Teratogenic Potential of Solenstemma Argel Extract in Wistar Albino Rats

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Teratogenic Potential of Solenstemma Argel Extract in Wistar Albino Rats

https://doi.org/10.21203/rs.3.rs-847368/v2

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The majority of people in Africa receive their basic health care through herbal treatments. Herbal medicine may negatively impact fetal development irreparably.

This study examined the teratogenic potential of Solenstemma argel extract in pregnant rats. Pregnant rats were treated with Solenstemma argel from 7th to 16th day of gestation. The dosage used was 250 mg/kg, intraperitoneal.

Solenstemma argel extract treated group showed fetal abnormalities appeared as body hemorrhage, limbs abnormalities and resorption of fetuses. These appears in 25% of the fetuses (P-value = 0.01) which is significantly differed from control group. Furthermore, histopathological findings of liver sections from fetuses of Solenstemma argel - treated mothers showed loose liver texture and hepatocytes hemorrhage.

In this study, we conclude that the use Solenstemma argel extract during the organogenesis period in pregnant rats has the potential to cause teratogenic effects, as well as abnormalities in liver histopathology.

Integrative & Complementary Medicine

Teratogenic

rats

Solenstemma argel

In Africa, nearly 80% of the population relies largely on herbal medicine to provide basic healthcare (1). Insufficient quality control and safety are the most common problems with using herbal medicine (2). Consequently, herbal remedies need to be standardized to ensure their safety and effectiveness (3).

Solenstemma argel is found in tropical Africa in the desert area of Mali, Niger, Chad and Sudan. It is also widely distributed in Algeria, Libya, Egypt and Saudi Arabia.

In Sudan, Solenstemma argel is cultivated under irrigation for the production of leaves. In northern and central Sudan, the flowering aerial parts are sold in the local markets for medicinal use.

Murwan and Murwa reported that S. argel contains various percentages of minerals, carbohydrates and proteins(4), together with a number of organic compounds including flavonoids, kaempferaol, quercetin, rutin, flavonols, flavanones, chalcones and alkaloids (5).

Phytochemical studies of the leaves, stems and flowers showed the existence of -amyrin and -amyrin, -sitosterol, 7- methoxy-3 -22 -dihydroxy-stigmastene, ethoxy derivative of vangurolic acid, an unidentified sterol, flavonoids and saponins in different organs, alkaloids and/or nitrogenous bases in the leaves, stems and flowers have been reported also (6).

Also, proteins, sugars, fiber, and vitamins are present with minerals Na⁺, K⁺, Ca⁺⁺, Ni⁺³, Mg⁺² and P⁺³ (7).

Solenstemma argel has a wide range of traditional uses with deep cultural believes in developing countries that it is safer to use them even during pregnancy, as well as a lack of access to conventional medication and health care, which promotes their use even more.

Solenstemma argel was used as antispasmodic (8), anti-inflammatory (8,9) and anti- oxidant(10). The plant was used for the treatment of diabetes mellitus (11), and cancer (12,13). Infusion of leaves of the plant was used for the treatment of gastrointestinal cramps, jaundice and urinary tract infections (10). It is also used as anti-colic and anti-syphilitic when used for prolonged period of 40 to 80 days (14,15). Leaves of the plant possess purgative properties (14) and in the crushed form used to treat suppurating wounds (10). The smoke of the plant is also considered useful for nasal congestion of common cold (14). S. argel was reported to reduce aluminum toxicity (16). Pregnane glycosides isolated from this plant were reported to reduce cell proliferation (17). The ethanolic extract of S.argel plant demonstrated presence of antibiotic substances. It was reported to have antibacterial and antioxidant activity (10).

Toxicological study conducted by Shyoub et al to evaluate the acute toxicity of S. argel and to determine the lethal doses of S. argel in albino Canadian rats and local species of rabbits using intra-peritoneal doses. The results showed that the mean lethal dose was 6.35g/kg for the rabbits and 5.49g/kg for the rats. Another toxicological parameter determined was the median lethal dose (LD50) was 5.0 g/kg in albino rats (6).

To the best of our knowledge, no studies have been conducted on the teratogenic potential of Solenstemma argel extract and among its wide range of traditional uses as herbal medicine, it has a high probability of being used by pregnant women. Therefore, our study aimed to investigate adverse effect on fetuses.

Experimental Animals

Healthy Female Wistar rats weighing 150–200g were used. The rats were housed in groups, under controlled conditions of temperature (22 ± 1 °C) and relative humidity (~ 50 %) as well as 12-hour light / dark cycle. Animals were allowed food (standard laboratory rodents chow) and water ad libitum.

the animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals.

Plant material

Solenstemma argel leaves were brought from Omdurman market, the plant then taxonomically authenticated at the herbarium of medicinal and aromatic plant research institute (MAPRI).

Preparation of the Solenstemma argel extract

Solenstemma argel plant sample was grounded using mortar and pestle and successively extracted by soaking 96 % ethanol for about seventy-two hours with daily filtration and evaporation. Solvents were evaporated under reduced pressure to dryness using rotary evaporator apparatus and the extracts were combined together. Extracts allowed to air till complete dryness (18).

Phytochemical screening tests

Test for unsaturated sterols and triterpenes

Ten milliliters of Solenstemma argel extract were evaporated to dryness on a water bath and the cooled residue was stirred several times with petroleum ether to remove most of the coloring materials. The residue was then extracted with 20 ml chloroform. The chloroform solution was dehydrated over sodium sulphate anhydrous. Portion of 5 ml of the chloroform solution was mixed with 0.5 ml of acetic anhydride followed by two drops of concentrated sulphuric acid. The gradual appearance of green, blue pink to purple color was taken as evidence of the presence of sterols (green to blue) and or triterpenes (pink to purple) in the sample.

Test for alkaloids

About 7.5 ml of the Solenstemma argel extract were evaporated to dryness on a water bath. 5 ml of 2N HCl was added and stirred while heating on the water bath for 10 minutes, cooled, filtered and divided into two test tubes.

Few drops of Mayer’s reagent were added to one test tube while in the other tube few drops of Valser’s reagent were added. A slight turbidity or heavy precipitate in either of the two test tubes was taken as presumptive evidence for the presence of alkaloids.

Tests for Flavonoids

A volume of 17.5 ml of the Solenstemma argel extract was evaporated to dryness on a water bath, cooled and the residue was defatted by several extractions with petroleum ether. The defatted residue was dissolved in 30 ml of 80% ethanol and filtered. The filtrate was then used for following tests:

One ml of 1% aluminum chloride solution in methanol was added to 3 ml of the filtrate. Formation of a yellow color indicated the presence of flavonoids, flavones or and chalcone.

One ml of 1% potassium hydroxide solution was added to 3 ml of the filtrate. A dark yellow color indicated the presence of flavonoids compounds (flavones or flavonenes), chalcone and or flavonols.

Tests for Tannins

Seven milliliters of the Solenstemma argel extract were evaporated to dryness on water bath. The residue was extracted several times with N-hexane and filtered. The insoluble residue was stirred with 10 ml of hot saline solution. The mixture was cooled, filtered and the volume of the filtrate was adjusted to 10 ml with more saline solution. The solution was then used for following tests:

few drops of gelatin salt reagent were added to 5 ml of the solution, Immediate formation of a precipitate was taken as evidence for the presence of tannins in the plant sample.

few drops of ferric chloride test reagent were added to 5 ml of the solution, The formation of blue, black or green was taken as evidence for the presence of tannins.

Test for Saponins

One gram of the original dried powder Solenstemma argel leaves was placed in a clean test tube. 10 ml of distilled water was added and the tube was stoppered and vigorously shaken for about 30 seconds. The tube was then allowed to stand and observed for the formation of honeycomb. The appearance of honeycomb, which persisted at least for an hour, was taken as evidence for the presence of Saponins.

Test for anthraquinone glycosides

Ten grams of the powdered Solenstemma argel leaves were boiled with 10 ml of 0.5 N KOH and 1 mL of 3% hydrogen peroxide solution. The mixture was extracted by shaking with 10 mL of benzene. 5 mL of the solution was shaken with 3 mL of 10% ammonium hydroxide solution and the two layers were allowed to separate. The presence of anthraquinone was indicated if the alkaline layer was found to have assumed pink or red color.

Test for cumarins

Three grams of the original powdered plant sample were boiled with 20 mL distilled water in test tube. A filter paper spotted with 0.5 N KOH was attached to the test tube. The filter paper was allowed to be saturated with vapor and then inspected under UV light. Absorption of UV light indicate the presence of cumarins.

Induction of pregnancy

Adult females Wistar rats weighing 150–200 g was allowed to mate with proven male rats in the ratio of 2 females (in estrus cycle) to 1 male/cage. Vaginal smear for sperm detection was taken as evidence for copulation and that day was designated as day 1 of pregnancy. We adopted that method which described by Al-Harbi et al and (19) and ElTahir et al (20), with slight modifications.

Experimental design and treatment protocol:

This study was carried out in accordance with ARRIVE guidelines for the reporting of animal experiments. and approved by the Ethical Research Board (ERB), Al- Neelain University.

The selected pregnant rats were divided into two groups of 7 animals each. The first group received normal saline and served as control. Rats in second group received Solenstemma argel extract intraperitoneal from day 6 to day 15 of pregnancy [ period of organogenesis in rats(20)].

Fetuses were removed on 21^st day of pregnancy by caesarian section under light diethyl ether anesthesia. Litter size, alive and dead fetus number of resorptions were recorded. Fetal and placental weights were also recorded. The fetuses were examined for external gross malformation , and histopathological examination of the liver.

Histopathological examination

Liver samples removed from the fetuses by dissection and immediately fixed in 10% neutral buffered formalin, and then embedded in paraffin wax, sectioned at 5µm and stained routinely with hematoxylin and eosin (H&E) (21).

Statistical work:

The results were analyzed using IBM SPSS Statistics version 24. Results summaries were presented as mean ± Standard Error of the Mean (S.E.M.). Differed statistical tests (t-test, and Chi square test,) were performed. Difference between groups was considered statistically significant at P-value < 0.05. Charts and graphs were drawn by graph pad prism version 9.2.0.

Phytochemical screening

A preliminary phytochemical analysis conducted on the S. argel extract revealed that the plant contains high content of flavonoids, tannins and steroids and moderate contents of Saponins with traces of triterpenes and cumarins, whereas Anthraquinone glycosides are completely absent (Table 1).

Maternal weight:

Maternal weight from day1 to day 21of gestation, in both control group and S. argel treated group, showed a significant increase (P-value = 0.010* and 0.007*) respectively, (Fig. 1).

Pregnancy percentages and litter size:

In both of control group and S. argel treated group 71.43% of the mated rats have life fetuses whereas 28.57% of the mated rats deprived of fetuses (no significance different in the pregnancy percentage between the control group and S. argel treated group. P-value =1.00), Fig. 2.

There was no significant effect of S. argel extract on maternal litter size percentages. P-value = 8.33 (Fig 3).

Fetal and placental weight:

There was no significant difference in fetal and placental weights between control and S. argel group. P-value = 0.722 & 0.917 respectively (Fig. 4).

Fetal abnormalities:

The percentage of abnormal to normal fetuses showed significant difference (P-value = 0.010) between the control group fetuses and S. argel treated group fetuses, However, 25% of the fetuses from the group of S. argel treated mothers for were abnormal. (Fig. 5).

Abnormalities of fetuses appeared in the form of upper or lower limb abnormalities or absent limbs, and body hemorrhage and subcutaneous bleeding. Resorbtion of fetuses was reported also. As presented on Fig. 6 (A.B, C, D, E, F and G),

Histopathological findings

Histopathological examination of the fetal liver sections in the control group showed dense liver texture (compact) with high erythropoietic activity and numerous megakaryocytes. Whereas liver sections from fetuses belong to S.argel treated mothers showed loose liver texture, hepatocyte vaculation, nuclear vesiculations, hepatocyte hemorrhage and erythropoietic activity observed (Fig 7. A, B C, D, E, and F).

Table 1 phytochemical screening of Solenstemma argel extract

Phytochemical component	Result
Alkaloids	+
Anthraquinone glycosides	-
Cumarins	+
Flavonoids	+ + +
Saponins	+ +
Tannins	+ +
Triterpenes	+
Steroids	+ +

(-) = Negative (+) = Trace (++) = Moderate (+++) = High

Herbal medicines are widely used as traditional remedies that have been consumed more frequently in recent decades (22). Herbal medicines can have negative impacts on the health of the pregnancy that may extent to teratogenicity in a dose related manner during specific gestational period (23). Due to these concerns, researchers have been more cautious when studying the effect of herbal extracts during pregnancy (23,24).

This study aimed to explore the teratogenic potential of the crude extract of S. argel in pregnant rats. Extract phytochemical screening revealed high flavonoid content and moderate saponin content. The results are consistent with the findings of Kamel Ms. and Shyoub et al (5,6). High contents of tannins and steroids, traces of triterpenes and cumarins were detected, where Anthraquinone glycosides are completely absent.

Studies on animal models often cannot distinguish between direct and indirect effects of substances on embryos. Developmental toxicity is frequently related to maternal toxicity in many cases(25).

In our study, administration of S. argel extract in dose of 250 mg/kg, intraperitoneally, did not exert any signs of maternal toxicity or maternal dietary restriction. This evident by the significant increase in maternal weight of the S. argel treated group during the gestation period from day 1 to day 21. Comparing the increase in maternal weight to the control group, this can be a predictor of normal pregnancy progression and fetal growth. Several studies have linked maternal dietary restriction to adverse newborn outcomes in pregnant animals. It has been reported that a 10% decrease in normal food intake is associated with an increased risk of maternal toxicity, as well as developmental problems(26).

According to pregnancy percentage for mated rats and litter size percentages for the S. argel treated group compared to the control group, it could be seen that both groups had the same percentage of pregnant rats carrying live fetuses with no significance difference in litter size. Based on these results, pregnancy index and litter size were not affected by extract administration. These results are in harmony with the findings by Weidner et al. (27) who investigated the teratogenic potential of Zingiber officinale extract in rats that share the phytochemical components of Saponins and flavonoids with S.argel (28).

The fetal and placental weight did not indicate abnormal patterns or significant differences from controls, reflecting normal development of the offspring of the treated group; this corresponds with the follow up of maternal weight during the gestation period mentioned above.

The gestation period between days 6 through to 15 is the period of organogenesis. It is more common for congenital anomalies to occur during this period of exposure to teratogens than spontaneous abortion or resorption of the embryo(29).

Our study found that 25% of the fetuses from the S. argel treated group were abnormal, which is significantly different from the control group. The most common abnormalities were hemorrhage in fetal bodies, external morphological abnormalities, and fetus resorption. A study on Melia azedarach L leaves extract on mice that share most of the active components of our studied plant such as alkaloid, flavonoid, Saponins, and steroid have identical traits in teratogens (30). Examination of fetal liver sections of S.argel treated mothers in our study, showed loose liver texture, hepatocyte vaculation, nuclear vesiculations and hepatocyte hemorrhage, that is totally in line with histopathological results of the study by Shyoub et al. (6) for evaluation of acute toxicity of S.argel on Nubian goats.

In summary administration of S. argel extract to female pregnant rats results in fetal hemorrhage, limbs abnormalities (missed limbs), resorbtion and loose liver texture with hepatocyte hemorrhage.

In conclusion, the use of S. argel extract during the organogenesis period of pregnancy in rats induced teratogenic effects, and liver histopathological abnormalities. We recommend, to conduct further studies to test which of the phytochemical components of the plant is responsible for the teratogenic effect.

S. argel: Solenstemma argel

H&E: hematoxylin and eosin.

ERB: Ethical Review Board

ARRIVE: Animal Research: Reporting of In Vivo Experiments

Authors’ contributions

Nazik Mohamed Elamin Suliman Mustafa carried out the experimental part and wrote the manuscript editing tables and figures.

Tarig Mohamed Hashim Elhadeyah conception design of the experiment revised the manuscript.

Nafisa Abo Obieda Osman carried out the statistical analysis of data.

Ahmed Abdel Rahim Gameel carried out the histopathology part of the experiment.

Shahenaz Satti wrote and revised the final manuscript.

All authors read and approved the final manuscript

Acknowledgements

Not applicable.

Availability of data and materials

All data analyzed in this study is available from the corresponding author on

reasonable request

Funding

The research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Ethics approval and consent to participate

The experimental procedures and methods were in accordance with the international guidelines of the care and use of laboratory animals in scientific investigations and Ethical approval for this study was obtained from the Ethical Review Board (ERB)- Al-Neelain University- Sudan.

All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments.

Consent for publication

All authors declare no conflict of interest.

Competing interests

The authors declare that they have no competing interests

Authors' information

¹Department of Pharmacology, Faculty of pharmacy, Al-Neelain University, Khartoum, Sudan, Email: [email protected].

²Department of pharmaceutical chemistry, Faculty of pharmacy, Al-Neelain University, Khartoum, Sudan

³Department of Physiology, Faculty of Medicine, Al-Neelain University, P.O. Box: 11121, Khartoum, 12702, Sudan

⁴Department of veterinary pathology, Faculty of veterinary Medicine, University of Khartoum, Khartoum, Sudan

⁵Department of Pharmacology, Faculty of pharmacy, International University of Africa.

1. Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bull World Health Organ. 1985;63(6):965–81.

2. James PB, Wardle J, Steel A, Adams J. Traditional, complementary and alternative medicine use in Sub-Saharan Africa: A systematic review. BMJ Glob Heal. 2018;3(5).

3. Sukandar EY, Safitri D. Evaluation of teratogenic effect of tempuyung (Sonchus arvensis) extract on wistar rats. Int J Pharmacogn Phytochem Res. 2016;8(5):761–6.

4. El-Kheir MKS, Murwa AM. Chemical composition, minerals, protein fractionation, and anti-nutrition factors in leaf of hargel plant (Solenostemma Argel). Eur J Sci Res. 2010;43(3):430–4.

5. Kamel MS. Acylated phenolic glycosides from Solenostemma argel. Phytochemistry. 2003;62(8):1247–50.

6. Shyoub Elhassan Mohammed; Haj Elamin. E. Azhari; Dr. Makawy. Abd. A; Dr. M. R. Rasha;Dr.Abd. A. Mona. Determination of acute Toxicity of Solenstemma argel and it is effect on the vital organs and hematological parameters. J Pharm Sci. 2013;3(2):11–7.

7. Ridgway K, Rupp R. The mixing of powder layers on a chute: The effect of particle size and shape. Powder Technol. 1971;4(4):195–202.

8. Innocenti G, Dall’Acqua S, Minesso P, Budriesi R, Micucci M, Chiarini A. Evaluation of muscarinic M3-receptor antagonism of Solenostemma argel leaves. Planta Med. 2010;76(12):634.

9. Innocenti G, Dall’Acqua S, Sosa S, Altinier G, Della Loggia R. Topical anti-inflammatory activity of Solenostemma argel leaves. J Ethnopharmacol. 2005;102(2):307–10.

10. Shafek R, Shaﬁk N, Michael H. Argel Anti Bactrial Activity.Pdf [Internet]. Vol. 11, Asian journal of plant sciences. 2012. p. 143–7. Available from: http://docsdrive.com/pdfs/ansinet/ajps/2012/143-147.pdf

11. Trojan-Rodrigues M, Alves TLS, Soares GLG, Ritter MR. Plants used as antidiabetics in popular medicine in Rio Grande do Sul, southern Brazil. J Ethnopharmacol [Internet]. 2012;139(1):155–63. Available from: http://dx.doi.org/10.1016/j.jep.2011.10.034

12. Nassr-Allah AA, Aboul-Enein AM, Aboul-Enein KM, Lightfoot DA, Cocchetto A, El-Shemy HA. Anti-cancer and anti-oxidant activity of some Egyptian medicinal plants. J Med Plants Res. 2009;3(10):799–808.

13. N. Hanafi and S.Z. Mansour. Antitumer efficacy of salennostemma Argel and/orᵧ-Irradiation Againist Ehrich Carcinoma. J Biol. 2010;10(6):468–79.

14. EL-KAMALI HH, KHALID SA. The most common herbal remedies in Central Sudan. Fitoterapia. 1996;67(4):301–6.

15. Floch L. Medicinal plants for North Africa. Vol. 31, Journal d’agriculture traditionnelle et de botanique appliquée. 1984. 127–127 p.

16. Osman HM, Shayoub M. the Effect of Solenostemma Argel Leaves Extract on Status of the Effect of Solenostemma Argel Leaves Extract on Status of Induced Lipid. 2015;5(March):3828–30.

17. Plaza A, Perrone A, Balestrieri ML, Felice F, Balestrieri C, Hamed AI, et al. New unusual pregnane glycosides with antiproliferative activity from Solenostemma argel. Steroids. 2005;70(9):594–603.

18. Sukhdev Swami Handa, Suman Preet Singh Khanuja, Gennaro Longo DDR. Extraction technologies for medicinal and aromatic plants (United Nations Industrial Development Organisation and the International Centre for Science and High Technology). Vol. 99, International Centre for Science and High Technology-United Nations Industrial Development Organization, area Science Park Padriciano. 2008. 34012 p.

19. Al-Harbi MM, al-Shabanah OA, al-Gharably NM IM. The effect of maternal administration of enalapril on fetal development in the rat. Res Commun Chem Pathol Pharmacol. 1992;77(3):347–58.

20. El Tahir, K.E.H., Al Tahir, A. Y. and Ageel A. Pharmacological studies on Sesame and Nigella sativa fixed oils: effects on the sensitivities of the adrenoceptors, baroreceptors, platelets and uterus of the rat. Saudi Pharm J,. 1999;7:205–15.

21. Mohammadi Z, Azarnia M, Mirabolghasemi G, Shiravi A. Histological changes in the liver of fetuses of pregnant rats following citalopram administration. indian J Pharmacol. 2013;45(5):517–22.

22. Mehrabani G, Aminian S, Mehrabani G, Rabiee M. Dietetic Plans within the Multiple Sclerosis Community: A Review. Int J Nutr Sci. 2019;4(March):14–22.

23. De Santis M, Cesari E, Cavaliere A, Ligato MS, Nobili E, Visconti D, et al. Paternal exposure and counselling: Experience of a Teratology Information Service. Reprod Toxicol. 2008;26(1):42–6.

24. Parivar K, Mohsenikouchesfahani H, MOhammadzadehasl B. Surveying Acyclovir side effects on developing embryos of Balb/C mice strain. Med Sci J Islam Azad Univesity-Tehran Med Branch. 2006;16(4):231–5.

25. Chahoud I, Ligensa A, Dietzel L, Faqi AS. Correlation between maternal toxicity and embryo/fetal effects. Reprod Toxicol. 1999;13(5):375–81.

26. Holson JF, Nemec MD, Stump DG, Kaufman LE, Lindstrom P VB. Significance, Reliability, and Inerpretation of developmental and reproductive toxicity study findings. 2nd ed. Hood RD E, editor. Paper Knowledge . Toward a Media History of Documents. Developmental and reproductive toxicology: a practical approach, 2nd ed. Boca Raton: Taylor & Francis Group.; 2006. 329–424 p.

27. Weidner MS, Sigwart K. Investigation of the teratogenic potential of a Zingiber officinale extract in the rat. Reprod Toxicol. 2000;15(1):75–80.

28. Osabor V, Bassey F, Umoh U. Phytochemical Screening and Quantitative Evaluation of Nutritional Values of Zingiber officinale (Ginger). Am Chem Sci J. 2015;8(4):1–6.

29. Moore KL, Persaud TVN. The Developing Human: Clinically Oriented Embryology [slides]. Saunders; 1993.

30. Sutomo AE, Sitorus TD, Pribadi A. The Teratogenic Effect of The Mindi (Melia azedarach L) Leaves Ethanol Extract on Mice (Mus musculus) Fetus. Althea Med J. 2015;2(2):221–5.

No competing interests reported.

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Teratogenic Potential of Solenstemma Argel Extract in Wistar Albino Rats

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