Appetite suppressing effect of Spinacia oleracea in rats

14 Pages·2017·2.39 MB·English

by  Vandana Panda

Accepted Manuscript
Appetite suppressing effect of Spinacia oleracea in rats: Involvement of the short term
satiety signal cholecystokinin
Vandana Panda, Priyanka Shinde
PII: S0195-6663(17)30278-7
DOI: 10.1016/j.appet.2017.02.030
Reference: APPET 3348
To appear in: Appetite
Received Date: 15 May 2016
Revised Date: 12 December 2016
Accepted Date: 20 February 2017
Please cite this article as: Panda V. & Shinde P., Appetite suppressing effect of Spinacia oleracea
in rats: Involvement of the short term satiety signal cholecystokinin, Appetite (2017), doi: 10.1016/
j.appet.2017.02.030.
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Control Rats Slow onset of satiety
Rat feed S
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Inhibitory effect
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Release of Cholecystokinin M Paraventricular
(CCK) CCK Receptors Nucleus (PVN)

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Quicker onset of Satiety
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Spinach Extract 400 mg/kg
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Feeling of Satiety
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1 Title: Appetite suppressing effect of Spinacia oleracea in rats: Involvement of the short
2 term satiety signal cholecystokinin
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4 Names of Authors: Vandana Panda*, Priyanka Shinde T
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6 Department: Department of Pharmacology & Toxicology
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8 Institution: Prin. K. M. Kundnani College of Pharmacy,
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9 Jote Joy Building,
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10 Rambhau Salgaonkar Marg,
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15 *Corresponding author: Dr. (Mrs.) Vandana S. Panda
16 Prin. K. M. Kun dnani College of Pharmacy,
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17 Jote Joy Building, Rambhau Salgaonkar Marg,
18 Cuffe ParaEde, Colaba, Mumbai 400005. India
19 Tel: +T91 22 22164387 Fax: [phone]
20 e-mail: [email protected]
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26 Running Title: Appetite suppressing effect of Spinacia oleracea
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32 ABSTRACT
33 Spinacia oleracea (spinach) is a green leafy vegetable rich in antioxidant phyto-constituents such
34 as flavonoids, polyphenols, carotenoids and vitamins. Fruits and vegetables rich in flavonoids are
35 known to prevent weight gain by inducing satiety. The present study evaluates the appetite
36 suppressing effect of a flavonoid rich extract of the spinach leaf (SOE) in rats. HPTLCT of SOE
37 was performed for detecting flavonoids. Rats were administered SOE (200 mg/kg and 400
38 mg/kg, p.o) and fluoxetine (6 mg/kg i.p) as a pre-meal for 14 days. Food intake anPd weight gain
39 was observed daily during the treatment period. Serum levels of the short term satiety signals
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40 cholecystokinin (CCK) and glucose were measured on the 7th and 14thdays at different time
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41 points after start of meal to study the satiety inducing effect of SOE. HPTLC showed the
42 presence of 14 flavonoids in SOE. SOE and fluoxetine treated rats showed a significant
43 reduction in food intake and weight gain when compared with the normaCl control rats. On the 7th
44 day of treatment, peak CCK levels were reached in 30 min after start of meal in fluoxetine
45 treated rats and in 60 min in the remaining rats. On the 14th day, CCSK peaking was observed in
46 30 min after start of meal in the fluoxetine as well as SOE 400 mg/kg treated rats. Peak glucose
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47 levels in all treatment groups were obtained in 60 min after start of feeding on both days of the
48 study. It maybe concluded that SOE exhibited a promising appetite suppressing effect by
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49 inducing a quicker than normal release of CCK, thus eliciting an early onset of satiety in rats.
50 This effect may be due to its high flavonoid content. A
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52 KEY WORDS: Spinacia oleracea; flavonoids; satiety; cholecystokinin; glucose

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65 INTRODUCTION
66 Energy homeostasis is a complex mechanism which maintains body weight by regulating energy
67 intake (1). Obesity occurs due to an imbalance in energy homeostasis. This imbalance is in the
68 form of increased food intake and reduced energy expenditure. Low metabolic rate, low
69 spontaneous physical activity, low sympathetic nervous system activity and low fat oxiTdation are
70 known to be the four main predisposing factors for weight gain (1). However, genetic,
71 environmental, metabolic and behavioral issues may also contribute to the developPment of
72 obesity.
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74 Figure1: Regulation of food inPtake by CNS and peripheral signals
75 The hypothalamus, a small aErea of the brain located just above the brainstem is the regulating
76 center of appetite and energy homeostasis (Figure 1). It consists of several interconnecting nuclei
77 including the arcuate nuCcleus (ARC) and the paraventricular nucleus (PVN). The ARC of the
78 hypothalamus is considered to be an area primarily sensing peripheral metabolic signals. It is an
79 aggregation of two Cdistinct populations of neurons, the AgRP/NPY expressing neurons and the
80 pro-opiomelanocortin (POMC) expressing neurons (2). The agouti-related peptide (AgRP) co-
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81 expressed with neuropeptide Y (NPY) is orexigenic and promotes weight gain by stimulating
82 appetite, while the POMC neurons which secrete α- melanocyte stimulating hormone (α-MSH)
83 and contain receptors for serotonin and leptin are anorexigenic and cause weight loss by
84 decreasing appetite and inducing satiety. POMC neurons are also present in the nucleus tractus
85 solitarius (NTS) in the medulla, which are involved in regulation of satiety. The orexigenic and
86 anorexigenic neurons integrate various nutritional, hormonal and neuronal signals to regulate
87 food intake and energy expenditure, thus helping to achieve energy homeostasis.
88 Nutrients and gastrointestinal peptide hormones such as cholecystokinin (CCK), glucagon like
89 peptide-1 (GLP-1) and peptide YY are involved in the short term regulation of food intake (3).
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90 These short term signals interact with long term regulators such as insulin, leptin and ghrelin to
91 maintain energy homeostasis.
92 In the ARC, glucose induced stimulation of the anorexigenic POMC neurons has been reported,
93 while the orexigenic AgRP/NPY neurons may be directly inhibited by glucose (4). Glucose may
94 thus play an important role in suppressing appetite and reducing food intake. T
95 Recently, much attention has been focused on plant foods that are effective in modifying release
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96 of the above mentioned short term satiety signals which regulate the balance between food intake
97 and energy expenditure to maintain body weight. Spinacea oleracea known as sIpinach is a green
98 leafy vegetable consumed by people across the globe in various culinary prepRarations. Spinach is
99 reported to possess antimicrobial, anticancer, antiulcer, antidiabetic, antihyperlipidemic and
100 antithrombotic activities by virtue of its numerous antioxidant phyto-conCstituents, together
101 termed as natural antioxidants, NAO (5). NAO comprises flavonols (spinacetin, patuletin,
102 jaceidin), polyphenols (quercetin, luteolin) and vitamins (A, B1, B2S, B3, B6, C, E, K and folic
103 acid), (6). This wide array of antioxidant phyto-constituents present in spinach renders it a potent
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104 ROS scavenger and antioxidant.
105 It is reported that fruits and vegetables which are rich in flaNvonoids particularly flavonols,
106 anthocyanins and flavones are associated with reduced weight gain (7).Chrysin, a naturally
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107 occurring flavone has been demonstrated to bring about reduction in weight gain by inducing
108 satiety (8). Recent studies have shown thylakoids, the internal membranes of chloroplasts in
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109 green leafy vegetables to induce satiety (9). Hence, we contemplated to study whether spinach
110 could curb food intake and reduce weight gain in rats by inducing satiety due to its high content
111 of thylakoids and flavonols. The present study evaluates the effect of a flavonoid- rich extract of
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112 Spinacea oleracea on food intake, body weight and the short term satiety markers
113 cholecystokinin and glucose.
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114 MATERIALS AND METHODS
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115 Drugs and chemicals
116 Fluoxetine was a gift sample froPm Macleods Pharmaceuticals Ltd., Mumbai, India. Methanol,
117 ethyl acetate, formic acid and glacial acetic acid were purchased from Merck® India Ltd. Rutin
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118 was purchased from Sigma Chemical Co., St Louis, MO, USA. All other chemicals were
119 obtained from local sources and were of analytical grade.
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121 Plant material
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122 Spinach was purchased from a farm on the outskirts of Mumbai, India and authenticated at the
123 Blatter HerbariuAm, St. Xavier’s College, Mumbai after matching with the existing specimen
124 (specimen no. TK-1).The leaves were washed, shade dried and ground to obtain a dry powder.
125 This powder was extracted using a mixture of methanol: water (70:30) v/v in a soxhlet apparatus.
126 This extract of Spinacea oleracea termed as “SOE” was dried and stored in a refrigerator for
127 further use. The yield of dried SOE from the leaves was 6.5 % w/w.
128 HPTLC fingerprinting of SOE for flavonoids
129 HPTLC of SOE for detecting flavonoids was performed on the CAMAG system using silica gel
130 60 F254 (20x10 cm) plates and a mobile phase comprising ethyl acetate: formic acid: glacial
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131 acetic acid: water [10:0.5:0.5:1.3 (v/v)]. Solutions of the reference standard rutin and SOE at
132 different concentrations were applied to the plates as bands of 8 mm each using the CAMAG
133 Linomat V applicator at a speed of 150 nL/s. The plates were then treated with a solution of 10%
134 methanolic sulfuric acid, heated at 110˚C and scanned using the CAMAG TLC scanner at 366
135 nm.
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136 Animals
137 Sprague Dawley female rats (150- 200g) were acquired from Glenmark PharmaceuPticals Ltd.,
138 India. They were housed in clean polypropylene cages under standard conditions of humidity (50
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139 ± 5%), temperature (25 ± 2°C) and light (12 h light/12 h dark cycle) and fed with a standard diet
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140 (Amrut laboratory animal feed, Pranav Agro Industries, India) and water ad libitum. All animals
141 were handled with humane care. Experimental protocols were reviewed and approved by the
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142 Institutional Animal Ethics Committee (Animal House Registration No. 25/1999/CPCSEA) and
143 conform to the Indian National Science Academy Guidelines for the Use and Care of
144 Experimental Animals in Research. S
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146 Preparation of test solutions
147 SOE was dissolved in distilled water and the fresh solution was used for administration.
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148 Fluoxetine was dissolved in distilled water and was administered intraperitoneal to the rats.
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150 Procedure
151 Animals, after acclimatization (6 –7 days) in the aniMmal quarters, were randomly divided into 4
152 groups of 6 animals each (Groups I to IV) and treated in the following way:
153 Group I (Normal control) - received drinking water (1 mL/kg, per oral) daily for 14 days, 20

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155 Group II (Reference standard) – received fluoxetine (6 mg/kg, intraperitoneal) daily for 14 days,
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157 Group III (SOE200) - received SOE (200 mg/kg, per oral) daily for 14 days, 20 minutes before
158 meal time. T
159 Group IV (SOE400) - received SOE (400 mg/kg, per oral) daily for 14 days, 20 minutes before
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161 Animals of all groups were given access to normal chow diet for 4h every day and drinking
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162 water ad libitum throughout the treatment period. Animals of the SOE and fluoxetine treatment
163 groups were administered SOE and fluoxetine 20 min prior to the feeding period. The effect of
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164 treatment was determined by monitoring changes in total food intake and body weight each day.
165 Blood was withdrawn on the 7th day and 14th day from the retro orbital plexus at different time
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166 points (0 min, 30 min, 60 min and 180 min) during the feeding period and a drop was used for
167 glucose measureAment. The remaining blood was allowed to clot for 30 min at room temperature
168 and serum was separated by centrifugation at 2500 rpm at 30˚C for 15 min and used for
169 estimation of CCK.
170 Determination of blood glucose levels
171 Blood glucose levels were measured using an automated digital glucometer (Accu-chek
172 Advantage Roche, USA) and expressed as mg/dL.
173 Determination of plasma cholecystokinin levels
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174 CCK levels were estimated using an ELISA kit supplied by Bioassay Technology Laboratory,
175 Korea. The assay is based on the biotin double antibody sandwich technology in which the
176 sample and standard solutions are reacted with CCK monoclonal antibodies. Chromogen
177 solutions A and B are added to form a blue color turning to yellow in presence of acid. The
178 absorbance of the yellow colored solution and the concentration of the rat CCK was positively
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180 Statistical analysis P
181 The results are expressed as mean ± SEM from 6 animals in each group. ResultIs were
182 statistically analyzed using one-way ANOVA, followed by the Tukey’s KramRer multiple
183 comparison test; P<0.05 was considered significant. Graphpad Prism version 5.00.288 of Graph
184 Pad Software Inc., San Diego, USA was the software used for statistical Canalysis.
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186 RESULTS
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187 HPTLC fingerprinting of SOE for flavonoids
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188 HPTLC showed clear separation of 14 flavonoids from SOE (Figure 2). Rf values of the
189 separated flavonoids were observed to be in the range oAf 0.03 to 0.93.
190 Figure2a. HPTLC fingerprint of SOE at 366 nm
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3 Extract 1.0
4 Extract 1.0
5 Rutin 2.0
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7 Extract 1.5
8 Extract 1.5
9 Extract 2.0
10 Extract 2.0
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193 Figure2b. HPTLC Chromatogram and Rf values of SOE at 366 nm
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198 Effect of SOE and fluoxetine on foTod intake and reduction in weight gain
199 The food intake of rats treated wPith SOE200, SOE400 and fluoxetine was significantly (p<0.001)
200 lower than that of the normal control rats indicating a distinct satiating effect of the treatments
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202 Rats treated with SOE40C0 and fluoxetine showed significant reduction in weight gain (p<0.05 for
203 SOE400 and p<0.01 for fluoxetine) when compared with rats of the normal control group at the
204 end of the study, refClective of a good satiating effect of SOE400 and fluoxetine (Table 1).
205 Table 1. Effect ofA SOE and fluoxetine on food intake and reduction in weight gain
Treatment Group Food intake (g) % Reduction in weight
gain

Normal control 9.204± 0.111 1.66 ± 1.17

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Fluoxetine 3.104± 0.298 (66.27) 11.32 ± 2.36**

(6mg/kg)
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SOE200 5.795± 0.279*** (37.03) 7.96 ± 1.94

SOE400 5.714± 0.249*** (37.92) 10.05 ± 1.76*

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207 All values are expressed as mean ± SEM; N = 6 in each group; Values in parenthesPis indicate %
208 reduction in food intake. One way ANOVA followed by Tukey-Kramer multipIle comparison
209 test is applied for statistical analysis *p< 0.05, **p< 0.01 and ***p< 0.001 wRhen experimental
210 groups compared with control group.
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211 Effect of SOE and fluoxetine on serum CCK
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212 The effect of SOE and fluoxetine on serum CCK levels at different time intervals (0 min, 30 min,
213 60 min and 180 min) after start of feeding is shown in Figure 3. UPeak levels of CCK were
214 reached in 30 min after start of feeding in fluoxetine treated animals and in 60 min in the
215 remaining animals on the 7th day of treatment. On the14th daNy, peaking of CCK levels was
216 observed in 30 min after start of feeding in the fluoxetine treated animals as well as the SOE400
217 animals. However, the SOE200 and normal control grouAp of animals exhibited peak CCK levels
218 only after 60 min of start of feeding.
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219 Figure3a. Effect of SOE and fluoxetine on serum CCK levels on day 7

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