Scholarly article on topic 'Effect of Aqueous and Ethanolic Extracts of Pimpinella anisum L. Seeds on Milk Production in Rats'

Effect of Aqueous and Ethanolic Extracts of Pimpinella anisum L. Seeds on Milk Production in Rats Academic research paper on "Veterinary science"

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{galactagogue / lactogenic / "milk production" / " Pimpinella anisum "}

Abstract of research paper on Veterinary science, author of scientific article — Hossein Hosseinzadeh, Mohsen Tafaghodi, Shirin Abedzadeh, Elahe Taghiabadi

Abstract Pimpinella anisum L. (P. anisum) is used as a galactagogue in traditional medicine; hence, the effect of aqueous and ethanolic extracts of P. anisum seeds on milk production in rats was evaluated. The milk production was assessed by measuring the pups' weights during the suckling period. The intraperitoneal LD50 values of P. anisum aqueous and ethanolic extracts were 4.93 and 3.77 g/kg, respectively. The aqueous (1 g/kg) and ethanolic extracts (1 g/kg) increased the milk production significantly (p < 0.001), with about 68.1% and 81% more milk being produced, respectively, than in the control group. The pups gained weight during the study period with the aqueous (0.5 and 1 g/kg, p < 0.05) and ethanolic (0.5 and 1 g/kg, p < 0.01) extracts. Thus, P. anisum aqueous and ethanolic extracts can increase milk production in rats.

Academic research paper on topic "Effect of Aqueous and Ethanolic Extracts of Pimpinella anisum L. Seeds on Milk Production in Rats"

J Acupunct Meridian Stud 2013;

ELSEVIER

I RESEARCH ARTICLE I

Effect of Aqueous and Ethanolic Extracts of Pimpinella anisum L. Seeds on Milk Production in Rats

Hossein Hosseinzadeh 1'*, Mohsen Tafaghodi2, Shirin Abedzadeh3, Elahe Taghiabadi3

1 Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Nanotechnology Research Center, Pharmaceutical Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

3 Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Available online ■ ■ ■

Received: May 29, 2013 Accepted: Sep 4, 2013

KEYWORDS

galactagogue; lactogenic; milk production; Pimpinella anisum

Abstract

Pimpinella anisum L. (P. anisum) is used as a galactagogue in traditional medicine; hence, the effect of aqueous and ethanolic extracts of P. anisum seeds on milk production in rats was evaluated. The milk production was assessed by measuring the pups' weights during the suckling period. The intraperitoneal LD50 values of P. anisum aqueous and ethanolic extracts were 4.93 and 3.77 g/kg, respectively. The aqueous (1 g/kg) and ethanolic extracts (1 g/kg) increased the milk production significantly (p < 0.001), with about 68.1% and 81% more milk being produced, respectively, than in the control group. The pups gained weight during the study period with the aqueous (0.5 and 1 g/kg, p < 0.05) and ethanolic (0.5 and 1 g/kg, p < 0.01) extracts. Thus, P. anisum aqueous and ethanolic extracts can increase milk production in rats.

* Corresponding author. Pharmaceutical Research Center, Pharmacodynamics and Toxicology Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail: hosseinzadehh@mums.ac.ir (H. Hosseinzadeh). Copyright © 2013, International Pharmacopuncture Institute pISSN 2005-2901 elSSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2013.10.004

ARTICLE IN PRESS

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1. Introduction

Breastfeeding is essential for the survival of newborns. Numerous women with deficient milk production use traditional plants to enhance milk production or yield. The useful activity of some herbs on milk production has been revealed in many reports. It was demonstrated that some plants increase the milk production because of the induction of the lactogenic hormones (prolactin), growth hormone, and casein accumulation in the mammary gland. Prolactin as a lactogenic hormone has a significant role in lactogenesis. In fact, prolactin induces the production of milk proteins in the epithelial cells and secretory tissue proliferation [1]. The milk production assessment in animals such as rats is hard. In several reports, the measurement of pup weight and weight gain is used to determine the milkyield and production. The pup weight and weight gain are usually utilized as indirect items for milk secretion. However, the direct determination of milk yield can be obtained by measuring pup weight during the suckling period that followed the separation time of dams and pups [2].

It was revealed that some plants increase the prolactin liberation, milk production, or growth of mammary gland [1]. P. anisum is one of the herbs is used in folk medicine for many beneficial effects, especially during the lactation time [3]. P. anisum (Umbelliferae family) is an annual plant with white flowers and small seeds, which is cultivated in Iraq, Turkey, Iran, India, and Egypt [4] and it is also a plant rich in volatile oils [5,6]. The seeds of the P. anisum fruit are commercially available and used for flavoring [7]. This plant is cultivated for its aromatic seeds, which is used in medicine as a mild expectorant [8]. Some beneficial and therapeutic activities including those on digestive disorders, gynecologic, and also anticonvulsant, antiasthma, and dyspnea have been shown for P. anisum in Iranian ancient medical books [9]. Many studies have been described that P. anisum or its constituents demonstrated some pharmacological effects such as fungicidal [10,11], antibacterial [12,13], anticonvulsant [4], antioxidant [7], and relaxant effects on the isolated tracheal muscles of guinea pig [14,15], antispasmodic and relaxant effects on rat ano-coccygeus smooth muscle [16], and anesthetic and hypothermic activities [17]. P. anisum seeds consist of several types of compounds, and the effects of this herb are related to these components. Some reports have shown that this plant contains anethole, estragole, methyl chavi-col and anisaldehyde, coumarins, scopoletin, umbellifer-one, sterols, and terpene hydrocarbons [7,18—20].

Owing to the application of P. anisum as a galactagogue in traditional medicine, and the lack of any report of its effect on milk production, the present study investigated the acute toxicity and effect of aqueous and ethanolic extracts of P. anisum seeds on milk production in rats.

2. Materials and methods 2.1. Animals

The study was performed on male and female albino mice, 25 ± 2 g for the acute toxicity study, and lactating rats, weighing 250 ± 25 g and suckling four to six pups each, for the 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 the aqueous and ethanolic extracts of P. anisum

P. anisum seeds, obtained from Razavi Khorasan, were purchased from a local market in Mashhad, Iran. Seeds were identified by Botanists in the Herbarium of Ferdowsi University, Mashhad, Iran, and the voucher sample of the plant is 7701 that was preserved for reference in the herbarium of Mashhad Pharmacy School, Mashhad university of Medical Sciences, Iran. P. anisum seeds were cleaned and dried in shadow and powdered by a mechanical grinder. For the aqueous extract, the seed powder (100 g) was added to 1000 mL of hot water, boiled for 15 minutes and filtered using a cloth. The filtrate was evaporated to dryness under reduced pressure to afford a viscous residue. The residue was suspended in normal saline. For the ethanolic extract, the seeds 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 vacuum at 40 °C. The residue was suspended in saline.

2.3. Acute toxicity

Different doses of the aqueous and ethanolic extracts of P. anisum were administered orally and intraperitoneally into groups of six mice. The number of deaths was counted at 48 hours after treatment. LD50 values and corresponding confidence limits were determined by the Litchfield and Wil-coxon method (PHARM/PCS Version 4).

2.4. The effect of the aqueous and ethanolic extracts of P. anisum extract on milk production

The method is based on Lompo-Ouedraogo et al modification [1]. In brief, 30 lactating dams (250 ± 25 g) at the start of lactation and suckling four to six pups were used for this experiment. Females were divided into five groups of six animals each. Group 1 was given orally 0.5 mL of saline as a 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 female rats were treated daily at 2:30 pm. The production of milk was measured 23 hours after gavage. The milk production was estimated from Day 3 to Day 15 of lactation. Milk yield and dams' weight, and the weight gain of pups were measured every day. During the test period, the pups were weighed daily at 8:30 am (w1) and then separated 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. Then, they were weighed (w3) at 1:30 pm. The milkyield 23 hours after the gavage was estimated as w3 - w2. The daily milk yield was corrected for loss of weight because of the metabolic processes occurring in the pup throughout suckling. 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

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Effect of Pimpinella anisum on milk production in rats

suckling gain. The daily weight gain of pups was measured from the weight of pups at w2.

2.5. Statistical analysis

The mean ± SEM were determined for each study group and tested with analysis of variance followed by the multiple comparison test of Tukey—Kramer. Discrepancies with p values <0.05 were considered significant.

3. Results

3.1. Acute toxicity

No mortality was observed with the aqueous and ethanolic extracts of P. anisum (0.5, 2, 8,16, and 32 g/kg, orally) after 24 and 48 hours of treatment. The intraperitoneal LD50 values of P. anisum aqueous and ethanolic extracts were 4.93 (95% CL: 2.89—8.38) and 3.77 (95% CL: 2.4—5.98) g/kg, respectively, and the maximum nonfatal doses were 2.2 g/kg.

3.2. The effect of the aqueous and ethanolic extracts of P. anisum extract on milk production

The milk production of the aqueous and ethanolic extracts groups was more than that of the control group (Figs. 1 and 2). The milk yield changed from 8.09 ± 0.2, 14.25 ± 0.4, and 14.87 ± 0.35 g/pup per day to 7.3 ± 0.71, 15.4 ± 0.7, and 13.1 ± 0.29 g/pup per day for the controls, and those treated with the aqueous extract at doses 0.5 and 1 g/kg, respectively. The milk yield changed from 8.09 ± 0.2, 11.1 ± 0.4 and 15.89 ± 0.35 g/pup perday to about 7.3 ± 0.71,12.1 ± 0.7, and 12.41 ± 0.26 g/pup per day for the controls, and those receiving the ethanolic extract at doses 0.5 and 1 g/kg, respectively. The milk yield in group received the ethanolic extract at a dose of 1 g/kg was significantly different from Day 3 to the end of test in comparison to the control group (p < 0.05) and in the group that received the ethanolic extract at a dose of 0.5 g/kg was significantly different at Days 12 and 14 of the test in comparison to the control group (p < 0.01) (Figs. 1Aand 2A).

The milk production data 23 hour after gavage showed that the milk production was significantly increased in groups that received the aqueous extract at a dose of 1 g/kg and ethanolic extract at doses 0.5 and 1 g/kg in comparison to the control group (p < 0.001). The mean milk yields for the control group was 7.68 ± 1.2 g/pup and for the groups that were given the aqueous (1 g/kg) and ethanolic (0.5 and 1 g/kg) extracts were 12.85 ± 1.02,10.35 ± 0.99, and 13.58 ± 1.01 g/pup, respectively. The aqueous extract at a dose of 1 g/kg and the etha-nolicextract at doses of0.5and 1 g/kgincreased thedailymilk production to about 68.1%, 37%, and 81.1%, respectively (Figs. 1B and 2B).

The weight of all pups increased during the experiment time (Figs. 3 and 4), and the weight gain in groups received the aqueous (0.5 and 1 g/kg, p < 0.05) and ethanolic extracts (0.5 and 1 g/kg, p < 0.01) showed significant increase in comparison to the control group. The aqueous (0.5 and 1 g/kg) extract induced an elevation in the body weight from 10.5 ± 0.85 to 20.3 ± 1.19 and 12.09 ± 0.87 to

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2 3 4 5 6 7 8 9 10 11 12 13 14 15 Day of lactation

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Figure 1 (A) The effect of aqueous extract of P. anisum at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as mean ± SEM (n = 6). Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). (B) Mean milk production per day. Values are mean ± SEM (n = 6). Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). *** p < 0.001. ANOVA = analysis of variance; SEM = standard error of the mean.

21.2 ± 0.57 g/pup per day, respectively, and the ethanolic (0.5 and 1 g/kg) extract increased the body weight from 11.8 ± 0.5 to 19.1 ± 0.4 and 12.45 ± 0.65 to 20.98 ± 0.87 g/ pup per day, respectively. The level of weight gain for control group was 10.1 ± 0.69 to 18.37 ± 0.68 g/pup per day (Figs. 3A and 4A). The daily weight gain was 14.25 ± 0.86 g/pup for control, 16.35 ± 1.1, and 16.58 ± 1.02 g/pup for the aqueous extract at doses 0.5 and 1 g/kg, respectively and 15.76 ± 1.1 and 16.78 ± 1.09 g/ pup for the ethanolic extract at doses 0.5 and 1 g/kg, respectively (Figs. 3B and 4B).

4. Discussion

P. anisum seeds have been utilized as flavoring and medicinal plants for many times [7]. P. anisum has been applied in traditional remedy for the treatment of some diseases and as a galactagogue [3,8,9]. The result of the

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■ Aqueous extract 1 g/kg

2 3 4 5 6 7 8 9 10 11 12 13 14 15 Day of lactation

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Figure 2 (A) The effect of ethanolic extract of P. anisum at doses 0.5 and 1 mg/kg on milk production 23 hours after gavage. Values are expressed as mean ± SEM (n = 6). Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). (B) Mean milk production per day. Values are mean ± SEM (n = 6). Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). * p < 0.05. **p < 0.01. ***p < 0.001. ANOVA = analysis of variance; SEM = standard error of the mean.

acute toxicity test showed that LD50 values of compounds are different according to species and administration routes. No mortality was observed with the aqueous and ethanolic extracts of P. anisum at doses of 0.5, 2, 8, 16, and 32 g/kg orally. The i.p. LD50 values of P. anisum aqueous and ethanolic extracts in mice were 4.93 and 3.77 g/kg, respectively. According to the toxicity classification, substances with LD50 values in the range of 1—5 g/kg are practically low toxic, and compounds with an LD50 value of more than 5.0 g/kg are considered practically nontoxic [21]. With regard to the LD50 value by the oral route (>5.0 g/kg), this study shows that P. anisum is practically nontoxic in acute oral treatment. The i.p. LD50 values of both extracts were in the range of 1—5 g/kg; thus, P. ani-sum should be considered practically low toxic in acute intraperitoneal administration, and data showed that the toxicity of the ethanolic extract is more than that of the aqueous extract.

The milk production measurements for rats by the weight of pup and weight gains have been used in some studies [2,22—24]. Data showed that the milk production in the female rats receiving P. anisum was considerably higher than in the controls. Besides, the milk yield significantly increased about 23 hours after the treatment of the both the extracts, and the pup growth level improved. In some reports, it was demonstrated that many plants increased milk production in animals through the induction of lacto-genic hormone (prolactin) [1,25]. Some plants contain es-trogenic substances such as anethole that increase the secretion of milk. Structurally, anethole is similar to dopamine and shows a competitive antagonism at the dopamine receptor site. Thus, it can increase the liberation of prolactin and induce the production of milk [26].

P. anisum seeds have many components such as t-anet-hole [18], and the lactogenic activity of this plant may be related to this constituent. In addition, the growth of pups and gain weight in treated groups may be attributed to induction of milk production and milk constituents. This

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Figure 3 (A) The effect of aqueous extract of P. anisum at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are mean ± SEM. Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). (B) Mean weight gain of the pup. Values are mean ± SEM. Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). *p < 0.05. **p < 0.01. ***p < 0.001. ANOVA = analysis of variance; SEM = standard error of the mean.

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Figure 4 (A) The effect of ethanolic extract of P. anisum at doses 0.5 and 1 mg/kg on pup weight 23 hours after gavage. Values are mean ± SEM. Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). (B) Mean weight gain of pup. Values are mean ± SEM. Statistically significant differences are given in comparison to the control group (ANOVA followed by Tukey—Kramer). *p < 0.05. **p < 0.01. ***p < 0.001. ANOVA = analysis of variance; SEM = standard error of the mean.

study indicated that the beneficial activity of P. anisum ethanolic extract on milk production was more than that of aqueous extract, and it is related to the existence of active constituents such as anethole in the ethanolic extract.

It is concluded that P. anisum aqueous and ethanolic extracts increase milk production. This research confirms the traditional use of P. anisum seeds as a lactogenic agent.

Acknowledgments

The authors are grateful for the financial support of School of Pharmacy, Mashhad University of Medical Sciences. This study was part of PharmD thesis.

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