Abstract

Introduction:Labisia pumila var alata or as known among Malay women as Kacip Fatimah (KF), has long being recognized for its medicinal value. Recently, researches have demonstrated its estrogenic activity.  Ovariectomy (surgical menopause) is known to cause weight gain and increase adiposity due to estrogen deficiency. Objective: This study was aimed to investigate the effect of Labisia pumila consumption and Estrogen Replacement Therapy (ERT) on the morphology of adipose tissue following ovariectomy. Methodology: 6 months old female (Sprague Dawley) rats were ovariectomized (bilaterally) under anesthesia using the ventral approach and randomly divided into OVXC (ovariectomized control), KF and ERT. KF and ERT received daily oral treatment of water extract of Labisia pumila (17.5 mg/kg/day), and ERT (64.5 µg/kg/day) respectively for a period of three months. A group of 9 normal rats was left intact and used as normal control (NOR). Rats were sacrificed by cervical dislocation and tissue samples from abdominal fat were collected and fixed immediately either in 4% paraformaldehyde for electron microscopy or in 10% formalin for light microscopy. Results: Light microscopy revealed hypertrophic growth (increase in the size of adipocytes) as well as increasing vasculature of adipose tissue in the OVX rats compared to the normal rats. Transmission electron microscopy (TEM) revealed thickening of the adipocyte membrane of the OVXC rats. KF and ERT-treatment were demonstrated to cause adipocytes membranes' breakage as shown by the fragmentation of the collagen bands. Conclusion: Results implied a possible role for Labisia pumila var alata in modulating postmenopausal adiposity through the initiation of the lipolysis process in adipose tissue.

INTRODUCTION

Labisia pumila var alata (Kacip Fatimah): Labisia pumila var alata or as locally known as Kacip Fatimah, has long being recognized by Malay women for its medicinal value. Usually the plant is boiled and the water extract is than taken to facilitate childbirth as well as a postpartum medicine. It is also believed to be beneficial in firming and toning of abdominal muscles, breasts and tighten vaginal muscles, anti-dysmenorrhoea; cleansing and avoiding painful or difficult menstruation, to treat dysentery, rheumatism and women's ailments associated with childbirth. Recently, researches have demonstrated the estrogenic activity of Labisia pumila and showed that it is possible that it acts as estrogen receptor modulators (SERMS) which is active on certain tissues, (Institute For Medical Research, [IMR] (2002). Husniza et al. (2000) as cited in (IMR, 2002), showed that water extracts of the KF were able to displace estradiol binding to antibodies raised against estradiol, making it similar to other estrogens such as estrone and estradiol. The extract has also been found to produce a dose-response effect on  the  reproductive  hormones  of  female  rats,  notably  on  the  estradiol  and  free testosterone levels, (IMR, 2002).

Adipose Tissue: Adipose tissue is "a specialized connective tissue that functions as a major storage site for fat in the form of triglycerides" (Albright & Stern, 1998). In mammals, there are two forms of adipose tissue, white adipose tissue (WAT) and brown adipose tissue (BAT). WAT stores energy as triglycerides whereas BAT specializes in adaptive thermogenesis (Tiraby & Langin, 2003). Our interest in this paper is WAT. The formation of WAT begins during late embryonic development, with rapid expansion shortly after birth caused by increased cell size and cell number (Jazet et al. 2003). In the past, adipose tissue was viewed only as an organ for storing excess energy in the form of triglycerides. It is now clear that adipose tissue is a complex and highly active metabolic and endocrine organ. In 1987, adipose tissue was identified as a major site for sex steroids metabolism and the production of adipsin, an endocrine factor that is markedly down-regulated in rodent obesity (Kershaw & Flier, 2004).

The morphology of WAT is well studied and described. The major constituent of adipose tissue is large lipid-filled cells called adipocytes that are held in a framework of collagen fibers (Albright & Stern, 1998). In addition to adipocytes, adipose tissue contains stromal-vascular cells, including fibroblastic connective tissue cells, immune cells (Kershaw & Flier, 2004) and pre-adipocytes (small adipocyte with no lipid accumulation) (Albright & Stern, 1998). The adipocyte is a mature white fat cell that is spherical in shape, measures up to 120 µm in diameter, has a single flattened nucleus and displaced cytoplasm by the lipid droplet that filled the adipocyte (Brooks & Perosio, 1997).  

The size of adipose tissue mass is defined by both adipocyte number and size. Adipose tissue can increase in size by the increase in adipocyte size (hypertrophic growth) or by the increase in adipocyte number (hyperplastic growth) (Albright & Stern, 1998). Metabolically active WAT is characterized by small numerous adipocytes that are connected to capillary network (Schling & Löffler, 2002). Unlike the hypertrophic growth, the hyperplastic growth is accompanied by temporal development of blood vessels (Crandall et al. 1997). 

Ovariectomy (surgical menopause) is known to cause weight gain and increase adiposity due to estrogen deficiency. The theory behind obesity is that initially adipocytes increase in size followed by increase in number which is associated with changes in the vasculature of the adipose tissue, (Albright and Stern 1998), (Stunkard & Wadden 1993).

Objective of the study: Labisia pumila is widely used among Malaysia women but up to date there is little scientific information available about its effects and mechanism of action. This study was aimed to investigate the effect of Labisia pumila consumption and Estrogen Replacement Therapy (ERT) on the morphology of adipose tissue following ovariectomy.

MATERIALS AND METHODS

Animal and Tissue Preparation: 35 Adult female (Sprague Dawley) rats, 6 months old were used in the experiment. The rats were housed at normal room temperature with adequate ventilation and normal 12-hours light-dark cycle with free access to food (commercial laboratory rat’s food) and water. They were divided into two main groups; Normal (NOR, n = 9) and ovariectomized (n = 26) which were further divided into; ovariectomized control (OVXC, n = 8) and the treatment groups. The treatment groups consisted of, Labisia pumila (KF, n = 9) and Estrogen Replacement therapy (ERT, n = 9). Rats were sacrificed by cervical dislocation. Tissue samples from abdominal adipose tissue were collected and fixed either in 4% paraformaldehyde for electron microscopy or in 10% formalin for normal histology.

Ovariectomy and Treatment: Bilateral ovariectomy (OVX) was performed under anesthesia using the ventral approach. Rats were anesthetized with IM injection of Zoletil 50 (Virbac Laboratories) 0.1 ml, Ketapex 0.1 ml, Xylazil 0.03 ml. After ovariectomy, rats were randomly allocated to the nominated groups. Treatment started one month after OVX in the following doses; ERT (120 µg/kg/day) and standardized water extract (patent pending) of Labisia pumila var alata (17.5 mg/kg/day), both given orally.

Electron Microscopy: Upon animal sacrificing, liver samples were immediately dissected into 1-mm cubes and placed into fresh fixative (4% paraformaldehyde) overnight at 4°C. Tissues were then washed in distilled water, post fixed in 2% osmium tetroxide for one hour at room temperature, washed in distilled water, treated with 10% uranyl acetate for 15 minutes, washed in distilled water, dehydrated through a graded series of alcohol, treated with two changes of 100% propylene oxide, infiltrated with propylene oxide and resin (1:1) for two hours and finally embedded in resin. Ultra thin sections were obtained and contrasted with 10% uranyl acetate in 75% methanol for 5 minutes and with lead citrate for 5 minutes. 

Verhoeff's Haematoxylin Stain: This staining procedure was described by Verhoeff's, (1908) for the staining of elastic tissue, (Drury and Wallington, 1980). Samples of adipose tissue were immediately fixed in 10% formalin for 24 hours and further processed for paraffin-embedded sections. Five-micron thick sections were obtained for the staining procedure. Sections were dewaxed in xylene, dehydrated in series of alcohol to water and then stained in a jar with Verhoeff's haematoxlin for 5 minutes. Sections were then differentiated in 2% ferric chloride. Differentiation was controlled by alteration between ferric chloride and a rinse in tap water. After reaching the optimal differentiation, sections were washed in tap water, treated with 95% alcohol for five minutes, washed in running tap water for five minutes and counter stained with Van Gieson for 30 seconds. Finally, sections were dehydrated in series of alcohol, cleared in xylene and mounted in Dibutyl Phathalate in Xylene (DPX).

Adipocyte Measurements: All measurements were conducted using the Image Analysis Software (Video Test). Cell size was measured as area and cell number as cell count in an image size of 608.8 µm x 487.06 µm.

RESULTS and DISCUSSION

In this paper we have demonstrated changes in the morphology of adipose tissue mediated by different factors. Light microscopy revealed hypertrophic growth (increase in the size of adipocytes) of the ovariectomized control rats (OVXC) and Labisia pumila-treated (Fig. 1) as well as increasing vasculature of adipose tissue in the OVXC compared to the normal rats (Fig.2). Transmission electron microscopy (TEM) also revealed thickening of the adipocyte membrane of these rats (Fig 3).  Ovariectomy or surgical menopause is known to induce estrogen deficiency which will lead to weight gain and increased adiposity. The current study showed that the average sizes of the adipocytes of the ovariectomized rats were bigger than those reported for the normal rats although the difference was not statistically significant. The study also demonstrated that the adipose tissue of OVXC rats consists of adipocytes of different sizes that are connected to an extensive capillary network. These features are characteristic of metabolically active adipose tissue (Crandall et al. 1997) which means that the tissue is in the process of continuous growth as demonstrated by the increase in cell size (hypertrophic growth) and number (hyperplastic growth) which requires the development of new blood vessels. This in turn explains the weight gain associated with ovariectomy.

In the treatments groups, hypertrophic growth was reported with no increase in the vasculature of the tissue with Labisia pumila treatment. Statistically, adipocyte size of the ERT-treated rats was found to be significantly (P<0.05) smaller when compared to the OVXC and Labisia pumila-treated groups but not when compared with the NOR group (Fig. 1 & 4). The effect seen with ERT treatment is known to be due to the influence of ovarian steroid hormones on the metabolism of adipose tissue (Krotkiewski, 1976, Hansen, et al. 1980, Dorai et al. 1990), which were suggested to play an important role in the regional specificities of adipose tissue localization and metabolism (Rebuffe-Scrive et al. 1985, Rebuffe-Scrive et al. 1987, Lacasa et al. 1991 and Lacasa et al. 1994). Our findings correspond with reports by (Krotkiewski 1976, Steingrimsdottir et al. 1980 and Wilson et al. 1976) where treating OVX rats with Estradiol (E2) were able reduce fat deposits in intact females following long-term administration.

The effect seen with Labisia pumila treatment could imply that initially after ovariectomy, the adipocytes started to increase in size but did not reach the stage of producing new pre-adipocytes and thus no additional blood vessels were acquired. On the other hand, both treatments (ERT and Labisia pumila) demonstrated that they were able to induce fragmentation of the framework of collagen fibers that hold the adipocytes together as well as were able to rupture the adipocytes' membrane (Fig. 5) and thus losing their contents. This observation provides another possible justification for the large sized adipocytes reported with Labisia pumila treatment and that which were observed under the light microscope is the fusion of more than one adipocyte as a result of membrane rupturing.

Fig. 1 Comparison of Adipocytes size (area) and number between normal rats (NOR), ovariectomized (OVXC), Labisia pumila-treated (KF) and Estrogen-treated (ERT). Data presented as mean ± standard error. Adipocyte of ERT-treated rats were significantly smaller (P<0.05) than those of OVXC and KF-treated rat.



 

Fig. 2 Micrographs (X 20, Verhoeff's Haematoxylin Stain) of adipose tissue of normal rats (NOR) and ovariectomized rats (OVXC) Showing increased vasculature of adipose tissue of the OVXC rats as a result of ovariectomy.



 

Fig. 3 Transmission Electron Micrographs (TEM) of adipocytes of normal rats (NOR) and ovariectomized rats (OVXC) showing the increased thickness of the adipocytes' membrane of the ovariectomized rats.





Fig. 4 Micrographs (x 20, Verhoeff's Haematoxylin Stain) of adipose tissue of (ERT) and Labisia pumila-treated Rats (KF). The first micrograph (KF) shows the hypertrophic growth of the adipocytes with no evidence for increased vasculature of the tissue.  The Second Micrograph (ERT) shows significantly smaller adipocytes.  



 

Fig. 5 Transmission Electron Micrographs (TEM) of adipocytes of ERT-treated Rats (ERT) and Labisia pumila-treated rats (KF) showing ruptured membrane (arrow) and fragmentation of collagen fibers (arrow heads).

CONCLUSION

Results implied a possible role for Labisia pumila var alata in modulating postmenopause adiposity in a manner similar to that reported for estrogen through the initiation of the lipolysis process in adipose tissue and thus a may have a possible effect on weight management.

ACKNOWLEDGEMENT

The Authors thank Mr. Santhanna Raj L and his team from Electron Microscopy unit, Institute for Medical Research – Kuala Lumpur. This research is supported by the Ministry of Science Technology and Environment, Malaysia, IRPA Grant No. BTK/ER/013 (06-05-002).

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