Phytochemical screening and hemolytique activity of some leaves extracts of Lantana camara L.


 Medicinal plants have several therapeutic properties; they have been used for a long time to treat different diseases. Lantana camara L. has been widely used by man for healing these diseases. In this study, four leaves extracts of L. camara were subjected to preliminary phytochemical screening to determine the presence and/or the absence of phytochemical constituents; In addition, they were tested for hemolytic activity on human erythrocytes. This activity is performed using the UV-Vis spectrophotometer method at 520 nm and at five different concentrations (125 µg/ml, 250 µg/ml, 500 µg/ml, 750 µg/ml, and 1000 µg/ml). The phytochemical screening showed the presence of various phytochemical groups such as phenolic compounds, saponins, sterols, tannins, flavonoids, reducing compounds and the absence of alkaloids in the four extracts. These same extracts showed average hemolytic activity sequentially: chloroformic extract, petroleum ether extract, aqueous extract and then methanolic extract. This activity is dependent on the concentration of the extract.


Introduction
For a long time, humans have used plants for therapeutic purposes to treat various diseases such as fever, cold, pain, headache and others [1]. This use is reported in the oldest literature [2]. All the different organs of the plant such as leaves, bark, roots and flowers are used in the treatment of common diseases such Corresponding author: f.talhi@centre-univ-mila.dz Full Paper DOI: 10.2478/asn-2021-0023 ©2021 Konstantin Preslavsky University of Shumen. All rights reserved and left in agitation for 24 h. The residue is re-extracted per 250 ml of methanol for 24 h under the same conditions. Each extraction step is repeated three times with solvent renewal. All three extracts were vacuumconcentrated on rotavapor at 40°C. The three extracts of each solvent were pooled and vacuum-concentrated on rotavapor at 40°C. The four obtained extracts were considered as the stock solution and a dilution series (125 µg/ml, 250 µg/ml, 500 µg/ml, 750 µg/ml, and 1000 µg/ml) was prepared for each.

Phytochemical screening
The chemical characterization tests covered the investigation of the different chemical compounds in all different extracts. These characterizations were made using mainly tube reactions. The results are classified into: • Very positive reaction +++ • Positive reaction + • Negative reaction -

1-Alkaloid test
Precipitation reaction was adapted for the detection of the alkaloids using Dragendorff reagent [19].
5ml of the extract was introduced in test tubes, and then added 2 ml of HCl and 1 ml of Dragendorff reagent.
The formation of a red or orange precipitate indicates the presence of alkaloids.

2-Flavonoid test
1ml of extract was added to NaOH. Let it work for 3 minutes. An intense yellow coloring indicates the presence or not of flavonoids [20].

3-Tannin test
The addition of 2% iron trichloride (FeCl 3 ) to the test tube with 2 ml of extract can detect the presence or the absence of tannins. The color turns black brown in the presence of gallic tannins (hydrolysable tannins) and heartworm blue in the presence of catechic tannins (condensed tannins) [21].

4-Steroid test
Dissolve the extracts in 1ml of chloroform and add 1 ml of concentrated sulfuric acid. A positive test is revealed by the appearance of a red top layer [21].

5-Reducing compounds
Their detection consists in treating 1ml of the extract with 2 ml of distilled water and 20 drops of Fehling's solution, then heating. A positive test is revealed by the formation of a red-brick precipitate [22].

6-Saponins test
Mix 1ml of the extract with a few drops of distilled water and then shake strongly the solution. The mixture is left for 15 min. The foam persistence of at least 1 cm indicates the presence of saponins [23].
Corresponding author: f.talhi@centre-univ-mila.dz Full Paper DOI: 10.2478/asn-2021-0023 ©2021 Konstantin Preslavsky University of Shumen. All rights reserved  phenolic compounds, tannins, terpenoids, sesquiterpenes, etc., which cause a definitive pharmacological action on the human body. The results obtained in this study are similar to those of Oyedara [25], Naeem [26] and are somewhat similar to those of Jo-Ann T. Salada [27] on the essential oils of the leaves of L. camara and those obtained with Tripathi [28].
The hemolytic activity of any compound is an indicator of general cytotoxicity to normal healthy cells [29]. The medium hemolytic effect of the four extracts indicates their medium cytotoxicity to human Corresponding author: f.talhi@centre-univ-mila.dz Full Paper DOI: 10.2478/asn-2021-0023 ©2021 Konstantin Preslavsky University of Shumen. All rights reserved erythrocytes [1]. This test is useful in determining whether cytotoxic activity is related to direct damage to the membrane or not. Red blood cells are among the most commonly used cells in the toxicity assessment because of their availability, and the ease of their monitoring during cell lysis through the release of hemoglobin.
These globules were used as a good cell model to assess the cytotoxicity of different molecules, particularly those isolated from medicinal plants, in order to determine the toxicity of these plants [30]. camara which acts as a secondary metabolite modifying the surface tension of the extracellular medium [31,32]. Since saponins are terpenic components, the hemolytic effect of this plant can be explained by the presence of these molecules, which have the ability to induce pore formation through cell membranes, resulting in haemolysis and release of hemoglobin into plasma [33].
The hemolytic activity of saponins is considered the result of affinity of the aglycone fraction of these molecules to membrane sterols, especially cholesterol by forming an insoluble complex (saponin-cholesterol micelles), which leads to the destabilization of the lipid bilayer with permanent pore formation in the membrane, and hence to cell lysis [34]. Medicinal plants, despite their therapeutic effects, should be used with the utmost caution as they may have a risk of toxicity [35].