Potential anti-adipogenic activity of Calligonum comosum cuminaldehyde on mouse 3T3-preadipocytes

Obesity is a medical condition associated with serious medical and psycho-social consequences and an augmented body fat mass. Several compounds were suggested to counteract obesity and fat accumulation with variable degrees of success. Searching for a safe and effective anti-adipogenic substance, we found that cuminaldehyde-rich essential oil extracted from Calligonum comosum potentially mediate activities. The results showed that C. comosum essential oil and its major component cuminaldehyde, selectively caused significant reduction in the viability of 3T3-L1 cells when compared to fibroblasts. Furthermore, cuminaldehyde caused significant reduction in the lipid content, glucose uptake and levels of both triglycerides and cholesterol in adipocytes. Moreover, the formation of 3D-adipocyte pellets in the presence of cuminaldeyde was affected. Adipocytes matured in the presence of cuminaldehyde have significant reduction in the expression of adipocyte-specific transcripts, CAAT-enhancer binding protein alpha (CEBPa) and Peroxisome proliferator-activated receptor gamma (PPARg). Taken together, these results demonstrate a potential inhibitory role of cuminaldehyde extracted from C. comosum oil on lipid accumulation. Consequently, cuminaldeyde can be considered as a new potential anti-adipogenic agent for prevention and treatment of obesity.


Introduction
Obesity is a medical condition in which the body fat mass is increased to a level that can adversely affect human health. According to the World Health Organization (WHO), obesity has tripled since 1975 and 39% of adults aged 18 years and above were overweight in 2020 and 13% were obese [1]. Obesity is considered as a risk factor for life-threatening conditions, including cardiovascular diseases [2], musculoskeletal disorders [3] and some types of cancer [4]. A fundamental cause of obesity is an energy imbalance between calories consumption and exhaustion [5]. Obesity is largely preventable and can be managed by applying several strategies including the use of therapeutic compounds [6]. A key strategy in reducing obesity is dampening adipocyte differentiation, reducing adipocyte hypertrophy and hyperplasia, as well as antagonizing fat accumulation [7]. Several therapeutic compounds were developed in order to control general body obesity, as well as localized fat accumulation [8]. On the other hand, local communities accumulated considerable experience for the use of native growing plants as a treatment ofadiposity. For example, compounds such as scoparone isolated from Chinese herb Artemisia scoparia [9], quinoa saponins [10], myricetin [11], 4-hydroxyderricin and xanthoangelol from Japanese Ashitaba (Angelica keiskei) [12] have shown significant interference with adipocyte functions. Calligonum comosum L. has been employed by local communities in the United Arab Emirates (UAE) for decades in their foods for the treatment of several gastric and skin illnesses [13,14]. Various pharmacological activities of the plant were also reported, including anti-ulcer, anti-inflammatory and anti-cancer activities [15,16]. C.
comosum is known as a source of volatile essential oil [15,17]. The purpose of this study was to investigate the anti-adipogenic activity of C. comosum oil and to determine a new lead compound that might interfere with adipocytes differentiation and metabolism.

Plant essential oil preparation and analysis
Air-dried plant stems were grinded, and the essential oil was extracted by steam distillation method according to [19,20]. Briefly, 200gm plant stem powder was extracted with water steam at 100 o C for 3 hr. The condensed oil was collected and dried over anhydrous sodium sulphate to yield 250 µL. The oil was diluted in chloroform in a ratio of 1:100 prior to GC-MS analysis. The composition of essential oil was analyzed and identified according to Soliman et al., 2017 [21] using the Agilent Gas Chromatography-Mass Spectrometry (GC-MS). The identified compounds including the target compound, cuminaldehyde, were described in Soliman et al., 2017 [19] in both tabular and chromatogram forms.

Fractionation-based bioassay and partial purification of cuminaldehyde
Identification of the major anti-adipogenic compound in the plant essential oil extract was performed according to Soliman et. al, [20]. Briefly, the plant essential oil was separated on 6 analytical TLC plates (Sigma-Aldrich, Germany) and developed by petroleum ether: acetone (5:1) as solvent system. The plate was visualized under UV lamb using short wavelength (250nm). The major bright bands were scrapped off the plate and each fraction was tested for its anti-adipogenic activity using 3T3 cells. The fraction (# 3) containing the active compound, cuminaldehyde, was confirmed by GC-MS. The percentage of cuminaldehyde in the fraction was > 50%. The fractionation TLC and corresponding GC-MS chromatogram was described in Soliman et al., 2020 [20].

Cell culture
The 3T3-L1 mouse pre-adipocyte cell line was originally developed by clonal expansion from murine Swiss 3T3 cells. Normal fibroblast cells line (F180) was employed as control. Both cell lines were cultured in the Dulbecco's Modified Eagle Media (DMEM; Sigma-Aldrich, Germany) supplemented with 25 mM glucose, 10% Fetal Bovine Serum (FBS; Sigma-Aldrich, Germany) and 1% penicillin-streptomycin (Sigma-Aldrich, Germany) and maintained at 37 o C in a humidified atmosphere of 5% CO 2 .

MTT cell viability assay and determination of LD 50
The reduction of yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich, Germany) was used to measure cellular metabolic activity as a proxy for cell viability [19,22]. To measure the lethal dose that is required to induce 50% death of the cells (LD50), 3T3 and F10 cell lines were cultured till reached 80% confluency. A 96-well plate was seeded with 4000 cells per 100μl media and incubated at 37 o C for 24 hrs. A serial dilution of C. comosum oil extract, its separated fractions, or cuminaldehydecontaining fraction were added onto the cells and incubated for further 24 hrs. Freshly prepared 7 MTT solution (5 mg/ml) was added to each well (20 μl per well) and followed by incubation for 2 hrs at 37 o C. The supernatant were then removed and 100 μl DMSO was added and incubated until formazan violet crystals were developed and the OD 540 was measured.

Pre-adipocyte cell differentiation
Differentiation of pre-adipocytic 3T3 cells was followed a published protocol by Vishwanath et.at., [23]. Briefly, 2x10 4 pre-3T3 cells were seeded in a 12-well plate till reached confluency level. The medium was then supplemented with 10 µM dexamethasone [Sigma-Aldrich, Germany], 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Calbiochem, La Jolla, CA), 100 µM indomethacin (Sigma-Aldrich, St. Louis, MO), and insulin-transferrin-sodium selenite (ITS) supplement to provide 10 µg/ml of insulin (Sigma-Aldrich, Germany). After three days, the media was replaced with fresh media supplemented with ITS only. The last step was repeated once more cycle after three day. Cultures were maintained in a humidified incubator containing 5% (v/v) CO 2 at 37℃. The accumulation of lipid droplets and adipocytes growth was monitored over the cultivation time.

Cholesterol, triglyceride and glucose-intake measurements
Post-differentiated adipocytes were incubated for 24 hrs with C. comosum cuminaldehyde fraction in 12-well plate at its LD 50 . Wells received no oil were employed as a negative controls. Cell lysates and culture supernatants were then obtained and stored at -80 o C prior to testing. Ezymatic colorimetric assays were used to measure cholesterol, triglycerides and glucose (using Diasys colorimetric kits, Diagnostic systems) in comparison with provided known standard (DIASYS) [24,25]. 8

Nile red stain and flow cytometry
To quantify the changes in intracellular lipid droplets accumulation in differentiated adipocytes following the treatment for 24 hrs, Nile red stain was employed. Nile red stock solution (Sigma-Aldrich, Germany) was prepared in acetone and diluted in PBS at concentration of 0.5 μg/mL. The cells were then fixed with 4% paraformaldehyde.

Pre-adipocyte pellet cultures
To evaluate the anti-adipogenic effects of cuminaldehyde on adipose tissue, 3T3 adipocytes were transferred to three dimensions as pellet culture, which provides a more physiological environment for cell to cell interaction and differentiation. The procedure of adipocyte pellet cultures preparation was performed according to [26,27]. Briefly, 1x10 6 3T3 cells/pellet were suspended in 1 ml media and centrifuged for 10 min at 400xg, followed by media change for three times per week. The pellets were then treated with cuminaldehyde at LD50 for 24 hours.

Statistical analysis
Values were presented as means ± standard error of mean (SEM). Experiments were repeated with 3-6 replicas per each experiment. Data was analyzed by T test using Graphpad Prism software. Statistical significance was considered at P-value < 0.05.

C. comosum essential oil showed potential and safe anti-adipogenic activity
The effect of C. comosum essential oil on the viability of pre-adipocyte mouse cell line (3T3-L1) and normal fibroblasts cell line (F180) were determined using MTT assay. The results showed that C. comosum oil caused selective inhibitory activities on 3T3 when compared to normal F180 cells. The oil at concentrations 0.4-0.8 μg/ mL caused significant reduction (~50%, P-value < 0.05) in the viability of 3T3 cells when compared to F180 (Figure 1). The results obtained indicated that C. comosum essential oil can be employed as potential anti-adipogenic and 0.4-0.8 μg/ mL is the concentration of choice for selective inhibition of 3T3 cells.

Cuminaldehyde was the oil major component responsible for the anti-adipogenic activity
The plant oil was subjected to fractionation-based 3T3 cell viability bioassay using MTT assay. The oil was fractionated on TLC into 5 fractions and each fraction was tested on 3T3 cells. Fraction number 3 that contained > 50% cuminaldehyde showed 90-100% killing activity when compared to other fractions, while its LD 50 was determined as 0.4 μg/ mL (Figure 1).
Cuminaldehyde was further tested on the intracellular lipid accumulation of 8-day postdifferentiated 3T3 adipocytes using Nile red staining. The results indicated that cuminaldehyde oil at the concentration of 0.4 μg/ mL caused significant reduction (25%, p-value =0.038) in the intracellular lipid content of 3T3 cells (Figure 2A-D). The concentration of both triglycerides and cholesterol from cell lysates and the level of glucose in media supernatants were measured.
The results showed non-significant reduction in the triglyceride content (~10% reduction, Pvalue =0.495), while showed 50% reduction in the cholesterol level contents at concentration 0.4 μg/ mL (p-value=0.009; Figure 3A). On the other hand, the glucose level in oil treatments was reduced by 40% (p-value=0.0012; Figure 3B).
Expression of the adipogenesis transcription factors CEBPa and PPARg is crucial for the adipocyte maturation [28]. Adding cuminaldehyde oil at concentration 0.4 μg/ mL caused significant reduction in the expression of CEBPα and PPAR by approximately 50% and 70%, (p-value=0.01 and 0.0001, respectively; Figure 4A).
Additionally, cuminaldehyde caused reduction by 30% in the weight of the 3D adipogenic pellet (P-value=0.044; Figure 4B). Testing the histological structure of the formed adipocytes pellets indicated an absence of intact inner center as compared to the untreated controls ( Figure   4C).

Discussion
Adiposity is one of the world's health problems that affects approximately third of the population all over the globe and can contribute to severity of many chronic diseases [29]. Several strategies and regimens have been established to control and reduce lipid accumulation, including those dependent on the inhibition of adipogenicity [30]. Strategies used to reduce adiposity can include controlling calories intake, induction of lipolysis, enhancement of adipocyte apoptosis and suppression of adipocyte differentiation [31]. Here in this study, we demonstrated the ability of pure cuminaldehyde and cuminaldehyde-rich oil isolated from, C. comosum as a natural product, to enhance adipocyte metabolism and hence induction of lipolysis.
General or local obesity can be a result of increase in the tissue fat mass which is primarily consists of mature differentiated adipocytes [32]. Thus, both lipolysis and inhibition of adipocyte differentiation can be considered as effective strategies in body weight loss or dissolution of local fat deposits. Consistently, our results indicated that cuminaldehyde caused cytotoxic effects on 3T3 cells. Inhibition of 3T3 is a known model associated with reduction in the tissues' fat mass [33]. On the other hand, the cytotoxic effects of cuminaldehyde on 3T3 were accompanied by enhanced glucose intake and lipid turnover, which could be considered as an indicator of enhanced metabolism and/ or cells metabolic stress. Enhanced metabolism can maintain the body energy stores and hence regulate the storage and mobilization of fat stores [34]. In this regard, cuminaldehyde can be considered as a hypoglycemic inducing factor. The significant reduction in fat globules (stained with Nile red) due to the effect of cuminaldehyde, could suggest that the compound interfered with fat formation by holding the adipogenesis or reflecting the dissolution of fat in adipocytes. A dampened adipogenesis would result in reduced fat production and formation of intact adipose tissue. This observation was supported by the significant reduction in the expression of transcripts known to control adipocyte maturation, as a result of treatment with the compound [28]. These results can indicate that cuminaldehyde antiadipogenic potential effects can be -at least partially-through antagonizing the expression of adipogenesis-related genes. The suppression effects of cuminaldehyde on adipocyte maturation are similar to inhibition effects of cocoa tea [35].
The process of adipogenesis is highly regulated by complex network including transcription factors, calories intake, fat used up and adipocyte maturation. C. comosum essential oil extract, and its major component cuminaldehyde, showed potential anti-adipogenic activities.
Cuminaldehyde increased the metabolic activities of adipocytes and hence the consumption of stored lipids for energy use along with suppression of pre-adipocytes differentiation. The aforementioned double activities of cuminaldehyde by reducing lipid content can be employed as a potential anti-adipogenic lead compound.

Conclusion
As concluding remarks, the results obtained demonstrated that cuminaldehyde and cuminaldehyde and cuminaldehyde-rich essential oil extracted from C. comosum essential oil exhibited both suppression in pre-adipocytes maturation and an increase in lipid turnover; indicative of a potential anti-adipogenic activity. This study can be considered, up to our best of knowledge, as the first report for the anti-adipogenic activities of C. comosum essential oil extract and cuminaldehyde. The extract from C. comosum and/ or its major component cuminaldehyde can be further investigated for the use as a potential therapeutic agent for the prevention and treatment of obesity.