and BMLE

on cytotoxicity in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with MLE (400 and 800 μg/mL), BMLE (400 and 800 μg/mL) or digitonin (100 μg/mL, positive control for cytotoxicity).

After 8 days of treatment, an MTT assay was performed. (c) Effects of MLE and BMLE on C/EBPα and PPARγ protein expression in 3T3-L1 preadipocytes and adipocytes. 3T3-L1 adipocytes were

treated with MLE (400 and 800 μg/mL) and BMLE (400 and 800 μg/mL) for 8 days. **P