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IMMUNOMODULATORY ACTIVITY OF LEAVES OF RUMEX VESICARIUS LINN. AND SYMPLOCOS RACEMOSA ROXB.

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IMMUNOMODULATORY ACTIVITY OF LEAVES OF RUMEX VESICARIUS LINN. AND SYMPLOCOS RACEMOSA ROXB.

J. Y. Manure* and N. S. Naikwade

Department of Pharmaceutical Chemistry, Appasaheb Birnale College of Pharmacy, Sangli - 416416, Maharashtra, India.

ABSTRACT: Objective: To determine the immunomodulatory activity of leaves of Rumex vesicarius Linn. and Symplocos racemosa Roxb. Methods: The ethyl acetate and ethanolic extract of Rumex Vesicarius Linn. and ethanolic and N-hexane extract of Symplocos racemosa Roxb. were administered orally at the dosage level of 200 mg/kg/day and 400 mg/kg/day each according to the body weight of Rat. The assessments of immunomodulatory activity were carried out by using Carbon Clearance Test and Delayed Type Hypersensitivity Test. Result: Oral administration of all the extract of Rumex vesicarius Linn. and Symplocos racemosa Roxb. significantly (P < 0.0001) showed an increase in phagocytic activity and DTH response in Rat at an experimental dose. The study also comprises the acute toxicity study and preliminary phytochemical screening. Conclusion: The study demonstrate that all the extract of Rumex vesicarius Linn. and Symplocos racemosa Roxb. shows the significant immunomodulatory effect on both humoral as well as cell-mediated immunity.

Keywords:

Rumex vesicarius Linn., Symplocos racemosa Roxb., Carbon Clearance Test, Delayed Type Hypersensitivity test

INTRODUCTION: The immune system is a remarkably versatile defense system that has evolved to protect animals from invading pathogenic microorganisms to eliminate the disease. It is able to generate a multifarious variety of cells and molecules capable of specifically recognizing and eliminating variety of foreign invaders 1. It is now being recognized that modulation of immunological response could provide an alternative to conventional chemotherapy for a variety of diseased conditions of impaired immune responsiveness or when a selective immunosuppression has to be induced in situations like autoimmune disorders and organ transplantation.

The modulation of the immune response by using Ayurvedic herbal medications as a possible therapeutic measure has now become a subject of scientific investigation. One of the therapeutic strategies in Ayurvedic medicines is to enhance the body’s overall natural resistance to the disease causing agent rather than directly neutralizing the agent itself 2. Immunomodulation is the process of modifying an immune response in a positive (immunostimulation) or negative manner (immunosuppressant) by administration of a drug or compound 3. 

There are many plants which are used as immunomodulators. Heterostemma tanjorense shows immunostimulant activity against Azathioprine administered rats 4. The methanolic root extract of Withania somnifera shows immunostimulatory activities in dexamethasone induced immunocompromised mice and in vitro model 5. The Methanolic leaf extract of Moringa oleifera shows an immunostimulatory effect on both the cell-mediated and humoral immune systems in the Wistar albino rats 6. The methanolic leaf extracts of Cameroonian medicinal plants possess immunomodulatory activity 7.The ethanolic extract of Sonerila tinnevelliensis showed a stimulatory effect on both humoral and cellular immune functions in animal models 8. Caesalpinia sappan shows the nonspecific immunomodulatory effect on murine peritoneal macrophages 9. The aqueous leaf extract of Ocimum basilicum Linn. is a potent immunostimulant, stimulating specific and nonspecific immune mechanisms. The immuno-stimulatory activity of O. basilicum is due to flavonoids (quercetin), alkaloids, tannins, saponin glycosides and phenolic compounds 10.

The methanolic extract of Trigonella foenum whole plant has shown significant immunostimulatory activity in various models of Drug-induced myelosuppression 11. The flavonoids isolated from freshly harvested leaves of Prosopis spicigera, Mimusops elengi and Terminalia arjuna showed dose dependent immunosuppressive activity 12. The crude terpenoids extract from the leaves of Emblica officinalis, Ficus racemosa and Strychnos nux-vomica on human whole blood stimulated with hepatitis B vaccine possess immunosuppressive activity 13.

The aqueous extract of Leucas aspera was evaluated in cyclophosphamide-induced immunosuppressive mice and it shows prominent immunostimulatory effect 14. The leaves of Calotropis gigantea, C. rotang and A. integrifolia have an immunosuppressive activity of the variable doses of crude saponin (0.625 – 2.5 mg) on lymphocytes, monocytes and granulocytes 15.

Triptolide, the active component of Tripterygium wilfordii can be used alone or in combination with existing therapeutic modalities as novel treatments for autoimmune disorders, cancers, and for immunosuppression 16. The ethanolic extracts from leaves of Rhaphidophora korthalsii stimulate immune cell proliferation, peripheral blood NK cell population 17. Cyclotides, ribosomally synthesized plant peptides have growth-inhibiting effects on primary cells of the human immune system 18. The isogarcinol, active compound from Garcinia mangostana L. inhibite Calcineurin unique protein phosphatase, plays an important role in immune regulation in a dose-dependent manner 19. Natural therapies help to regulate the immune system’s aggressive behavior without suppressing necessary defenses 20.

For this study we have selected two plants, Rumex vesicarius Linn. and Symplocos racemosa Roxb.

Rumex vesicarius Linn. (Chooka) belongs to perennial herbs to the family Polygonaceae. The plant is an erect usually with a long taproot. Traditionally the plant is used as stomachic, Diuretic, used for the disorders of the lymphatic and glandular system, for bronchitis, asthma, constipation, dyspepsia and the diseases of the liver. Plant leaves are rich in ascorbic acid, citric acid and tartaric acid, it also contains glycoside, alkaloid, flavonoids, tannins and phenolic compounds 21, 22.

Symplocos racemosa Roxb. (Lodhra) belongs to the family Symplocaceae, is a small evergreen tree upto 6 m tall. In traditional system it is mainly used as cardiotonic, antipyretic, antihelmintic and laxative properties. It is beneficial in billow fever, urinary discharge; pharmacologically it is used as antimicrobial, antidiarrhoeal, spasmogenic and heart depressant. The plant mainly contains monomethyl pelargonidin gluocosides, loturidine also contain oxalic acid, phytosterol, ellagic acids and oleanolic acid 23, 24, 25.

MATERIALS AND METHODS:

Plant Material: The fresh leaves of Rumex vesicarius and Symplocos racemosa Roxb. used in this study, collected at the flowering stage (Month: August - November) from the local area of Sangli and Satara, Maharashtra state, India respectively and authenticated by Botanical Survey of India, Pune, Maharashtra. (BSI/WRC/Iden./2015 dated 4-12-2015)

Extraction: The leaves were separated from fresh stems and dried under shade at room temperature until it becomes completely dry. After drying leaves were subjected to size reduction. The shade-dried coarsely powdered leaves (500 g) were subjected to Soxhlet extraction.

A) Rumex vesicarius leaves were subjected to Soxhlet extraction with 95% ethanol and ethyl acetate to obtain ethanolic and ethyl acetate extract respectively.

B) Symplocos racemosa leaves were subjected to Soxhlet extraction with 95% ethanol and N-hexane to obtain ethanolic and N-hexane extract respectively. The extracts obtained were subjected to the Rotary flash evaporator to remove excess of solvent and dried extracts were stored in a cool place in tight pack container for further use.

Animals: All the experiments were carried out using male albino rats of wistar strain. Weight around 150 - 200 gm. The animals are free to access of food and water, and they were housed in a natural (12 h each) light-dark cycle. The animals were acclimatized for at least 5 days to the laboratory conditions before the experiment. The experimental protocol was approved by the institutional animal ethics committee (IAEC/ ABCP/09/2016-17) and the care of laboratory animal was taken as per the guidelines of CPCSEA, the ministry of forests and environment government of India.

Preliminary Phytochemical Screening: All the extracts were subjected to preliminary phytochemical screening using the method described by Kokate, Trease and Evans for the detection of various plants constituents. Test were carried out for the presence or absence of Phytoconstituents like glycosides, flavonoids, saponins, alkaloids, carbohydrates, sterols, phenolic compound and reducing compounds 26, 27, 28.

Drugs and Chemicals: All the drugs and Chemical were of analytical grade while the other drugs were procured from - Levamisole (Johnson and Johnson Ltd.,) Cyclophosphamide (Biochem pharmaceutical industries Ltd.,) Colloidal carbon (Indian ink, Camel India Pvt. Ltd.,).

Selection of doses: Acute Toxicity studies were performed according to the organization for economic cooperation and development (OECD) guideline (425). Form acute toxicity study there was no mortality and physical/behavioral changes showed after administration of all the extract over 14 days at the dose of 2000 and 5000 mg/kg to a different group of rat weight around 150 - 200 gm. The experiments were performed after the experimental protocols had been approved by the institutional animal ethical committee.

Pharmacological Screening: The immuno-modulatory activity is carried out by using Carbon clearance test. (Test for Phagocytosis) and Delayed-Type Hypersensitivity Reaction.

Carbon Clearance Test: (Test for Phagocytosis):

Procedure:

  • In this test, animals were divided into 11 group comprising 6 animals in each.

TABLE 1: TREATMENT GROUPS OF CARBON CLEARANCE TEST

Groups Treatment Dose and route of administration
Group I Vehicle 10 ml/kg P.O.
Group II Standard drug (Cyclophosphamide) 50 mg/kg P.O.
Group III Standard drug ( Levamisole) 2.5 mg/kg P.O
Group IV Ethyl acetate extract of leaves of  Rumex vesicarius L. (EARV-400) 400 mg/kg P.O.
Group V Ethyl acetate extract of leaves of  Rumex vesicarius L. (EARV-200) 200 mg/kg P.O.
Group VI Ethanolic extract of leaves of  Rumex vesicarius L. (ERV-400) 400 mg/kg P.O.
Group VII Ethanolic extract of leaves of  Rumex vesicarius L. (ERV-200) 200 mg/kg P.O.
Group VIII Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-400) 400 mg/kg P.O.
Group IX Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-200) 200 mg/kg P.O.
Group X n-hexane extract of leaves of Symplocos racemosa Roxb.(NSR-400) 400 mg/kg P.O.
Group XI n-hexane extract of leaves of Symplocos racemosa Roxb.(NSR-200) 200 mg/kg P.O.
  • Carbon ink suspension was injected via tail vein to each rat 48 hours after the five days treatment
  • Blood samples (25 μl) were then withdrawn from the retro-orbital plexus with mild ether anesthesia at 5 and 15 min after injection of colloidal carbon ink lysed in 0.1% sodium carbonate solution (3 ml).
  • The optical density was measured spectrophotometrically at 660 nm.
  • The phagocytic activity was calculated using the following formula 29, 30.

K = Log OD1 – Log OD2

t2 – t1

Where ODI and OD2 are the optical density at time t1 and t2, respectively.

Preparation of Carbon Ink Suspension: Camlin ink was diluted eight times with saline and used for carbon clearance test in a dose of 10 μl/gm body weight of rat 31.

Statistical Analysis: The result was expressed as mean value ± SEM. The variation in a set of data has been estimated by performing one-way analysis of variation (ANOVA). Individual comparison of group mean value were done using Dunnet’s test. The P value < 0.05, were considered statistically significant.

Delayed Type of Hypersensitivity Reaction:

Procedure:

  • In this test, animals were divided into 11 group comprising 6 animals in each.

TABLE 2: TREATMENT GROUPS OF DELAYED TYPE HYPERSENSITIVITY

Groups Treatment Dose and route of administration
Group I Vehicle 10 ml/kg P.O.
Group II Standard drug (Cyclophosphamide) 50 mg/kg P.O.
Group III Standard drug ( Levamisole) 2.5 mg/kg P.O
Group IV Ethyl acetate extract of leaves of  Rumex vesicarius L. (EARV-400) 400 mg/kg P.O.
Group V Ethyl acetate extract of leaves of  Rumex vesicarius L. (EARV-200)  200 mg/kg P.O.
Group VI Ethanolic extract of leaves of  Rumex vesicarius L. (ERV-400) 400 mg/kg P.O.
Group VII Ethanolic extract of leaves of  Rumex vesicarius L. (ERV-200) 200 mg/kg P.O.
Group VIII Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-400) 400 mg/kg P.O.
Group IX Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-200) 200 mg/kg P.O.
Group X N-hexane extract of leaves of Symplocos racemosa Roxb. (NSR-400) 400 mg/kg P.O.
Group XI N-hexane extract of leaves of Symplocos racemosa Roxb. (NSR-200) 200 mg/kg P.O.
  • Immunized Rat with 0.1ml of 20% SRBCS in normal saline intraperitoneally on 14th day of the study. On day 21st, animals from all groups get challenge with 0.03 ml of 1% SRBCs in sub plantar region of the right hind paw. Footpad reaction was assessed after 24 hrs e. on the 22nd day. Increase in foot pad edema was measured with the help of vernier caliper 29.

Antigenic Material:

Preparation of Sheep RBCs: Sheep blood was collected in sterile Alsever’s solution in 1:1 proportion, Alsever’s solution (freshly prepared) blood was kept in the refrigerator and processed for the preparation of SRBCs batch, by centrifugation at 2000 rpm for 10 min and washing with physiological saline 4 - 5 times and then suspending into buffered saline for further use 31.

Composition of Alsever’s Solution:

TABLE 3: COMPOSITION OF ALSEVER’S SOLUTION

Chemicals Quantity(g/L)
Sodium Chloride 4.2
Sodium Citrate 8.0
Citric acid anhydrous 0.55
Glucose 20.5
Distilled water q.s. 1000 ml

Statistical Analysis: The result was expressed as mean value ± SEM. The variation in a set of data has been estimated by performing one-way analysis of variation (ANOVA). Individual comparison of group mean value were done using Dunnett’s test. The P value < 0.05, were considered statistically significant.

RESULT:

Acute Oral Toxicity Study: Acute oral toxicity was carried out by the up-down method. It is found that all extract (EARV, ERV, ESR and NSR) were safe at limit dose 4000 mg/kg and 2000 mg/kg, with no mortality and physical/behavioral changes. 1/10th of this dose i.e. 400 mg/kg and 200 mg/kg were used in the subsequent study.

Preliminary Phytochemical Study: The presence of various phytoconstituents of the extract was detected by phytochemical screening. The EARV found to contains Alkaloids, Flavonoids, Tannins, Sterols, Carbohydrate and Vitamin C. ERV contains Alkaloids, Flavonoids, Carbohydrate and Vitamin C. ESR found to contain Cardiac glycoside, Flavonoids, Alkaloids, Tannins and Carbohydrate. NSR contains cardiac glycoside, alkaloids and steroids.

Carbon Clearance Test: Effect of EARV, ERV, ESR, and NSR on the phagocytic activity by the carbon clearance test is shown in Table 4. The phagocytic activity of the reticuloendothelial system is generally measured by the rate of removal of carbon particles from the bloodstream. In carbon clearance test EARV, ERV and ESR, treated all groups exhibited significantly high phagocytic index (P < 0.0001) when compared with control group. While NSR treated group showed a small increase in their phagocytic index when compared with control group. This indicates stimulation of the reticuloendothelial system.

TABLE 4: RESULT OF CARBON CLEARANCE TEST

S. no. Groups Treatments Dose and route of administration Phagocytic index

(Mean ± SEM)
1 I Control 10 ml/kg (P.O.) 0.0312±0.0005
2 II Standard (Cyclophosphamide) 50 mg/kg (P.O.) 0.0206±0.0005****
3 III Standard (Levamisole) 2.5 mg/kg (P.O.) 0.0573±0.0003****
4 IV Ethyl acetate extract of leaves of Rumex vesicarius L. (EARV-400) 400 mg/kg (P.O.) 0.0530±0.0004****
5 V Ethyl acetate extract of leaves of Rumex vesicarius L. (EARV-200) 200 mg/kg (P.O.) 0.0492±0.0004****
6 VI Ethanolic extract of leaves of Rumex vesicarius L. (ERV-400) 400 mg/kg (P.O.) 0.04566±0.0005****
7 VII Ethanolic extract of leaves of Rumex vesicarius L. (ERV-200) 200 mg/kg (P.O.) 0.0414±0.0003****
8 VIII Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-400) 400 mg/kg (P.O.) 0.0391±0.0003****
9 IX Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-200) 200 mg/kg (P.O.) 0.0363±0.0004****
10 X N-hexane extract of leaves of Symplocos racemosa Roxb. (NSR-400) 400 mg/kg (P.O.) 0.0350±0.0002****
11 XI N-hexane extract of leaves of Symplocos racemosa Roxb. (NSR-200) 200 mg/kg (P.O.) 0.0327±0.0002

Values are expressed as (Mean ±SEM). N = 6 ****P < 0.0001. Statistically significant when compared with control group by ANOVA followed by Dunnett’s test.

FIG. 1: GRAPHICAL REPRESENTATION OF CARBON CLEARANCE TEST

Delayed Type of Hypersensitivity Reaction: Effect of EARV, ERV, ESR and NSR on the cell-mediated immune response by DTH induce footpad edema is shown in Table 5. All treated group EARV, ERV and ESR showed the significantly (p < 0.0001) potentiating DTH response in terms of increase in the mean difference of paw edema when compared with control group. It indicates activation of the cellular immune system. While NSR treated group showed a small increase in footpad oedema when compared with control group. Cyclo-phosphamide treated group showed a significant decrease in the mean difference of paw edema when compared with control group.

TABLE 5: RESULT OF DTH

S. no. Groups Treatments Dose and route of administration Mean Difference in Paw edema

(Mean ± SEM)
1 I Control 10 ml/kg (P.O.) 1.508±0.0316
2 II Standard (Cyclophosphamide) 50 mg/kg (P.O.) 0.59±0.0513****
3 III Standard (Levamisole) 2.5 mg/kg (P.O.) 4.56±0.0594****
4 IV Ethyl acetate extract of leaves of Rumex vesicarius L. (EARV-400) 400 mg/kg (P.O.) 4.20±0.0545****
5 V Ethyl acetate extract of leaves of Rumex vesicarius L. (EARV-200) 200 mg/kg (P.O.) 3.66±0.123****
6 VI Ethanolic extract of leaves of Rumex vesicarius L. (ERV-400) 400 mg/kg (P.O.) 3.22±0.0519****
7 VII Ethanolic extract of leaves of Rumex vesicarius L. (ERV-200) 200 mg/kg (P.O.) 2.51±0.0586****
8 VIII Ethanolic extract of leaves of Symplocos racemosa Roxb. (ESR-400) 400 mg/kg (P.O.) 2.29±0.0152****
9 IX Ethanolic extract of leaves of Symplocos racemosa Roxb (ESR-200) 200 mg/kg (P.O.) 2.08±0.0345****
10 X N-hexane extract of leaves of Symplocos racemosa Roxb (NSR-400) 400 mg/kg (P.O.) 1.90±0.0085****
11 XI N-hexane extract of leaves of Symplocos racemosa Roxb. (NSR-200) 200 mg/kg (P.O.) 1.74±0.02986*

Values are expressed as (Mean ± SEM). n = 6 ****P < 0.0001. Statistically significant when compared with control group by ANOVA followed by Dunnett’s test.

FIG. 2: GRAPHICAL REPRESENTATION OF DTH

DISCUSSION: The immune system is a remarkably versatile system that has evolved to defend itself against a vast range of harmful agents. It is able to generate an enormous variety of cell and molecules capable of specifically recognizing and eliminating a variety of foreign invaders. Immunomodulators are a natural or synthetic substance that helps to regulate or normalize the immune system. Immunomodulators correct immune systems that are out of balance. And immunomodulation is a process which can alter the immune system specifically immunostimulation and immunosuppressant.

The present study was designed to explore the immunomodulatory activity of Rumex vesicarius Linn. and Symplocos racemosa Roxb. In this study Carbon Clearance Test and Delayed Type Hypersensitivity test were selected for evaluation of immunomodulatory activity of Rumex vesicarius Linn. and Symplocos racemosa Roxb.

According to Smrithi Tripathi et al., the Phagocytic activity of reticuloendothelial system was assayed by carbon clearance test phagocytic index was calculated as the rate of carbon elimination of reticuloendothelial system.

In the present study after oral administration of all the extract (EARV, ERV, ESR and NSR) at an experimental dose (400 mg/kg and 200 mg/kg) showed a significant increase in the phagocytic index (P < 0.0001) when compared with control group. Increase in phagocytic activity indicates that there was stimulation of reticuloendothelial system.

According to N. L. Dashputre et al., Cell-mediated immunity involves the interaction of effectors mechanism carried out by T lymphocytes and their products (lymphokines). DTH required specific recognition of antigen by activated T lymphocytes, which subsequently proliferate and release cytokines. These in turn increase vessel permeability cause vasodilatation, macrophage accumulation, and activation promoting phagocytic activity and increased concentration of lytic enzyme for more effective killing. The delay in the onset of the response reflects the time required for the cytokines to induce the recruitment and activation of macrophages. Therefore increase in DTH response after oral administration of all the extract (EARV, ERV, ESR and NSR) at experimental dose (400 mg/kg and 200 mg/kg) showed a significant increase in footpad edema (P < 0.0001) when compared with control. It is indicate stimulation of the cell-mediated immunity.

The Present study revealed that ethyl acetate extract of Rumex vesicarius Linn. showed highest immunomodulatory activity.

In this study, the overall order of immuno-modulatory activity was established as EARV > ERV > ESR > NSR.

CONCLUSION: The present study demonstrates that Ethyl acetate and Ethanolic extract of leaves of Rumex vesicarius Linn. and ethanolic extract of leaves of Symplocos racemosa Roxb. showed significant immunomodulatory effect on both humoral as well as cell-mediated immunity which is due to

  • Activation of T-cell which mediated DTH response.
  • By the activation of the reticuloendothelial system.
  • Enhance the capacity of monocytes Macrophages system.

Further among the all extract (EARV, ERV, ESR and NSR) Ethyl acetate extract of leaves of Rumex vesicarius Linn. (EARV) at experimental dose shows more immunomodulatory activity than ERV, ESR and NSR. The overall order of immunomodulatory activity is EARV > ERV > ESR > NSR. A detailed investigation may be carried out to ascertain its exact mechanism of immunomodulatory action.

ACKNOWLEDEMENT: The authors thank Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra for providing lab facilities.

CONFLICT OF INTEREST: The authors declare that they have no conflict of interests.

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    How to cite this article:

    Manure JY and Naikwade NS: Immunomodulatory activity of leaves of Rumex vesicarius linn. and Symplocos racemosa roxb.. Int J Pharm Sci Res 2018; 9(4): 1537-44.doi: 10.13040/IJPSR.0975-8232.9(4):1537-44.

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Article Information

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1537-1544

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English

IJPSR

J. Y. Manure* and N. S. Naikwade

Department of Pharmaceutical Chemistry, Appasaheb Birnale College of Pharmacy, Sangli, Maharashtra, India.

javeedmanure.98600@yahoo.com

28 July, 2017

22 September, 2017

20 October, 2017

10.13040/IJPSR.0975-8232.9(4).1537-44

01 April, 2018

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