Abstract

Purpose: To study the effect of the methanolic extract of Urena lobata Linn. on invitro immunostimulant activity.Method: Different concentrations (5-100µg/ml) of methanolic extract ofUrena lobata was subjected for its immunomodulatory activity using human blood by phagocytosis.Result: This study revealed that the methanolic extract of Urena lobata Linn. has phagocytosis and intracellular killing potency of human neutrophils at different concentration.Conclusion:Urena lobata Linn posses’ immunomodulatory property.

Introduction

Urena lobata Linn. (Malvaceae) is otherwise called as Caesar weed, is an erect herbaceous or semi woody, tomentose, under shrub growing 60-250cms or more height and up to 7cm in basal diameter ¹ .Various extracts of the leaves and roots are used in herbal medicine to treat diverse ailments .The traditional uses of the plant were found to be diuretic, febrifuge and on the treatment of rheumatism. It is also used for gonorrhea, wounds toothache and also used for food for animals as well as humans ^2. Previous research by other workers on the aerial part of the plant yield mangeferin and quercetin.Triglycerids were isolated from the plant ³ .and imperatorin and a furocoumarin was isolated from roots of Urena lobata⁴

Immunology is thus probably one of the most rapidly developing areas of biomedical research and has great promises with regard to the prevention and treatment of a wide range of disorders. Inflammatory diseases of the skin, gut, respiratory tract, joints and central organs In addition, infectious diseases are now primarily considered immunological disorders, while neoplastic diseases and organ transplantation and several autoimmune diseases may involve in an immunosuppressive state The immune system is known to be involved in the etiology as well as the path physiologic mechanism of many diseases

Macrophages are a corn stone of the innate immune system. White blood cells are the mainstay of immune system. Some white blood cells known as macrophage play a function in innate immunity by surrounding ingesting and destroy invading bacteria and other foreign organisms in a process called phagocytosis. Macrophages play an important role in adaptive immunity in that they attach to invading antigens and deliver them to destroy by other components of adaptive immunity.Phagocytosis is the cellular process of engulfing solid particles by the cell membrane to form phagosome or “food vacuole”. Phagocytosis is involved in the acquisition of nutrients for some cells, and in the immune system is a major mechanism used to remove pathogens and cell debris; bacteria, dead tissue cell and small mineral particles are all examples of objects that may be phagocytosed.

Neutrophil granulocytes usually make up to 80% of white blood count. They have multilobed nuclei and lightly staining granules. They assist in destruction of foreign particles by immune system. The use of variety of agents to enhance immunological and non-specific host defenses and thus to modify the defenses and thus to modify the defense favorable is an exciting development in immunopharmacology.Such agents may act by increasing the humoral antibody responses, by enhancing the phagocytic activity of macrophages or by modifying the cell mediated immune response ⁵ Thus in our present study, we have attempted to evaluate the immunomodulatory potency to the methanolic extract of Urena lobata using invitro immunostimulant activity by slide method.

Materials And Methods

Urena lobata, methanol. Phosphate buffer solution, Disodium hydrogen phosphate, sodium chloride, distilled water, micro slide, normal saline, Candida albicans suspension, Sabrauds broth, test tube, electron microscope, human blood

Plant material

The plant Urena lobata Linn. (Malvaceae) were collected from Herbal Garden Division of Kerala Ayurveda Limited, Aluva and authentified at the Department of Botoany,KAPL and voucher specimen of the plant were deposited at college herbarium for future references.

The freshly collected plant was washed and sun-dried and coarsely powdered until able to pass through sieve number 40

Preparation of Extract

The standardized coarse powder of plant material was subjected to soxhlet extraction using methanol as the solvent. The dark green filtrate obtained was concentrated. The crude methanolic extract was used for the presence studies and the extract also subjected to preliminary phytochemical investigations.

Preparation of the test sample ⁶

Sample for invitro study was prepared by dissolving 2.0gm of crude extract in 20ml of distilled water and to obtain a solution of 100mg/ml. From this stock solution, different working dilution were prepared to get concentration of 5,10,20,50,100µg/ml. Neutrophils of the blood withdrawn from healthy human volunteers were used to study the activity. Normal saline was used as control

Preparation of the Phosphate Buffer Solution (PBS) ⁷

2.38 gm of disodium hydrogen phosphate was dissolved in 0.19gm of potassium dihydrogen phosphate and 8gm of sodium chloride and made up to 1000ml with distilled water and pH was adjusted to 7.4.

Preparation of Candida albicans suspension

Candida albicans suspension was incubated in the Sabradous broth for 3-7 days. After incubation the cells were centrifuged and the supernatant was discarded. Pellet was re –suspended in PBS and centrifuged again for 3-4 times. The final cell button was re-suspended in PBS and adjusted to a concentration of 2 x10 ⁶ /ml

Slide preparation

Human blood (2 drops) obtained by finger prick method was collected on to a sterile glass slide. The slide was kept on a cotton pad in a sterile Petri plate and incubated at 37°Cfor 25 minutes. After incubation the clot was removed very gently and the slide was slowly drained with sterile normal saline, taking care not wash the adhered neutrophils (invisible).The slide was flooded with pre determined concentrations (5-100µg/ml) of sample and incubated at 37°C for onre hour. The slide was drained, fixed with suspension of Candida albicans and incubated at 37°C for one hour. The slide drained, fixed with methanol and stained with Giesma stain. Positive control was tested by preparing the slide in the same way with pooled with normal human blood.

Evaluation of Phagocytosis

The mean number of Candida cells phagocytosed on the slide was determined microscopically for 100 granulocytes using morphological criteria. This number was taken as a phagocytic index (PI) and was compared with basal PI of control. The procedure was repeated for different concentration (5, 10, 20, 50, 100µg/ml) of test sample.Immunostimulation in % was calculated by using the following equation. The results were tabulated respectively in table -1and a diagrammatic representation of neutrophil phagocytosis on Figure (a)

Results And Discussion

Immunomodulatory agents of plant and animal origin increase the immune responsiveness of the body against pathogens by activating the non-specific immune system. However; there is a need to subject such medicinal plants to systematic studies to substantiate the therapeutic claims made with regard to their clinical utility ⁸ .

Recently, there is an enthusiasm towards exploration of a novel group of compounds from natural sources that modulate the immune response of living systems and influence the disease process. ⁹

In the present study, the preliminary phytochemical investigation on Urena lobata reveals the presence of flavonoids saponins, tannins, and glycosides .The effect of methanolic extract of Urena lobata on neutrophil phagocytosis at concentration ranging from (5-100µg/ml) was depicted in table -1.

Table 1: Effect of the methanolic extract of Urena lobata Linn. on neutrophil phagocytosis by slide method

Since Urena lobata has been reported to contain flavonoid it is expected that Urena lobata immuno potentiating activity. Presence of hydroxyl group in flavonoid structure can affect the enzyme or electron transferring system resulting in immuno-modulatory property.

The effect of methanolic extracts of Urena lobata on neutrophil phagocytosis at concentration range -100µg/ml was represented diagrammatically in Fig (a).

Effect of the methanolic extract ofUrena lobata Linn on neutrophil phagocytosis

Figure 1: Phagocytosis by neutrophil

The Urena lobata extract has significantly increased the neutrophil chemotactic movement indicated by the increase in the number of cell reached by the micro-organism after slide method which provides a rapid and simple means of assessing the overall phagocytic process by the neutrophils. The methanolic extract of Urena lobata has significantly increased the ingestion of Candida albicans by neutrophils The plant extract showed maximum activity at 100µg/ml.Depending upon the parameters in the present study has been concluded.

Conclusion

From the results obtained, it can be concluded that, methanolic extract of Urena lobata significantly increased the phagocytic function of human neutrophils when compared to control indicating the possible immunomodulatory effect. Thus, the plant can be further explored for its phytochemical profile to identify the active constituents responsible for the above mentioned activities.

Acknowledgement

The authors wish to thanks Mrs.Kavitha (chairperson) and Mrs. Anitha Prasad (member of management) of Gautham College of Pharmacy, Bangalore, India for providing facilities to carry out the research work

Correspondence to

Rinku Mathappan
Lecturer
Gautham College of Pharmacy
Bangalore, India
e- mail:prasanthunni@gmail.com

rinkumathappan@gmail.com
prasanthunni@gmail.com

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