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Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats
J Acupunct Meridian Stud 2013;6(1):18e23
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- RESEARCH
ARTICLE
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Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats Hossein Hosseinzadeh 1,*, Mohsen Tafaghodi 2, Mojdeh Jalal Mosavi 3, Elahe Taghiabadi 3 1
Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 2 Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 3 Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran Available online 11 August 2012 Received: Feb 22, 2012 Accepted: May 11, 2012 KEYWORDS black cumin; galactagogue; herbal; lactogenic; milk production; Nigella sativa
Abstract Nigella sativa L. is used as a galactagogue in traditional medicine. Hence, the effects of aqueous and ethanolic extracts of N. sativa seeds on milk production in rats were evaluated. The measurement of milk production was by measuring pup weight during suckling period. The intraperitoneal LD50 values of aqueous and ethanolic extracts of N. sativa were 4.23 and 4.9 g/kg, respectively. The aqueous (0.5 g/kg) and ethanolic extracts (1 g/kg) increased milk production significantly (p < 0.001), producing about 31.3% and 37.6% more milk than control, respectively. During the study period, the pups gained weight with the aqueous (0.5 g/kg, p < 0.01) and ethanolic extracts (1 g/kg, p < 0.05). It is concluded that aqueous and ethanolic extracts of N. sativa can stimulate milk production in rats.
1. Introduction Breastfeeding is very necessary for the survival of newborns. Many women with problems in milk production
use traditional herbs to increase their milk production or yield. Some studies have reported the beneficial effects of many plants on milk production. Several herbs have been shown to increase the production of milk due to induction
* Corresponding author. Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, P. O. Box: 1365-91775, Mashhad, Islamic Republic of Iran. E-mail: [email protected] Copyright ª 2013, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2012.07.019
Effect of Nigella sativa on milk production of the lactogenic hormones (prolactin), growth hormone, and casein accumulation in the mammary gland. Prolactin has important functions and roles in lactogenesis. Indeed, prolactin stimulates the production of milk proteins in the epithelial cells and induces the proliferation of secretory tissue [1]. Estimation of milk production in animals such as rats is difficult. In some studies, measurements of pups’ weight and their weight gain have been used to measure the milk yield and production. The weight of pups and their weight gains are usually applied as an indirect factor for secretion of milk. But milk yield can be directly measured by measuring pup weight during suckling period that followed separation time between dams and pups [2]. A few herbs have been shown to increase the release of prolactin, production of milk, or growth of mammary gland [1]. Nigella sativa has been used in folk medicine for its many useful effects especially during lactation time. N. sativa is an annual plant of the Ranunculaceae family and is found in various countries bordering the Mediterranean Sea, Pakistan, India, and Iran [3,4]. Also its seeds, which are commonly known as black seeds, are eaten with honey, sweet foods, bread, and cheese. In Arabic countries, the black seeds are known as black caraway seeds, Habbatu Sawda, and Habatul Baraka [5,6]. N. sativa seeds have been used in traditional medicine to treat asthma, cough, bronchitis, headache, rheumatism, fever, influenza, and eczema and as a diuretic, galactagogue, and vermifuge [5,7]. It has been reported that N. sativa or its constituents such as thymoquinone demonstrated several pharmacological activities such as anti-inflammatory [8], anti-ischemic [9e13], antioxidant and antieicosanoid [7,13e15], anticonvulsant [10,16], antibacterial [9,17], antihistaminic [18,19], antitussive [20], antihypertensive [21,22], hypoglycemic [23,24], antinociceptive [25], hepatoprotective [26], immunopotentiating [27], and calcium channel blocking [28] effects. N. sativa seeds possess many kinds of compounds, which account for the various pharmacological effects of this plant. Seeds of N. sativa contain fixed oils (about 30%) and volatile oils (average 0.5%, maximum 1.5%); this plant is also a rich source of unsaturated fatty acids, amino acids and proteins, carbohydrates, quinones (such as thymoquinone, nigellone, and thymohydroquinone), alkaloids and terpenoids, carvacrol, t-anetholet, crude fiber, and minerals such as calcium, iron, sodium, and potassium [3,6]. Due to the use of this plant as a galactagogue in traditional medicine and the lack of any report on its effect on milk production, the present study investigated the acute toxicity and effect of aqueous and ethanolic extracts of N. sativa seeds on the production of milk in rats.
2. Materials and methods 2.1. Animals The study was performed on male and female albino mice, weighing 25 2 g, for investigating the acute toxicity, and on lactating rats, weighing 250 25 g and suckling four to six pups each, for evaluation of milk production. Animals were housed in a ventilated room under a 12/12-hour light/ dark cycle at 24 2 C and had free access to water and
19 food. All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee Acts.
2.2. Preparation of aqueous and ethanolic extracts of N. sativa N. sativa seeds, obtained from the south parts of Khorassan, were purchased from a local market in Mashhad, Iran. Seeds were identified by Mr Ahi from Pharmacy School of Mashhad, and a voucher sample (Voucher no. 247-1419-1) was preserved for reference in the herbarium of Mashhad Pharmacy School, Mashhad university of Medical Sciences, Iran. The seeds were cleaned and dried in shadow and powdered using a mechanical grinder. For the aqueous extract, the seed powder (100 g) was added to 1000 mL hot water, boiled for 15 minutes, and filtered through a cloth. The filtrate was evaporated to dryness under reduced pressure to obtain a viscous residue. The residue was suspended in normal saline. For the ethanolic extract, the seed powders (100 g) were defatted with petroleum ether (40e60 C) using the Soxhlet apparatus. Then, the powder was macerated in 800 mL ethanol (80%, v/v) for 72 hours, and the mixture was subsequently filtered and concentrated in vacuo at 40 C. The residue was suspended in saline solution.
2.3. Acute toxicity Different doses of the aqueous and ethanolic extracts of N. sativa were administrated orally and intraperitoneally to groups of six mice. The number of deaths was counted at 48 hours after the treatment. LD50 values and corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS version 4).
2.4. Effect of the aqueous and ethanolic extracts of N. sativa extract on milk production The method is a modification of Lompo-Ouedraogo et al’s method [1]. Briefly, 30 lactating dams (weighing 250 25 g), which were at the starting of lactation and suckling four to six pups each, were used for this test. Females were divided into five groups of six animals each. Group 1 received orally 0.5 mL of saline (control group), and groups 2e5 received orally 0.5 and 1 g/kg of the aqueous and ethanolic extracts per 0.5 mL saline. All animals were treated daily at 2:30 PM. The production of milk was estimated 23 hours after gavage. Milk production was measured from Day 3 to Day 15 of lactation. Milk yield and dams’ weight, and weight gain of pups were calculated every day. During the test time, the pups were weighed daily at 8:30 AM (w1) and then isolated from their mothers for 4 hours. At 12:30 PM, the pups were weighed (w2), returned to their mother, and permitted to feed for 1 hour. They were weighed again (w3) at 1:30 PM. The milk yield 23 hours after gavage was estimated as w3 e w2. The daily milk yield was corrected for the loss of weight caused by the metabolic processes in the pups during the suckling period. The value used was (w2 e w1)/4. Next, this value
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2.5. Statistical analysis The mean standard error of the mean (SEM) were determined for each study group and tested using analysis of variance followed by the multiple comparison test of TukeyeKramer. Discrepancies with p < 0.05 were considered significant.
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Milk Yield (g/pup/day)
was multiplied by the amount of suckling hours per day and added to the daily suckling gain. Every day, gain in pups’ weight was measured from w2.
H. Hosseinzadeh et al. No r m al sal i ne Aque o us e xtr ac t 0 .5 g /kg Aque o us e xtr ac t 1 g /kg 14 * * ** * ** * ** * * ***
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3. Results
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No mortality was seen in mice given the aqueous and ethanolic extracts of N. sativa (0.5, 2, 8, 16, and 32 g/kg, orally) after 24 and 48 hours of treatment. The intraperitoneal LD50 values of aqueous and ethanolic extracts of N. sativa were 4.23 (95% CL: 3.45e5.18) and 4.9 (95% CL: 4.16e5.19) g/kg, respectively, and the maximum non-fatal doses were 2.5 and 3.1 g/kg, respectively.
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Milk production of the aqueous and ethanolic extracts groups was more than the control group (Figs. 1 and 2). The milk yield increased from 4.8 0.1, 4.84 0.16, and 5.76 0.34 g per pup per day to 6.92 0.46, 8.07 0.23, and 10.84 0.18 g per pup per day for the controls, those treated with aqueous extract at a dose of 0.5, and those treated with aqueous extract at a dose of 1 g/kg, respectively. The milk yield elevated from 4.8 0.1, 4.75 0.1, and 6.2 0.15 g per pup per day to about 6.92 0.46, 8.27 0.25, and 11.58 0.2 g per pup per day for the controls, those receiving ethanolic extract at a dose of 0.5, and those receiving ethanolic extract at a dose of 1 g/kg, respectively. The milk yield, from Day 8 to the end of test, of the groups that were given aqueous (0.5 g/kg) and ethanolic (1 g/kg) extracts was significantly different from that of the control group (p < 0.05) (Figs. 1A and 2A). The milk production data 23 hours after gavage showed that milk production was significantly increased in groups receiving the aqueous extract at a dose of 0.5 g/kg and ethanolic extract at a dose of 1 g/kg compared to the control group (p < 0.001). The mean milk yield for the control group was 6 0.2 g per pup, and for the groups that were given aqueous (0.5 g/kg) and ethanolic (1 g/kg) extracts were 8.8 0.6 and 9.64 0.64 g per pup, respectively. The aqueous extract at a dose of 0.5 g/kg and ethanolic extract at a dose of 1 g/kg increased the daily milk production by about 31.3% and 37.6%, respectively (Figs. 1B and 2B). Weights of all pups increased throughout the test period (Figs. 3 and 4), and the weight gain for the groups receiving aqueous (0.5 g/kg, p < 0.01) and ethanolic extracts (1 g/kg, p < 0.05) showed a significant increase in comparison to the control group. Aqueous (0.5 g/kg) and ethanolic (1 g/kg)
Treatment
Figure 1 (A) The effect of aqueous extract of N. sativa at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *p < 0.05, **p < 0.01, ***p < 0.001. (B) Mean milk production per day. Values are means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *** p < 0.001. ANOVA Z analysis of variance; SEM Z standard error of the mean.
extracts increased the body weight from 13.2 0.8 to 26 1.2 and from 12 0.08 to 23.18 0.45 g per pup per day, respectively, and the body weight in control group increased from 10.4 0.4 to 19.2 0.35 g per pup per day (Figs. 3A and 4A). The daily weight gains for the control group was 14.7 0.86 g per pup, for the group given aqueous extract (0.5 g/kg) was 20 0.1 g per pup, and for the group given ethanolic extract (1 g/kg) was 19.1 0.65 g per pup (Figs. 3B and 4B).
4. Discussion N. sativa seeds have been used as a food additive and medicinal herb for many years [3,4]. Black seeds, which have shown galactagogue effect, have been used in traditional medicine for the treatment of several health problems [5,7]. The results of the acute toxicity test demonstrated that LD50 values of compounds vary depending on the species
Effect of Nigella sativa on milk production
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Figure 2 (A) The effect of ethanolic extract of N. sativa at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Mean milk production per day. Values are means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *** p < 0.001. ANOVA Z analysis of variance; SEM Z standard error of the mean.
and ways of administration. The aqueous and ethanolic extracts of N. sativa (0.5, 2, 8, 16, and 32 g/kg, orally) did not cause any mortality. The i.p. LD50 values of N. sativa aqueous and ethanolic extracts in mice were 4.23 and 4.9 g/kg, respectively. According to the toxicity classification, compounds with an LD50 value in the range of 1e5 g/ kg are considered slightly toxic and those with an LD50 value of more than 5.0 g/kg may be considered practically nontoxic [29]. According to the LD50 values ( g/kg, oral route), the present study indicates that N. sativa is practically nontoxic in acute oral administration. The i.p. LD50 values of both extracts were in the range of 1e5 g/kg; thus, N. sativa should be considered slightly toxic in acute intraperitoneal route, and data showed that the toxicity of aqueous extract is more than that of ethanolic extract. Determination of the milk yield in animals is difficult. Milk production measurement for rats from the weight of pups and their weight gains has been applied in some reports [2,30e32]. According to data, production of milk in
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Figure 3 (A) The effect of aqueous extract of N. sativa at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). * p < 0.05. (B) Mean weight gain of pup. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). ** p < 0.01. ANOVA Z analysis of variance; SEM Z standard error of the mean.
the animals treated with N. sativa was significantly more than in the control group from Day 8. Also, the milk yield increased considerably about 23 hours after treatment with both extracts, and the pup growth level was improved. In some study, it was indicated that several plants influenced milk production in animals through the stimulation of lactogenic hormone (prolactin) [1,33]. Fennel (Foeniculum vulgare) and anise (Pimpinella anisum), which contain estrogenic constituents such as anethole, increase milk secretion, promote menstruation, and facilitate birth. Structurally, anethole is similar to dopamine and exerts a competitive antagonism at the dopamine receptor site. Thus, it may stimulate prolactin release and increase milk production [34].
22
H. Hosseinzadeh et al.
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ethanolic extract was observed in high dose (1 g/kg). Regarding the LD50 value of these extracts, the toxicity of aqueous extract is more than that of ethanolic extract. The aqueous extract may induce chronic toxic effect and muscle relaxant effect at high dose [37,38]. Thus, the beneficial effect of aqueous extract was demonstrated at a lower dose. It is concluded that aqueous and ethanolic extracts of N. sativa stimulates milk production. This research confirms the traditional use of N. sativa seeds as a lactogenic agent.
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Acknowledgments
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The authors wish to thank School of Pharmacy, Mashhad University of Medical Sciences, for the financial support. This study was part of a PharmD thesis.
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References
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Figure 4 (A) The effect of ethanolic extract of N. sativa at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). (B) Mean weight gain of pup. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *p < 0.05. ANOVA Z analysis of variance; SEM Z standard error of the mean.
One of the various constituents of N. sativa seeds, t-anethole (1e4%) [35], accounts for the lactogenic activity of this plant. In addition, the growth and weight gain of pups in treated groups could be related to increase of milk production and milk components. In this study, it was shown that the milk production and milk yield increased considerably from about Day 8 after treatment with both the extracts of N. sativa. With regard to the fact that the pups can take a feed from Days 10e12 [36], the growth and weight gain of pups might be also due to the effect of this plant on their nutrition and feeding. Therefore, evaluation of milk component, and microscopic examinations of mammary glands and pituitary prolactin contents are needed to clarify the exact mechanism of the lactogenic activity of this plant. The results of this study showed that the beneficial effect of N. sativa aqueous extract on milk production was observed in lower dose (0.5 g/kg), whereas that of the
1. Lompo-Ouedraogo Z, van der Heide D, van der Beek EM, Swarts HJ, John AM, Mattheij JA, et al. Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat. J Endocrinol. 2004;182:257e266. 2. Sampson DA, Jansen GR. Measurement of milk yield in lactating rat from pup weight and weight gain. J Pediatr Gastroenterol Nutr. 1984;3:613e617. 3. Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine. 2004;11:56e64. 4. Jansen PCM. Spices, Condiments and Medicinal Plants in Ethiopia, Their Taxonomy and Agricultural Significance. Addis Ababa: Center for Agricultural Publishing and Documentation; 1981. 5. Bourgou S, Pichette A, Marzouk B, Legault J. Bioactivities of black cumin essential oil and its main terpenes from Tunisia. S Afr J Bot. 2010;76:210e216. 6. Gali-Muhtasib H, El-Najjar N, Schneider-Stock R. The medicinal potential of black seed (Nigella sativa) and its components. Adv Phytomed. 2006;2:133e153. 7. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14:323e328. 8. Mutabagani A, El-Mahdy SA. Study of the anti-inflammatory activity of Nigella sativa L. and thymoquinone in rats. Saudi Pharm J. 1997;5:110e113. 9. Hosseinzadeh H, Fazly Bazzaz BS, Haghi MM. Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in mice. Pharmacologyonline. 2007;2:429e435. 10. Hosseinzadeh H, Moghim FF, Mansouri SMT. Effect of Nigella sativa seed extracts on ischemia-reperfusion in rat skeletal muscle. Pharmacologyonline. 2007;2:326e335. 11. Hosseinzadeh H, Montahaei R. Protective effect of Nigella sativa L. extracts and thymoquinone, its active constituent, on renal ischemia-reperfusion-induced oxidative damage in rats. Pharmacologyonline. 2007;1:176e189. 12. Hosseinzadeh H, Taiari S, Nassiri-Asl M. Effect of thymoquinone, a constituent of Nigella sativa L., on ischemia-reperfusion in rat skeletal muscle. Naunyn-Schmiedeberg Arch Pharmacol. 2012;385:503e508. 13. Hosseinzadeh H, Jaafaria MR, Ali Reza Khoei AR, Rahmania M. Anti-ischemic effect of Nigella sativa L. seed in male rats. Iran J Pharmaceut Res. 2006;1:53e58. 14. Houghton PJ, Zarka R, de las Heras B, Hoult JR. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med. 1995;61:33e36.
Effect of Nigella sativa on milk production 15. Ismail M, Al-Naqeep G, Chan KW. Nigella sativa thymoquinonerich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Rad Biol Med. 2010;48:664e672. 16. Hosseinzadeh H, Parvardeh S, Nassiri Asl M, Mansouri MT. Intracerebroventricular administration of thymoquinone, the major constituent of Nigella sativa seeds, suppresses epileptic seizures in rats. Med Sci Monit. 2005;11:106e110. 17. Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant bacteria. Acta Microbiol Pol. 2000;49:63e74. 18. Chakravarty N. Inhibition of histamine release from mast cells by nigellone. Ann Allergy. 1993;70:237e242. 19. Mahfouz M, El-Dakhakhny M. The isolation of a crystalline active principle from Nigella sativa L. seeds. J Pharm Sci United Arab Republic. 1960;1:9e19. 20. Hosseinzadeh H, Eskandari M, Ziaee T. Antitussive effect of thymoquinone, a constituent of Nigella sativa seeds, in guinea pigs. Pharmacologyonline. 2008;2:480e484. 21. El-Tahir KE, Ashour MM, Al-Harbi MM. The cardiovascular actions of the volatile oil of the black seed (Nigella sativa) in rats: elucidation of the mechanism of action. Gen Pharmacol. 1993;24:1123e1131. 22. Zaoui A, Cherrah Y, Lacaille-Dubois MA, Settaf A, Amarouch H, Hassar M. Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Therapie. 2000;5:379e382. 23. Al-Hader AA, Aqel MB, Hasan ZA. Hypoglycemic effects of the volatile oil of Nigella sativa seeds. Int J Pharmacogn. 1993;31: 96e100. 24. Mathura ML, Gaura J, Sharma P, Haldiya KR. Antidiabetic properties of a spice plant Nigella sativa. J Endocrinol Metab. 2011;1:1e8. 25. Abdel-Fattah AM, Matsumoto K, Watanabe H. Antinociceptive effects of Nigella sativa oil and its major component, thymoquinone in mice. Eur J Pharmacol. 2000;400:89e97.
23 26. Daba MH, Abdel-Rahman MS. Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol Lett. 1998; 16:23e29. 27. Swamy SM, Tan BK. Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L seeds. J Ethnopharmacol. 2000;70:1e7. 28. Gilani AH, Aziz N, Khurram IM, Chaudhary KS, Iqbal A. Bronchodilator, spasmolytic and calcium antagonist activities of Nigella sativa seeds (kalonji): a traditional herbal product with multiple medicinal uses. J Pak Med Assoc. 2001;51:115e120. 29. Loomis T. Essential of toxicology. Philadelphia: Lea and Febiger; 1968. 30. Kamani HT, Karunanayake EH, Mahindartna MPD. Evaluation of galactoguic activity of Asparagus falcatus. Ceylon J Med Sci. 1987;30:63e67. 31. Kim SH, Moon YS, Keller WL, Park CS. Compensatory nutritiondirected mammary cell proliferation and lactation in rats. Br J Nutr. 1998;79:177e183. 32. Morag M, Popliker F, Yagil R. 1975 Effect of litter size on milk yield in the rat. Lab Anim. 1975;9:43e47. 33. Patel AB, Kanitkar UK. Asparagus racemosus Willd form. bordi as a galactogogue in buffaloes. Indian Vet J. 1969;46:718e721. 34. Lis-Balchin M. Aromatherapy science: a guide for healthcare professionals. London: Pharmaceutical Press; 2006. 35. Khader M, Bresgen N, Eckl PM. In vitro toxicological properties of thymoquinone. Food Chem Toxicol. 2009;47:129e133. 36. Hall WG, Jay S, Rosenblantt JS. Development of nutritional control of food intake in suckling rat pups. Behav Biol. 1978;24: 413e427. 37. Aqel M, Shaheen R. Effect of the volatile oil of Nigella sativa seeds on the uterine smooth muscle of rat and guinea pig. J Ethnopharmacol. 1996;52:23e26. 38. Zaoui A, Cherrah Y, Mahassini N, Alaoui K, Amarouch H, Hassar M. Acute and chronic toxicity of Nigella sativa fixed oil. Phytomedicine. 2002;9:69e74.
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com
- RESEARCH
ARTICLE
-
Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats Hossein Hosseinzadeh 1,*, Mohsen Tafaghodi 2, Mojdeh Jalal Mosavi 3, Elahe Taghiabadi 3 1
Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 2 Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran 3 Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Islamic Republic of Iran Available online 11 August 2012 Received: Feb 22, 2012 Accepted: May 11, 2012 KEYWORDS black cumin; galactagogue; herbal; lactogenic; milk production; Nigella sativa
Abstract Nigella sativa L. is used as a galactagogue in traditional medicine. Hence, the effects of aqueous and ethanolic extracts of N. sativa seeds on milk production in rats were evaluated. The measurement of milk production was by measuring pup weight during suckling period. The intraperitoneal LD50 values of aqueous and ethanolic extracts of N. sativa were 4.23 and 4.9 g/kg, respectively. The aqueous (0.5 g/kg) and ethanolic extracts (1 g/kg) increased milk production significantly (p < 0.001), producing about 31.3% and 37.6% more milk than control, respectively. During the study period, the pups gained weight with the aqueous (0.5 g/kg, p < 0.01) and ethanolic extracts (1 g/kg, p < 0.05). It is concluded that aqueous and ethanolic extracts of N. sativa can stimulate milk production in rats.
1. Introduction Breastfeeding is very necessary for the survival of newborns. Many women with problems in milk production
use traditional herbs to increase their milk production or yield. Some studies have reported the beneficial effects of many plants on milk production. Several herbs have been shown to increase the production of milk due to induction
* Corresponding author. Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, P. O. Box: 1365-91775, Mashhad, Islamic Republic of Iran. E-mail: [email protected] Copyright ª 2013, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2012.07.019
Effect of Nigella sativa on milk production of the lactogenic hormones (prolactin), growth hormone, and casein accumulation in the mammary gland. Prolactin has important functions and roles in lactogenesis. Indeed, prolactin stimulates the production of milk proteins in the epithelial cells and induces the proliferation of secretory tissue [1]. Estimation of milk production in animals such as rats is difficult. In some studies, measurements of pups’ weight and their weight gain have been used to measure the milk yield and production. The weight of pups and their weight gains are usually applied as an indirect factor for secretion of milk. But milk yield can be directly measured by measuring pup weight during suckling period that followed separation time between dams and pups [2]. A few herbs have been shown to increase the release of prolactin, production of milk, or growth of mammary gland [1]. Nigella sativa has been used in folk medicine for its many useful effects especially during lactation time. N. sativa is an annual plant of the Ranunculaceae family and is found in various countries bordering the Mediterranean Sea, Pakistan, India, and Iran [3,4]. Also its seeds, which are commonly known as black seeds, are eaten with honey, sweet foods, bread, and cheese. In Arabic countries, the black seeds are known as black caraway seeds, Habbatu Sawda, and Habatul Baraka [5,6]. N. sativa seeds have been used in traditional medicine to treat asthma, cough, bronchitis, headache, rheumatism, fever, influenza, and eczema and as a diuretic, galactagogue, and vermifuge [5,7]. It has been reported that N. sativa or its constituents such as thymoquinone demonstrated several pharmacological activities such as anti-inflammatory [8], anti-ischemic [9e13], antioxidant and antieicosanoid [7,13e15], anticonvulsant [10,16], antibacterial [9,17], antihistaminic [18,19], antitussive [20], antihypertensive [21,22], hypoglycemic [23,24], antinociceptive [25], hepatoprotective [26], immunopotentiating [27], and calcium channel blocking [28] effects. N. sativa seeds possess many kinds of compounds, which account for the various pharmacological effects of this plant. Seeds of N. sativa contain fixed oils (about 30%) and volatile oils (average 0.5%, maximum 1.5%); this plant is also a rich source of unsaturated fatty acids, amino acids and proteins, carbohydrates, quinones (such as thymoquinone, nigellone, and thymohydroquinone), alkaloids and terpenoids, carvacrol, t-anetholet, crude fiber, and minerals such as calcium, iron, sodium, and potassium [3,6]. Due to the use of this plant as a galactagogue in traditional medicine and the lack of any report on its effect on milk production, the present study investigated the acute toxicity and effect of aqueous and ethanolic extracts of N. sativa seeds on the production of milk in rats.
2. Materials and methods 2.1. Animals The study was performed on male and female albino mice, weighing 25 2 g, for investigating the acute toxicity, and on lactating rats, weighing 250 25 g and suckling four to six pups each, for evaluation of milk production. Animals were housed in a ventilated room under a 12/12-hour light/ dark cycle at 24 2 C and had free access to water and
19 food. All animal experiments were carried out in accordance with Mashhad University of Medical Sciences, Ethical Committee Acts.
2.2. Preparation of aqueous and ethanolic extracts of N. sativa N. sativa seeds, obtained from the south parts of Khorassan, were purchased from a local market in Mashhad, Iran. Seeds were identified by Mr Ahi from Pharmacy School of Mashhad, and a voucher sample (Voucher no. 247-1419-1) was preserved for reference in the herbarium of Mashhad Pharmacy School, Mashhad university of Medical Sciences, Iran. The seeds were cleaned and dried in shadow and powdered using a mechanical grinder. For the aqueous extract, the seed powder (100 g) was added to 1000 mL hot water, boiled for 15 minutes, and filtered through a cloth. The filtrate was evaporated to dryness under reduced pressure to obtain a viscous residue. The residue was suspended in normal saline. For the ethanolic extract, the seed powders (100 g) were defatted with petroleum ether (40e60 C) using the Soxhlet apparatus. Then, the powder was macerated in 800 mL ethanol (80%, v/v) for 72 hours, and the mixture was subsequently filtered and concentrated in vacuo at 40 C. The residue was suspended in saline solution.
2.3. Acute toxicity Different doses of the aqueous and ethanolic extracts of N. sativa were administrated orally and intraperitoneally to groups of six mice. The number of deaths was counted at 48 hours after the treatment. LD50 values and corresponding confidence limits were determined by the Litchfield and Wilcoxon method (PHARM/PCS version 4).
2.4. Effect of the aqueous and ethanolic extracts of N. sativa extract on milk production The method is a modification of Lompo-Ouedraogo et al’s method [1]. Briefly, 30 lactating dams (weighing 250 25 g), which were at the starting of lactation and suckling four to six pups each, were used for this test. Females were divided into five groups of six animals each. Group 1 received orally 0.5 mL of saline (control group), and groups 2e5 received orally 0.5 and 1 g/kg of the aqueous and ethanolic extracts per 0.5 mL saline. All animals were treated daily at 2:30 PM. The production of milk was estimated 23 hours after gavage. Milk production was measured from Day 3 to Day 15 of lactation. Milk yield and dams’ weight, and weight gain of pups were calculated every day. During the test time, the pups were weighed daily at 8:30 AM (w1) and then isolated from their mothers for 4 hours. At 12:30 PM, the pups were weighed (w2), returned to their mother, and permitted to feed for 1 hour. They were weighed again (w3) at 1:30 PM. The milk yield 23 hours after gavage was estimated as w3 e w2. The daily milk yield was corrected for the loss of weight caused by the metabolic processes in the pups during the suckling period. The value used was (w2 e w1)/4. Next, this value
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2.5. Statistical analysis The mean standard error of the mean (SEM) were determined for each study group and tested using analysis of variance followed by the multiple comparison test of TukeyeKramer. Discrepancies with p < 0.05 were considered significant.
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was multiplied by the amount of suckling hours per day and added to the daily suckling gain. Every day, gain in pups’ weight was measured from w2.
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No mortality was seen in mice given the aqueous and ethanolic extracts of N. sativa (0.5, 2, 8, 16, and 32 g/kg, orally) after 24 and 48 hours of treatment. The intraperitoneal LD50 values of aqueous and ethanolic extracts of N. sativa were 4.23 (95% CL: 3.45e5.18) and 4.9 (95% CL: 4.16e5.19) g/kg, respectively, and the maximum non-fatal doses were 2.5 and 3.1 g/kg, respectively.
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Milk production of the aqueous and ethanolic extracts groups was more than the control group (Figs. 1 and 2). The milk yield increased from 4.8 0.1, 4.84 0.16, and 5.76 0.34 g per pup per day to 6.92 0.46, 8.07 0.23, and 10.84 0.18 g per pup per day for the controls, those treated with aqueous extract at a dose of 0.5, and those treated with aqueous extract at a dose of 1 g/kg, respectively. The milk yield elevated from 4.8 0.1, 4.75 0.1, and 6.2 0.15 g per pup per day to about 6.92 0.46, 8.27 0.25, and 11.58 0.2 g per pup per day for the controls, those receiving ethanolic extract at a dose of 0.5, and those receiving ethanolic extract at a dose of 1 g/kg, respectively. The milk yield, from Day 8 to the end of test, of the groups that were given aqueous (0.5 g/kg) and ethanolic (1 g/kg) extracts was significantly different from that of the control group (p < 0.05) (Figs. 1A and 2A). The milk production data 23 hours after gavage showed that milk production was significantly increased in groups receiving the aqueous extract at a dose of 0.5 g/kg and ethanolic extract at a dose of 1 g/kg compared to the control group (p < 0.001). The mean milk yield for the control group was 6 0.2 g per pup, and for the groups that were given aqueous (0.5 g/kg) and ethanolic (1 g/kg) extracts were 8.8 0.6 and 9.64 0.64 g per pup, respectively. The aqueous extract at a dose of 0.5 g/kg and ethanolic extract at a dose of 1 g/kg increased the daily milk production by about 31.3% and 37.6%, respectively (Figs. 1B and 2B). Weights of all pups increased throughout the test period (Figs. 3 and 4), and the weight gain for the groups receiving aqueous (0.5 g/kg, p < 0.01) and ethanolic extracts (1 g/kg, p < 0.05) showed a significant increase in comparison to the control group. Aqueous (0.5 g/kg) and ethanolic (1 g/kg)
Treatment
Figure 1 (A) The effect of aqueous extract of N. sativa at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *p < 0.05, **p < 0.01, ***p < 0.001. (B) Mean milk production per day. Values are means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *** p < 0.001. ANOVA Z analysis of variance; SEM Z standard error of the mean.
extracts increased the body weight from 13.2 0.8 to 26 1.2 and from 12 0.08 to 23.18 0.45 g per pup per day, respectively, and the body weight in control group increased from 10.4 0.4 to 19.2 0.35 g per pup per day (Figs. 3A and 4A). The daily weight gains for the control group was 14.7 0.86 g per pup, for the group given aqueous extract (0.5 g/kg) was 20 0.1 g per pup, and for the group given ethanolic extract (1 g/kg) was 19.1 0.65 g per pup (Figs. 3B and 4B).
4. Discussion N. sativa seeds have been used as a food additive and medicinal herb for many years [3,4]. Black seeds, which have shown galactagogue effect, have been used in traditional medicine for the treatment of several health problems [5,7]. The results of the acute toxicity test demonstrated that LD50 values of compounds vary depending on the species
Effect of Nigella sativa on milk production
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Figure 2 (A) The effect of ethanolic extract of N. sativa at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Mean milk production per day. Values are means SEM, n Z 6. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *** p < 0.001. ANOVA Z analysis of variance; SEM Z standard error of the mean.
and ways of administration. The aqueous and ethanolic extracts of N. sativa (0.5, 2, 8, 16, and 32 g/kg, orally) did not cause any mortality. The i.p. LD50 values of N. sativa aqueous and ethanolic extracts in mice were 4.23 and 4.9 g/kg, respectively. According to the toxicity classification, compounds with an LD50 value in the range of 1e5 g/ kg are considered slightly toxic and those with an LD50 value of more than 5.0 g/kg may be considered practically nontoxic [29]. According to the LD50 values ( g/kg, oral route), the present study indicates that N. sativa is practically nontoxic in acute oral administration. The i.p. LD50 values of both extracts were in the range of 1e5 g/kg; thus, N. sativa should be considered slightly toxic in acute intraperitoneal route, and data showed that the toxicity of aqueous extract is more than that of ethanolic extract. Determination of the milk yield in animals is difficult. Milk production measurement for rats from the weight of pups and their weight gains has been applied in some reports [2,30e32]. According to data, production of milk in
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Figure 3 (A) The effect of aqueous extract of N. sativa at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). * p < 0.05. (B) Mean weight gain of pup. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). ** p < 0.01. ANOVA Z analysis of variance; SEM Z standard error of the mean.
the animals treated with N. sativa was significantly more than in the control group from Day 8. Also, the milk yield increased considerably about 23 hours after treatment with both extracts, and the pup growth level was improved. In some study, it was indicated that several plants influenced milk production in animals through the stimulation of lactogenic hormone (prolactin) [1,33]. Fennel (Foeniculum vulgare) and anise (Pimpinella anisum), which contain estrogenic constituents such as anethole, increase milk secretion, promote menstruation, and facilitate birth. Structurally, anethole is similar to dopamine and exerts a competitive antagonism at the dopamine receptor site. Thus, it may stimulate prolactin release and increase milk production [34].
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ethanolic extract was observed in high dose (1 g/kg). Regarding the LD50 value of these extracts, the toxicity of aqueous extract is more than that of ethanolic extract. The aqueous extract may induce chronic toxic effect and muscle relaxant effect at high dose [37,38]. Thus, the beneficial effect of aqueous extract was demonstrated at a lower dose. It is concluded that aqueous and ethanolic extracts of N. sativa stimulates milk production. This research confirms the traditional use of N. sativa seeds as a lactogenic agent.
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Acknowledgments
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The authors wish to thank School of Pharmacy, Mashhad University of Medical Sciences, for the financial support. This study was part of a PharmD thesis.
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References
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Figure 4 (A) The effect of ethanolic extract of N. sativa at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). (B) Mean weight gain of pup. Values are means SEM. Statistically significant differences are given compared to the control group (ANOVA followed by TukeyeKramer). *p < 0.05. ANOVA Z analysis of variance; SEM Z standard error of the mean.
One of the various constituents of N. sativa seeds, t-anethole (1e4%) [35], accounts for the lactogenic activity of this plant. In addition, the growth and weight gain of pups in treated groups could be related to increase of milk production and milk components. In this study, it was shown that the milk production and milk yield increased considerably from about Day 8 after treatment with both the extracts of N. sativa. With regard to the fact that the pups can take a feed from Days 10e12 [36], the growth and weight gain of pups might be also due to the effect of this plant on their nutrition and feeding. Therefore, evaluation of milk component, and microscopic examinations of mammary glands and pituitary prolactin contents are needed to clarify the exact mechanism of the lactogenic activity of this plant. The results of this study showed that the beneficial effect of N. sativa aqueous extract on milk production was observed in lower dose (0.5 g/kg), whereas that of the
1. Lompo-Ouedraogo Z, van der Heide D, van der Beek EM, Swarts HJ, John AM, Mattheij JA, et al. Effect of aqueous extract of Acacia nilotica ssp adansonii on milk production and prolactin release in the rat. J Endocrinol. 2004;182:257e266. 2. Sampson DA, Jansen GR. Measurement of milk yield in lactating rat from pup weight and weight gain. J Pediatr Gastroenterol Nutr. 1984;3:613e617. 3. Hosseinzadeh H, Parvardeh S. Anticonvulsant effects of thymoquinone, the major constituent of Nigella sativa seeds, in mice. Phytomedicine. 2004;11:56e64. 4. Jansen PCM. Spices, Condiments and Medicinal Plants in Ethiopia, Their Taxonomy and Agricultural Significance. Addis Ababa: Center for Agricultural Publishing and Documentation; 1981. 5. Bourgou S, Pichette A, Marzouk B, Legault J. Bioactivities of black cumin essential oil and its main terpenes from Tunisia. S Afr J Bot. 2010;76:210e216. 6. Gali-Muhtasib H, El-Najjar N, Schneider-Stock R. The medicinal potential of black seed (Nigella sativa) and its components. Adv Phytomed. 2006;2:133e153. 7. Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14:323e328. 8. Mutabagani A, El-Mahdy SA. Study of the anti-inflammatory activity of Nigella sativa L. and thymoquinone in rats. Saudi Pharm J. 1997;5:110e113. 9. Hosseinzadeh H, Fazly Bazzaz BS, Haghi MM. Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in mice. Pharmacologyonline. 2007;2:429e435. 10. Hosseinzadeh H, Moghim FF, Mansouri SMT. Effect of Nigella sativa seed extracts on ischemia-reperfusion in rat skeletal muscle. Pharmacologyonline. 2007;2:326e335. 11. Hosseinzadeh H, Montahaei R. Protective effect of Nigella sativa L. extracts and thymoquinone, its active constituent, on renal ischemia-reperfusion-induced oxidative damage in rats. Pharmacologyonline. 2007;1:176e189. 12. Hosseinzadeh H, Taiari S, Nassiri-Asl M. Effect of thymoquinone, a constituent of Nigella sativa L., on ischemia-reperfusion in rat skeletal muscle. Naunyn-Schmiedeberg Arch Pharmacol. 2012;385:503e508. 13. Hosseinzadeh H, Jaafaria MR, Ali Reza Khoei AR, Rahmania M. Anti-ischemic effect of Nigella sativa L. seed in male rats. Iran J Pharmaceut Res. 2006;1:53e58. 14. Houghton PJ, Zarka R, de las Heras B, Hoult JR. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med. 1995;61:33e36.
Effect of Nigella sativa on milk production 15. Ismail M, Al-Naqeep G, Chan KW. Nigella sativa thymoquinonerich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Rad Biol Med. 2010;48:664e672. 16. Hosseinzadeh H, Parvardeh S, Nassiri Asl M, Mansouri MT. Intracerebroventricular administration of thymoquinone, the major constituent of Nigella sativa seeds, suppresses epileptic seizures in rats. Med Sci Monit. 2005;11:106e110. 17. Morsi NM. Antimicrobial effect of crude extracts of Nigella sativa on multiple antibiotics-resistant bacteria. Acta Microbiol Pol. 2000;49:63e74. 18. Chakravarty N. Inhibition of histamine release from mast cells by nigellone. Ann Allergy. 1993;70:237e242. 19. Mahfouz M, El-Dakhakhny M. The isolation of a crystalline active principle from Nigella sativa L. seeds. J Pharm Sci United Arab Republic. 1960;1:9e19. 20. Hosseinzadeh H, Eskandari M, Ziaee T. Antitussive effect of thymoquinone, a constituent of Nigella sativa seeds, in guinea pigs. Pharmacologyonline. 2008;2:480e484. 21. El-Tahir KE, Ashour MM, Al-Harbi MM. The cardiovascular actions of the volatile oil of the black seed (Nigella sativa) in rats: elucidation of the mechanism of action. Gen Pharmacol. 1993;24:1123e1131. 22. Zaoui A, Cherrah Y, Lacaille-Dubois MA, Settaf A, Amarouch H, Hassar M. Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Therapie. 2000;5:379e382. 23. Al-Hader AA, Aqel MB, Hasan ZA. Hypoglycemic effects of the volatile oil of Nigella sativa seeds. Int J Pharmacogn. 1993;31: 96e100. 24. Mathura ML, Gaura J, Sharma P, Haldiya KR. Antidiabetic properties of a spice plant Nigella sativa. J Endocrinol Metab. 2011;1:1e8. 25. Abdel-Fattah AM, Matsumoto K, Watanabe H. Antinociceptive effects of Nigella sativa oil and its major component, thymoquinone in mice. Eur J Pharmacol. 2000;400:89e97.
23 26. Daba MH, Abdel-Rahman MS. Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol Lett. 1998; 16:23e29. 27. Swamy SM, Tan BK. Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa L seeds. J Ethnopharmacol. 2000;70:1e7. 28. Gilani AH, Aziz N, Khurram IM, Chaudhary KS, Iqbal A. Bronchodilator, spasmolytic and calcium antagonist activities of Nigella sativa seeds (kalonji): a traditional herbal product with multiple medicinal uses. J Pak Med Assoc. 2001;51:115e120. 29. Loomis T. Essential of toxicology. Philadelphia: Lea and Febiger; 1968. 30. Kamani HT, Karunanayake EH, Mahindartna MPD. Evaluation of galactoguic activity of Asparagus falcatus. Ceylon J Med Sci. 1987;30:63e67. 31. Kim SH, Moon YS, Keller WL, Park CS. Compensatory nutritiondirected mammary cell proliferation and lactation in rats. Br J Nutr. 1998;79:177e183. 32. Morag M, Popliker F, Yagil R. 1975 Effect of litter size on milk yield in the rat. Lab Anim. 1975;9:43e47. 33. Patel AB, Kanitkar UK. Asparagus racemosus Willd form. bordi as a galactogogue in buffaloes. Indian Vet J. 1969;46:718e721. 34. Lis-Balchin M. Aromatherapy science: a guide for healthcare professionals. London: Pharmaceutical Press; 2006. 35. Khader M, Bresgen N, Eckl PM. In vitro toxicological properties of thymoquinone. Food Chem Toxicol. 2009;47:129e133. 36. Hall WG, Jay S, Rosenblantt JS. Development of nutritional control of food intake in suckling rat pups. Behav Biol. 1978;24: 413e427. 37. Aqel M, Shaheen R. Effect of the volatile oil of Nigella sativa seeds on the uterine smooth muscle of rat and guinea pig. J Ethnopharmacol. 1996;52:23e26. 38. Zaoui A, Cherrah Y, Mahassini N, Alaoui K, Amarouch H, Hassar M. Acute and chronic toxicity of Nigella sativa fixed oil. Phytomedicine. 2002;9:69e74.