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ORIGINAL ARTICLE
Year : 2012  |  Volume : 2  |  Issue : 2  |  Page : 121-131

Anti-obesity effect of Stellaria media methanolic extract in the murine model of cafeteria diet induced obesity


1 Department of Pharmacology, Shree H. N. Shukla Institute of Pharmaceutical Education and Research, Rajkot, India
2 Department of Pharmacognosy and Phytochemistry, SAL Institute of Pharmacy, Ahmedabad, India
3 Shri G M Bilakhia College of Pharmacy, Vapi, Gujarat, India
4 Sudhakarrao Naik Institute of Pharmacy, Pusad, Maharashtra, India
5 Department of Pharmaceutics, Maliba Pharmacy College, Bardoli, Gujarat, India
6 Department of Pharmaceutical Chemistry, Maliba Pharmacy College, Bardoli, Gujarat, India

Date of Submission15-Jun-2011
Date of Acceptance30-Aug-2011
Date of Web Publication9-May-2012

Correspondence Address:
Vijay R Chidrawar
Department of Pharmacology, Shree H. N. Shukla Institute of Pharmaceutical Education and Research, c/o B. M. Kiyada Campus, B/H Marketing Yard, Nr. Lalpari Lake, Amargadh-Bhichari, Rajkot - 360 001
India
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DOI: 10.4103/2231-0738.95963

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   Abstract 

Context: The whole plant of Stellaria media Linn (family Caryophyllaceae) is used by the local people of Dibrugarh district of Assam state, India, as a dietary supplement for the treatment of overweight and is also mentioned in the traditional system of Indian medicine as a remedy for obesity. Moreover phytoconstituents like flavonoid, saponin, and sitosterol have already been reported for their anti-obesity activity, and the extract of S. media also contains the same constituents in its extracts. With this background, this study was carried out. Objective: The anti-obesity activity of the alcoholic and methanolic extracts of Stellaria media was evaluated against the cafeteria diet-induced obesity model in female Wistar rats. Materials and Methods: Obesity was induced by feeding a cafeteria diet for 48 days to female Wistar rats, although one group was kept on a normal chow diet to evaluate the effect of Stellaria media on body weight changes, exploratory behavior, theromogenesis, lipid metabolism, effect on White Adipose Tissue (WAT), and histology of the fat pad. Results: Among these two extracts, the methanolic extract of Stellaria media (MESM) has shown a strong anti-obesity effect compared to the alcoholic extract of Stellaria media (AESM); may be because of its multiple mechanisms. The LD 50 value was found to be more than 5000 mg/kg. Discussion and Conclusion: These findings suggest that the anti-obesity activity produced by MESM may be because of the flavonoid and saponin contents, which have thermogenic and appetite-suppressant properties or it may be due to the β-sitosterol content. With this study we conclude that MESM is beneficial for the suppression of obesity and its associated complications.

Keywords: Adiposity index, fat cell isolation, Stellaria media, thin layer chromatography, Wistar rats, β-sitosterol


How to cite this article:
Chidrawar VR, Patel KN, Bothra SB, Shiromwar SS, Koli AR, Kalyankar GG. Anti-obesity effect of Stellaria media methanolic extract in the murine model of cafeteria diet induced obesity. Int J Nutr Pharmacol Neurol Dis 2012;2:121-31

How to cite this URL:
Chidrawar VR, Patel KN, Bothra SB, Shiromwar SS, Koli AR, Kalyankar GG. Anti-obesity effect of Stellaria media methanolic extract in the murine model of cafeteria diet induced obesity. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2014 Nov 26];2:121-31. Available from: http://www.ijnpnd.com/text.asp?2012/2/2/121/95963


   Introduction Top


Obesity is operationally defined by using a relationship of height and weight called the body mass index. Obesity is increasing at a high rate in developed and developing countries. At the beginning of the Twenty-first Century, obesity has become the leading metabolic disease in the World. So much so, that the World Health Organization refers to obesity as the global epidemic. In fact, obesity is a common disease affecting not only affluent societies, but also developing countries. Currently 300 million people can be considered as obese, and due to the rising trend in obesity prevalence, this figure could double by the year 2025, if no action is taken against this threat. Central obesity is a strong predictor of a higher prevalence of Type 2 diabetes, dyslipedimia, hypertension, coronary artery disease, and other cardiovascular comorbidities. To date, pharmacological treatments do not appear to be effective in producing sustained long-term weight loss. [1],[2] Therefore, future research is necessary to discover new drug therapies that can be used to reduce the prevalence of obesity. For the present study we have chosen Stellaria media, to evaluate its anti-obesity and related complications like type-2 diabetes mellitus (T2DM) against cafeteria diet-induced obesity, in Wistar rats. As per the scientific literature, flavonoids, sitosterols, and saponines have shown promising effects to tackle obesity by various mechanisms. The selected plant has shown the presence of triterpenoids, flavonoids, saponins, and so on, in their extract. [3] Moreover, the traditional Indian medicine system also claims its anti-obesity activity. With this back ground we have selected this plant for its phytochemical analysis and for screening its anti-obesity activity. [4]

Stellaria media Linn (family Caryophyllaceae) is a small shrub 20 - 30 cm tall, found in the Dibrugarh district, situated in the eastern part of Assam state, India, with the total area of 3381 sq km surrounded by the Brahmaputra river in the north. As per the ethnobotanical claims S. media is used as an astringent, carminative, anti-asthmatic, demulcent , depurative, diuretic, emmenagogue, expectorant, and galactogogue, as also for kidney complications, inflammation in rheumatic joints, wounds, and ulcers. It is also used in treatment of various kinds of skin diseases, bronchitis, rheumatic pains, arthritis, and period pain. A poultice of chickweed can be applied to cuts, burns, and bruises. [4]

Cafeteria-diet animal models of obesity have been reported to bear a close resemblance to human obesity. [5] It is well-known that high-fat intake and a sedentary lifestyle, white collar jobs, lack of exercise, and so forth cause fat accumulation and an increase in body weight. A cafeteria diet is the combination of different compositions like highly palatable supermarket food. Cafeteria diets have been previously reported to increase energy intake and cause obesity in humans as well as animals. [6],[7] With this setting we have selected the cafeteria diet-induced model, to evaluate the anti-obesity activity of S. media in female Wistar rats.


   Materials and Methods Top


Materials

Remi research centrifuge (R-24), Soxhlet extractor, OLYMPUS iNEA 5X, 10X/0.2; India, and 100X/1.25 oil India, HPTLC (CAMAG, Switzerland), the Shimadzu UV-visible Spectrophotometer (UV1800), microtone, Open field model was fabricated based on earlier standard literature, STAT Fax autoanalyzer (2000), Afcoset digital balance (ER-180A), 250 μ pone nylon mesh, micro-pipette, and so on.

Chemicals

Sibutramine and Orlistat were gifted by the Ranbaxy Laboratory Ltd, Devas, MP (India), Bovine Serum Albumin, Collagenase Type-1, Methylene blue, Oil red O, Trypsin, biochemical kits of Span diagnostics. All the chemicals used in the study were of analytical grade.

Preparation of extract

Plant authentication Plant samples of the S. media (L) were collected in July-August, 2007, from the Dibrugrah district of Assam state, India, and verified by Prof. (Dr.) H.B. Singh, Head, Raw Materials Herbarium and Museum, NISCAIR, New Delhi, India. Duplicate herbariums were also retained in the Department of Pharmacognosy of Shree H. N. Shukla Institute of Pharmaceutical Education and research, Rajkot, for future reference, the voucher No. of specimen was HNSIPER/herb/09.

The collected plant was dried in a shadow, powered, and then passed through sieves # 40 and used for the extraction process. Successive solvent extraction was carried out; a dried material was extracted with different solvents, starting from the solvent of low polarity first with alcohol and then methanol. After extraction by one solvent, the material was removed from the thimble, dried, and recharged, and extracted with a solvent of successively high polarity. Successive solvent extraction was done by using alcohol and methanol as prescribed by Kokate, 1994, to get thecoholic extract of S. media (AESM) and the methanolic extract of S. media (MESM) (Kokate, 1994).

The extract was filtered and concentrated by using the rotary flash vacuum evaporator (ROTEVA, EQUITRON, Mumbai, India). The extract was dried in a vacuum drier and stored below 10°C.

Toxicity study

Procedure: Acute toxicity studies were performed according to the Organization for Economic Cooperation and Development (OECD) 423 guidelines category IV substance (acute toxic class method). Albino mice (n=3) of either sex selected by the random sampling technique were employed in this study. The animals were fasted for four hours with free access to water only. The plant extracts of S. media were administered orally with an initial dose of 1000 mg/kg body weight. The mortality was observed for three days. If mortality was observed in two out of three or three out of three animals, then the dose administered was considered to be a toxic dose. However, if mortality was observed in only one mouse out of the three animals then the same dose was repeated again to confirm the toxic effect. If mortality was not observed, the procedure was then repeated with a higher dose. [8]

Phytochemical investigation

Phytochemical investigation was carried out as per the method prescribed by Kokate. [9]

Estimation of total flavonoid content

Flavonoid concentration was determined as per Chang, et al. [10] A known volume of methanolic extract was diluted with 80% aqueous ethanol (0.9 ml). An aliquot of 0.5 ml was added to the test tube containing 0.1 ml of 10% aluminum nitrate, 0.1 ml 1 M aqueous potassium acetate, and 4.3 ml of 80% ethanol. After 40 minutes at room temperature the absorbance was determined at 415 nm with a UV spectrophotometer. The total flavonoid content was calculated according to a standard curve established with Quercetin.

Estimation of total saponin glycosides

The total saponin content in both the extracts was established by using a method prescribed by Rajpal. [11]

The total Saponin content was found to be 0.121% w/w and 0.108% w/w for methanolic and ethanolic, respectively.

Thin-layer chromatography study of the methanolic extract of Stellaria media

The stationary phase and mobile phase set for β-sitosterol as per the Harborne JB

Thin-Layer Chromatography (TLC) for β-sitosterol

Plate dimension : 5 × 15 cm.

Stationary phase : Silica gel G for TLC

Sample preparation :

5 mg methanolic extract was dissolved in 5 ml of acetone

Mobile phase :

Various solvent systems have been tried for optimization of a better resolution, mainly using chloroform and ethyl acetate as the methanol extracts contain sterols, which are non-polar to semi-polar in nature

Visualization :

Spraying with 20% sulfuric acid in methanol

Treatment after

spraying :

Heated in oven at 105 o C for two to five minutes

Animals

Forty-five, five month-old Wistar female rats weighing around 80 to 120 g were used in this study. The rats were bred at the Central Animal House, Shree H. N. Shukla Institute of Pharmaceutical Education and Research, Rajkot, Gujarat, and were used in this experiment. The animals were housed in a standard controlled animal care facility, in cages (five rats/cage), and maintained in a temperature-controlled room (22-25°C, 45% humidity) on a 12:12-hour dark-light cycle. The animals were maintained under standard nutritional and environmental conditions throughout the experiment. All the experiments were carried out between 9:00 and 16:00 hours, at ambient temperature. The Nations Control and Supervision of Experiments on Animals (CPCSEA) guidelines were strictly followed and all the studies were approved by the Institutional Animal Ethical Committee (IAEC), (Ref: IAEC/HNSIPER/RJK/05/2009) of the Shree H. N. Shukla Institute of Pharmaceutical Education and Research, Rajkot, Gujarat.

Induction of experimental obesity

The cafeteria diet consisted of three diets (condensed milk, 48 g + bread, 48 g), (chocolate, 18 g + biscuits, 36 g + dried coconut, 36 g), (cheese, 48 g + boiled potatoes, 60 g). The three diets were presented to a group of six rats on days one, two, and three, respectively, and then repeated in the same succession for 48 days. The diet was provided in addition to the normal pellet chow. The test drugs were administered half an hour earlier to the cafeteria-diet presentation. The control animals received only the vehicle in the same volume. [12]

Test drug preparation

The MESM, AESM, and standard Sibutramine are soluble in water, so distilled water was used as a media to dissolve. All the drug concentrations were prepared freshly, just before administration. All the test drugs including the standard were given by oral gavages via the p.o. route.

Evaluation

Body weight

The body weight of rats (g) was recorded every week for 48 days for each group, just before dosing, by using a precision balance of 10 mg sensitivity.

Exploratory behavior

The exploratory behavior was recorded on day 48, using an open field behavior test apparatus, 30 minutes after test drug administration to the treatment groups. The open field test was performed by placing the rat in the center and recording the ambulatory activity (squares crossed by horizontal movement), the frequency of rearing (standing up vertically), and grooming (face washing and repetitive licks directed to the body). The rat was placed in the center of the field and observed, and its movements were recorded using a Sony handy camcorder for five minutes.The animals were kept under laboratory conditions for one hour prior to the test. Their behavior was recorded using a Sony handy camcorder for noting the exact reading and storage of data. Between the trials, the field was cleaned after every reading with a wet sponge and tissue papers.

Body temperature

The body temperature of the rats was recorded on day 49 using a rectal telethermometer, before and after drug administration, at one and two hours. After measuring the body weight, each animal was placed in a specially designed restrictor, to measure the rectal temperature. A Yellow Spring Instrument telethermometer with a series 500 probe was used. The probe was lubricated using petroleum jelly prior to use and was inserted between 2.0 and 2.5 cm into the rectum and held in position for 10 seconds before the temperature was determined. Measurements were made once for each animal and were conducted during a two-hour period, four hours before light offset.

Biochemical parameters

Preparation of serum Twenty-four hours (forty-ninth day of study) after the last administration of the test drug, the animals were anesthetized under light ether anesthesia and blood for serum preparation was collected via a retro-orbital puncture, using a 10 μl x 20 mm (L) x 0.8 mm (2R) glass capillary, into a sterile EDTA-coated tube (3 mg/ml), for estimation. The blood was kept in wet ice for 30 minutes, centrifuged for five minutes at 4000 rpm, at 4 o C (REMIMAK, India), and plasma was aspirated out for the analysis of lipid profile and glucose. The serum was stored in the refrigerator for analysis of the biochemical parameters. All analyses on the serum were completed within 24 hours of sample collection. The serum samples were analyzed for glucose, triglycerides, and total cholesterol, using biochemical kits of Span diagnostics Glucose: Glucose oxidase-peroxidase (GOD/POD) method; [13] Cholesterol: One step method of Wybenga and Pilleggi; [14] Triglycerides: glycerol-3-phosphate oxidase - phenol aminophenazone (GPO-PAP), end-point method, [15] and high density lipoprotein cholesterol (HDL-C) by the cholesterol oxidase-phenol aminophenazone (CHOD-PAP) [14] method.

Effect on white adipose tissue

The rats were euthanized by ether overdose on day 50 and then the white adipose tissues (WAT, periovarian, perirenal, and mesenteric fat pad) were isolated. After weighing the fat content it was preserved for further study. The adiposity index, a quantitative measure of the total fat mass was calculated by using the previously determined equation derived by Gregoire, et al. [16]

Adiposity Index (%) = [Σ (fat pad)/Body weight x 100]

Isolation of fat pads and experimental procedure

The four regions of the adipose tissue that were carefully dissected were:

  1. The periovarian fat: The ovaries were taken out by gentle squeezing from the peripheral fat, and then by a horizontal cut from all sides, the fat was isolated; care was taken that too much traction was avoided on the ovaries and fat.
  2. The retroperitoneal fat: This was obtained by first separating the perirenal fat and then dissecting the retroperitoneal pad in toto.
  3. The mesenteric fat: All fat found along the mesentery starting at the lesser curvature of the stomach and ending at the sigmoid colon was considered as mesenteric fat. It was obtained by cutting the intestine below the duodenal-jejunal junction and stripping the fat by gently pulling the intestinal loops apart.
  4. The Omental fat: This was obtained by separating its large fold that hung down from the stomach and extended from the stomach to the posterior abdominal, without causing any harm to the peripheral portion of the tissue.
Isolation and sizing of fat cells

Adipocyte isolation The Periovarian adipocytes were isolated using trypsin and were centrifuged as per the previous method described by Honnor et al. with slight modifications. [17] The samples were provided with (5% CO 2 and 95% O 2 ), capped and incubated at 37°C, with shaking, until digestion was complete (30-40 minutes).

Sizing of fat cells

About 0.2 to 0.4 ml aliquots of the stirred suspension of stained cells were placed on a siliconized glass slide and examined with a Zeiss microscope, equipped with a Polaroid camera attachment. The insertion of a micrometer disk in a focusing eyepiece placed in the phototube of the camera attachment produced a projected caliper scale. At a magnification of ×200, the caliper scale was calibrated, so that the unit marks had a constant interval of 7 μ. The free fat cells, floating on the surface of the medium, were recognized by the spherical shape, the stained nucleus with one or two nucleoli, and the stained cytoplasm; the latter features clearly distinguished the fat cells from the occasional droplets of floating lipid. One hundred cells, one by one, from the same population, were brought in the caliper field with a systematic motion of the stage control knobs. The cells were aligned on the caliper scale, the equatorial plane of the cells was brought into focus and the fat cell diameter was determined with accuracy. The sizing and grouping of 100 fat cells was performed by one observer in approximately 15-20 minutes. From this, the mean diameter and the standard deviation of the mean could be rapidly calculated using the usual formulas [Figure 1].
Figure 1: Basic microscopic determination of fat cell diameter. Stained fat cells, floating on the surface of the medium are aligned on a caliper scale, brought into focus, and the transverse diameter was recorded in units. Magnification x200, Free fat cells seen in this photograph are derived from the prioverian fat pads of a 150-g rat

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Histology of the fat pad

The periovarian fat was selected for histological study. The periovarian fat of each group was excised and rinsed in 0.9% saline, and was then blotted dry of saline and excess blood. It was fixed in 12% formalin for 24 hours. The tissues, after fixation, were washed in water to remove the excess fixative. The washed tissues were then dehydrated through a graded series of ethyl alcohol, cleared with xylene, and embedded in paraffin wax. Sections were cut at 3 μm with a microtone blade and mounted on a clean glass slide. The sections were routinely stained with hemotoxyllin and eosin. The stained slides were observed (X 200) through a research microscope and photographed.

Statistical analysis

The results were expressed as mean±SEM. Comparisons between the treatment groups and positive control,and positive control and control, were performed by one way analysis of variance (ANOVA), followed by the Dunnett's t-test. In all the tests the criterion for statistical significance was P<0.05 (95% level). The analysis was performed by using GraphPad Prism 4.

A P value of P<0.05, was considered as significant *P<0.05, **P<0.01.


   Results Top


Toxicity study

The plant extracts of Stellaria media did not show any mortality and toxicity even at the highest dose of 5000 mg/kg body weight employed, so the LD 50 value was expected to exceed 5000 mg/kg body weight.

The best resolution and separation were observed with chloroform and ethyl acetate (8:0.6). The TLC profile revealed the presence of seven spots after spraying with 20% methanolic sulfuric acid.

Feeding with CD caused a significant increase in the glucose, insulin, SGOT, SGPT, TG, TC, and LDL-C levels in plain CD-treated group animals as compared to normal control animals, which was significantly decreased by the co-administration of MESM 200 and 400 mg/kg, compared to the disease control and normal control groups. Treatment with medium and high-dose of MESM also improved the HDL-C level compared with plain CD feed rats. Sibutramine was the most significant in this regard.


   Discussion Top


Obesity is a chronic metabolic disorder that results from the imbalance between energy intake and energy expenditure, characterized by enlarged fat mass and elevated lipid concentration in blood. Obesity is also a primary risk factor for CVD (cardiovascular disorders). It has reached epidemic proportions globally, with approximately 1.6 billion persons (aged 15 years old and above) being overweight. [18] Many attempts have been made to correct the metabolic disparity of the obesity condition, producing a number of reagents, including fibrates, Sibutramine (an anorectic or appetite suppressant), and Orlistat, but no one is free from the severe side effects. [19],[20],[21] At present, because of dissatisfaction with high costs and potentially hazardous side-effects, the potential of natural products for treating obesity is under exploration and this may be an excellent alternative strategy for developing future effective, safe anti-obesity drugs. A variety of natural products including crude extracts and isolated compounds from plants can induce body weight reduction and prevent diet-induced obesity. Therefore, they have been widely used in treating obesity. [22],[23]

Several infusions or decoctions of plants used in traditional medicine to reduce obesity could be utilized to delete the clinical side effects of the current chemically formulated anti-obesity agents; the examples include Camellia sinensis (L.) Kuntze (Theaceae), Chlorella pyrenoidosa Chick. (Oocystaceae), Citrus aurantium L. (Rutaceae), Garcinia cambogia L. (Clusiaceae), Lagerstroemia speciosa (L.) Pers. (Lythraceaea), Panax ginseng C.A. Meyer (Araliaceae), Salix matsudana Koidzumi (Salicaceae), Nelumbo nucifera Gaertn. (Nymphaeaceae), and Hibiscus sabdariffa L. (Malvaceae). [24],[25],[26]

Stellaria media, commonly known as chickweed, is found throughout the Himalayas up to an altitude of 4300 m and is found in the Dibrugarh district, situated in the eastern part of the Assam state, in India. It is reported to be very useful in the treatment of inflammations of the digestive, renal, respiratory, and reproductive tracts. The plant is employed in plasters used for broken bones and swellings. It also possesses diuretic, expectorant, and anti-asthmatic properties. Some phenolic acids, flavones, fatty esters, and gypsogenin have been reported earlier from this species. [27] The traditional system of Indian medicine also reports its anti-obesity activity. [4]

In the present study we have studied the effects of methanolic and alcoholic extracts of S. media for 48 consecutive days, by p.o. administration, on body weight changes, exploratory behavior, change in body temperature, blood lipid levels, adiposity index, and sizing of fat cells in normal and CD fed rats. The treatment and grouping is as explained in [Table 1].
Table 1: Animal grouping and diet

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In our study, initially a pharmacognostic and phytochemical analysis was carried out in the phytochemical investigation. It was clear that S. media contained triterpenoids, glycoside, carbohydrates, proteins, flavonoids, anthocynidine, and saponins. The results of the extractive values and phytochemical analysis are shown in [Table 2] and [Table 3], respectively. Some chemical constituents like saponins, flavonoids, and some triterpenoids have been noted for their anti-obesity effect in various plants. [3] Based on this phytochemical study and ethnobotanical claims, this plant was selected to carry out this study.
Table 2: Extractive values

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Table 3: Phytochemical investigation

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Rats that are fed cafeteria diet (CD) are a widely used model for obesity. The so-called 'cafeteria diet' involves feeding experimental animals a mixture of palatable commercially available supermarket foods, to stimulate energy intake. [7] Characteristic for such diets is the combination of the high fat content with high carbohydrate content. Furthermore, the components of the cafeteria diet are a variety of foods high in fat and sugar, but usually low in protein, vitamins, and minerals. Such diets have pronounced implications in the development of obesity, leading to significant body weight gain, fat deposition, and also insulin resistance, resembling that in human beings. [28] It has been suggested that rats become more obese with cafeteria diets than with pure high-fat diets and normal chow diet, indicating a greater hyperphagia arising from the food variety, texture, and palatability. [29] On account of the above-mentioned facts the 'cafeteria diet' model is considered for the study.

Our results demonstrated that feeding with CD had caused a significant increase in weight gain compared with the normal chow diet-feed rats, which was significantly decreased by the co-administration of MESM, in a dose-dependent manner. The Sibutramine-treated group, along with CD, had shown significant reduction in weight gain compared with the plain CD and normal control groups, as shown in [Figure 2] and [Figure 3]. This increase in weight gain in the CD-treated group was probably due to more palatability. [29]
Figure 2: Animals fed with cafeteria diet showed significant increase in the body weight as compared to normal control animals which was significantly decreased in MESM 100, 200 and 400 mg/kg in dose dependent manner and most significantly by Sibutramine compared to normal control and disease control animals. *Comparison of test and disease control with normal control, # Comparison test with disease control

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Figure 3: Animals fed with cafeteria diet showed significant increase in the body weight as compared to normal control animals which was significantly decreased in MESM 100, 200 and 400 mg/kg in dose dependent manner and most significantly by Sibutramine compared to normal control and disease control animals. *Comparison of test and disease control with normal control, # Comparison test with disease control

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Reduction in weight gain by co-administration of MESM, in medium and high doses, may be due to its crude saponin and flavonoid content; these phytoconstituents are present in abundant quantity in this extract, which is confirmed by the total saponin and total flavonoid content in the extract, as given in [Table 4]. Crude saponin and flavonoid have been reported for their appetite-suppressant property. [3] From this study we are predicting that saponin and flavonoids, after absorption from the gastrointestinal tract (GIT) cross the Blood Brain Barrier (BBB) and enter the brain, and amplify signaling in the basal hypothalamus energy sensing function, which is the master regulator of food intake and energy expenditure or it may also be possible that saponin inhibits the re-uptake of 5-HT in the hypothalamus. Some flavonoids also activate β-adrenergic receptors, which are involved in the burning of fats.[30]
Table 4: Total flavonoids and saponin content

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It is well known that an increase in food ingestion results in the activation of both heat production and deposition of reserves, mainly fat. [31] Consumption of CD results in overweight animals, but also increases heat production through diet-induced thermogenesis, so measurement of rectal temperature recording was considered one of the parameters in this study. [32] The drugs that demonstrate such changes indicate the thermogenic property of the drugs. In the present study, as presented in [Figure 4], administration of a high dose of MESM has shown a significant rise in body temperature after one and two hours of administration, than that in the normal control and plain CD-fed rats. A medium dose of MESM has shown a rise in body temperature after one and two hours of administration compared to normal control rats. Treatment with standard Sibutramine has shown the most significant result in the rise in body temperature compared to the normal control and plain CD-treated groups.
Figure 4: Administration of MESM have shown significant rise in body temperature after 1 hr and 2 hr of administration then normal control and plane cafeteria diet feed rats. Medium dose of MESM have shown rise in body temp. after 1 hr and 2 hr of administration compared to only normal control rats. Treatment with standard sibutramine has shown most significant result in rise in body temp compared with normal control and plane CD feed rats. *Comparison of test and disease control with normal control, # Comparison test with disease control

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Exploratory behavior was performed on day 48 of the study. Administration of a high dose of MESM and standard Sibutramine have shown a significant increase in ambulation and grooming compared to plain CD-fed and normal control rats. Administration of a low and medium dose of MESM has shown a significant rise in rearing and grooming compared to normal control rats. The increases in rectal temperature and ambulatory activity by MESM may be attributed to the overall stimulant and thermogenic property of the phytoconstituents in the extracts, [Figure 4] and [Figure 5].
Figure 5: Administration high dose of MESM and standard Sibutarmine have shown significant increase in ambulation and grooming behavior compared to plane CD feed and normal control rats. Administration of low and medium dose of MESM have shown significant rise in rearing and grooming compared with normal control rats. *Comparison of test and disease control with normal control, #Comparison test with disease control

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Feeding with CD caused a significant increase in the serum glucose, insulin, Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate Pyruvate transaminase (SGPT), Triglycerides (TG), Total cholesterol (TC), and Low density lipoprotein cholesterol (LDL-C), and a fall in the HDL-C levels as compared to normal control animals, which was significantly decreased by the administration of MESM 200 and 400 mg/kg. Sibutramine was most significant in this case. Increase in the glucose as well as serum insulin level indicated obesity-induced insulin resistance DM (T2DM) [Table 5].
Table 5: Effect of Stellaria media on biochemical parameters in cafeteria diet-fed Wistar rats

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Several studies show that an increase in HDL cholesterol is associated with a decrease in cardiovascular risk, which is a major complication of obesity-associated dyslipidemia, and most of the drugs that decrease total cholesterol also decrease HDL cholesterol. [33],[34] However, in the present study the extract decreased the total cholesterol and LDL cholesterol, and enhanced the HDL cholesterol significantly. This is an important advantage in the treatment of hypercholesterolemia, especially among Indians, where low HDL cholesterol is the prevalent lipoprotein abnormality. [35],[36]

The decrease in serum TG level is an important finding of this experiment. Recent studies have shown that triglycerides are independently related to obesity-induced cardiovascular complications [37],[38] and most of the anti-hypercholesterolemic drugs (can also be used to correct obesity-associated dyslipidemia) do not decrease the triglyceride levels, but MESM 400 mg/kg has lowered it significantly, by 21.83%, when compared to the CD group.

There was a significant increase in the WAT, idiposity index, and adiposite diameter in the CD-treated animals, which was significantly decreased by the co-administration of MESM, in a dose-dependent manner, and standard Sibutramine was the most significant in this view, but no significant effect was observed in the AESM-treated animals [Figure 6]. From the histological study it is clear that the adipocyte diameter is clearly reflected high in plain CD-fed rats, when compared to the normal control group. Treatment with MESM (200 mg/kg and 400 mg/kg) along with CD has shown a smaller size of adipocytes, when compared with those in rats fed with plain CD. Standard Sibutramine has shown the best protection in this regard [Figure 7].
Figure 6: Feeding with CD caused a significant increase in WAT, adiposity index, and adiposity diameter compared to the normal control animals, which was significantly decreased by the co-administration of MECP 200 and 400 mg/kg, compared to the disease control and normal control group. Sibutramine was the most significant in this regard. *Comparison of test and disease control with normal control, #Comparison test with disease control

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Figure 7: Light micrography of perioverian adipocytes in rats treated with CD (×200). Representative pictures of haematoxylin and eosin-stained sections of perioverian adipocytes from rats. CD treated group supplemented with extracts of S. media at various doses. Plane CD induced group have shown larger size of adipocytes compared to normal control diet group. Treatment with MESM (400 mg/kg), along with CD has shown smaller size of adipocytes than rats treated with plane CD. Standard Sibutramine have shown smaller adiposities compared with plane CD and MESM treated group, (a) Treatment with Vehicle and Normal Standard diet, (b) Treatment with Vehicle and CD (c) Treatment with MECP (400 mg/kg) and CD, (d) Treatment with Sibutramine (10 mg/kg) and CD

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Correction in lipid profile, decrease in adiposite diameter, and adiposity index by MESM may be because of the β-sitosterol content, which has already been reported and further confirmed by the TLC study[27] [Table 6]. Stigmasterols and β-sitosterol are plant sterols, with a structure similar to that of cholesterol. Among that β-sitosterol compounds having more comparable except for the substitution of an ethyl group at C-24 of its side chain and it is cholesterol lowering agent. [39] β-sitosterol reduced absorption of cholesterol by 42% in a meal containing 500 mg of cholesterol. [40]
Table 6: Description of TLC pattern for methanolic extract of S. media

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Furthermore, MESM contains saponin in large quantity and some past reports have shown that several saponins inhibit pancreatic lipase activity. [3] Pancreatic lipase, which is the most important enzyme of the human lipases for digesting fats, is responsible for the hydrolysis of 50 - 70% of the total dietary fats. Verger (1984) reported that dietary fat was hydrolyzed during digestion by pancreatic lipase. The two main products formed by the hydrolysis of pancreatic lipase are fatty acids and 2-monoacylglycerols. [41] These lipolytic products are mixed with bile salts, dispersed as micelles and carried in this form to the site of fat absorption. Lipid absorption takes place in the apical part of the plasma membrane of epithelial cells or enterocytes lining the gut, so inhibition of pancreatic lipase may causes a decrease in fat absorption. Saponins, flavonoids, and β-sitosterol are the bioactive phytoconstituents in

S. media, which may be responsible for its anti-obesity activity by multiple mechanisms, as explained earlier.


   Conclusion Top


Administration of the extracts corrected the level of the circulating lipids as well as the size of adipocyte diameter, resulting in the decrease of body weight in female Wistar rats. The extracts appear to show such activities maybe because of their thermogenic property, modulating lipid metabolism through the decreased absorption of dietary fats, and maybe because of the inhibition of the pancreatic lipase activity. In comparison, between these two extracts, MESM has shown promising results compared to AESM, maybe because of its multiple targets as mentioned earlier. From this we also propose that the use of MESM is beneficial for the management of obesity.

 
   References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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