Anti-Adipogenic Activity of Fractions of Guazuma ulmifolia Leaf

Nuri

1,2*

, Bambang Prajogo

, Sukardiman

Doctoral Program of Pharmaceutical Sciences, Departement of Pharmacognosy and Phytochemistry Faculty of Pharmacy,

Airlangga University, Surabaya, Indonesia

Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Jember, Indonesia,

Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia.

Keywords: Guazuma ulmifolia leaf, Fractions, Anti-adipogenic, Preadipocytes

Abstract: Objective: This study purposed to investigate the anti-adipogenic activity of the chloroform, ethyl

acetate, and residual ethanol fractions of Guazuma ulmifolia leaf extract. Methods: The

fractionation of ethanol extract was carried out by solvent-solvent partition using chloroform

and ethyl acetate. Inhibition of fractions to the proliferation and differentiation of primary

cultures of rat preadipocytes were tested to investigate the anti-adipogenic activity. Results:

Separation of ethanol extract yielded three fractions, i.e. fraction of chloroform, fraction of ethyl

acetate, and fraction of residual ethanol. The results of anti-proliferation and anti-differentiation

activity test showed that the highest activity was demonstrated by ethyl acetate fraction, followed

by residual ethanol fraction and chloroform fraction. The highest total flavonoid content was also

shown by the fraction of ethyl acetate. Conclusion: The fraction of ethyl acetate showed the

highest anti-adipogenic activity and the highest total flavonoid content

1 INTRODUCTION

Nowadays obesity is not only seen as a performance

problem but has become a pandemic and a major

health problem resulting in increased risk of

comorbidity that contributes significantly to

mortality (Castanon, Lasselin and Capuron, 2014).

Obesity has become a major risk for diabetes,

hypertension, coronary heart disease, dyslipidemia,

and certain tumors (Pi-Sunyer, 2002). The World

Health Organization (WHO) defines obesity as an

abnormal or excessive accumulation of fat that

harms the health of human (Word Health

Organization, 2018). In fact, obesity can be

controlled by reducing the fat content of food

accompanied by an increase in physical exercise.

However, it is estimated that over 90% of people

who lose weight with diet and increase physical

exercise, within 2-5 years will return to their original

weight. Increased adipose tissue mass involves an

increase in the number of adipocytes formed from

precursor cells, which in turn occurs enlargement of

adipocyte size. The formation of the adipocytes from

precursor cells and enlargement of their size is a life

cycle of adipocyte, and treatment that can regulate

adipocyte counts and measures can be used as a

therapeutic approach to treat obesity (Rayalam,

Della-Fera, and Baile, 2008).

Flavonoids, polyphenol compounds, are widely

present in plants and are known to inhibit

proliferation in some cell cultures and have anti-

adipogenic effects on 3T3-L1 line cells. The results

of Park et al (2009) research show that luteolin

inhibits the differentiation of preadipocyte and

regulates the early stages of adipogenesis. Other

flavonoids myricetin, a flavonoid found in various

foods, can inhibit adipogenesis as indicated by

decreased intracellular lipid droplet accumulation

(Bin and Choi, 2012). Flavonoids present in Citrus

aurantium L. suppress adipogenesis by decreasing

Nuri, ., Prajogo, B. and Sukardiman, .

Anti-Adipogenic Activity of Fractions of Guazuma ulmifolia Leaf.

DOI: 10.5220/0008360902550260

In Proceedings of BROMO Conference (BROMO 2018), pages 255-260

ISBN: 978-989-758-347-6

255

PPARg and C/EBPa expression (Kim et al., 2012).

The anti-adipogenic effects of this 3T3-L1 cell,

coupled with anti-proliferative activity, proposes the

presumption that flavonoids may inhibit the increase

of adipocytes or signals that promote adipogenesis.

G. ulmifolia is a plant originating from tropical

America and is a plant belonging to the family

Sterculiaceae. In Indonesia, this plant commonly

called Jati belanda and traditionally has been used to

lose weight and reduce excessive fat content

(Mardisiswojo and Rajakmangunsudarso, 1985).

The leaf of G. ulmifolia contain a number of

phytochemical constituents i.e. colistin, colatannins,

caffeine, tartaric acid, theobromine, xanthan gum,

catechins, kaempferol, some of procyanidin

(procyanidin B-2, B-5, and C-1), and tiliroside

(Sharma and Prasad, 2014; Departement of Health

Republic of Indonesia, 2008). Among these

compounds, namely catechins, kaempferol,

prosianidin and tiliroside including flavonoid

derivatives compounds. The presence of this

compounds gives the estimate of G. ulmifolia leaf as

anti-obesity by inhibiting adipogenesis.

In this study, the ethanolic extract of G. ulmifolia

leaf was fractionated by means of the liquid-liquid

partition using chloroform, ethyl acetate. The

obtained fractions (chloroform, ethyl acetate, and

last remaining ethanol) were tested for inhibiting the

proliferation and differentiation of rat preadipocytes.

2 MATERIALS AND METHODS

2.1 Materials

Organic solvents of n-hexane, chloroform, ethyl

acetate and ethanol were in proanalytical grade

(Merck), TLC plate (Merck), collagenase type I

(Sigma), culture media DMEM, HEPES, NaHCO

biotin, D-pantothenate, FBS, Penicillin and

Streptomycin (Sigma), differentiation induction

materials insulin, dexamethasone, IBMX (Sigma).

2.2 Collection and Drying of G.

ulmifolia Leaf

Leaf of G. ulmifolia was collected from Meru Betiri

National Park with an altitude of 900 - 1,223 m asl

and an average rainfall of 2,300 mm/year in

October 2016. Prior to collection, the plants were

determined in LIPI Botanical Gardens, Purwodadi,

East Java. The leaf was sorted, i.e. removed the

damaged leaf and other impurities then washed

with running water. The clean leaf was dried and

then pulverized (grounded) to powder.

2.3 Extraction and Fractionation

G. ulmifolia leaf powder weighing 800 g was

defatted with n-hexane four times (each 1000 mL).

The residue was collected, air dried, and macerated

in 70% ethanol (2000 mL) for 24 hours. This

procedure was repeated three times using the same

powdered leaf. The filtrate then concentrated by

using a rotary evaporator at 45°C under reduced

pressure to obtain a less 70% ethanol extract.

Successively, the extract was fractionated using

chloroform and ethyl acetate (3 x 350 mL of each

solvent) to obtain chloroform, ethyl acetate, and

residual 70% ethanol fraction. The fractions solvent

was completely removed under the vacuum to

obtain dry fractions and preserved in vials and kept

at 4 °C before use.

2.4 Determination of Total Flavonoid

Content

Total flavonoid content was measured by the

aluminum chloride colorimetric assay. An aliquot

(150 μL) of fractions or standard solution of

quercetin (5, 10, 20, 40, 60, 80 and 100 mg/L) was

added to 1.5 ml cuvet containing 0.4 ml of aqua

distilled water. To the cuvet was added 0.03 mL 5

% NaNO

and 0.03 mL 10 % AlCl

. After 6 min,

0.2 mL 1 N NaOH and 0,24 of mL distilled water

were added. The solution was stirred until

homogeneous, then the absorbance was measured at

415 nm. Total flavonoid content of fraction was

expressed as mg quercetin equivalents (QE)/g

fraction. Samples were analyzed in triplicates

(Ratnadewi et al., 2018).

2.5 Preparation of Cell Culture

Preadipocytes were isolated from mice adipose

tissue aged 4-8 weeks. The visceral fat tissue was

sliced in a sterile condition and cleaned as much as

possible from surrounding tissues. The tissue was

washed with PBS and chopped into small pieces.

Chopped tissue was digested by type I of

collagenase at 37 °C for an hour. After that, the

suspension was filtered through 250 µm nylon

mesh. The suspension containing isolated cells

were centrifuged at 1000 rpm for 7 minutes, and the

two types of cells were separated. Mature

adipocytes were found at the top layer of the

suspension and the pellet at the bottom of a tube

containing preadipocytes cells. Furthermore, the

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

256

pellet was resuspended in culture medium

containing FBS 10%, homogenized, and plated on

plate culture, then incubated at 37˚C, 5% CO

(Duarte, et al., 2012). After two days,

differentiation was induced by the addition of

induction medium ((DMEM/F12 added by 66 mM

insulin, 100 nM dexamethasone, 0.5 mM 3-

isobutyl-1-methylxanthine) and incubated at 37°C

in a 5% CO

incubator for 24 hours.

The cell culture was incubated with chloroform,

ethyl acetate, and ethanol fractions of G. ulmifolia

leaf fraction for up to 24 hours (Lin, Della-Fera,

and Baile, 2005). All experiments were performed

at least in triplicate at concentrations of 500 ppm.

2.6 MTT Assay

Cell proliferation was examined by MTT assay. In

brief, 20μl assay medium containing MTT was

added to each well of 96 well plates. The

incubation continued at 37 ℃ for 4 hours , adding

150 μl DMSO to dissolved the colored formazan.

The absorbance of each sample was measured by a

microplate reader at 490 nm.

2.7 Determination of The Cells

Differentiation

After the treatment with fractions, the amount of

differentiated and non-differentiated cells were

calculated under the microscope at a 400

magniﬁcation (Hemmrich et al., 2005). Cells were

calculated at 25 fields of view. Cell differentiation

was calculated based on the number of cells

undergoing morphological changes in adult

adipocytes.

2.8 Oil-Red-O Staining

Cells were fixed in 10% formalin and washed a

moment with running tap water. After rinsed with

propylene glycol, the freshly prepared Oil-Red-O

working solution was added with agitation for 7

minutes. Then, the cells were rinsed with 85%

propylene glycol and stained with hematoxylin.

Finally, cells were washed with running tap water,

dried, and observed under the microscope at 400

magnification (Lin, Della-Fera, and Baile, 2005).

3 RESULTS

3.1 Extraction and Fractionation

The yields of all the fractions corresponding to the

initial dry leaf material are shown in Table 1. The

extractive yield varied among the solvents used.

Chloroform and ethyl acetate fractions showed less

extractive yield as compared to residual 70%

ethanol fraction.

Table 1: Fractions yield (%) of 800 g G. ulmifolia leaf powder in the different solvent

Fractions

Extractive value (g)

% Yield

Chloroform Fraction

3,61

0,45

Ethyl acetate Fraction

5,98

0,75

Ethanol Fraction

48,41

6,05

3.2 Total Flavonoid Content

The results obtained in the estimation of flavonoid

content (Table 2) showed that all fractions had a

certain amount of total flavonoid content. The

fraction of ethyl acetate showed the highest total

flavonoid content (314,50 ±4,50 mg QE/g fraction).

The total flavonoid contents exhibited the

descending order with fractions of ethyl acetate >

chloroform > ethanol.

Table 2: Total flavonoid content of fractions

Fractions

Total flavonoid content (mg QE/g Fraction) ± SD

Chloroform Fraction

109,92 ± 2,15

Ethyl acetate Fraction

314,50 ± 4,50

Ethanol Fraction

10,22 ± 1,08

Anti-Adipogenic Activity of Fractions of Guazuma ulmifolia Leaf

257

3.3 Anti-proliferation and Anti-

differentiation Activities of

Guazuma ulmifolia Leaf Fractions

Anti-proliferation activity indicates the ability of

fractions to inhibit the proliferation of preadipocyte

cells. Anti-proliferation activity of each fraction is

shown in Figure 1. Cell differentiation is

characterized by changes in adipocyte morphology.

Inside the cell, there is a lipid drop as shown in

Figure 2. The anti-differentiation activity showed

the ability to inhibit preadipocyte cell changes in

adipocyte mature cells. The anti-differentiation

activity of the fractions is shown in Figure 3.

Figure 1: Anti-proliferation activity of G. ulmifolia leaf fractions

Figure 2: Preadipocyte morphology after incubation with G. ulmifolia leaf fractions for 24 hours. D = Differentiated cells,

U = Undifferentiated cells

Control

Chloroform Fraction

Ethyl acetate Fraction

Ethanol Fraction

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

258

Figure 3: Anti-differentiation activity of G. ulmifolia leaf fractions

4 DISCUSSION

Fractionation of ethanol extract of G. ulmifolia leaf

using chloroform and ethyl acetate showed the most

yield was the residual 70% ethanol fractions (Table

1). These results indicated that most of the extracted

content was polar compounds.

The leaf of G. ulmifolia contain flavonoids, i.e.

catechins, kaempferol, procyanidin B-2, procyanidin

B-5, procyanidin C-1 and tiliroside. Numerous

studies reported that flavonoids inhibited

adipogenesis (Park et al., 2009; Bin and Choi, 2012;

Hsu and Yen, 2007; Chien et al., 2005)(5,6,15,16).

The fractionation of ethanol crude extract resulted in

fractions with different total flavonoid content. The

sequence of the fractions based on total flavonoid

content detected were ethyl acetate fraction >

chloroform fraction > ethanol fraction (Table 2).

This results indicated that flavonoids compound

which was found in the leaf of G. ulmifolia prefer to

be extracted with solvent possessing moderate

polarity degrees (semi-polar) such as ethyl acetate

and chloroform than solvents with strong polarity

(ethanol) as seen in Table 2. Based on the principle

of like dissolves like, it was thought that most

flavonoids were in the form of aglycons (Sarker,

Latif, and Gray, 2006).

Anti-proliferation activity of ethyl acetate fraction

was higher than other fractions (Figure 1). This

result was consistent with the total flavonoid content

of the ethyl acetate fraction as mentioned above, that

higher than other fractions. Other studies have

shown that flavonoid of G. ulmifolia leaf (tiliroside)

can inhibit the proliferation of T47D and MCF7

cancers cell lines (Da’i et al., 2016).

Cells differentiation were indicated by preadipocytes

morphological changes into mature adipocytes that

were shown by the formation of fat droplets in the

adipocytes. Fat droplets can be observed with Oil-

Red-O staining. This staining has been widely used

to exhibit the differentiation of preadipocytes to

adipocytes because it is positively correlated with

the amount of lipid stored in the cell. Hence it is

used to indicate potential anti-obesity effects of

natural products (Poudel et al., 2015). Figure 2

showed the morphology of adipocytes after the

treatment with chloroform, ethyl acetate, and

residual ethanol fractions for 24 hours. The

differentiated cells (D) were characterized by orange

lipid droplets and otherwise, these droplets were not

observed in undifferentiated cells (U). The ethyl

acetate fraction also showed the highest anti-

differentiation activity among the other fractions

(Figure 3).

In the case of anti-proliferation activity of

chloroform fraction and residual ethanol fraction,

ethanol fraction showed higher activity than the

chloroform fraction. Similarly, by anti-

differentiation activity, ethanol fraction showed

higher activity than chloroform fraction. This may

be due to only certain flavonoids that can inhibit

proliferation and differentiation of preadipocytes.

For example, ganistein may inhibit the

differentiation of preadipocytes of the 3T3-L1 cell

line, but naringenin had no effect on this process

(Harmon and Harp, 2001).

Anti-Adipogenic Activity of Fractions of Guazuma ulmifolia Leaf

259

5 CONCLUSION

Based on the result of above research can be

concluded that the ethyl acetate fraction contains

the highest total flavonoid contents among other

fractions. The ethyl acetate fraction also showed the

highest anti-proliferation and anti-differentiation of

rat preadipocytes.

CONFLICT OF INTEREST

The authors declare that there is no conflict of

interest.

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