- Email: [email protected]
Antidepressant activity and HPTLC fingerprinting of stearic acid in different days of wheat seedlings
Grain & Oil Science and Technology 2 (2019) 6–10
Contents lists available at ScienceDirect
Grain & Oil Science and Technology journal homepage: http://www.keaipublishing.com/en/journals/grain-and-oilscience-and-technology/
Antidepressant activity and HPTLC fingerprinting of stearic acid in different days of wheat seedlings Palanisamy Ravikumar, Muthusamy Jeyam
⁎
Biochematics Lab, Department of Bioinformatics, Bharathiar University, Coimbatore 641046, India
A R T I C L E
I N F O
Article history: Received 19 February 2019 Received in revised form 20 April 2019 Accepted 21 April 2019
Keywords: Depression Forced swimming test Tail suspension test Stearic acid HPTLC fingerprinting 3-d-old wheat seedlings
A B S T R A C T
Depression is a chronic, recurring and potentially life threatening illness and affects up to 20% of the world population and in the year 2020, depression will become the second most common disease in the world. To find the remedy from nutraceuticals, the present study was designed to evaluate the antidepressant activity of stearic acid (SA) and to quantify its maximum content in different dayold wheat seedlings and wheat grains. Forced swimming test (FST) and tail suspension test (TST) were done to evaluate the antidepressant activity of SA. HPTLC fingerprinting of SA was done in different days (3 , 5 and 7 d) of wheat seedlings and wheat grains to quantify its maximum content. In the antidepressant study, when compared with the control (326.67 ± 3.02 s), SA showed potential antidepressant activity in TST (131.67 ± 2.60 s) and SA also showed very good antidepressant activity in FST (124.83 ± 5.37 s) when compared with the control (215.83 ± 6.64 s). In HPTLC fingerprinting, the maximum content of SA was identified in 3-d-old wheat seedlings (89.43 μg) when compared to wheat grains (84.69 μg), 5-d-old (86.43 μg) and 7-d-old (85.32 μg) wheat seedlings. Hence, the present study concludes that SA has a potential antidepressant activity and 3-d-old wheat seedlings are the essential sources of SA among the different dayold wheat seedlings.
1. Introduction Depression is a chronic, recurring and potentially life threatening illness which affects up to 20% of the world population [1,2]. Depression is associated with poor health behaviors such as smoking, physical inactivity, caloric intake which also increase the risk of type 2 diabetes [3]. Mental depression affects a person's mood, behavior and general health. The report of WHO states that depression will become the second most common disease worldwide in the year 2020 [4]. In India, the occurrence of depression has increased from 49.93 cases per 1000 people to 73.97 cases per 1000 [5] and 50 million people had been affected by depression and anxiety [6]. Both male and female are affected by depression but the depression ratio for female is 50% higher than males, and for women, depression is the leading cause of diseases in high, low as well as middle income countries [7]. In the developing countries, poor growth in young children is due to maternal depression [8]. Further, the poor maternal mental health in low-income countries affects the growth of children and depression affects not only on the current generation but also the next. Stearic acid (Octadecanoic acid) is one of the major saturated fatty acid derived from animal and vegetable fats and oils. It is a unique sat-
⁎ Corresponding author. E-mail address: [email protected]. (M. Jeyam).
http://dx.doi.org/10.1016/j.gaost.2019.04.002 2590-2598/© 2019 Henan University of Technology. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
urated fatty acid among all the other fatty acids because it reduces plasma cholesterol concentration in the body [9–11]. Studies in rats revealed that dietary stearic acid would inhibit cholesterol absorption [12,13]. A review by Grundy [14] states that several human and animal studies have been conducted for confirming the “neutral” or hypocholesterolemic effect of dietary stearic acid. Schneider et al. [15] reported that the hamsters fed with high stearic acid (18:0) had a reduced concentration of plasma cholesterol which might be attributed to reduced cholesterol absorption and increased excretion of endogenous cholesterol. The report of Lipid research clinics program [16] also proposes that the reduction of 1% total plasma cholesterol may induce 2% reduction of coronary heart disease (CHD) in humans. Wheat (Triticum aestivum) belonging to the family of Poaceae is the most important cereal and food ingredient across the world and it is the second vital food grain in south India. Some wheat by-products exclusively exhibit functional and nutritional properties related to color and cooking performance [17]. Wheat bran extracts have been demonstrated to scavenge free radicals of DPPH, ABTS+ and peroxide anion (O− 2 ) [18] and to inhibit phospholipid liposomes and hydrogen peroxide [19] and also the peroxidation of human LDL cholesterol [20]. Hence, the present study has been designed to evaluate the antidepressant activity of stearic acid using forced swimming test, tail suspension test and quantification and variation of maximum contents of stearic acid in different days (3 , 5 , and 7 d) of old wheat seedlings and wheat grains using HPTLC analysis.
P. Ravikumar, et al.
Grain & Oil Science and Technology 2 (2019) 6–10
2. Materials and methods
was performed according to Steru et al. [23]. Antidepressant-like behavior was identified by the decreased immobility of the mice. Briefly, 30 min after drug administration, the animals were suspended by the tail from an edge with adhesive tape (approximately 1 cm from the tip of the tail). The distance between the tip of the nose of the mouse and the floor was approximately 50 cm. Animals were partitioned to avoid interference during the test. Immobility was defined as the absence of movement and was scored over a 6 min trial by an observer blinded to the drug treatment. Further, percentage (%) of reduction in immobility was calculated using the following formula.
2.1. Chemicals All the solvents (Petroleum ether, Hexane, Chloroform, Ethyl acetate and Methanol), Silica Gel (100–200 mesh size) and Stearic acid used for this study were purchased from Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India. 2.2. Collection of plant material
Percentage of reduction in immobility ð%Þ reduction in immobility time of sample 100% ¼ immobility time of control
Wheat (Triticum aestivum) seeds (Variety: HD2833 - Pusa Tripti) were procured from the Indian Agricultural Research Institute (IARI), Regional station, Wellington, Nilgiris, Tamil Nadu, India. Six hundred grams (600 g) of wheat seeds were taken separately as four sets and washed with distilled water, in which, one set was shade dried and then homogenized to fine powder. Remaining, three sets were soaked in water for 12 h, then the seeds were taken out and kept for 3 , 5 and 7 d germination, respectively, in a pure cloths in different trays. The seeds were monitored carefully by spraying water frequently until the completion of germination. After germination, all the seeds transformed into seedlings and those wheat seedlings were collected, shade dried and then homogenized to fine powder. These powders were stored in airtight bottles for further processes.
where, reduction in immobility time of sample = immobility time of control – immobility time of sample. 2.4.3. Forced swimming test (FST) Swiss albino male mice (25–30 g) were divided into 4 groups of 6 animals each (n = 6). In the test experiment, group I served as control group. Group II animals received the standard drug imipramine (15 mg/kg BW). Groups III and IV were orally administered with SA at concentrations of 25 mg/kg BW and 50 mg/kg BW, respectively. The test was performed according to the method described by Porsolt et al. [24]. Decreased immobility (improved mobility) of the mice was identified as the antidepressant-like behavior. Thirty minutes after the drug administration, each mouse was placed in an open cylindrical container (total volume of 1000 mL, 21 cm in height and 12 cm in diameter) filled with water up to 12 cm height (room temperature). A mouse was judged to be immobile when it remained floating in the water, making only the necessary movements to keep its head above water. Duration of immobility of the last 4 min of the total 6 min of swimming time was recorded. The water was changed after each mouse was tested. The animals were constantly watched to ensure that there was no contact between their paws and the base of the cylinder during FST [25]. Further, percentage (%) of reduction in immobility was calculated using the above mentioned formula.
2.3. Preparation of extract Two hundred grams of wheat grains, 3-d-old, 5-d-old, and 7-d-old wheat seedlings powders were taken and extracted separately with 5 organic solvents viz. petroleum ether, hexane, chloroform, ethyl acetate and methanol in the order of increasing polarities using Soxhlet apparatus, where the quantity of each solvent was 700 mL. The filtered extracts were then concentrated with the help of Rotavapor (Buchi-R210, Switzer land) and stored in airtight bottles. High contents of reactive oxygen species (ROS) and reactive nitrogen species (RNS) play major roles in causing depression. Antioxidants are useful to remove high content of ROS and RNS [21]. In addition, Ravikumar et al. [22] have reported a high antioxidant activity for the ethyl acetate extract of 3-d-old wheat seedlings. Based on that report, in the present study, ethyl acetate extract of wheat grains (WHGE), 3-d-old (3DE), 5-d-old (5DE) and 7-d-old (7DE) old wheat seedlings were subjected to HPTLC analysis.
2.5. Quantification study using HPTLC fingerprinting Stearic acid in different days of old wheat seedlings and wheat grains were quantified using HPTLC fingerprinting technique. To quantify SA, three different concentrations (5, 10 and 15 μL) of standard SA solution and samples (10 μL) from each ethyl acetate extract (5 mg/mL) of WHG, 3DE, 5DE and 7DE were loaded in 4 mm band length of 5 × 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG Linomat 5 instrument. The loaded plate was kept in TLC twin trough developing chamber (after saturated with solvent vapor) with respective mobile phase and the plate was developed in the respective mobile phase up to 90 mm out of 100 mm plate. The developed plate was dried to evaporate solvents from the plate. The plate was kept in the photo-documentation chamber (CAMAG Reprostar 3) and the images were captured at UV 254 nm and UV366 nm. The developed plate was sprayed with the respective spray reagent (iodine chamber). After derivatization, the plate was fixed in the scanner stage (CAMAG TLC SCANNER 3) and scanning was done at 500 nm. The peak table, peak display and peak densitogram were noted.
2.4. Antidepressant study Antidepressant activity of SA was evaluated using FST and TST. This study was approved by institutional animal ethical committee and the approval number is ML-EA-CPCSEA/09-2013/01. 2.4.1. Experimental animals Male Swiss albino mice (25–30 g) used in the present study were procured from the small animals breeding station, Mannuthy, Kerala, India. They were housed in polypropylene cages (38 cm × 23 cm × 10 cm) with not more than six animals per cage and maintained under standard environmental conditions (14 h dark/10 h light cycles; temp (25 ± 2)°C; 35%–60% humidity and air ventilation). The animals were fed with a standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai, India) and fresh water ad libitum. The animals were acclimatized to the environment for two weeks prior to experimental use. Animals were fasted over night before the experimental schedule but had access to water ad libitum.
3. Results
2.4.2. Tail suspension test (TST) Swiss albino male mice (25–30 g) were divided into 4 groups of 6 animals each (n = 6). In the test experiment, group I served as the control group. Groups II animals received the standard drug imipramine (15 mg/kg BW). Groups III and IV were orally administered with SA at concentrations of 25 mg/kg BW and 50 mg/kg BW, respectively. TST
3.1. Antidepressant study Antidepressant activity of SA was evaluated using TST and FST and the results are shown in Table 1 and 2. TST results showed that the immobility time of standard drug imipramine was 58.33 ± 3.94 s, immobility time for treatment with the high dose of SA was 131.67 ± 2.60 s 7
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Grain & Oil Science and Technology 2 (2019) 6–10
and wheat grains (Fig. 1). Rf value was 0.55 and this value was almost similar for wheat grains and all the different days of wheat seedlings. 3D display of SA for all the sample tracks, peak densitogram display of SA in wheat grains, peak densitogram display of SA in 3-d-old, 5-d-old, and 7-d-old wheat seedlings and peak densitogram display of SA (standard) are shown in Fig. 2 and 3. Each of all the peaks exhibited the maximum contents of stearic acid in the ethyl acetate extracts of wheat grains and wheat seedlings. The area and quantity of SA in all the samples are also given in Table 3.
when compared to 326.67 ± 3.02 s for control which confirmed the potential antidepressant activity of SA. FST results showed that the immobility time of standard drug imipramine was 81.67 ± 5.43 s, immobility time for treatment with the high dose of SA was 124.83 ± 5.37 s when compared to 215.83 ± 6.64 s for control which confirmed good antidepressant activity of SA. Further, when compared to high doses, immobility time was low for low doses of SA in both the tests. 3.2. Quantification study using HPTLC fingerprinting Stearic acid in the ethyl acetate extracts of wheat grains and different days (3 , 5 , and 7 d) old wheat seedlings were quantified using HPTLC finger printing technique. n-hexane: ethyl acetate: methanol (2.5:0.5:5 drops, V/V/V) was used as the mobile phase. While performing analysis, white colored bands were observed under UV at 366 nm in the chromatogram after derivatization, which confirmed the presence of stearic acid in the ethyl acetate extracts of different day old wheat seedlings
4. Discussion The report of Killen et al. [26] says that stearic acid is used in the production of pharmaceutical products. In addition, because of its inert, inexpensive, biocompatible and less toxic nature, it is used in the development of drug delivery systems. Jubie et al. [27] have reported the antidepressant and antimicrobial activities of some novel stearic acid analogues. Previously, it was reported that the stearic acid derivative from Stemodia foliosa had antibacterial activities against the gram-positive bacteria such as, B. cereus and B. subtilis [28]. Besides, in the present study, the antidepressant activity of stearic acid was evaluated. TST and FST results showed that, immobility time for treatment with the high dose of SA was 131.67 ± 2.60 s and 124.83 ± 5.37 s when compared to 326.67 ± 3.02 s and 215.83 ± 6.64 s for control respectively, which confirmed the potential antidepressant activity of SA. Further, when compared to high doses, immobility time was low for low doses of SA in both the tests. Hence, the present study concludes that SA has a potential antidepressant activity. Several fatty acids are used to reduce the growth of unnecessary microorganisms in food additives [29] and in the treatment of neuropsychological disorders such as depression and schizophrenia [30]. Due to lower melting point and potential oxidative stability of stearic acid, the researchers have focused to work on stearic acid [31]. Solubility of stearic acid in different organic solvents has been reported by Heryanto et al. [32]. In addition, in the present study, for the first time, the quantity of stearic acid in different day old wheat seedlings has been reported. From the HPTLC analysis results, the maximum content of SA was identified in 3-d-old wheat seedlings (89.43 μg) when compared to wheat grains (84.69 μg), 5-d-old (86.43 μg), and 7-d-old (85.32 μg) wheat seedlings. Previously, Ravikumar et al. [22,33] reported only the antioxidant activity of wheat grains and different day old wheat seedlings. Previous studies on the phytochemical profiles of wheat varieties were mostly about phenolic contents [34–38] and the present study has focused on stearic acid and it is concluded that
Table 1 Antidepressant activity of stearic acid (SA) using TST. Group
Dose (mg/kg P.O)
Immobility timea (s)
Reduction in immobility (%)
Control Standard-imipramine SA (low dose) SA (high dose)
– 15 25 50
326.67 ± 3.02 58.33 ± 3.94 183.50 ± 6.73 131.67 ± 2.60
– 82.14 43.83 59.69
Note: a The values are expressed as mean ± S.E.M. (n = 6).
Table 2 Antidepressant activity of (SA) using FST. Group
Dose (mg/kg P.O)
Immobility timea (s)
Reduction in immobility (%)
Control Standard-imipramine SA (low dose) SA (high dose)
– 15 25 50
215.83 ± 6.64 81.67 ± 5.43 165.67 ± 6.37 124.83 ± 5.37
– 62.16 23.24 42.16
Note: a The values are expressed as mean ± S.E.M. (n = 6).
Fig. 2. 3D display of stearic acid in all the samples track @ 366 nm. The color peaks respective to wheat grains (black), 3-d-old (pink), 5-d-old (violet), and 7-d-old (blue) wheat seedlings and stearic acid (green).
Fig. 1. HPTLC of stearic acid in wheat grains, 3-d-old, 5-d-old, and 7-d-old wheat seedlings. 8
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Grain & Oil Science and Technology 2 (2019) 6–10
Fig. 3. Peak densitogram display of SA. (a): wheat grains(WHGE); (b): 3DE; (c): 5DE; (d): 7DE; (e): standard. 9
P. Ravikumar, et al.
Grain & Oil Science and Technology 2 (2019) 6–10 [12] E.B. Feldman, B.S. Russell, F.H. Schnare, B.C. Miles, E.A. Doyle, I. Moretti-Rojas, Effects of tristearin, triolein and safflower oil diets on cholesterol balance in rats, J Nutr 109 (1979) 2226–2236. [13] E.B. Feldman, B.S. Russell, F.H. Schnare, I. Moretti-Rojas, B.C. Miles, E.A. Doyle, Effects of diets of homogeneous saturated triglycerides on cholesterol balance in rats, J Nutr 109 (1979) 2237–2246. [14] S.M. Grundy, Influence of stearic acid on cholesterol metabolism relative to other longchain fatty acids, Am J Clin Nutr 60 (1994) 986S–990S. [15] C.L. Schneider, R.L. Cowles, C.L. Stuefer-Powell, T.P. Carr, Dietary stearic acid reduces cholesterol absorption and increases endogenous cholesterol excretion in hamsters fed cereal-based diets, J Nutr 130 (2000) 1232–1238. [16] Research Clinics Program, Lipid. The LRC-CPPT results: II, The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J Am Med Assoc 251 (1984) 365–374. [17] J.E. Dexter, D.G. Martin, G.T. Sadaranganey, J. Michaelides, N. Mathieson, J.J. Tkac, et al., Preprocessing: effects on durum wheat milling and spaghetti-making quality, Cereal Chem 71 (1994) 10–15. [18] K. Zhou, L. Su, L. Yu, Phytochemicals and antioxidant properties in wheat bran, J Agric Food Chem 52 (2004) 6108–6114. [19] M. Martínez-Tomé, M.A. Murcia, N. Frega, S. Ruggieri, A.M. Jiménez, F. Roses, et al., Evaluation of antioxidant capacity of cereal brans, J Agric Food Chem 52 (2004) 4690–4699. [20] L.L. Yu, K. Zhou, J.W. Parry, Inhibitory effects of wheat bran extracts on human LDL oxidation and free radicals, LWT Food Sci Technol 38 (2005) 463–470. [21] M. Maes, P. Galecki, Y.S. Chang, M. Berk, A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro) degenerative processes in that illness, Prog Neuropsychopharmacol Biol Psychiatry 35 (2011) 676–692. [22] P. Ravikumar, G. Shalini, M. Jeyam, Wheat seedlings as food supplement to combat free radicals: an in vitro approach, Indian J Pharm Sci 77 (2015) 592–598. [23] L. Steru, R. Chermat, B. Thierry, P. Simon, The tail suspension test: a new method for screening antidepressants in mice, Psychopharmacol 85 (1985) 367–370. [24] R.D. Porsolt, M.L. Pichon, M. Jalfre, Depression: a new animal model sensitive to antidepressant treatments, Nature 266 (1977) 730–732. [25] I. Lucki, A. Dalvi, A.J. Mayorga, Sensitivity to the effects of pharmacologically selective antidepressants in different strains of mice, Psychopharmacol 155 (2001) 315–322. [26] B.U. Killen, O.I. Corrigan, Factors influencing drug release from stearic acid based compacts, Int J Pharm 228 (2001) 189–198. [27] S. Jubie, P.N. Ramesh, P. Dhanabal, R. Kalirajan, N. Muruganantham, A.S. Antony, Synthesis, antidepressant and antimicrobial activities of some novel stearic acid analogues, Eur J Med Chem 54 (2012) 931–935. [28] L.L.D. Silva, M. Nascimento, D.H.S. Silva, M. Furlan, V.S. Bolzani, Antibacterial activity of a stearic acid derivative from Stemodia foliosa, Planta Med 68 (2002) 1137–1139. [29] C.J. Zheng, J.S. Yoo, T.G. Lee, H.Y. Cho, Y.H. Kim, W.G. Kim, Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids, FEBS Lett 579 (2005) 5157–5162. [30] O. Yamima, R.H. Belmaker, Omega-3 fatty acids in depression: a review of three studies, CNS Neurosci Ther 15 (2009) 128–133. [31] L.N. Helen, O.D. Robert, Synthesis and physical properties of isostearic acids and their esters, Eur J Lipid Sci Technol 113 (2011) 180–188. [32] R. Heryanto, M. Hasan, E.C. Abdullah, A.C. Kumoro, Solubility of stearic acid in various organic solvents and its prediction using non ideal solution, Sci Asia 33 (2007) 469–472. [33] P. Ravikumar, G. Shalini, M. Jeyam, Phytochemical analysis and antioxidant activity of wheat grains and seedlings, J Pharm Sci Innov 3 (2014) 319–326. [34] E. Labat, M.H. Morel, X. Rouau, Wheat gluten phenolic acids: occurrence and fate upon mixing, J Agric Food Chem 48 (2000) 6280–6283. [35] H. Zieliński, H. Kozłowska, Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions, J Agric Food Chem 48 (2000) 2008–2016. [36] E.S. Abdel-Aal, P. Hucl, F.W. Sosulski, R. Graf, C. Gillott, L. Pietrzak, Screening spring wheat for midge resistance in relation to ferulic acid content, J Agric Food Chem 49 (2001) 3559–3566. [37] L. Yu, S. Haley, J. Perret, M. Harris, J. Wilson, M. Qian, Free radical scavenging properties of wheat extracts, J Agric Food Chem 50 (2002) 1619–1624. [38] K.K. Adom, R.H. Liu, Antioxidant activity of grains, J Agric Food Chem 50 (2002) 6182–6187.
Table 3 Quantification report of stearic acid (SA). Samples
Sample code
Rf
Area
Wheat grains 3-d-old wheat seedlings 5-d-old wheat seedlings 7-d-old wheat seedlings SA (standard)
WHGE 3DE 5DE 7DE SA
0.57 0.56 0.55 0.54 0.55
3595.06 3796.36 3668.74 3621.89 2122.48
Quantity (μg) 84.69 89.43 86.43 85.32 50.00
wheat seedlings are also the major sources for fatty acids, mainly stearic acid. 5. Conclusions The present study concludes that the high content of SA is present in 3d-old wheat seedlings when compared to wheat grains, 5-d-old, and 7-d-old wheat seedlings. In addition, this study concludes that SA has a potential antidepressant activity. Thus, according to the current study results and previous reports, the present study proposes that regular consumption of 3-d-old wheat seedlings may reduce the risk of depression as well as heart diseases in humans. Declaration of Competing Interest The authors don't have any conflict of interest to publish this paper in Grain & Oil Science and Technology. Acknowledgement The authors are very grateful to PSG College of Pharmacy, Coimbatore, Tamil Nadu, India for providing HPTLC facility. References [1] E.J. Nestler, M. Barrot, R.J. Dileone, A.J. Eisch, S.J. Gold, L.M. Monteggia, Neurobiology of depression, Neuron 34 (2002) 13–25. [2] C.F. Gillespie, C.B. Nemeroff, Hypercortisolemia and depression, Psychosom Med 67 (2005) S26–S28. [3] T.W. Strine, A.H. Mokdad, S.R. Dube, L.S. Balluz, O. Gonzalez, J.T. Berry, et al., The association of depression and anxiety with obesity and unhealthy behaviors among community-dwelling US adults, Gen Hosp Psychiatry 30 (2008) 127–137. [4] J.H. Kim, S.Y. Kim, S.Y. Lee, C.G. Jang, Antidepressant-like effects of Albizzia julibrissin in mice: involvement of the 5-HT1A receptor system, Pharmacol Biochem Behav 87 (2007) 41–47. [5] D.N. Nandi, G. Banerjee, S.P. Mukherjee, A. Ghosh, P.S. Nandi, S. Nandi, Psychiatric morbidity of a rural Indian community changes over a 20 year interval, Br J Psychiatry 176 (2000) 351–356. [6] S. Rao, N. Ramesh, Depression, anxiety and stress levels in industrial workers: a pilot study in Bangalore, India, Ind Psychiatry J 24 (2015) 23–28. [7] A.J. Baxter, T. Vos, K.M. Scott, A.J. Ferrari, H.A. Whiteford, The global burden of anxiety disorders in 2010, Psychol Med 44 (2014) 2363–2374. [8] A. Rahman, V. Patel, J. Maselko, B. Kirkwood, The neglected ‘m’ in MCH programmes – why mental health of mothers is important for child nutrition, Trop Med Int Health 13 (2008) 579–583. [9] E.H. Ahrens, W. Insull, R. Blomstrand, J. Hirsch, T.T. Tsaltas, M.L. Peterson, The influence of dietary fats on serum-lipid levels in man, Lancet 1 (1957) 19–57. [10] A. Keys, J.T. Anderson, F. Grande, Serum cholesterol response to changes in the diet: IV. particular saturated fatty acids in the diet, Metabolism 14 (1965) 776–787. [11] D.M. Hegsted, R.B. McGandy, M.L. Myers, F.J. Stare, Quantitative effects of dietary fat on serum cholesterol in man, Am J Clin Nutr 17 (1965) 281–295.
10
Contents lists available at ScienceDirect
Grain & Oil Science and Technology journal homepage: http://www.keaipublishing.com/en/journals/grain-and-oilscience-and-technology/
Antidepressant activity and HPTLC fingerprinting of stearic acid in different days of wheat seedlings Palanisamy Ravikumar, Muthusamy Jeyam
⁎
Biochematics Lab, Department of Bioinformatics, Bharathiar University, Coimbatore 641046, India
A R T I C L E
I N F O
Article history: Received 19 February 2019 Received in revised form 20 April 2019 Accepted 21 April 2019
Keywords: Depression Forced swimming test Tail suspension test Stearic acid HPTLC fingerprinting 3-d-old wheat seedlings
A B S T R A C T
Depression is a chronic, recurring and potentially life threatening illness and affects up to 20% of the world population and in the year 2020, depression will become the second most common disease in the world. To find the remedy from nutraceuticals, the present study was designed to evaluate the antidepressant activity of stearic acid (SA) and to quantify its maximum content in different dayold wheat seedlings and wheat grains. Forced swimming test (FST) and tail suspension test (TST) were done to evaluate the antidepressant activity of SA. HPTLC fingerprinting of SA was done in different days (3 , 5 and 7 d) of wheat seedlings and wheat grains to quantify its maximum content. In the antidepressant study, when compared with the control (326.67 ± 3.02 s), SA showed potential antidepressant activity in TST (131.67 ± 2.60 s) and SA also showed very good antidepressant activity in FST (124.83 ± 5.37 s) when compared with the control (215.83 ± 6.64 s). In HPTLC fingerprinting, the maximum content of SA was identified in 3-d-old wheat seedlings (89.43 μg) when compared to wheat grains (84.69 μg), 5-d-old (86.43 μg) and 7-d-old (85.32 μg) wheat seedlings. Hence, the present study concludes that SA has a potential antidepressant activity and 3-d-old wheat seedlings are the essential sources of SA among the different dayold wheat seedlings.
1. Introduction Depression is a chronic, recurring and potentially life threatening illness which affects up to 20% of the world population [1,2]. Depression is associated with poor health behaviors such as smoking, physical inactivity, caloric intake which also increase the risk of type 2 diabetes [3]. Mental depression affects a person's mood, behavior and general health. The report of WHO states that depression will become the second most common disease worldwide in the year 2020 [4]. In India, the occurrence of depression has increased from 49.93 cases per 1000 people to 73.97 cases per 1000 [5] and 50 million people had been affected by depression and anxiety [6]. Both male and female are affected by depression but the depression ratio for female is 50% higher than males, and for women, depression is the leading cause of diseases in high, low as well as middle income countries [7]. In the developing countries, poor growth in young children is due to maternal depression [8]. Further, the poor maternal mental health in low-income countries affects the growth of children and depression affects not only on the current generation but also the next. Stearic acid (Octadecanoic acid) is one of the major saturated fatty acid derived from animal and vegetable fats and oils. It is a unique sat-
⁎ Corresponding author. E-mail address: [email protected]. (M. Jeyam).
http://dx.doi.org/10.1016/j.gaost.2019.04.002 2590-2598/© 2019 Henan University of Technology. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
urated fatty acid among all the other fatty acids because it reduces plasma cholesterol concentration in the body [9–11]. Studies in rats revealed that dietary stearic acid would inhibit cholesterol absorption [12,13]. A review by Grundy [14] states that several human and animal studies have been conducted for confirming the “neutral” or hypocholesterolemic effect of dietary stearic acid. Schneider et al. [15] reported that the hamsters fed with high stearic acid (18:0) had a reduced concentration of plasma cholesterol which might be attributed to reduced cholesterol absorption and increased excretion of endogenous cholesterol. The report of Lipid research clinics program [16] also proposes that the reduction of 1% total plasma cholesterol may induce 2% reduction of coronary heart disease (CHD) in humans. Wheat (Triticum aestivum) belonging to the family of Poaceae is the most important cereal and food ingredient across the world and it is the second vital food grain in south India. Some wheat by-products exclusively exhibit functional and nutritional properties related to color and cooking performance [17]. Wheat bran extracts have been demonstrated to scavenge free radicals of DPPH, ABTS+ and peroxide anion (O− 2 ) [18] and to inhibit phospholipid liposomes and hydrogen peroxide [19] and also the peroxidation of human LDL cholesterol [20]. Hence, the present study has been designed to evaluate the antidepressant activity of stearic acid using forced swimming test, tail suspension test and quantification and variation of maximum contents of stearic acid in different days (3 , 5 , and 7 d) of old wheat seedlings and wheat grains using HPTLC analysis.
P. Ravikumar, et al.
Grain & Oil Science and Technology 2 (2019) 6–10
2. Materials and methods
was performed according to Steru et al. [23]. Antidepressant-like behavior was identified by the decreased immobility of the mice. Briefly, 30 min after drug administration, the animals were suspended by the tail from an edge with adhesive tape (approximately 1 cm from the tip of the tail). The distance between the tip of the nose of the mouse and the floor was approximately 50 cm. Animals were partitioned to avoid interference during the test. Immobility was defined as the absence of movement and was scored over a 6 min trial by an observer blinded to the drug treatment. Further, percentage (%) of reduction in immobility was calculated using the following formula.
2.1. Chemicals All the solvents (Petroleum ether, Hexane, Chloroform, Ethyl acetate and Methanol), Silica Gel (100–200 mesh size) and Stearic acid used for this study were purchased from Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India. 2.2. Collection of plant material
Percentage of reduction in immobility ð%Þ reduction in immobility time of sample 100% ¼ immobility time of control
Wheat (Triticum aestivum) seeds (Variety: HD2833 - Pusa Tripti) were procured from the Indian Agricultural Research Institute (IARI), Regional station, Wellington, Nilgiris, Tamil Nadu, India. Six hundred grams (600 g) of wheat seeds were taken separately as four sets and washed with distilled water, in which, one set was shade dried and then homogenized to fine powder. Remaining, three sets were soaked in water for 12 h, then the seeds were taken out and kept for 3 , 5 and 7 d germination, respectively, in a pure cloths in different trays. The seeds were monitored carefully by spraying water frequently until the completion of germination. After germination, all the seeds transformed into seedlings and those wheat seedlings were collected, shade dried and then homogenized to fine powder. These powders were stored in airtight bottles for further processes.
where, reduction in immobility time of sample = immobility time of control – immobility time of sample. 2.4.3. Forced swimming test (FST) Swiss albino male mice (25–30 g) were divided into 4 groups of 6 animals each (n = 6). In the test experiment, group I served as control group. Group II animals received the standard drug imipramine (15 mg/kg BW). Groups III and IV were orally administered with SA at concentrations of 25 mg/kg BW and 50 mg/kg BW, respectively. The test was performed according to the method described by Porsolt et al. [24]. Decreased immobility (improved mobility) of the mice was identified as the antidepressant-like behavior. Thirty minutes after the drug administration, each mouse was placed in an open cylindrical container (total volume of 1000 mL, 21 cm in height and 12 cm in diameter) filled with water up to 12 cm height (room temperature). A mouse was judged to be immobile when it remained floating in the water, making only the necessary movements to keep its head above water. Duration of immobility of the last 4 min of the total 6 min of swimming time was recorded. The water was changed after each mouse was tested. The animals were constantly watched to ensure that there was no contact between their paws and the base of the cylinder during FST [25]. Further, percentage (%) of reduction in immobility was calculated using the above mentioned formula.
2.3. Preparation of extract Two hundred grams of wheat grains, 3-d-old, 5-d-old, and 7-d-old wheat seedlings powders were taken and extracted separately with 5 organic solvents viz. petroleum ether, hexane, chloroform, ethyl acetate and methanol in the order of increasing polarities using Soxhlet apparatus, where the quantity of each solvent was 700 mL. The filtered extracts were then concentrated with the help of Rotavapor (Buchi-R210, Switzer land) and stored in airtight bottles. High contents of reactive oxygen species (ROS) and reactive nitrogen species (RNS) play major roles in causing depression. Antioxidants are useful to remove high content of ROS and RNS [21]. In addition, Ravikumar et al. [22] have reported a high antioxidant activity for the ethyl acetate extract of 3-d-old wheat seedlings. Based on that report, in the present study, ethyl acetate extract of wheat grains (WHGE), 3-d-old (3DE), 5-d-old (5DE) and 7-d-old (7DE) old wheat seedlings were subjected to HPTLC analysis.
2.5. Quantification study using HPTLC fingerprinting Stearic acid in different days of old wheat seedlings and wheat grains were quantified using HPTLC fingerprinting technique. To quantify SA, three different concentrations (5, 10 and 15 μL) of standard SA solution and samples (10 μL) from each ethyl acetate extract (5 mg/mL) of WHG, 3DE, 5DE and 7DE were loaded in 4 mm band length of 5 × 10 Silica gel 60F254 TLC plate using Hamilton syringe and CAMAG Linomat 5 instrument. The loaded plate was kept in TLC twin trough developing chamber (after saturated with solvent vapor) with respective mobile phase and the plate was developed in the respective mobile phase up to 90 mm out of 100 mm plate. The developed plate was dried to evaporate solvents from the plate. The plate was kept in the photo-documentation chamber (CAMAG Reprostar 3) and the images were captured at UV 254 nm and UV366 nm. The developed plate was sprayed with the respective spray reagent (iodine chamber). After derivatization, the plate was fixed in the scanner stage (CAMAG TLC SCANNER 3) and scanning was done at 500 nm. The peak table, peak display and peak densitogram were noted.
2.4. Antidepressant study Antidepressant activity of SA was evaluated using FST and TST. This study was approved by institutional animal ethical committee and the approval number is ML-EA-CPCSEA/09-2013/01. 2.4.1. Experimental animals Male Swiss albino mice (25–30 g) used in the present study were procured from the small animals breeding station, Mannuthy, Kerala, India. They were housed in polypropylene cages (38 cm × 23 cm × 10 cm) with not more than six animals per cage and maintained under standard environmental conditions (14 h dark/10 h light cycles; temp (25 ± 2)°C; 35%–60% humidity and air ventilation). The animals were fed with a standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai, India) and fresh water ad libitum. The animals were acclimatized to the environment for two weeks prior to experimental use. Animals were fasted over night before the experimental schedule but had access to water ad libitum.
3. Results
2.4.2. Tail suspension test (TST) Swiss albino male mice (25–30 g) were divided into 4 groups of 6 animals each (n = 6). In the test experiment, group I served as the control group. Groups II animals received the standard drug imipramine (15 mg/kg BW). Groups III and IV were orally administered with SA at concentrations of 25 mg/kg BW and 50 mg/kg BW, respectively. TST
3.1. Antidepressant study Antidepressant activity of SA was evaluated using TST and FST and the results are shown in Table 1 and 2. TST results showed that the immobility time of standard drug imipramine was 58.33 ± 3.94 s, immobility time for treatment with the high dose of SA was 131.67 ± 2.60 s 7
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and wheat grains (Fig. 1). Rf value was 0.55 and this value was almost similar for wheat grains and all the different days of wheat seedlings. 3D display of SA for all the sample tracks, peak densitogram display of SA in wheat grains, peak densitogram display of SA in 3-d-old, 5-d-old, and 7-d-old wheat seedlings and peak densitogram display of SA (standard) are shown in Fig. 2 and 3. Each of all the peaks exhibited the maximum contents of stearic acid in the ethyl acetate extracts of wheat grains and wheat seedlings. The area and quantity of SA in all the samples are also given in Table 3.
when compared to 326.67 ± 3.02 s for control which confirmed the potential antidepressant activity of SA. FST results showed that the immobility time of standard drug imipramine was 81.67 ± 5.43 s, immobility time for treatment with the high dose of SA was 124.83 ± 5.37 s when compared to 215.83 ± 6.64 s for control which confirmed good antidepressant activity of SA. Further, when compared to high doses, immobility time was low for low doses of SA in both the tests. 3.2. Quantification study using HPTLC fingerprinting Stearic acid in the ethyl acetate extracts of wheat grains and different days (3 , 5 , and 7 d) old wheat seedlings were quantified using HPTLC finger printing technique. n-hexane: ethyl acetate: methanol (2.5:0.5:5 drops, V/V/V) was used as the mobile phase. While performing analysis, white colored bands were observed under UV at 366 nm in the chromatogram after derivatization, which confirmed the presence of stearic acid in the ethyl acetate extracts of different day old wheat seedlings
4. Discussion The report of Killen et al. [26] says that stearic acid is used in the production of pharmaceutical products. In addition, because of its inert, inexpensive, biocompatible and less toxic nature, it is used in the development of drug delivery systems. Jubie et al. [27] have reported the antidepressant and antimicrobial activities of some novel stearic acid analogues. Previously, it was reported that the stearic acid derivative from Stemodia foliosa had antibacterial activities against the gram-positive bacteria such as, B. cereus and B. subtilis [28]. Besides, in the present study, the antidepressant activity of stearic acid was evaluated. TST and FST results showed that, immobility time for treatment with the high dose of SA was 131.67 ± 2.60 s and 124.83 ± 5.37 s when compared to 326.67 ± 3.02 s and 215.83 ± 6.64 s for control respectively, which confirmed the potential antidepressant activity of SA. Further, when compared to high doses, immobility time was low for low doses of SA in both the tests. Hence, the present study concludes that SA has a potential antidepressant activity. Several fatty acids are used to reduce the growth of unnecessary microorganisms in food additives [29] and in the treatment of neuropsychological disorders such as depression and schizophrenia [30]. Due to lower melting point and potential oxidative stability of stearic acid, the researchers have focused to work on stearic acid [31]. Solubility of stearic acid in different organic solvents has been reported by Heryanto et al. [32]. In addition, in the present study, for the first time, the quantity of stearic acid in different day old wheat seedlings has been reported. From the HPTLC analysis results, the maximum content of SA was identified in 3-d-old wheat seedlings (89.43 μg) when compared to wheat grains (84.69 μg), 5-d-old (86.43 μg), and 7-d-old (85.32 μg) wheat seedlings. Previously, Ravikumar et al. [22,33] reported only the antioxidant activity of wheat grains and different day old wheat seedlings. Previous studies on the phytochemical profiles of wheat varieties were mostly about phenolic contents [34–38] and the present study has focused on stearic acid and it is concluded that
Table 1 Antidepressant activity of stearic acid (SA) using TST. Group
Dose (mg/kg P.O)
Immobility timea (s)
Reduction in immobility (%)
Control Standard-imipramine SA (low dose) SA (high dose)
– 15 25 50
326.67 ± 3.02 58.33 ± 3.94 183.50 ± 6.73 131.67 ± 2.60
– 82.14 43.83 59.69
Note: a The values are expressed as mean ± S.E.M. (n = 6).
Table 2 Antidepressant activity of (SA) using FST. Group
Dose (mg/kg P.O)
Immobility timea (s)
Reduction in immobility (%)
Control Standard-imipramine SA (low dose) SA (high dose)
– 15 25 50
215.83 ± 6.64 81.67 ± 5.43 165.67 ± 6.37 124.83 ± 5.37
– 62.16 23.24 42.16
Note: a The values are expressed as mean ± S.E.M. (n = 6).
Fig. 2. 3D display of stearic acid in all the samples track @ 366 nm. The color peaks respective to wheat grains (black), 3-d-old (pink), 5-d-old (violet), and 7-d-old (blue) wheat seedlings and stearic acid (green).
Fig. 1. HPTLC of stearic acid in wheat grains, 3-d-old, 5-d-old, and 7-d-old wheat seedlings. 8
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Fig. 3. Peak densitogram display of SA. (a): wheat grains(WHGE); (b): 3DE; (c): 5DE; (d): 7DE; (e): standard. 9
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Grain & Oil Science and Technology 2 (2019) 6–10 [12] E.B. Feldman, B.S. Russell, F.H. Schnare, B.C. Miles, E.A. Doyle, I. Moretti-Rojas, Effects of tristearin, triolein and safflower oil diets on cholesterol balance in rats, J Nutr 109 (1979) 2226–2236. [13] E.B. Feldman, B.S. Russell, F.H. Schnare, I. Moretti-Rojas, B.C. Miles, E.A. Doyle, Effects of diets of homogeneous saturated triglycerides on cholesterol balance in rats, J Nutr 109 (1979) 2237–2246. [14] S.M. Grundy, Influence of stearic acid on cholesterol metabolism relative to other longchain fatty acids, Am J Clin Nutr 60 (1994) 986S–990S. [15] C.L. Schneider, R.L. Cowles, C.L. Stuefer-Powell, T.P. Carr, Dietary stearic acid reduces cholesterol absorption and increases endogenous cholesterol excretion in hamsters fed cereal-based diets, J Nutr 130 (2000) 1232–1238. [16] Research Clinics Program, Lipid. The LRC-CPPT results: II, The relationship of reduction in incidence of coronary heart disease to cholesterol lowering, J Am Med Assoc 251 (1984) 365–374. [17] J.E. Dexter, D.G. Martin, G.T. Sadaranganey, J. Michaelides, N. Mathieson, J.J. Tkac, et al., Preprocessing: effects on durum wheat milling and spaghetti-making quality, Cereal Chem 71 (1994) 10–15. [18] K. Zhou, L. Su, L. Yu, Phytochemicals and antioxidant properties in wheat bran, J Agric Food Chem 52 (2004) 6108–6114. [19] M. Martínez-Tomé, M.A. Murcia, N. Frega, S. Ruggieri, A.M. Jiménez, F. Roses, et al., Evaluation of antioxidant capacity of cereal brans, J Agric Food Chem 52 (2004) 4690–4699. [20] L.L. Yu, K. Zhou, J.W. Parry, Inhibitory effects of wheat bran extracts on human LDL oxidation and free radicals, LWT Food Sci Technol 38 (2005) 463–470. [21] M. Maes, P. Galecki, Y.S. Chang, M. Berk, A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro) degenerative processes in that illness, Prog Neuropsychopharmacol Biol Psychiatry 35 (2011) 676–692. [22] P. Ravikumar, G. Shalini, M. Jeyam, Wheat seedlings as food supplement to combat free radicals: an in vitro approach, Indian J Pharm Sci 77 (2015) 592–598. [23] L. Steru, R. Chermat, B. Thierry, P. Simon, The tail suspension test: a new method for screening antidepressants in mice, Psychopharmacol 85 (1985) 367–370. [24] R.D. Porsolt, M.L. Pichon, M. Jalfre, Depression: a new animal model sensitive to antidepressant treatments, Nature 266 (1977) 730–732. [25] I. Lucki, A. Dalvi, A.J. Mayorga, Sensitivity to the effects of pharmacologically selective antidepressants in different strains of mice, Psychopharmacol 155 (2001) 315–322. [26] B.U. Killen, O.I. Corrigan, Factors influencing drug release from stearic acid based compacts, Int J Pharm 228 (2001) 189–198. [27] S. Jubie, P.N. Ramesh, P. Dhanabal, R. Kalirajan, N. Muruganantham, A.S. Antony, Synthesis, antidepressant and antimicrobial activities of some novel stearic acid analogues, Eur J Med Chem 54 (2012) 931–935. [28] L.L.D. Silva, M. Nascimento, D.H.S. Silva, M. Furlan, V.S. Bolzani, Antibacterial activity of a stearic acid derivative from Stemodia foliosa, Planta Med 68 (2002) 1137–1139. [29] C.J. Zheng, J.S. Yoo, T.G. Lee, H.Y. Cho, Y.H. Kim, W.G. Kim, Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids, FEBS Lett 579 (2005) 5157–5162. [30] O. Yamima, R.H. Belmaker, Omega-3 fatty acids in depression: a review of three studies, CNS Neurosci Ther 15 (2009) 128–133. [31] L.N. Helen, O.D. Robert, Synthesis and physical properties of isostearic acids and their esters, Eur J Lipid Sci Technol 113 (2011) 180–188. [32] R. Heryanto, M. Hasan, E.C. Abdullah, A.C. Kumoro, Solubility of stearic acid in various organic solvents and its prediction using non ideal solution, Sci Asia 33 (2007) 469–472. [33] P. Ravikumar, G. Shalini, M. Jeyam, Phytochemical analysis and antioxidant activity of wheat grains and seedlings, J Pharm Sci Innov 3 (2014) 319–326. [34] E. Labat, M.H. Morel, X. Rouau, Wheat gluten phenolic acids: occurrence and fate upon mixing, J Agric Food Chem 48 (2000) 6280–6283. [35] H. Zieliński, H. Kozłowska, Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions, J Agric Food Chem 48 (2000) 2008–2016. [36] E.S. Abdel-Aal, P. Hucl, F.W. Sosulski, R. Graf, C. Gillott, L. Pietrzak, Screening spring wheat for midge resistance in relation to ferulic acid content, J Agric Food Chem 49 (2001) 3559–3566. [37] L. Yu, S. Haley, J. Perret, M. Harris, J. Wilson, M. Qian, Free radical scavenging properties of wheat extracts, J Agric Food Chem 50 (2002) 1619–1624. [38] K.K. Adom, R.H. Liu, Antioxidant activity of grains, J Agric Food Chem 50 (2002) 6182–6187.
Table 3 Quantification report of stearic acid (SA). Samples
Sample code
Rf
Area
Wheat grains 3-d-old wheat seedlings 5-d-old wheat seedlings 7-d-old wheat seedlings SA (standard)
WHGE 3DE 5DE 7DE SA
0.57 0.56 0.55 0.54 0.55
3595.06 3796.36 3668.74 3621.89 2122.48
Quantity (μg) 84.69 89.43 86.43 85.32 50.00
wheat seedlings are also the major sources for fatty acids, mainly stearic acid. 5. Conclusions The present study concludes that the high content of SA is present in 3d-old wheat seedlings when compared to wheat grains, 5-d-old, and 7-d-old wheat seedlings. In addition, this study concludes that SA has a potential antidepressant activity. Thus, according to the current study results and previous reports, the present study proposes that regular consumption of 3-d-old wheat seedlings may reduce the risk of depression as well as heart diseases in humans. Declaration of Competing Interest The authors don't have any conflict of interest to publish this paper in Grain & Oil Science and Technology. Acknowledgement The authors are very grateful to PSG College of Pharmacy, Coimbatore, Tamil Nadu, India for providing HPTLC facility. References [1] E.J. Nestler, M. Barrot, R.J. Dileone, A.J. Eisch, S.J. Gold, L.M. Monteggia, Neurobiology of depression, Neuron 34 (2002) 13–25. [2] C.F. Gillespie, C.B. Nemeroff, Hypercortisolemia and depression, Psychosom Med 67 (2005) S26–S28. [3] T.W. Strine, A.H. Mokdad, S.R. Dube, L.S. Balluz, O. Gonzalez, J.T. Berry, et al., The association of depression and anxiety with obesity and unhealthy behaviors among community-dwelling US adults, Gen Hosp Psychiatry 30 (2008) 127–137. [4] J.H. Kim, S.Y. Kim, S.Y. Lee, C.G. Jang, Antidepressant-like effects of Albizzia julibrissin in mice: involvement of the 5-HT1A receptor system, Pharmacol Biochem Behav 87 (2007) 41–47. [5] D.N. Nandi, G. Banerjee, S.P. Mukherjee, A. Ghosh, P.S. Nandi, S. Nandi, Psychiatric morbidity of a rural Indian community changes over a 20 year interval, Br J Psychiatry 176 (2000) 351–356. [6] S. Rao, N. Ramesh, Depression, anxiety and stress levels in industrial workers: a pilot study in Bangalore, India, Ind Psychiatry J 24 (2015) 23–28. [7] A.J. Baxter, T. Vos, K.M. Scott, A.J. Ferrari, H.A. Whiteford, The global burden of anxiety disorders in 2010, Psychol Med 44 (2014) 2363–2374. [8] A. Rahman, V. Patel, J. Maselko, B. Kirkwood, The neglected ‘m’ in MCH programmes – why mental health of mothers is important for child nutrition, Trop Med Int Health 13 (2008) 579–583. [9] E.H. Ahrens, W. Insull, R. Blomstrand, J. Hirsch, T.T. Tsaltas, M.L. Peterson, The influence of dietary fats on serum-lipid levels in man, Lancet 1 (1957) 19–57. [10] A. Keys, J.T. Anderson, F. Grande, Serum cholesterol response to changes in the diet: IV. particular saturated fatty acids in the diet, Metabolism 14 (1965) 776–787. [11] D.M. Hegsted, R.B. McGandy, M.L. Myers, F.J. Stare, Quantitative effects of dietary fat on serum cholesterol in man, Am J Clin Nutr 17 (1965) 281–295.
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