Effect of Pimpinella anisum and Salvia officinalis Extracts on Developing Liver of Chick Embryo: 14-Day Incubation

 

 

 

 

 

*E-mail: [email protected]

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Abstract

The present study was designed to evaluate the effects of P. anisum and S. officinalis extracts on the liver of chicken embryos. Eighty fertilized chicken eggs were divided into eight groups.  The first group (C) served as a control group. The second, third, and fourth groups (P1, P2, and P3) of high, medium, and low concentrations of P. anisum aquatic extracts. The fifth, sixth, and seventh groups (S1, S2, and S3) of high, medium, and low concentrations of S. officinalis aquatic extracts. The eighth group (M) of medium concentrations of P. anisum and S.officinalis aquatic extracts. All groups were injected in ovo (0.1 ml/egg) of aquatic extracts before incubation and the eighth day of incubation. After 14 days of incubation, the eggs were opened and biochemical assays in liver homogenate were made. Also, histological studies were made in the liver. Results revealed that the activity of liver homogenate levels of SOD, GSH, and CAT significantly decreased in P1, S1, and M-treated chicken embryos, while significantly increasing in P2, P3, S2, and S3-treated chicken embryos. A significant increase in liver homogenate levels of MDA was observed in all treatment groups. Histological studies of the liver in medium and low concentrations showed normal structures. Furthermore, the liver sections in P1, S1, and M-treated chicken embryos showed some damage. In conclusion, high concentrations of P. anisum and S.officinalis and the combined group have negative effects on the embryo chick liver. While medium and low concentrations have positive effects due to their antioxidant constituents.

Keywords: Chick embryo liver, Pimpinella anisum, Salvia officinalis, Oxidative stress markers

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Introduction

Herbs are widely used for a variety of therapeutic purposes all over the world (Faizi & Kazmi, 2017). According to a World Health Organization (WHO) survey, roughly 70-80 percent of the global population relies on non-conventional treatment, primary herbal sources (Zaitoun et al., 2019; Galea-Holhoș et al., 2023). The incidence of herbal medicine use among Middle Eastern pregnant women ranged from 22.3 percent to 82.3 percent (John & Shantakumari, 2015). In numerous countries, traditional medicine has provided an enticing supply of natural medicines for the treatment of chronic disorders (Asadollahpoor et al., 2017; Popa-Nedelcu et al., 2020).

Anise (Pimpinella anisum) belongs to the Umbelliferae family (Shojaii & Abdollahi, 2012). P. anisum is an annual grassy plant and has a sweet, spicy, and aromatic flavor. It is native to the Near East and commonly farmed in countries along the Mediterranean Sea (Mohamed et al., 2015; Vartolomei et al., 2022).  Extract of P. anisum seed contains trans-anethole as the major compound, estragole, eugenol, pseudo isoeugenol, methyl chavicol, o-isoeugenol, α-zingiberene, Cis-anethole, γ-himachalene, anisaldehyde, Octanol, terpene hydrocarbons, polyenes and polyacetylenes as the principal chemical components (Müller-Fabian et al., 2018; Alafaria et al., 2020).  P. anisum is used to treat dyspeptic symptoms, spasmodic gastrointestinal bloating, effect on menopausal hot flashes, and upper respiratory catarrh (Al-Halaseh, 2013; Jayavel et al., 2022). The aqueous extract of P. anisum may have a protective effect against neurological problems, especially during development (Bekara et al., 2015; Singh et al., 2022). This impact could be attributed to the antioxidant capacity of P. anisum, which was linked to their amount of bioactive chemicals (Shojaii & Abdollahi 2012). The effect of P. anisum can sometimes substitute estrogen in the body, resulting in cause hormone sensitivity and increased risk of cancer (Albini et al., 2014; Akatli et al., 2022). The harmful effect of an excessive amount of anise consumption causes hormonal imbalances and lowers the number of sperm in males (Helal et al., 2019; Asfahani, 2022).

Sage (Salvia officinalis) is a perennial round shrub and belongs to Labiatae/Lamiaceae (Ghorbani & Esmaeilizadeh, 2017). S. officinalis is native to the Mediterranean; it is aromatic and has wonderfully colored leaves (Hamidpour et al., 2014; Çakar et al., 2022). The primary compounds found in S. officinalis flowers, leaves, and stems are eucalyptol (1,8-cineole), α-thujone, β-thujone, camphor, β-caryophyllene, α-caryophyllene (α-humulene), tannic acid, viridiflorol, manool, estrogenic substances, caffeic acid, niacin,12-methoxycarnosinic acid, oleic acid, rosmarinic acid, cornsolic acid, ursolic acid, nicotinamide, chlorogenic acid, malic acid, ursonic acid, succinic acid, fumaric acid, tartaric acid, cornsole, cítric acid and Luteolin-7-O-glucoronide (Garcia et al., 2016).

S. Officinalis is used to treat anxiety, excessive lactation, and perspiration (Lopresti, 2017). Excessive use of S. officinalis might result in a warm feeling, disorientation, tachycardia, and convulsions that are like epilepsy. S. officinalis raises the risk of drug interactions with diabetes and blood pressure medications (Kargozar et al., 2017).

Pregnant and breastfeeding moms who use herbal remedies without understanding the risks to both mother and fetus or child may put themselves and their fetus or their infant in grave danger, including abortion, early birth, uterine hemorrhage, and physical and mental retardation fetus (Eid & Jaradat, 2020).

The liver is the body's biggest organ It is situated between the portal and the general circulation. The liver is in charge of a variety of processes including detoxification, metabolism, immunity, digestion, and vitamin storage (Ramadori et al., 2008).

Materials and Methods

The principles of animal care and use were carefully followed in this study following the guide to the care and use of laboratory animals approved by the College of Science, University of Jeddah, Saudi Arabia.

Eggs

Fertilized chicken eggs were obtained from a local farm in Jeddah City, Makkah region, Saudi Arabia.

Incubation

Eighty eggs with average weight (60-62 g) were weighed in the lab and incubated at 37 °C and 65% humidity, the incubator automatically rotates the eggs once every 90 minutes (El-Borm et al., 2019).

Injection Method

Eggs were washed under high sterilization conditions and sterilized with 70% alcohol, and two holes were made parallel to each other with a sterile needle. Then, the eggs were injected with saline solution, P. anisum aquatic extract, S. officinalis aquatic extract, and their combination. Finally, holes were closed with paraffin wax.

Preparation of Plant Aquatic Extracts

P. anisum Aquatic Extract

1. The P. anisum seeds were washed with water to get rid of dust and left to dry for two days at room temperature with constant stirring so as not to rot.
2. The P. anisum seeds were ground using an electric grinder to get a fine powder.
3. 10 g of P. anisum seeds powder was placed into 250 ml of water and soak it for 24 hours.
4. The extract was filtered using filter paper (90 mm diameter, grade 2).

The preparation of the P. anisum aqueous extract was according to Alsalhi et al. (2016).

S. Officinalis Aquatic Extract

1. Washed the S. officinalis leaves to get rid of dust and let them dry.
2. 250 ml of boiling water was poured over 10 g of S. officinalis leaves and covered for 5 minutes.
3. The solution was filtered through a strainer.

The extract was made as a tea according to its traditional use, according to Salah et al. (2016).

 

The method in which different concentrations were made from both P. anisum and S. officinalis aquatic extracts is the dilution law for the solutions to make three concentrations for each extract (Almakhzumi et al., 2015).

 

V1C1=V2C2 V1=V2C2/C1

(1)

 

High Concentration

 

V1=10 ml*(100%)/100%= 10 ml

(2)

 

Medium Concentration

 

V1=10 ml*(50%)/100%= 5 ml

(3)

 

Low Concentration

 

V1=10 ml*(10%)/100%= 1 ml

(4)

 

Experimental Design

Eighty fresh fertilized chicken eggs (Gallus domesticus) were divided into 8 groups, each containing 10 eggs, and all received repeated doses of different concentrations of P. anisum and S.officinalis aquatic extracts before incubation and the eighth day of incubation. A total of 80 fertilized eggs were included.

1. Group C was injected in ovo (0.1 ml/egg) of saline solution (Control group).
2. Group P1 was injected in ovo (0.1 ml/egg) of high concentration of P. anisum aquatic extract.
3. Group P2 was injected in ovo (0.1 ml/egg) of medium concentration of P. anisum aquatic extract.
4. Group P3 was injected in ovo (0.1 ml/egg) of law concentration of P. anisum aquatic extract.
5. Group S1 was injected in ovo (0.1 ml/egg) of high concentration of S.officinalis aquatic extract.
6.  Group S2 was injected in ovo (0.1 ml/egg) of medium concentration of S.officinalis aquatic extract.
7. Group S3 was injected in ovo (0.1 ml/egg) of law concentration of S.officinalis aquatic extract.
8. Group M was injected in ovo (0.1 ml/egg) of medium concentrations of P. anisum and S.officinalis aquatic extracts.            

 

Collecting Embryos and Specimens

The eggs designated for the study were opened on the 14^th day of incubation, and the live embryos were anesthetized using ether and dissected to remove the liver. A portion of liver tissue from all groups was collected and fixed in 10% formalin for histological studies. Also, a portion of liver tissue from all groups was frozen at -80 °C for the preparation of tissues homogenate with 0.1 M sodium phosphate buffer (pH 7.4). The frozen liver was homogenized for biochemical studies, then centrifuged for 15 min at 10,000 revolutions per minute (rpm) at 4°C. After the supernatant was separated and aliquoted, it was stored at -80 °C for biochemical assay.  

Liver Oxidative Markers Estimation

Fluorometric Assay kits were used to measure malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), and catalase (CAT) were measured according to methods of Ohkawa et al. (1979), Jing and Zhao (1995), Ellman (1959), Aebi (1984), respectively.

Histological Studies

After fixation of liver tissues in formalin saline (10%) for 24 hours, they were subjected to serial alcohol dilution for dehydration. Liver tissues were immersed in paraffin, cut into 5 µm thick sections, and stained in hematoxylin and eosin (H&E) for examination by a light microscope (Humason, 1979).

Statistical Analysis

The statistical analysis was made using Statistical Package for Social Science program, (SPSS version 25). Values expressed as mean +/- standard error. The difference between experimental groups was made using One Way ANOVA (Tukey test).  P-value < 0.05 was recognized as a significant value.

Results and Discussion

 

Liver Oxidative Stress Markers

The levels of MDA in the liver homogenate were significantly increased in P1, P2, P3, S1, S2, S3, and M groups compared with the control group (P <0.05) (Figure 1). The levels of SOD, GSH, and CAT in the liver homogenate were significantly increased in P2, P3, S2, and S3 groups compared with the control group (P <0.05) and was significantly decreased in P1, S1, and M groups compared with control group (P<0.05) (Figures 1 and 2).

a)

b)

Figure 1. Effects of Pimpinella anisum and Salvia officinalis aquatic extracts on MDA levels (µmol/mg) in liver homogenate and SOD levels (U/mg) in liver homogenate of 14-day-old chick embryos of different groups.

 

a)

b)

Figure 2. Effects of Pimpinella anisum and Salvia officinalis aquatic extracts on GSH levels (U/mg) in liver homogenate and CAT levels (U/mg) in liver homogenate of 14-day-old chick embryos of different groups.

 

Histological Studies

 

Liver

 

Control Group

Light microscopic observation of the control liver of 14-day-old chick embryos (C group) showed normal structure. The hepatic tissue appeared dense. The hepatic cords appeared radially arranged and convergent around the central veins. The central veins are interspersed with narrow sinusoids with voids and irregular edges. The sinusoids are lined by endothelial cells and Kupffer cells. Also, some red blood cells were seen (Figure 3).

Combined Group

Comparing the histological structure of the liver of chick embryos injected with repeated doses of mid concentrations of P. anisum and S.officinalis (M group) before incubation and the eighth day of incubation revealed that some hepatocytes appear healthy and the others damaged. Furthermore, relative deformation of the cells surrounding the central veins was seen. The sinusoids were narrow and lined by endothelial cells and Kupffer cells. Also, some red blood cells were seen inside the sinuses. In addition, some hepatocytes showed degeneration (Figure 3).

P.anisum Groups

The chick embryos injected with repeated doses of high concentration of P. anisum (P1 group) before incubation and on the eighth day of incubation showed hemolytic changes. The hepatocytes were radically arranged around the central vein. Red blood cells were seen inside the central vein. The sinusoids are lined by endothelial cells and Kupffer cells with relative degeneration. Moreover, lipid droplets were seen (Figure 3).

On the other hand, the histological structure of the liver of the groups treated with mid and low concentrations of P. anisum (P2 and P3 groups) before incubation and the eighth day of incubation revealed that the liver structure appeared similar to the control group. In the P2 group, relative degeneration and deformation of blood sinusoids and separation of cell lining of the sinusoids and lipid droplets were seen (Figure 3). While in the P3 group, relative degeneration and deformation of blood sinusoids were seen (Figure 3).

S.officinalis Groups

The histological structure of the liver of the group treated with a high concentration of S.officinalis (S1 group) before incubation and the eighth day of incubation showed that the tissue was affected and differed in its shape compared to the control group. There was congestion in the central veins. Moreover, blood sinusoids appeared congested, and the separation of cells lining the sinusoids and lipid droplets was seen. Also, the tissue revealed degeneration and pyknotic in hepatocytes (Figure 3). On the other hand, the histological structure of the liver of the groups treated with mid and low concentrations of S.officinalis (S2 and S3 groups) before incubation and the eighth day of incubation revealed that the liver structure appeared similar to the control group. Furthermore, relative degeneration and separation of the cell lining of the sinusoids and lipid droplets were seen (Figure 3).

a)

b)

c)

d)

e)

f)

g)

h)

Figure 3. A photomicrograph of a transverse liver section in the 14-old chick embryo (H&E40x). C: (control group) The hepatic cords, consisting of solid and lumenated cords, were radially arranged and convergent around the central veins, surrounded by narrow blood sinusoids and endothelial and Kupffer cells. M: The study reveals unclear types of hepatic chords, with some hepatocytes appearing healthy while others degenerated, and blood sinusoids were narrow with red blood cells. P1: The hepatic tissue showed hemolytic changes and degeneration, while lipid droplets and red blood cells were observed in the central veins. P2: Some hepatocytes appeared healthy, while others degenerated, causing blood sinusoids to deform, cell lining separation, and the presence of lipid droplets. P3: Some hepatocytes appeared healthy, and others relatively degenerated (D). Also, some deformation of blood sinusoids was seen.S1:Hepatic tissue exhibited hemolytic changes, including hepatocyte degeneration and pyknosis, congestion (ç) in the central vein and blood sinusoids, and lipid droplets (0) In some cells. S2 and S3: The hepatic tissue resembled the control group, with some sinusoids lining cells separating (é) and hepatocytes degeneration and lipid droplets observed.

Chick embryos were chosen in this study due to the lack of research on the effect of P.anisum and S.officinalis extracts on embryos in terms of morphology, histology, and physiology. The selection of chick embryos has several reasons, most notably the ease of access, incubation, and determining age (Dakhel et al., 2021). The embryos were opened at the age of 14 days, and the reason for this is that from the age of 12 days, all organs have been fully developed (Abdrabou et al., 2022).

Oxidative stress is caused by an imbalance between antioxidants and free radicals, which cause cellular damage (Surgucheva et al., 2020). In the current study, MDA in the embryos of the experimental groups increased at age (14) days. P1 and S1-treated chicken embryos had the highest MDA levels, followed by M-treated chicken embryos. Then P2, P3, and S2, S3 treated chicken embryos. The SOD, GSH, and CAT's antioxidant levels decreased in P1, S1, and M-treated chicken embryos. Excessive ROS production that is not carefully controlled by antioxidant "detoxifying" mechanisms can potentially harm every part of the cell.  MDA is a result of polyunsaturated fatty acid peroxidation brought on by ROS and is considered a typical indicator of oxidative stress (Chen et al., 2015). Baldissera et al. (2017) reported that levels of antioxidant enzymes SOD, GSH, and CAT decreased, while MDA levels increased due to the effect of monoterpene doses in a study conducted on rat livers.

In the current study, the levels of MDA, SOD, GSH, and CAT increased at age (14) days in the P2, P3, and S2, S3 treated chicken embryos compared to the control group. Several studies reported that P.anisum and S.officinalis extracts contain flavonoids and phenolic acids compounds, which have antioxidant properties (Picon et al., 2010, Sharma et al., 2019). The antioxidant effects of phenolic and flavonoid molecules are mediated by the following: removing reactive nitrogen species (RNS) and ROS and preventing the creation of ROS/RNS by blocking certain enzymes or chelating trace metals involved in free radical production, and enhancing or protecting antioxidant defense (Ghasemzadeh & Ghasemzadeh, 2011).

Furthermore, our results were confirmed by histological examination that revealed the presence of an apparent effect of varying intensity in the liver sections of the chick embryos of the experimental groups. P1 and S1-treated chicken embryos had the most damage rate, followed by M-treated chicken embryos. On the other hand, the damage was minimal in the P2, P3, and S2, S3 treated chicken embryos compared to the control.

The results of a light microscopic examination of the control group showed that the liver is composed of hepatic cords arranged radially around the central vein. The central vein is interspersed with blood sinusoids lined by endothelial cells and Kupffer cells. These results were identical to previous studies (Al-Qudsi & Al-Jahdali, 2012).

Changes in the architecture of the liver are signs of toxicity and harmful effects on the organ (Singh et al., 2016). In the current investigation, P1-treated chicken embryos displayed histological changes in the liver tissue portrayed by a loss in its typical structure, hepatocytes appearing distorted and undifferentiated, degeneration and pyknosis, and congested central vein. On the other hand, P2 and P3-treated chicken embryos had significantly milder effects than P1-treated chicken embryos. These results were in agreement with a previous study (by Latifa and Mustapha, 2021), which observed hepatocyte hypertrophy with cytoplasmic vacuolation and dilation of sinusoids after injection of male mice with different doses of P. anisum extract (250 – 500 – 1000 mg/kg).

In the current study, S1-treated chicken embryos displayed histological changes represented by the widening of blood sinuses, blood congestion, degeneration, pyknosis, and the presence of lipid droplets. On the other hand, S2 and S3-treated chicken embryos displayed fewer histological changes than S1-treated chicken embryos. El-Ghareeb et al. (2016) stated that S.officinalis extract induces several hemorrhagic spots in rat embryos, and the major vein widens with rupture in the endothelial cells lining it, also the increase of macrophages and red blood cells. Monoterpenes are present in the S. officinalis oil and cause alterations in the activities of the liver and kidneys (Wojtunik-Kulesza, 2022).

Cellular degeneration can occur because of inflammatory and immunological responses by chemical compounds, parasitic and viral infections, and damaging agents against the body's cells and tissues (Al-Ghamdi et al., 2017). Furthermore, lipid droplets are formed to counteract cytotoxicity (Zhang & Liu, 2018).

Conclusion

This study revealed that excessive consumption of P.anisum and S.officinalis extracts during pregnancy can have negative effects on the embryo's chick liver. Also, the effect of herbs depends on the time of the dose and the duration of its stay inside the tissue. On the other hand, low concentrations had a positive effect on antioxidant enzyme activities. It is recommended not to overconsume herbs during pregnancy. Moreover, it can be argued that the combination of P. anisum and S.officinalis extracts causes a multiplier effect, it is preferred to use the herbs separately.

Acknowledgments: This review article was part of a master thesis of N.A. The authors thank the College of Science at the University of Jeddah for their support.

Conflict of interest: None

Financial support: None

Ethics statement: Ethical was granted from the Department of Biology, College of Science, University of Jeddah, KSA ethical committee (ethical approval number: HAP-02-J-094).