INTRODUCTION:

The immune system is a system of biological structures and processes within an organism that protects against diseases. Disorders of the immune system can result in autoimmune diseases, inflammatory diseases, cancer and immunodeficiency¹. Immunomodulation is a procedure which can alter the immune system of an organism by interfering with its functions; if it results in an enhancement of immune reaction, it is named as an immunostimulative drug which primarily implies stimulation of nonspecific system. Immunosuppressant implies mainly to reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factors.

Immunostimulation and immunosuppression both need to be considered in order to regulate the normal immunological functioning. Hence both the immunostimulating and immunosuppressing agents have their own standing, so search for better agents exerting these activities is becoming the field of major interest all over the world. A number of Indian medicinal plants and their products have been claimed to possess immunomodulatory activity². The use of plant products as immunomodulators is still in a developing stage. A traditional and folklore medicine plays an important role in the health services around the globe. About three quarters of the world population relies on the plants and plant extracts for healthcare.

The immunomodulators have ability to stimulate natural and adaptive defense mechanisms, such as cytokines, which enables the body to help itself. The natural immunomodulators act to strengthen weak immune systems and to moderate immune systems that are overactive. There are several herbs used in indigenous system for immune modulatory effect³. A variety of plant-derived materials such as polysaccharides, lectins, peptides, flavonoids, terpenoids and tannins have been reported to modulate the immune system⁴. Synthetic drugs are used as immunosuppressive and immunostimulating agents, but there are major limitations to the general use of these agents such as an increased risk of therapeutic and generalized effect throughout life⁵. Ayurveda, the Indian traditional system of medicine, lays emphasis on promotion of health; a concept of prevention of diseases and strengthening of both physical as well as mental health. Ayurvedic medicine thus constitutes rich sources of active substances for immunomodulation based on herbal preparations. Among the wide range of plants claimed to possess the rasayana effect, the most popularly used are Withania Somnifera, Ocimum sanctum, Azadiracta indica, Curcuma longa, and Tinospora cordifolia, etc^6,7.

One such plant, Aquilaria malaccensis (Agar wood) belongs to the family Thymelaeaceae, locally known as Eagle wood distributed in India, Burma, Malaysia, Philippines and Indonesia. Agar wood leaves are reported to possess antidiabetic, anti-inflammatory, antioxidant, antibacterial, antidepressant and antiviral activities^8-10. Further the agar wood leaves are reported to possess anticancer and radioprotective activities^11,12. Agar Wood leaves are reported to contain several bioactive metabolites like alkaloids, flavonoids, glycosides, saponins, steroids, terpenoids and tannins. Some of its traditional uses, including treatment of tumours, rheumatism, hypoglycemic effect, anti-inflammatory, free radical scavenging potential and antiviral effects and its bioactive components suggest that the leaves of the Aquilaria malaccensis might possess immunomodulatory activity. However, the leaves of the plant have not yet been explored for its immunomodulatory activity. Hence the present study was designed to evaluate the immunomodulatory potentials of the ethanolic extract of the leaves of Aquilaria malaccensis.

MATERIALS AND METHODS:

Plant Material and Extraction

The leaves of A. malaccensis were collected from the local areas and authenticated by Taxonomist. The collected leaf materials were cleaned and shade dried. The coarse leaf powder was exhaustively extracted with ethanol for 2 days at room temperature. The extraction of grounded leaves was further repeated with ethanol (twice). The entire filtrate was subjected to evaporate under reduced pressure using rotary flash evaporator to give concentrated crude extracts. The dried extract was kept in a glass container until further use.

Experimental Animals:

Wistar albino rats (150-200 g) of either sex maintained under standard room conditions with free access to rodent pellet diet and water were used. The protocol was approved by the Institutional Animal Ethics Committee with the reference number SCP/IAEC/F150/P20/2015.

Preliminary Phytochemical Analysis:

Extract was subjected to phytochemical screening for the identification of various phytoconstituents like alkaloids, glycosides, steroids, flavonoids, tannins, etc.

Acute Oral Toxicity Study:

The extract was subjected to oral toxicity studies using female Wistar albino rats as per OECD guidelines¹³. The animals were fasted overnight and the extract administered orally with a starting dose of 2000 mg/kg, to different groups of animals. Animals were observed continuously for first 3h and monitored for 14 days for any mortality and general behavior of animals, signs of discomfort and nervous manifestations.

Cyclophosphamide induced immune response:

Wistar albino rats (150 – 200 g) of either sex (6 animals in each group) were selected for the study. The different groups were assigned as follows,

Group I: Normal Control

Group II: Toxic Control (Cyclophosphamide 30 mg/kg, p.o.)

Group III: Extract (200 mg/kg) + Cyclophosphamide

Group IV: Extract (400 mg/kg) + Cyclophosphamide

On first day blood was withdrawn through ritro-orbital plexus and haematological parameters (Hb, RBC and WBC count) were assessed. Drugs were administered from 1^st to 13^th day. cyclophosphamide (30 mg/kg, p.o.) was given to all groups except normal group on 11^th, 12^th and 13^th day. On 14^th day blood was withdrawn from all groups and subjected to assessment of hematological parameters and SGOT and SGPT^14,15.

Sheep RBC induced immune response:

Fresh blood was collected from a sheep in a sterile bottle containing Alsver’s solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid and 0.42% sodium chloride). The sheep red blood cells (SRBC) were thoroughly mixed and washed three times with normal saline and each time centrifuged at 3000 rpm for 5 min. The supernatant was then discarded. The SRBC got were washed again with sterilized phosphate buffer saline (pH 7.2). The total SRBC was counted using Neubauer chamber and finally 1x108 SRBC (1.5 ml) were injected intraperitoneally for sensitization and challenging the rats to study humoral antibody response and sheep erythrocyte agglutination reaction¹⁶.

Wistar albino rats (150 – 200 g) of either sex (6 animals in each group) were selected for the study. The different groups were assigned as follows,

Group I: Normal control

Group II: Sheep RBC control

Group III: Extract (200 mg/kg) + Sheep RBC

Group IV: Extract (400 mg/kg) + Sheep RBC

All the animals were treated orally with respective drugs for 10 days. 0.25 ml of 5x109 sheep RBC/ml was administered intraperitoneally on 6^th, 8^th and 10^th day to all the groups except normal control group. On 11^th day blood was collected through ritro-orbital plexus and serum was subjected to biochemical investigation, agglutination reaction and for anti-body titer¹⁷. The serum was diluted serially with normal saline in separate test tubes. Dilutions were made i.e. 20, 40, 60 up to 1280. To this 50 μ l of SRBC antigen solution was added and incubated at 37 °C for 18 h. All the tubes were subjected to examine agglutination visually and compared with control.

Statistical Analysis:

The data were expressed as mean value ± SEM, n=6 and significance was analyzed by one way ANOVA.

RESULTS:

Preliminary Phytochemical Analysis

Phytochemical evaluation of A. malaccensis leaves extract showed positive result for saponins, alkaloids, flavonoids, terpenoids, tannins, steroids, glycosides, coumarin, emodins, anthraquinones, and resins.

Acute Toxicity Study:

The oral acute toxicity study was found to be safe up to 2000 mg/kg. There was no mortality and no signs of toxicity observed. Therefore two dose levels i.e., 200 mg/kg and 400 mg/kg per body weight were selected for the present study.

Cyclophosphamide induced immune responseL

Cyclophosphamide treatment for the period of 3 days showed significant reduction in Hb, RBC and WBC count (p<0.05) and thereby exerted immunosuppressant effect when compared to control animals. Treatment of extract at both doses showed restoration of Hb, RBC and WBC count when compared to cyclophosphamide treated group. The ethanolic extract of A. malaccensis was found to be significant in increase the Hb, RBC and WBC counts. Suppressive effect of cyclophosphamide was protected by animals pretreated with extract and has shown restoration of haematological parameters on 14^th day of study. The extract showed dose dependent increase in hematological parameters. Results shown in the Table 1 have revealed that administration of extract of leaves could stimulate the haemopoietic system. Further, the findings revealed that the extract also increased the level of SGPT and SGOT dose dependently (Table 2).

Sheep RBC induced immune response:

Increase in humoral immunity was observed in animals pretreated with 200 and 400 mg/kg (p<0.05). Dose dependant increase in haemagglutination antibody (HA) titre value was observed with ethanolic extract (Table 3). Studies also suggested that animals pretreated with ethanolic extract increased the level of SGPT and SGOT in sheep RBC induced immune response.

Table 1: Effect of extract on hematological profile against cyclophosphamide induced immune response

Groups	Hb (g/dl)		RBC (x10⁶/mm³)		WBC (x10³/mm³)
Groups	0^th day	14^th day	0^th day	14^th day	0^th day	14^th day
Normal	11.21±0.21	11.34±1.15	7.07±0.16	7.18±0.57	9.55±0.73	9.52±0.71
Toxic control	11.28±0.71	6.97±0.30	7.13±0.28	5.37±0.31	9.41±0.28	3.55±0.28
Extract (200mg/kg)	11.15±0.11	8.12±1.95^*	7.25±0.23	6.31±0.38^**	9.56±0.26	6.62±0.60^**
Extract (400mg/kg)	11.16±0.12	9.13±0.81^**	7.14±0.24	6.89±0.73^***	9.66±0.25	8.75±1.17^***

One way ANOVA, mean ± SEM, n=6, ^*P< 0.05, ^**P< 0.01, ^***P< 0.001 when compared with toxic control group.

Table 2: Effect of extract on biochemical parameters against cyclophosphamide and sheep RBC induced immune response

Groups	Cyclophosphamide		Sheep RBC
Groups	SGPT (IU/L)	SGOT (IU/L)	SGPT (IU/L)	SGOT (IU/L)
Normal	59.78±1.73	63.87±2.73	58.08±2.13	61.24±2.78
Toxic control	113.49±2.83	115.43±3.88	102.91±3.42	110.41±2.61
Extract (200mg/kg)	95.75±2.58^*	87.15±3.58^*	99.55±2.89^*	93.15±3.41^*
Extract (400mg/kg)	71.95±1.73^***	75.95±2.71^***	88.26±2.34^**	78.05±3.36^**

One way ANOVA, mean ± SEM, n=6, ^*P< 0.05, ^**P< 0.01, ^***P< 0.001 when compared with toxic control group.

Table 3: Effect of extract on antibody titer value and agglutination against sheep RBC induced immune response

Groups	Antibody Titer	Agglutination
Normal	80.40±3.15	No agglutination
Toxic control	202.97±6.73	Agglutination
Extract (200mg/kg)	170.25±5.52^*	Agglutination
Extract (400mg/kg)	145.68±7.15^**	Agglutination

One way ANOVA, mean ± SEM, n=6, ^*P< 0.05, ^**P< 0.01 when compared with toxic control group.

DISCUSSIONS:

Stimulation or suppression of the immune response through may help in maintaining disease-free state. Agents that activate host defense mechanisms in the presence of an impaired immune responsiveness can provide supportive therapy to conventional chemotherapy¹⁸. Immunostimulation in a drug-induced immunosupression and immunosupression in an experimental hyper-reactivity model by the same preparation can be said to be true immunomodulation¹⁹. The presence of immunostimulant compounds in higher plants has been extensively reviewed but only a limited amount of immunosuppressive products of plant origin have been reported. Such products, if well tolerated by the patient may be developed into alternative co adjuvants in the treatment of disorders caused by an exaggerated unwanted immune response, such as in autoimmune diseases, allergies, glomerulonephritis, chronic hepatitis, etc.²⁰.

Immunomodulation using medicinal plants can provide an alternative to conventional chemotherapy for a variety of diseases especially when host defence mechanism has to be acquired under the conditions of impaired immune responsiveness²¹.

In the present study treatment with extract at both doses showed restoration of Hb, RBC and WBC count when compared to cyclophosphamide treated group. The ethanolic extract of A. malaccensis was found to be significant in increase the Hb, RBC and WBC counts. Suppressive effect of cyclophosphamide was protected by animals pre-treated with extract and has shown restoration of haematological parameters. The extract showed dose dependent increase in hematological parameters revealed that administration of extract of leaves could stimulate the haemopoietic system. Further, the findings revealed that the extract also increased the level of SGPT and SGOT dose dependently. Increase in humoral immunity was observed in animals pre-treated with 200 and 400 mg/kg. Dose dependant increase in haemagglutination antibody (HA) titre value was observed with ethanolic extract. Studies also suggested that animals pre-treated with ethanolic extract increased the level of SGPT and SGOT in sheep RBC induced immune response.

This immune modulation effect is probably attributed to the presence phytoconstituents in leaf extract of Aquilaria malaccensis including saponins, alkaloids, flavonoids, terpenoids, tannins, steroids, glycosides, coumarin, emodins, anthraquinones, and resins.

The above observations support the findings that, the extract of Aquilaria malaccensis leaves was able to offer significant protection against cyclophosphamide and sheep RBC induced immune response probably by the presence of array of phytoconstituents and might be beneficial in the treatment of immune suppression related disorders.

ACKNOWLEDGEMENT:

The authors are thankful to the Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore for financial assistance to this work.

REFERENCES:

1. Ukpo G, Ogbonnia S, Ehianeta T, Bashir S. Immunostimulatory a biochemical effects of ethanolic extract of Mangifera indica stem bark on dexamethasone induced immunosuppressed male rats. Int J Pharm Pharm Sci. 2013; 5(2):569-72.

2. Kuttan G. Immunomodulatory effect of come naturally occurring sulphur containing compounds. J Ethnopharmacol. 2000; 72:93-9.

3. Bodhankar S, Makare N, Rangari V. Immunomodulatory activity of alcoholic extract of Mangifera indica L. in mice. J Ethnopharmacol. 2001; 78:133-7.

4. Pezzuto JM. Plant-derived anticancer agents. Biochem Pharmacol. 1997; 53:121-33.

5. Diasio RB, LoBuglio AF. The Pharmacological Basis of Therapeutics. In Goodman, Gilman’s, editors, McGraw-Hill (N Y), 1996; 12:1291-307.

6. Sai Ram M, Sharma SK, Ilavazhagan G, Kumar D, Selvamurthy W. Immunomodulatory effect of NIM-76, a volatile fraction from neem oil. J Ethnopharmacol. 1997; 55:133-9.

7. Mediratta PK, Sharma KK, and Singh S. Evaluation of immunomodulatory potential of Ocimum sanctum seeds oil and its possible mechanism of action. J Ethnopharmacol. 2002; 80:15-20.

8. Sannigrahi S, Mazuder U K, Parida S, et al. Antioxidant potential of crude extract and different fractions of Enhydra fluctuans Lour. Iranian J Pharma Res. 2010;9(1):75-8.

9. Agrawal P K. Carbon-13NMR of flavonoids. Elsevier Science Publishers, Amsterdam.1989.

10. Gunasekera S P, Kinghorn A D, Cordell G A, et al. Plant Anticancer Agents. XIX. Constituents of Aquilaria malaccensis. J Nat Prod.1981; 44(5):569-72.

11. Karunakar Hegde, Fathima Jazeela M A, Vijetha Poojary K, Satish S. Anti-cancer potentials of the plant Aquilaria malaccensis leaves. Indian J Pharmacy Pharmacol. 2018; 5(3):135-40.

12. Karunakar Hegde, Manisha Mascarenhas J A, Rohan Joseph D Silva. Radio protective activity of the plant Aquilaria malaccensis leaves. Am J PharmTech Res. 2018; 8(6):240-8.

13. OECD, Guidelines for testing of chemicals, Acute oral toxicity, Environmental Health and Safety Monograph Series on Testing and Adjustment No. 425, 2001, 1.

14. Benaceraff B. A hypothesis to relate the specificity of T lymphocytes and the activity of I region specific Ir genes in macrophages and B lymphocytes. J Immunology. 1978; 120:1809-12.

15. Sudha P, Asaq SB, Dhamingi SS, Chandrakala GK. Immunomodulatory activity of methanolic leaf extract of Moringa oleifera in animals. Indian J Physiol Pharmacol. 2010; 54:133–40.

16. Dash S, Nath LK, Bhise S, Kar P, Bhattacharya S. Stimulation of immune function activity by the alcoholic root extract of Heracleum nepalense D. Don. Indian J Pharmacol. 2006; 38:336-40.

17. Puri A, Sahai R, Singh KL, Saxena R, Tandon J, Saxena K. Immunostimulant activity of dry fruits and plant materials used in Indian traditional medical system for mothers after child birth and invalids. Journal of Ethnopharmacol. 200;71: 89-92.

18. Wagner H, Proksch A. Immunomodulatory drugs of fungi and higher plant in economic and medicinal plant research. Academic Press, London.1983; 1: 113.

19. Labadie RP, Van der Nat JM, Simons JM, Kroes BH, Kosasi S, Van den Berg AJ, et al. An ethanopharmacognostic approach to the search for immunomodulators of plant origin. Planta Med. 1989; 55:339-48.

20. Rossi-Bergmann B, Costa S S, Borges M B S, Da Silva S A, Noleto G, Souza M L M, et al. Immunosuppressive effect of the aqueous extract of Kalanchoe pinnata in mice. Phytother Res. 1994; 8:399-402.

21. Mahadevan N, Shivali, Kamboj P. Hibiscus sabdariffa Linn: An overview. Nat Prod Radiance. 2009; 8(1):77-83.

Received on 26.12.2018 Modified on 12.01.2019

Res. J. Pharmacology and Pharmacodynamics.2019; 11(1):32-36.

DOI: 10.5958/2321-5836.2019.00007.7