Immunomodulatory Activity of Methanolic and Aqueous Extracts of Whole Plant of Achyranthes Aspera Linn. in Swiss Albino Mice and Wistar Albino Rats

Introduction

Achyranthes aspera Linn. belongs to the family Amaranthaceae commonly known as apamarg or uttrani in Kannada. It grows in the wild on field boundaries, and as a weed throughout India, up to an altitude of 2100 meters and in the South Andaman Islands.¹ It is also found in Baluchistan, Ceylon, Asia, Africa, Australia, and America. The different parts of the plant are used in native, traditional remedies as ingredients in combination with more active remedies. Apamarg is being used in traditional system of medicines as a laxative, and is considered useful in treating dropsy, piles, boils, skin eruptions etc. The dried parts of the plant are given to children for colic, gonorrhea, and the water extract is used in the treatment of pneumonia. In Las Bela, the plant is used as a cure for coughs and rheumatism.²

In most recent studies, Apamarg is widely studied for its medicinal properties and has been reported to have spermicidal activity³ , postcoital antifertility activity,⁴ antiparasitic activity,⁵ hypoglycaemic activity,⁶ antioxidant activity,^7,8 chemopreventive action,⁹ hepatoprotective activity,¹⁰ analgesic and antipyretic^11,12,13, anti-inflammatory activity,¹⁴ mild to moderate antibiotic activity against B. subtilis, E. coli, and P. aeruginosa,¹⁵ nephroprotective activity,¹⁶ anti-depressant effect,¹⁷ diuretic effect,¹⁸ bronchoprotective effect,¹⁹ and immunostimulatory properties.^20,21 Achyranthes aspera leaf extract has also been reported to show both thyroidstimulating and antiperoxide activities.²²

The plant is widely used in the treatment of various ailments and is a constituent of many formulations. Therefore, the primary objective of this study was to evaluate the immunomodulatory activity of Achyranthes aspera Linn. as MEAA and AEAA, in rats and also to compare the potency of these extracts.

Materials And Methods

Collection of plant material

Achyranthes aspera Linn. plants were collected from the local area of Kalaburagi, Karnataka, India. The plant was authenticated by botanist Dr. Pratibha Sanghapurkar, Professor and HOD, Department of Botany, H.K.E.S’s V. G. College for Women, Kalaburagi, Karnataka. Ref No.: HKES/VGC/BOT/08/2018-2019

Preparation of different extracts

Methanolic extract

The shade dried powder of the whole plant was extracted with methanol using continuous Soxhlet apparatus, at 40˚ C for 48 h to obtain the methanolic extract of the plant. The filtrates of the extract were concentrated, until dry, at temperature 40˚C.

Aqueous extract

About 200 g of the powder was taken in a round bottom flask and dissolved in 1000 ml of distilled water and 10 ml of chloroform (preservative) for 24 h with occasional shaking in a closed vessel. Marc was removed by filtering the extract and concentrated on a water bath at 40˚C to get a semi-solid mass. These extracts were stored in airtight containers and refrigerated at 10˚C.

Preparation of test samples

From previous literature of acute toxicity study the methanolic extract of whole plant of Achyranthes aspera Linn. when administered, 250 and 500 mg/kg by oral route to young and adult Swiss Albino mice, did not show any toxic symptoms and mortality.³⁵ In another toxicity study conducted on Swiss Albino mice it was found that ethanolic extract of Achyranthes aspera Linn. was found to be safe at a limited dose of 2000 mg/kg.³⁴

Acute oral toxicity study of aqueous extract of Achyranthes aspera Linn. using Swiss Albino mice reported that AEAA did not show any signs or symptoms of toxicity or mortality during the observation period, at the dose of 2000 mg/kg.²⁹

Oral toxicity studies of both alcoholic and aqueous extract of the plant are found to be safe at the dose of 2000 mg/kg, therefore 1/10th of this dose, which is 200 mg/kg was used in a subsequent study.

Methanolic extract was prepared to attain suitable dosage forms by suspending in 1% sodium methyl cellulose,²³ and aqueous extracts were prepared to attain suitable dosage forms by suspending in water for injection.

Animals: Swiss Albino mice (male) weighing between 25-30 g and Wistar Albino rats (male) weighing between 220-250 g, were procured from the Central Animal House, HKES MTRIPS, Kalaburagi. The animals were acclimatized for one week to laboratory conditions. They were maintained in standard conditions of light, temperature, and humidity, which were fed with standard rodent diet and water ad libitum throughout the course of study. The animals were fasted for 12 h prior to the experiment. The study protocol was duly approved by the Institutional Animal Ethical Committee (IAEC), Ref. No. HKES/MTRIPS/IAEC/102/2018-19.

I. Cyclophosphamide induced myelosuppression in Swiss albino mice

Cyclophosphamide (Cycloxan –200 mg powder for intravenous use, Biochem Pharmaceutical Industries Ltd., Mumbai) was used as a standard immunosuppressant, which was procured from authorized pharmacy and dilutions were made using sterile water for injection as instructed on the label of the product.

Procedure

Swiss Albino mice were divided into four groups containing six mice each.

Group 1- Control, received vehicle (1% sodium CMC), orally, for 13 days.

Group 2- Received vehicle orally for 13 days and was injected with cyclophosphamide by tail, intravenously on the 11th, 12th, and 13th days.

Group 3- Received AEAA orally for 13 days and were injected with cyclophosphamide by tail, intravenously on the 11th, 12th, and 13th day, 1 h after the administration of respective doses.

Group 4- Received MEAA, orally for 13 days and were injected with cyclophosphamide by tail, intravenously on the 11th, 12th, and 13th day, 1 h after the administration of respective doses. Blood samples were collected on the 14th day of the experiment by retro-orbital puncture and hematological tests were done for red blood corpuscles count (RBC), percentage of hemogloblin (Hb%), platelets count, total white blood corpuscles (WBC) and differential count (DLC).²⁴

II. Arthus type immediate hypersensitivity (ATIH)

Preparation of Sheep Red Blood Cells (SRBC)

The sheep blood freshly collected from the slaughterhouse was mixed with sterile Alsever’s solution (1:1) and then centrifuged at 3000 rpm for 5 minutes. SRBC were washed 3-4 times with a large quantity of sterile saline solution. The SRBC cell concentration (1 x 108 cells) was adjusted.²⁵

Procedure

The Wistar Albino rats were divided into 5 groups with 6 rats in each group.

All the animals except group 5 were sensitized by intramuscular administration of 0.5 ml of SRBC (sheep red blood cells) suspension 7 days prior to the start of the experiment. Twenty-four hours and one hour prior to induction of Arthus reaction the animals received the treatment as follows,

Group 1: SRBC sensitized animals received solvent alone and served as positive control.

Group 2: SRBC sensitized animals received standard prednisolone p.o. (30 mg/kg) and served as the active control.

Group 3: SRBC sensitized animals received AEAA orally.

Group 4: SRBC sensitized animals received MEAA orally.

Group 5: Nonsensitized animals that received solvent alone served as negative control.

After 24 hours and one hour of the respective treatment, animals were challenged by injection of 0.1 ml of 0.04% solution of purified SRBC solution in the left hind paw. The footpad thickness was measured by plethysmometer at 1, 2, 3 and 5 h after challenging with 0.1 ml of 0.04% solution of purified SRBC solution.²⁶

Results

I. Cyclophosphamide induced myelosuppression in Swiss Albino mice.

Animals treated with cyclophosphamide at the dose of 30 mg/kg by tail intravenously showed significant reduction in total WBC count, DLC, and platelets along with a marginal reduction in RBC and Hb% as compared to the control group. Both AEAA and MEAA at the dose of 200 mg/kg showed statistically significant (P<0.05 and P<0.01) protection from myelosuppression. The mice treated with AEAA and MEAA had higher total WBC and DLC counts along with marginally higher counts in RBC, platelets and Hb% compared to cyclophosphamide alone treated group. However, MEAA showed greater efficacy (P<0.01) than AEAA (P<0.05).

II Arthus type immediate hypersensitivity

The results obtained are summarized in Table 3. Methanolic and aqueous extracts of Achyranthes aspera Linn. produced marked decrease in paw edema which indicates that both extracts are effective in protecting from ATIH reaction. AEAA (P< 0.05) and prednisolone (P< 0.05) were equally potent when compared to the control group. However, methanolic extract of Achyranthes aspera Linn. showed significantly higher (P< 0.01) protection from ATIH reaction when compared to prednisolone (P< 0.05).

Discussion

Alteration of immune system of an organism by any substance is known as immunomodulation. The alteration of immune system can either be specific or nonspecific in nature or sometimes both. If a substance enhances immunity it is an immunostimulant and if it suppresses immunity it is an immunosuppressant.²⁷ However, some substances alter the immune system by normalizing or modulating pathophysiological processes without being specifically stimulative or suppressive and are hence simply named as immunomodulatory agents.²⁸

The chemical constituents found in the methanolic extract of Achyranthes aspera are carbohydrates, steroids, phenolic compounds, tannins, alkaloids, saponins, flavonoids, proteins, and glycosides.^30,36 The chemical constituents found in the aqueous extract of Achyranthes aspera are alkaloids, carbohydrates, tannins, saponins, glycosides, triterpenoids and flavonoids.²⁹

Cyclophosphamide, an immunosuppressive agent, acts at various levels on cells involved in defense mechanism, against various invaders by inhibiting both cellmediated and humoral immunity.³¹ Cyclophosphamide significantly decreases the Hb, RBC, and WBC counts. Cyclophosphamide treatment for the period of 3 days showed a significant reduction in Hb, total WBC count and RBC count and thereby exerted immunosuppressant effect when compared to control animals. Treatment with the extracts after administration of myelosuppressive drug i.e., Cyclophosphamide resulted in significantly higher counts on various hematological parameters when compared to the group that was administered only cyclophosphamide. Methanolic extract showed significantly higher (P< 0.01) total WBC and DLC count when compared to the aqueous extract.

Arthus reaction is the type III hypersensitivity reaction caused by deposition of immune complex on the tissues followed by activation of the complement system and inflammatory reactions, resulting in cell injury. First exposure to the antigen results in weak antigen-antibody reactions. Exposure to the same antigen results in chronic antigen-antibody complex formation which our immune system fails to eliminate from body fluids leading to deposition in the tissues and inflammation.³² In the present study, animals were immunized with SRBC seven days prior to start of the experiment. After 24 h and one hour of treatment, animals were challenged by SRBC, the footpad thickness was then measured by plethysmometer at 1, 2, 3 and 5 h. Both AEAA and MEAA showed statistically significant protection from ATIH reaction in Wistar Albino rats. AEAA (P< 0.05) and prednisolone (P< 0.05) were equally potent when compared to the control group. However, MEAA demonstrated greater protection (P< 0.01) from Arthus reaction when compared to AEAA and the standard drug, prednisolone.

Conclusions

Both AEAA and MEAA have shown statistically significant higher count of hematological parameters when compared to the untreated cyclophosphamide group, using cyclophosphamide induced myelosuppression method in Swiss Albino mice. MEAA showed higher efficacy when compared to AEAA. Both AEAA and MEAA demonstrated statistically significant protection from ATIH reaction in Wistar Albino rats. AEAA and prednisolone were equally potent when compared to the untreated control group and MEAA showed higher efficacy than the standard drug, prednisolone.

The activity may be due to the presence of alkaloids, steroids, glycosides, triterpenoids and flavonoids.^{29, 30, 36, 33} The results suggest that MEAA is a potent immunomodulator than that of AEAA in the first model and in the second model MEAA has shown better protection when compared to AEAA and standard drug, prednisolone from ATIH reaction. This could be due to the presence of β-sitosterol in MEAA.²⁹ β-sitosterol, is a type of steroid found in methanolic extract of Achyranthes aspera Linn.³⁷ which has been reported to have immunomodulatory and antiinflammatory activity. β-sitosterol and its glycosides seem to target specific T-helper lymphocytes and natural killer cells activity and also a dampening effect on overactive antibody responses has also been reported.³⁸ Future investigations should be planned to evaluate the exact mechanism of action of immunomodulatory activity of MEAA.

Conflicts of interest

None.

Acknowledgments

We are thankful to the authorities of H.K.E.S’s MTRIPS, Kalaburagi, for providing the facilities needed for this study