Aiming for high anti-cellulite action

It is an accumulation of fat which triggers local inflammation, causing water retention in the adipose tissue. The swollen adipose cells compress the blood and lymph vessels, blocking fluid circulation. At the same time, the connective tissue structures that partition the adipose compartment are distorted under the pressure from the lipid-gorged cells. As the fatty lobules expand they push the overlying tissues towards the skin surface (protrusion), causing the lumpy appearance of the skin (Fig. 1). What mediators cause this inflammatory state at the origin of the loss of homeostasis and function of the adipose tissue?

The adipose tissue is no longer considered as just a storage tissue for fat reserves, but rather as a complex endocrine organ playing an active role in metabolic, endocrine, immune and cardiovascular regulation. Adipose tissue secretes many substances, called adipocytokines or adipokines, which act either by endocrine, autocrine or paracrine pathways. The expression of these adipokines is altered when the fat mass varies, leading to major changes within the adipose tissue. For example, blood microcirculation is altered in cellulite tissue, which leads to tissue hypoxia.

In response to this stress, adipose tissue secretes various mediators such as:

Playing a messenger role, these adipokines make a substantial contribution to the inflammation related to excess fat accumulation. The homeostasis and function of the adipose tissue are altered, and the energy metabolism resulting from the equilibrium between the processes of lypolysis and lipogenesis is perturbed.

Recent studies have identified a hormone, adiponectin, secreted by adipocytes, capable of regulating local inflammation of adipose tissue (Fig. 2). In response to adiponectin, the expression of genes linked to inflammation is modified substantially: the production of pro-inflammatory factors by adipocyte cells is reduced and replaced by antiinflammatory molecules such as IL-10 and IL-1ra. Adiponectin also improves vascular quality (Clément et al., 2004) and reduces the induction of endothelial adhesion molecules, thus limiting the number of infiltrated macrophages (Fantuzzi 2005; Cancello et al., 2006). Lastly, it has an inhibitory effect on the secretion of proinflammatory factors by blocking the activity of nuclear factor kB (NF-kB), a major transcriptional regulator of pro-inflammatory cytokine expression (Guerre-Millo, 2001; Ajuwon et al., 2005). It has been shown that adiponectin expression, reduced in overweight individuals, is restored in the case of caloric restriction and reverses the inflammation linked to fat accumulation (Laquemant et al., 2003; Fantuzzi et al., 2005).

Researchers have also recently identified a protein that apparently plays a key role in obesity. Called SIRT-1, it is believed to have the capacity to detect the state of activation of the metabolism of the cell and to intervene in consequence in the mechanisms that control the accumulation of lipid reserves. SIRT-1 is a nuclear enzyme with a deacetylase activity dependent on a cofactor, NAD (nicotinamide adenine dinucleotide), associated with many metabolic enzymes. In periods of even relative shortage, this SIRT-1 protein is activated and stimulates the mobilisation of the fats contained in the adipose tissue, leading to beneficial effects in terms of health (Picard et al., 2004). The mechanism of action of SIRT-1 consists in limiting adipocyte differentiation but also triggers lipolysis, leading to the emptying of mature adipocytes. The development of adipose tissue is also accompanied by major remodelling of the ECM, a process regulated in part by the MMPs and their inhibitors the TIMPs. Excessive degradation of the matrix structures leaves the field clear for the development of blood vessels, which also enables the adipose tissue to be wellnourished and to extend. Adipose tissue expresses all the MMPs (except MMP-8).

Their synthesis and secretion are increased during the adipocyte differentiation process. MMP-2 and MMP-9, belonging to the gelatinase subgroup, seem to be particularly involved in adipogenesis (Croissandeau et al., 2002). Inhibition of their activity using reference molecules such as captopril and batimastat leads to a dose-dependent reduction of adipogenesis (Bouloumié et al., 2001). Magnetic resonance imaging studies have also shown that women with a high body mass index or excess cellulite had fewer and slacker adipose connective structures with diminished viscoelastic properties (Callaghan et al., 2005; Terranova et al., 2006).

Although cellulite at a moderate level is physiologically normal (adequate energy availability during pregnancy and breastfeeding), most women consider it to be an unacceptable cosmetic and aesthetic problem. Men, for their part, avoid this phenomenon, thanks to the different structure of their subcutaneous adipose tissue. They are not entirely spared, however, developing “love handles” and little bulges located predominantly above the abdominal muscles.

Recent studies, highlighting the complex role of adipokines in inflammation and in macrophage recruitment, suggest new pathways for blocking the inevitable sequence of fat accumulation followed by inflammation and then water retention. These approaches take into account the inflammation and vascularisation of the adipose tissue and the fibrous structures that compartment it. Silab has developed an active ingredient, extracted from Nelumbo nucifera (lotus) and rich in flavonols. This lotus extract has a triple action in adipose tissue – the extract:

Material and methods

Cell culture
3T3 F44-2A preadipocytes were inoculated in DMEM (Dulbecco’s modified Eagle’s medium) (Gibco, Cat. No. 31966) supplemented with 10% donor calf serum (DCS) (Gibco, Cat. No. 16030). The cells were then incubated for 4 days at 37°C in an incubator containing 5% CO2. Differentiation of preadipocytes was induced by replacing the preadipocyte culture medium with DMEM supplemented with 10% fetal calf serum (FCS) (Gibco, Cat. No. 10270), antibiotics, 50 nM insulin (Sigma, Cat. No.I-5500), 10-6 M biotin (Sigma, Cat. No. B-4639) and 1% (v/v) antibiotics (streptomycinpenicillin) (Gibco, Cat. No. 15070). The cells were then incubated at 37°C in an incubator containing 5% CO2. The medium was renewed every other day, and daily if there was acidification.

Lipolytic activity of mature adipocytes
After 8 days of culture (complete differentiation), the medium was discarded and replaced with DMEM containing 2% FCS and antibiotics but without insulin. The next day, the cells were rinsed with PBS without Ca2+ and Mg2+ and the medium was replaced with 2 ml of KRBA solution (Krebs-Ringer-Bicarbonate- Albumin) per well. The plates were incubated for 15 minutes at 37°C in an incubator containing 5% CO2. The lotus extract (0.10%, 0.25%, 0.50% and 1%) or caffeine (37.5 g/l at 2%) used as positive control was added to the corresponding wells and incubated for 120 minutes at 37°C in an incubator containing 5% CO2. Non-esterified fatty acids (NEFA) were assayed at 550 nm with the NEFA C colorimetric kit (Wako, Cat. No. FR46551).

SIRT-1 and adiponectin synthesis
Treatment of cells
The cells were treated with 0.25%, 0.50% lotus extract and then incubated for 3 days at 37°C in an incubator containing 5% CO2. The treatment was repeated and after 2 days of incubation, the cell-free extracts were recovered and stored at –80°C before assaying total proteins with a BCA kit (Sigma, cat. No. BCA1). SIRT-1 and adiponectin proteins were then assayed by Western blot.

SIRT-1: Electrophoresis was on a 12% polyacrylamide/SDS gel (15 µg of proteins deposited). Transfer was to an Immobilon P membrane (Millipore, IPVH 15150). The membrane was incubated with a murine anti-SIRT-1 antibody, then HRP-coupled anti-murine IgG antibody. Adiponectin: Electrophoresis was on a 15% polyacrylamide/SDS gel (30 µg of proteins deposited). Transfer was to an Immobilon P membrane (Millipore, IPVH 15150). The membrane was incubated with a rat anti-adiponectin antibody, then HRP-coupled anti-IgG rat antibody. The visualisation system was peroxidase substrate and chromogen solution. Bands were semi-quantified by densitometry after image analysis with BIO PROFIL software (Bio1D, Vilber Lourmat France).

MMP activities
The cells were treated with 0.25%, 0.50% and 1% lotus extract or 1mM captopril used as positive control and then incubated for 3 days at 37°C in an incubator containing 5% CO2. The treatment was repeated and after 2 days of incubation, the cell-free extracts were recovered and stored at -80°C before assaying total proteins with a BCA kit (Sigma, cat. No. BCA1). MMP-2 and MMP-9 activities were assayed by zymography. An 8% polyacrylamide/SDS gel containing 1 mg/ml of gelatin was prepared. Samples (about 35 µg of proteins) were mixed (1/1, v/v) with Laemmli buffer, deposited on the gel and separated by electrophoresis. After incubation in 2.5% (v/v) Triton X-100 buffer, the gel was incubated at 37°C in 50 mM Tris-HCl buffer, pH 7.6, containing 5 mM CaCl2, 200 mM NaCl and 0.02% Brij 35. The gel was then stained with Coomassie Blue G250 (Sigma, Ref. B-1131) and destained by successive baths in an acetic acid/methanol/water mixture (10/20/70). Gelatinolytic activity is shown by clear bands (lysis of gelatin) at about 64-72 kDa for MMP-2 and about 92 kDa for MMP-9, while non-hydrolysed gelatin remains blue. These lysis bands were semi-quantified by densitometry after image analysis with BIO PROFIL software (Bio1D, Vilber Lourmat-France).

Recombinant MMP-2 (R&D Systems, Cat. No. 902.MP010) and recombinant MMP-9 (R&D Systems, Cat. No. 911.MP010) were used as controls.

Study of the slenderising effect in vivo
The slenderising effect of the lotus extract formulated at 4% in an emulsion or the placebo was determined by measuring thigh circumference after 28 days and abdomen and hip circumference after 56 days of twice-daily applications. The study was conducted on 20 healthy female volunteers (mean age 43 ± 11 years) for thigh circumference and on 41 volunteers (mean age 41 ± 10 years) for abdomen and hip circumference divided into two groups, having applied either the lotus extract or the placebo emulsion. Volunteers were selected according to their body mass index (BMI), that had to be between 21 and 26, and the visual presence of cellulite for thigh measurement.

Study of the anti-cellulite effect in vivo
The effect of lotus extract formulated at 4% on cellulite and on the surface irregularities it caused was studied vs. placebo by scoring with a photographic scale (Fig. 3) and by subjective evaluation. Photographs of a zone on the exterior of the thigh were taken before and after 28 days of twice-daily treatment. In order to accentuate surface irregularities caused by the cellulite, a system of controlled pinching was developed in order to apply a standard pressure on the studied zone. The sensations felt when the volunteers used lotus extract or the placebo were then gathered using a self-evaluation questionnaire. The study was conducted on 20 healthy female volunteers between 25 and 63 years of age (mean age 43 ± 11 years) selected according the two criteria described above.

Results

Increased lipolytic activity of mature adipocytes
Tested from 0.1% to 1%, the lotus extract significantly increased the lipolytic activity of fat cells with a dose-dependent effect (Fig. 4). At 0.25%, lotus extract significantly favoured the lipolytic activity by 821% that was comparable to the effect of caffeine (+678%). Lotus extract thus increased the hydrolysis of triglycerides in the lipid compartment of differentiated adipocytes thereby promoting the elimination of lipids stored in adipose cells.

Reduction of adipogenesis via the stimulation of SIRT-1 synthesis
The capacity of the lotus extract to inhibit the process of preadipocyte differentiation into mature adipocytes was determined by assaying the synthesis of SIRT-1, the calorie restriction gene. The addition of 0.25% and 0.50% lotus extract to the preadipocyte culture medium during their differentiation increased the synthesis of SIRT-1 by respectively 6% and 22%. The lotus extract thus limited the conversion of preadipocytes into mature adipocytes that can accumulate lipids in their lipid compartment.

Reduction of the inflammation of the adipose tissue via the stimulation of adiponectin synthesis
The capacity of the lotus extract to limit the inflammatory profile of the adipose tissue was investigated by studying the synthesis of adiponectin, an antiinflammatory hormone secreted by adipose cells. The addition of 0.25% and 0.50% lotus extract to the preadipocyte culture medium during their differentiation increased significantly the synthesis of adiponectin by respectively 16% and 33% thereby reducing the inflammatory state of the adipose tissue.

Reduction of the degradation of the adipose tissue matrix by limiting MMP-2 and MMP-9 activities
Tested at various concentrations, the lotus extract tended to reduce MMP-2 activity and significantly decreased MMP-9 with a dose-dependent effect (Fig. 5). Tested at 1% on preadipocytes during their differentiation process, the lotus extract decreased the activity of MMP-2 and MMP-9 by respectively 27% and 73%. These effects were compared to that of captopril, a well-known inhibitor of MMPs that reduced respectively the MMP-2 and MMP-9 activities by 74% and 40%. The lotus extract thus limited the degradation of the connective tissue of the hypodermis.

Favouring a reduction in thigh, abdomen and hip circumference
After 28 days of twice-daily applications without losing weight, the volunteers lost an average of 0.4 cm (P=0.0493) on the thigh treated with lotus extract formulated at 4% and of 0.1 cm (P=0.3292) on the side of placebo. After 56 days of twiceapplications, the abdominal circumference was significantly reduced by 1.6 cm (P=0.0001) for the volunteers applying the lotus extract treatment while it increased by 0.4 cm (P=0.1723) for the volunteers applying the placebo. Finally, the hip circumference of the volunteers testing the lotus extract formula was also significantly reduced (-1.3 cm, P=0.0021) while it was only reduced by 0.4 cm (P=0.0738) after the placebo treatment. All the circumference variations observed after the lotus extract treatment were significant in comparison with those obtained after the placebo treatment. The maximal reduction observed was 2 cm for thighs, 5 cm for the abdomen and 4.5 cm for the hips (Table 1).

Anti-cellulite properties presented
Scoring with the photographic scale
In the conditions of this study, after 28 days of twice-daily applications, lotus extract formulated at 4% vs. placebo led to a significant reduction (-19%, P=0.0041) of surface irregularities resulting from the presence of underlying cellulite. This effect was observed in 68% of the volunteers and it is illustrated in Figure 6. Lotus extract treatment thus improved the visual appearance of the skin. Subjective evaluation After 1 month of twice-daily use, the volunteers felt that their skin was significantly smoother and that the orange peel skin appearance as well as the cellulite decreased on the thigh treated with lotus extract formulated at 4% in comparison with the side treated with the placebo (Fig. 7).

Conclusion

Tested in vitro on preadipocytes during their differentiation into mature adipocyte, lotus extract presented a powerful lipolytic activity. It also stimulated the synthesis of SIRT-1 (+22%), the calorie restriction gene that limits the adipogenesis process. The capability of lotus extract to inhibit MMP-2 (-27%) and MMP-9 (-73%) activities allowed a reduction of adipose connective tissue degradation and a limitation of the matrix and vascular remodelling necessary for adipose tissue development. Finally, lotus extract significantly stimulated adiponectin synthesis (+33%) thereby reducing the inflammatory state of adipose tissue. By promoting the reduction of stored fat and restoring the homeostasis of the adipose tissue, lotus extract had an overall slimming effect.

Tested directly on volunteers for 28 days of twice-daily treatment, lotus extract formulated at 4% significantly reduced surface irregularities (-19%, P=0.0041) linked to the presence of underlying cellulite. This anti-cellulite effect was perceived significantly by the volunteers in a subjective assessment of the product against placebo and confirmed by clinical assessment. Furthermore, a contact thermography study of thermal variations in cellulite areas showed that lotus extract at 4% promoted the drainage of cellulite areas by reducing the oedematous areas by 29% (P=0.0029) after 28 days of twice-daily treatment and in comparison with the placebo treatment (results not shown). Finally, it promoted a significant reduction of thigh circumference (-0.4 cm on average) after 28 days of twice-daily treatment, of abdomen circumference (-1.6 cm on average) and hip circumference (-1.3 cm on average) after 2 months of treatment. These effects were significantly different from those of the placebo.

Thanks to its anti-inflammatory properties and its capacity to restore the homeostasis of adipose tissue, the lotus extract improved adipose tissue function and limited water retention thereby improving the visual appearance of the skin orange peel effect.

References

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