Medicinal plants represent an important source of medically important compounds; since ancient times, medicinal plantshave been used to cure several health problems. Systemic analysis of these plants provides a variety of bioactive molecules for the development of newer pharmaceutical products.^[1]LCis a native of tropical America but now occurs throughout the Philippines in thickets and wastelands at low and medium altitudes.^[2]LC was probably introduced into India before 19^th century, currently LC is found throughout India and known by different name viz, Raimunivya(Hindi), Chaturangi and Vanacehedi(Sanskrit), Arippu and Unnichidi(Tamil), Aripoov, Poochedi, Konginipoo and Nattachedi (Malayalam), Thirei, Samballei and Nongballei(Manipuri), Tantani and Ghaneri (Marathi), Pulikampa(Telegu), Kakke and Natahu (Kannada).^[3-4]

LC is a flowering ornamental plant.It is used in several traditional medicinal preparations and is well-knownfor curing several diseases. From ancient times, flowers were used as pectoral for children, leaves and fruits of that plant can be used externally for various skin diseases, cuts and wounds, and stems and roots are used for gargles and toothaches as a toothbrush.^[5-7]It was traditionally used diaphoretic, carminative, Antispasmodic, tonic, and Antiemetic to treat respiratory infections and disorders (cough, cold, asthma and bronchitis) in the treatment of tetanus, epilepsy, dysentery and gas trophy. ^[8] LC belongs to the family Verbenaceae. It is also known as wild sage,Surinam, Tea plant, Spanish flag and West Indian Lantana.Differentparts of LC are reported to possess essential oil, phenolic compounds, flavonoids, carbohydrates, proteins, alkaloids, glycosides, phenyl ethanoic, oligosaccharides, quinine, saponins, steroids, triterpenes, sesquiterpenoids and tannin as major phytochemicalgroups.^[9-12]Parasitic worms(helminths) of the gastrointestinal tract are pathogens of paramount global importance. Over a billion people, mainly in developing countries, are transmitted to be infected with soil-transmitted helminths. At the same time, helminth infection is also a serious problem in livestock production worldwide, causing significant economic losses and threatening food security.^[13]

The Word “anthelmintic” is from the Greek word “anti,” which means “against,” and helminths means “worm,” which means to kill or wipe out worms or parasites. Anthelmintics are drugs that either kill (vermicide) or expel (vermifuge) infesting helminths. Helminthiasis or helminth infection is a parasitic disease of humans and other animals in which part of the body is infected with parasitic worms. They often live in the gastrointestinal tract of their hosts but may also stay in other organs.Scientific studies have shown that many plants used in human ethnomedicinal practice showed huge pharmacological activities. The human body, GIT, is the abode of many helminths, but some also live in tissues,or their larvae migrate into tissues. They harm the host by depriving them of food, causing blood loss, injury to organs, intestinal or lymphatic obstruction, and secreting toxins.^[14]

The toxicity of the active molecule is a key factor in the design, and hemolytic activity represents a useful starting point in this regard; it provides primary information on the interaction between molecules and biological entities at the cellular level. The haemolytic activity of any compound is an indicator of general cytotoxicity towards normal healthy cells.^[15] Usually, saponins present in the plants showed hemolytic activity by creating changes in theerythrocyte’smembrane.^[16]

Plant extracts can positively affect the red cell membrane, and many plants have serious adverse effects, which include induction of hemolytic anaemia. Therefore, many commonly used plants must be evaluated for their potential hemolytic activity.

Aim and Objectives

Aim: This study aims to investigate the L. camara Leaves for theiranthelmintic and hemolytic activity using Earthworm (Eisenia Fetida) and human erythrocytes, respectively.

Objective:

• To present the Anthelmintic activities and potential health benefits of Lantana camara.
• To perform the Hemolytic activity of Lantana camara.

To provide an overview of the chemical composition of Lantanacamara.

Plant Profile

       
           
       
Fig .1. Lantana camara

Synonym: Spanish Flag, Tick berry, Shrub Verbena, Kakke,      Chadurangi

Biological source:

It is an ornamental flowering plant of Lantana camara Linn belonging to the Verbenaceae family.^[17]

Botanical classification ^[18]     

Kingdom: Plante

Subkingdom: Tracheobionta

Super division: Spermotophyat

Division: Magnoliophyta

 Class: Magnoliopsida

 Subclass: Asteridae

 Order: Lamiales

 Family: Verbenaceae

 Genus: Lantana

 Species: L. camara

Eisenia fetida

       
           
       
Fig No. 3

Scientific classification

Domain: Eukaryote

Kingdom: Animalia

Phylum: Annelida

Clade: Pleistoannelida, Sedentaria.

Class: Clitellates

Order: Opisphopora

Family: Lumbricidae

Genus: Eisenia

Species: E. fetida

Synonyms: Eisenia foetida (older spelling)

 Eisenia fetida, known under various names such as manure worm, redworm, brandling worm, panfish worm, trout worm, tiger worm, red wiggler worm, etc.,^[28] is an earthworm adapted to decaying organic material. These worms thrive in rotting vegetation, compost, and manure.The red wiggler is reddish-brown, has small rings around its body, and has a yellowish tail.^[29] Groups of bristles (called setae) on each worm segment move in and out to grip nearby surfaces as it stretches and contracts its muscles to push itself forward or backward. E. fetida worms are native to Europe but have been introduced (both intentionally and unintentionally) to every other continent except Antarctica.^[30]

Life span

The lifespan of E. fetida under controlled conditions varies between one and five years.^[31]

MATERIALS AND METHODS

Plant material: In July, the plant was collected from Chadachana in Vijayapura district -586204, Karnataka. The plant is authenticated by Dr. Ajit Lingayat, Director of Shri B.M.K Ayurveda Mahavidyalaya. After authentication, fresh leaves material was collected, cleaned, and shade dried. 

Worm collection: Indian earthworms E. Fetidawere used to study anthelmintic activity. The earthworms were collected from Patil Organic Manure, Earthworms and Dairy Farms and washed with water. Earthworms of 3-5 cm in length and 0.1-2cm in width were used due to their anatomical and physiological resemblance with the intestinal roundworm parasite of human beings. Extract preparation: The leaves were pulverized by a mechanical grinder and passed through a 20-mesh sieve. The powdered leaves were extracted successively using chloroform (Soxhlet), ethanol, and aqueous (maceration) extract. The extracts were filtered through a cotton plug, followed by Whatman filter paper. The extract was evaporated under reduced pressure using a Rota evaporator at a low temperature of 40^0- 50⁰c until all the solvent had been removed to give an extract sample. Then the weight of each residue was recorded.^[17] Phytochemical screening: Phytochemical screening was carried out for all the extracts per the standard protocol. Plants were screened for alkaloids, carbohydrates, Flavonoids, oil and fats, proteins and tannins.

1. Alkaloids: 20 ml of distilled water was added to 2 gm of the powdered plant sample, boiled in a water bath, and filtered. About 10 ml of this filtrate was mixed with 5 ml of Wagner’s reagent and shaken vigorously for a stable, persistent froth. The Reddish Brown precipitate indicates the presence of alkaloids.
2. Carbohydrates: 100 mg of the plant extract was dissolved in 5 ml of water and filtered. To 0.5 ml of the filtrate, 0.5 ml of Benedict’s reagent was added and heated in a boiling water bath for 3 minutes. A characteristic colour indicates the presence of carbohydrates.
3. Flavonoids: To a portion of the filtrate, 10 ml of ethyl acetate was added and heated in a water bath and filtered. 1 ml of dilute ammonia solution was added to the filtrate and shaken well. A yellow coloration indicates the presence of flavonoids.
4. Oil and fats: A small extract was pressed between the two filter papers. Oil stain on the filter papers indicates the presence of oils and fats.
5. Proteins: The plant powder was extracted in different solvents, and solvent-free plant extract was mixed with a few ml of diluted HCl and filtered. Two drops of ninhydrin solution (10 mg of ninhydrin in 200 ml of Acetone) were added to the filtrate. The mixture was mixed properly. The purple color indicates the presence of Proteins and Amino acids.
6. Tannins: The powdered plant sample pinch was mixed with 5 ml of water. The mixture was boiled in a water bath and then filtered. A few drops of 0.1?rric chloride were added to the filtrate. Brownish green or a blue-black coloration indicates the presence of tannins.^[18]

Anthelmintic activity

The different concentrations (250µg/ml, 500µg/ml, and 750µg/ml) of Chloroform, ethanol extract and aqueous extract were prepared. All the earthworms were washed into a standard saline solution. At room temperature, two or three nearly equal-sized earthworms were placed in each Petri dish. All test, standard and control dilutions were placed in the three petri dishes. Observations were made for the time taken to paralyze (paralysis was said to occur when earthworms did not revive in normal saline) and death (death was conducted when earthworms lost their motility and followed by their body colors fading away).^[19]

Hemolytic activity

The heparinized human red blood 250 µL was utilized for the assay. Washed with saline (0.9%) and then centrifuged at 2000 rpm for 10 min to remove plasma. Precisely 40% v/v suspension was made with isotonic phosphate buffer at pH 7.4, and it was used as an RBC stock solution for further hemolytic assay. The Hemolysis assay was followed (Henkelman, 2009) with modifications per previous studies (Lakshmegowda et al., 2020). In the reaction mixture, 10% erythrocytes hemolysis was induced by an equal volume of 100 mM AAPH containing samples 50 µg/mL, without samples serves as the negative control, and normal erythrocytes, i.e., without AAPH or extracts, serves as Blank. Similarly, 0.2% Triton (in PBS) is taken for 100% hemolysis as a reference control. The results were compared with standard L-ascorbic acid as a positive control. The reaction mixture was incubated for two h at 37°C and, at the end of incubation, diluted with eight volumes of PBS and centrifuged at 2000 rpm for 10 min. The supernatant was collected, and the absorbance was read at 540 nm; the percentage of hemolysis was calculated. ^[20]

RESULTS

Percentageyield:50 gm of dried leaves powder of L. camera was extracted in sterilized distilled water to obtain the test extract. After drying, the filtrate yielded 3.05 gm of extract, which is 6.10% of the initial plant powder (50 gm)

Preliminary phytochemical determination

Table .1: Phytochemicals present and absent in various solvents of LC

Here, +: present, -: Absent

Table.2: Estimation of anthelmintic activity

       
           
       
   Fig. Albendazole Suspension
       
           
       
    Fig. Chloroform Extract

Hemolytic activity

Table 3:  Estimation of Hemolytic properties of the sample

       
           
       
Fig.     Hemolytic Activity Graph