Phytochemical Analysis and Acute Toxicity/ Lethality Study of Ethanol Extract of Eugenia uniflora Pulp.

Onwudiwe N.N.*, Njoku O.U. and Joshua P.E.

Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria

ABSTRACT:

The phytochemical analysis and acute toxicity (LD50) studies were carried out on the ethanol extract of Eugenia uniflora pulp. The phytochemical results showed the presence of alkaloids, glycosides, flavonoids, tannins, saponins and terpenoids. Results of the acute toxicity study gave LD50 value of 2408.3mg/kg.

KEYWORDS: Phytochemical analysis, Acute Toxicity, Eugenia uniflora, Ethanol.

INTRODUCTION:

Eugenia uniflora is an evergreen shrub that is about 6-8m high. It is a hardy species than can thrive in a variety of habitats both in its native and introduced forms. Eugenia uniflora can quickly reach thick densities which affect light penetrations, subsequently changing microenvironments. It is also known to host recognized pests and pathogens¹.

Eugenia uniflora is considered to be effective in treating many diseases and are popularly used in the Brazilian medicine. A leaf infusion of pitanga is used in Brazil for stomach pain, and as an astringent. In Surinam, the leaf decoction is taken as a cold remedy and, in combination with lemongrass, as a febrifuge. In Java, fruits are used to reduce blood pressure^2,3.

Extract from pitanga leaves are considered to be effective against many diseases and are therefore used in popular Brazilian medicines. In a study, where the volatile constituents of pitanga fruits (Eugenia uniflora L) were trapped on to porapak-Q and eluted with ethyl acetate, and the chemical composition of the extract was analysed by gas chromatography/mass spectrometry. Fifty four compounds were detected, and twenty-nine of those were identified by close matches with standard Ms Spectra⁴. Monosterpenes (75.3% in mass) were found to comprise the largest class of the pitanga fruit volatiles, and some therapeutic constituents of pitanga leaf extract, such as selina-1,3,7(11)-trien-8-one were also found to be present in the fruit volatile extract, suggesting that the fruit may display therapeutic properties similar to those of the leaf extract⁵.

Generally, Eugenia uniflora is economically important in its endemic range as pioneer specie in the resting ecosystem⁶. They prefer fertile, moist soils and partial shade⁷. They are grown in Brazil for its edible fruit which can be consumed fresh as food or made into pie or sauce or preserved whole in syrup. They are often made into Jam, Jelly, relish or pickles. Brazilians ferment the Juice into vinegar or wine⁷.

The leaves could be used as poison; the crushed leaves release pungent oil which is used as insect repellant⁸. Eugenia uniflora is appreciated in ice creams and liquids and are also used as phytocosmetic by the Brazilian cosmetics industry to develop shampoos, hair conditioners, face and bath soaps and perfumes⁹. The bark contains 20 to 28.5% tannins and can be used for tanning leather. The seeds are extremely resinous and toxic and should not be eaten¹⁰. However, sesquiterpenes are identified as the main class of compounds in Eugenia uniflora⁹.

Phytochemicals are naturally and biologically active plant compounds that provide health benefits¹¹. They are found in plant foods and they work together with nutrients and dietary fiber to protect against disease¹². Current studies on phytochemicals and nutrients found in fruits, vegetables and nuts, show that they help in aging process and also reduce the risk of many diseases, like cancer, heart disease, stroke, high blood pressure, cataracts, osteoporosis and urinary tract infection¹³.

Phytochemicals have complement any overlapping mechanisms of action in the body, including antioxidant effects, stimulation of the immune system, modulation of hormone metabolism, as well as antibacterial and antiviral effects¹⁴.

New phytochemicals are being discovered on a daily basis, and plants contain hundreds of thousands of different phytochemicals¹¹. Researchers have long known that phytochemicals provide health benefits for plants, but it is only recently that certain phytochemicals have been recommended for the purpose of disease prevention and treatment for humans¹⁵.

There a literally thousands of different phytochemicals found throughout the plant kingdom¹². These phytochemicals exhibit a wide range of biological activities, arising mainly from their antioxidant properties, anti-inflammatory strengths, and ability to boost, the body’s natural detoxification system. They have been recognized to exert anti-bacterial, anti-viral, anti-cardiovascular disease and anti-cancer activities as well as analgesic, anti-allergic, liver protective, estrogenic and anti-estrogenic effects¹⁶.

Hundreds of phytochemicals are currently being studied for their human health benefits. To better understand the scope of these studies, it might be helpful to know that carrots, orange juice and apples contain 217,170, and 150 phytochemicals in their disease fighting make up. Scientific research is helping us to understand how, and why, the phytochemicals found in color-laden produce, herbs and super foods have such positive effect on health¹⁷.

The Phytochemical database states, “Cancer, in many cases, is a deficiency of antioxidants¹⁸. So is heart disease. Scientist is starting to think of these diseases as a shortage of phytochemicals.

This study aims at determining the phytochemical constituents and the acute toxicity analysis of ethanol extract of Eugenia uniflora fruit pulp.

MATERIALS AND METHODS:

Plant Material:

Fresh fruits of Eugenia uniflora plant were obtained from Anambra State, and identified by Mr Ozioko of the Department of Botany, University of Nigeria Nsukka.

Animals:

Eighteen (18) experimental mice of between 8 and 12 weeks old with average weight range of 30 – 45 g were used. The mice were purchased from the animal house of the Faculty of Biological Sciences, University of Nigeria, Nsukka.

Chemicals/Reagents:

All chemicals used in this study were of analytical grade and products of Sigma GMb’H, England and Merck, Darmstadt, Germany.

Extraction Procedure:

The fresh fruit pulps of Eugenia uniflora were separated carefully from the seed. The pulps were grounded using mortar. A weighed quantity, 174 g were then Macerated in absolute ethanol, which was left to stand for 24 hours, after which the extractive was filtered out with cheese cloth and filter paper. The resulting ethanol extract was concentrated to obtain a semi-solid extract using a rotary evaporator at an optimum temperature of between 40 to 45°C to avoid denaturation of the active ingredients. The weight of the semi-solid extract was determined and the extract subjected to phytochemical and toxicological analyses.

Determination of Yield of Extract:

The percentage yield of the extract was determined by weighing the Eugenia uniflora pulp extract before extraction and after concentration and then calculated using the formula.

Percentage (%) yield =

Phytochemical Analysis:

The Phytochemical tests were carried out based on the procedures outlined by Harborne¹⁹; Trease and Evans²⁰.

Acute toxicity and lethality (LD₅₀) Test:

Investigation on the acute toxicity study (LD₅₀) of the ethanol extract was determined using the Lorke²¹ method.

RESULTS:

Percentage Yield of the Extract:

Table 1: The percentage yield of the alcoholic extract from the Eugenia uniflora pulp.

Initial weight of extract (g)	Final weight of Extract (g)	Percentage (%)
174	25.32	14.55

From the result in Table 1 the (%) yield of the ethanol extract of Eugenia uniflora pulp was found to be 14.55%.

Phytochemical Analysis of the Extract:

The results of the phytochemical analysis show that the alcoholic Eugenia uniflora pulp extract contains alkaloids, glycosides, flavonoids, tannins, saponins, terpenoids and reducing sugar. Resins were not detected during the test.

Table 2: Results of the phytochemical analysis

Constituents	Observation
Reducing sugar	++
Alkaloids	+
Resins	ND
Glycosides	+++
Tannins	+
Saponins	+
Terpenoids	+
Steroids	+
Flavonoids	++

Key: +++ = Relative abundance of compound.

++ = Moderate abundance of compound.

+ = Relative low presence of compound

ND = Not detected.

Acute Toxicity Studies of Eugenia uniflora Pulp

The acute toxicity studies (LD₅₀) of the ethanol extract of E. uniflora showed toxicity at 2408.3mg/kg.

Table 3: Results of the acute toxicity (LD₅₀) of Eugenia uniflora Pulp.

Dose (mg/kg body weight)	No of animals before Administration	No of deaths after Administration
10	3	–
100	3	–
1000	3	–
1600	3	–
2000	3	–
2900	3	1

DISCUSSION:

The reactive oxygen species (ROS) formed in the body is due to exogenous and endogenous factors. They are found to be responsible for many diseases²². Current research is revealing the potentials of phytochemical antioxidants as health benefactors due to their ability to neutralize the oxidant responsible for the onset of cell damage²³. Synthetic antioxidants are harmful to the body and most of the natural antioxidants from plants sources are safer to health and have better antioxidant activity.

The phytochemical screening of the alcoholic E. uniflora pulp extract revealed the presence of alkaloids, glycosides, tannins, saponins, flavonoids, terpenoids and steroids. Flavonoids and glycosides are the main constituent found from the alcoholic extract while alkaloids, tannins, saponins and terpenoids were also found to be present in little quantity. This result compared well with the phytochemical screening found on Eugenia jambolana another member of the Myrtaceae family. From the study it was found out that Eugenia jambolana contains flavonoids, glycosides, alkaloids, saponins, steroids tannins and terpenoids²⁴. Saponins cause heamolysis of the red blood cell and their medicinal value is due to their expectorant effect. They have been found to have a hypercholesterolemic and anti-diabetic property²⁵. Tannins are often perceived as detrimental because of their potential to affect protein digestibility or on metal ion availability. Current studies have shown that saponins, terpenoids, flavonoids, tannins, steroids and alkaloids have anti-inflammatory effect^{26- 28}. Steroids and saponins are equally responsible for central nervous system activities²⁹. The terpenoids have also been shown to decrease blood sugar level in animal studies³⁰. While steroids and triterpenoids show some analgesic properties³¹. From these findings, the extract of Eugenia uniflora fruit pulp could have various medicinal values such as anti-inflammatory, anti-diabetic and analgesic activities and also exhibit central nervous system activity since they contain saponins, flavonoids, tannins, alkaloids, steroids and terpernoids.

From the result of the toxicity screening E. uniflora pulp in mice established an LD₅₀ of 2408.3mg/kg. These LD₅₀ levels are relatively low. It could be suggested that, the alcoholic pulp extract of E. uniflora could be generally regarded as safe²¹. However further studies are needed to ascertain prolonged feeding effect on the organs and of tissues of experimental animals. This again may suggest why people who take cherry do not suffer from any known toxicity.

In conclusion, the results of this research, has shown that the alcoholic extract of E. uniflora fruit pulp has tremendous phytochemicals that have a lot of bio-pharmacological functions. It could be inferred that the alcoholic pulp extract of E. uniflora could be generally regarded as safe as a result of its relatively low LD₅₀ value.

REFERENCES:

1. Global Biodiversity Information Facility (GBIF) (2006). Species: Eugenia uniflora Avaliable on line at http://www.europe.gbif.net/portal/ecat_browser. JSP?Taxon key = 267855.

2. Morton, J. (1987). Eugenia uniflora. In: Fruit of Warm Climates. Morton, J. F. (ed), Julia, F. and Miami, F. L. National biological information infrastructure and invasive species specialist group. Pp.386-388. Available online at http://www.hort.purdue.edu/ newcrop/morton/surinam_cherry.html.

3. Pheloung, P. O., Williams, P. A. and Halloy, S.R. (1999). A weed risk assessment model for use as a biosecurity tool evaluating plant introductions. Journal of Environmental Management. 57: 239 - 251.

4. Alessandra, L. O., Roberta, B. L., Fernando, A. C and Marcos, N. E. (2005). Volatile compounds from pitanga fruit (Eugenia uniflora L).Plant Resources of Brazilian edible fruits. Pirassununga, 225:13635-900.

5. Marcos, N. E., Fernando, A. and Cabral, C. (2005).Volatile compounds from pitanga fruit (Eugenia uniflora L).Plant Resources of Brazilian edible fruit. Pirassununga, sp, Brazil, 225:13635-900.

6. Daehler, C. C., Denslow, L. S., An Sari, and Kuo, A. (2004). A risk assessment system for screening out harmful Invasive Pest Plant Hawaii”s and other Pacific Islands Conservation. Biology, 18: 360 – 368.

7. Csurhes, S. and Edwards, R. (1998). Potential environmental weeds in australia: candidate species for preventative control. canberra, australia. Biodiversity group, environment Australia. Springer Netherlands, 3 :75-88.

8. Kline, W. N. and Duquesnel, J. G. (1996). Management on inclusive Exotic Plants with herbicides in Florida. Down to Earth. 51(2): 1727-46.

9. Rosineia, M. M., Vivian, F. S. C., Ana, C. L. A., Ana, L. P. M. and Claudia, M. R. (2007). Identification of impact aroma compounds in Eugenia uniflora .L (Brazilian pitanga) leaf essential oil. Journal of the Brazilian Chemical Society, 18(1):5053-20.

10. Rifai, M. A. (1992). Eugenia uniflora L.; In coronel, r. e and verheji; e.w. m. (eds). plant resources of south-east asia. no.2; edible fruits and nuts. Prosea Foundation, Bogor, Indonesia. Pp.165 – 166.

11. Conn, E. (1995).The world of phytochemicals. Proceeding of the Tenth Annual Penn State Symposium in Plant Physiology, 1:1-14.

12. Polk, M. (1996). Feast on Phytochemicals. AICR Newsletter. Issues. 54(2):532-40.

13. Smith, T. J. (1998). Renewal: The anti–aging revolution. Emmaus, PA: Rodale Press. Pp 55-56.

14. Craig, W. (1996). Phytochemicals: Guardian of our health. The Journal of the American Dental Association, 97(10):S199 - S204.

15. Fahey, J. and Talalay, P. (1995). The role of crucifers in cancer chemo protection. in phytochemicals and health, D. L. Gustine, H.E. Flores, eds. Rock Ville, M. D: American Society of Plant Physiologists, 89:10377-10389.

16. Fahey, J., Clevidence, B. and Russell, M. (1999). Method of assessing the biological effects of specific plants components. Nutrition Reviews, 57(2):9-16.

17. Joseph, J. A. and Nadeau, D. A. (2002). The colour code, NY: Hyperion Book. 10: 214-215.

18. James, D. (2002). Phytochemical and ethaobotanical database, Avaliable on line a http://www.arsgrin.gov/egi -bin/duke/ farmacy 2. pl.

19. Harborne, J. B. (1984). Phytochemical Methods. A guide to modern technology of plant analysis, 2^nd edition. Chapman and Hall, New York. Pp 88-185.

20. Trease, G.E and Evans, W.C. (1989). Pharmacognosy. 13^th ed. Bailliene Tindall Book Publishers by Cas sell and Collines Macmillian Publishers Ltd London. Pp 1-805.

21. Lorke, D. (1984). A new approach to practical acute toxicity testing. Archives Toxicology, 53: 275-287.

22. Kehier, J. P. (1993). Free radical as mediators of tissue-injury and disease. Critica Review in Toxicology, 23:21-48.

23. Timothy, W. M., Charles, D. J. and David, M. B. (2003). Reaction as OH-Radical with H₂ in Sub-Critical Water Chemical Physics. Letters, 7:144-149.

24. Ruspasinghe, H. P., Jackson, C. J. V., Poysa, C., Di Benado, J. D. and Bewley, J. (2003). Soyasapogenol A and B distribution in Soyabean (Glycine Max L. Merr) in relation to seed physiology genetics variability and growing location. Journal of Agricultural food chemistry, 55 (30) 5894 - 80.

25. Kumar, R., Havarasan, T., Jayachandran, M., Decaraman, P., Aravindhan, N., Padmanabhan and Krishnan, M. R.V. (2009). Pyhtochemicals investigation on a tropical plant, Syzygium cumini from Kattuppalayam, Erode District, Tamil Nadu,South India. Pakistan Journal of Nutrition, 8(1):83-85.

26. Manach, C. F., Regerat, F. and Texier, O. (1996). Bioavailability, metabolism and Physiological impact of 4-oxo-flavonoids. Nutritional Research, 16:517-544.

27. Liu, R. H. (2003). Health benefits of fruit and vegetables that are additive and synergistic combinations of phytochemicals. .American Journal of Clinical Nutrition, 78:517S-520S.

28. Akindele, A. J. and Adeyemi. (2007). Anti-inflammatory activities of the aqueous leaf extract of Byrsocarpus coccineus, Fitoterap. Biochemical Pharmacology, 78: 25-28.

29. Argal, A. and Pathak, A. K. (2006). CNS activities of Calotropis gigantean roots. Journal of Ethnapharmacoloogy, 106:142-145.

30. Luo, J., Cheung, A. and Yevich, E. (1999). Novel terpenoids type quinines isolated from Pycnanthu angolensis of potential utility in the treatment of type 2 diabetes. Journal of Pharmacological and Experimental Therapy, 288:529-534.

31. Malairajan, P., Geetha, G., Jessi, K. V. and Nurasimban, S. (2006). Analgesic activities of some India medicinal plant. Journal of Ethnopharmacology, 19:425-428.

Received on 08.05.2010

Accepted on 10.08.2010

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 2(4): July-Aug. 2010, 336-339