Scholarly article on topic 'Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats'

Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats Academic research paper on "Veterinary science"

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{"black cumin" / galactagogue / herbal / lactogenic / "milk production" / " Nigella sativa "}

Abstract of research paper on Veterinary science, author of scientific article — Hossein Hosseinzadeh, Mohsen Tafaghodi, Mojdeh Jalal Mosavi, Elahe Taghiabadi

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.

Academic research paper on topic "Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats"

J Acupunct Meridian Stud 2013;6(1):18—23

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Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies

journal homepage: www.jams-kpi.com

I RESEARCH ARTICLE |

Effect of Aqueous and Ethanolic Extracts of Nigella sativa Seeds on Milk Production in Rats

Hossein Hosseinzadeh 1,*) Mohsen Tafaghodi2, Mojdeh Jalal Mosavi3, Elahe Taghiabadi3

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: hosseinzadehh@mums.ac.ir Copyright © 2013, International Pharmacopuncture Institute pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2012.07.019

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 [9—13], antioxidant and antieicosanoid [7,13—15], 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 thymohy-droquinone), alkaloids and terpenoids, carvacrol, t-ane-tholet, 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

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 Khor-assan, were purchased from a local market in Mashhad, Iran. Seeds were identified by Mr Ahi from Pharmacy School ofMashhad, 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 (40—60°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 2—5 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 — 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 — w1)/4. Next, this value

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.

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 Tukey—Kramer. Discrepancies with p < 0.05 were considered significant.

3. Results

3.1. Acute toxicity

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: 3.45—5.18) and 4.9 (95% CL: 4.16—5.19) g/kg, respectively, and the maximum non-fatal doses were 2.5 and 3.1 g/kg, respectively.

3.2. Effect of the aqueous and ethanolic extracts of N. sativa extract on milk production

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)

- Normal saline -Aqueous extract 0.5 g/kg -Aqueous extract 1 g/kg

a ■a 12-

u ft 10-

lie 6-

—I— 11

—I—

—I—

Day of lactation

I Normal saline ¡Aqueous extract 0.5 g/kg 3 Aqueous extract 1 g/kg

£ 6-=

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 = 6. Statistically significant differences are given compared to the control group (ANOVA followed by Tukey—Kramer). *p < 0.05, **p < 0.01, ***p < 0.001. (B) Mean milk production per day. Values are means ± SEM, n = 6. Statistically significant differences are given compared to the control group (ANOVA followed by Tukey—Kramer). *** p < 0.001. ANOVA = analysis of variance; SEM = 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

Normal saline -Ethanolic extract 0.5 g/kg -Ethanolic extract 1 g/kg

a TS 12-

jä 8-

lie 6-

—i— 11

—i—

—i—

Day of lactation

I Normal saline

I-1 Ethanolic extract 0.5 g/kg

^^ Ethanolic extract 1 g/kg

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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 = 6. Statistically significant differences are given compared to the control group (ANOVA followed by Tukey—Kramer). * p < 0.05,

** p < 0.01, *** p < 0.001. (B) Mean milk production per day. Values are means ± SEM, n = 6. Statistically significant differences are given compared to the control group (ANOVA followed by Tukey—Kramer). *** p < 0.001. ANOVA = analysis of variance; SEM = 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 1—5 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 1 —5 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 [2,30—32]. According to data, production of milk in

Normal saline ■ Aqueous extract 0.5 g/kg " Aqueous extract 1 g/kg

* 10 a

Day of Lactation

a, 20'

^ 15' f 10'

Normal saline Aqueous extract 0.5 g/kg

Aqueous extract 1 g/kg

Treatment

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 Tukey—Kramer). * 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 Tukey—Kramer). ** p < 0.01. ANOVA = analysis of variance; SEM = 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].

Normal saline 1— Ethanolic extract 0.5 g/kg >— Ethanolic extract 1 g/kg

a 30 1 a

^ 25 H

20 -15 -

S 10 i CL

■¡а

Day of lactation

I Normal Saline F^l Ethanolic extract 0.5 g/kg OS] Ethanolic extract 1 g/kg

Treatment

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 Tukey—Kramer). (B) Mean weight gain of pup. Values are means ± SEM. Statistically significant differences are given compared to the control group (ANOVA followed by Tukey—Kramer). *p < 0.05. ANOVA = analysis of variance; SEM = standard error of the mean.

One of the various constituents of N. sativa seeds, t-anethole (1—4%) [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 10—12 [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

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.

Acknowledgments

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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