INTRODUCTION:

Nature has provided an excellent storehouse of remedies to cure all the ailments of mankind. Modern research is now focusing greater attention on the generation of scientific validation of herbal drugs based on their folklore claim¹ Natural products and folklore medicines are the main contributors of the leads in the design and development of therapeutic agents²

Herbal medicines derived from plant extracts are being increasingly utilized to treat a wide variety of clinical diseases, though relatively little knowledge about their mode of action is available³ Herbal drugs constitute a major part in all the traditional system of medicine. Herbal medicine is a triumph of popular therapeutic diversity⁴ Therefore, in recent time, more interest is shown in alternative and nature drugs for the treatment of various diseases, but there is a lack of proper scientific evidence⁵The system is biological structures and processes within an organism that protects against invading pathogens microorganisms eliminating infective agent and diseases. The disorder of the system may result in inflammatory diseases, autoimmune diseases, immunodeficiency, and cancer⁶ Abnormalities of the immune system that result in defective immune responses make individuals susceptible to infections by viruses, bacteria, fungi, and parasites⁷.Environmental pollutants and dietary habits cause disturbances in immune activities and diet containing micronutrients and antioxidants are known to prevent these alterations⁸ An immunomodulator may be a substance which will alter the system of an organism by interfering with its functions; if it leads to an activate or induce of an immune response, it's called as an immunostimulant drug which primarily implies stimulation of nonspecific system, Immunostimulation constitutes an alternative to conventional chemotherapy and prophylaxis of infection especially when the host defense mechanism has to be activated under conditions of impaired immune responsiveness⁹Immunosuppressant implies mainly to suppress resistance against infections, stress and may even be utilized in the treatment of hypersensitivity and autoimmune diseases and should occur on account of environmental or chemotherapeutic factors. Immunosuppression and immunostimulation both activities got to be considered to manage the traditional immunological functioning. Thereby both immunosuppression agents and immunostimulating agents have their significance, so look for better agents exerting these activities is becoming the sector of major interest everywhere the planet.¹⁰ The use of plants and their products as immunomodulators remains during a developing stage. These are several herbs utilized in the indigenous system. A difference of plants and there derived materials like alkaloids, monoterpenoids, glycosides, phenolic compounds, polysaccharides, lectins, peptides, flavonoids, and tannins are reported to modulate the system.¹¹ Several diseases are treated by the administration of plant extracts and that they are derived from supported traditional medicine.¹²Natural adjuvant, synthetic agents, antibody reagents are used as immunosuppressive and immunostimulatory agents. But there's a serious limitation to the overall use of those agents like the increased risk of infection and generalized effect throughout the system.¹³ The advantages of immunomodulators stem from their ability to stimulate natural and adaptive defense reaction, like cytokines, which enables the body to assist itself. The natural immunomodulatory act to the moderate system that's overactive. Natural immunomodulators found in some fruits and vegetables and therefore the algae, Several plants are used folklore medicine^.14 Pavonia odorata family (Malvaceae) is understood as sugandhibala in native Indian sub-tropical areas, scientifically referred to as Pavonia odorata¹⁵ The roots and shoots of this plant are extremely aromatic. Ayurveda, the oldest of all healing science has recorded the utilization of sugandhibala herb and its extract as cooling, demulcent, carminative, diaphoretic, and fever¹⁶Previously reported from this plant, the presence of sesquiterpenes alcohol called as pavonenol (C15H24O; mp 52-550C). The reported of Pavonia odorata roots yield an important oil that different sort of chemical contain like isovaleric acid, isovaleraldehyde, aromadendrene, pavonene, α-terpinene, azulene and pavonenol^.17 Immunomodulators could also be synthetic drugs or herbal origin. thanks to the severe side effects associated with synthetic drugs, Immunomodulation using herbal drugs can provide an alternative to standard chemotherapy for a spread of diseases, especially when the host defense reaction has got to be activated under the condition of an impaired immune reaction. Herbal drugs are easily affordable and fewer potent than synthetic. Immunomodulators and are less likely to cause side effects. Therefore, there's a requirement to look for a plant with immunomodulatory activity to supply a unique approach for the treatment of communicable diseases. this work was aimed toward the evaluation parameters of a specific plant utilized in the treatment of immune disease. Immune diseases serious crippling diseases within the world resulting in huge economic losses.

MATERIAL AND METHOD:

Plant Material:

Roots of Pavonia odorata were collected from the local market Indore city, Madhya Pradesh, India. The plant was identified and authenticated by Dr. S K Mahajan, Ex-Professor Botany Department of Govt. P G College, Khargone, (M.P.). A voucher specimen (Ref. No. SKM/PGC/Herbarium/2017/A-2) has been deposited at the departmental herbarium.

Preparation of Extracts:

About 500g dried of roots powder of Pavonia odorata was placed within the soxhlet apparatus (Perfit, India) and subjected to extraction using methanol. Extracts were filtered and therefore the filtrate was evaporated employing a vacuum evaporator (Perfit, India) under reduced pressure at ≤ 50°C temperature. The crude extract obtained after evaporation stored in desiccators. After extraction with solvent, the remaining residue of the basis was discarded and therefore the extract was weighed

Experimental Animals:

Albino mice (Swiss) of either sex were utilized in this study. The animals were fed with standard pellet diet, water ad lib, and maintained under atmosphere condition employed. Animals were hosed under standard conditions (22±5^oC with 12 h of light/dark cycle). All experimental protocol has been approved by Institutional Animal Ethical Committee of B N College of Pharmacy, Bhupal Nobles University, Udaipur (Reg No. 870/PO/Re/S/05/CPCSEA) (Approved Number of IAEC 15/BNCP/IAEC/2018)

Antigen:

Fresh Sheep blood was collected from a local slaughterhouse in sterile Alsevar’s solution (1:1 proportion). Sheep red blood cells (SRBCs) were washed thrice in pyrogens free traditional saline and centrifuged at 2500-3000rpm for 10 minutes. The supernatant was removed with pasture pipette and suspended in normal saline. The concentration of 0.1ml containing 1x 108/mm3 cells was adjusted by using an improved Neubauer chamber for immunization and challenge.

Chemicals and reagents:

Cyclophosphamide (Khandelwal Laboratory Ltd, Mumbai) and all the solvent used for experimental work was analytical grade

Acute toxicities test:

Acute oral toxicity studies of methanolic extract and isolated compound of Pavonia odorata was carried out as per the OECD guideline no. 423. As per Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA), Ministry of Social Justice and Empowerment, Govt of India.¹⁸ Administration of methanolic extract of P odorata 50 mg/kg body weight up to the dose 2000mg/kg body weight and administration of isolated compound 50 mg/kg body weight up to 3700mg/kg body weight caused no considerable signs of the toxicity in the tested animals.

Experimental design:

Delayed-Type Hypersensitivity (DTH) in mice:¹⁹

Mice of either were divided into four, groups (n = 6), Group On 0 day, all groups were sensitized with 0.1ml of SRBC containing 1×108 cell i.p. Group, I- Control, 1% gum acacia suspension in saline Group II received 30mg/kg bd wt of standard cyclophosphamide, p.o. respectively (1 tp 7 days), Group III received 200mg/kg bd wt of Methanolic extract of Pavonia odorata (MEPO) respectively (1 to 7 days). Group IV received 370mg/kg bd wt of isolated compound of Pavonia odorata (ICPO). On the 7th day before injection, right hind footpad thickness was measured with digital vernier calipers. Teen animals were challenged by injecting 1% SRBC (20 µl) into the right hind footpad. On the 8^th and 9 th day, footpad thickness was again measured. The observed difference between prior and post-challenge footpad thickness was reported as a DTH response.

Carbon Clearance Test for Phagocytes Activity²⁰

Mice were divided into four groups (n = 6). Control group-I vehicle only. group II - received cyclophosphamide. While group III- treatment of MEPO and group IV-ICPO were given test extract in 1% tween 80 daily for 5 days, Cyclophosphamide was given to group II by a route at day 0 only. All the groups were given 0.1ml of carbon ink suspension through the tail vein. After 48 h of 5 days treatment. A blood sample was collected from the retro-orbital plexus at 0 min and 15 min after the injection of carbon suspension. Blood (25 µl) was lysed with 2ml of 0.1% sodium carbonate and absorbance was measured by spectrophotometrically at 675nm for determination of optical densities.

The rate of carbon clearance (Phagocytic Index) (K), was calculated by using equations;

K = (In OD1-In OD2)/t2-t1

Where OD1 and OD2 are the optical densities at times t1 and t2 respectively.²¹

Cyclophosphamide induced immunosuppression in mice:²²

Mice were divided into four groups (n=6), Group I: Control 1% gum acacia suspension in saline, Group II: Received cyclophosphamide (CP) 30Mg/k i.p. for 10 days, Group III: Received MEPO 200mg/kg orally daily for 14 days and CP (30mg/kg i.p.) for 10 days. Respectively. Group IV: Received ICPO 370mg/kg, orally daily for 14 days and CP (30mg/kg i.p.) for 10 days, respectively, On day 14, blood was collected by retro-orbital plexus under mild ether anesthesia. Total RBC, Hb, Platelets, and WBC were determined in the laboratory.

Nitroblue Tetrazolium NBT dye Test:²³

Preparation of test solution:

The stock solution of in vitro studies was prepared by dissolving methanolic extract of Pavonia odorata and isolated compound in 0.5 dimethyl sulfoxide DMSO and with phosphate buffer salt solution according to concentration range from 5,10, 25, 50 and 100µg/ml.

Evaluation of Nitroblue test:

The leukocyte suspension (5×10⁶ ml) of 0.5ml and 0.25 ml of freshly prepared NBT 90.15% were incubated at 37⁰C in an incubator for 20 min. After incubation, the leukocyte suspension centrifuged gently at 400rpm for 3 minutes. The supernatant has discarded the precipitate of cells was resuspended with phosphate buffer saline pH 7.2. The film was prepared on a microscope slide to fix gently by heating and counterstain with dilute corbol fusion. Then slides were washed with running water. The percentage of nitroblue tetrazolium positive cell counting blue deposition was counted. This procedure was repeated for different concentrations of the extracts in the given table. For control, 0.25ml of leukocytes suspension and .02ml of freshly prepared nitro blue tetrazolium (0.15%) were incubated.

Statistical Analysis:

The data expressed as mean±standard error mean (SEM). The significance of difference among the groups was assessed using one-way analysis of variance (ANOVA) by prism software the test was followed by Dunnett's *P < 0.5, **P < 0.01, ***P < 0.001 were considered as significant.

RESULTS:

Effect of methanolic extract and isolated compound of Pavonia odorata on foot paw edema in DTH model:

The results obtained in the DTH Model given in table 1

The immune response was determined by DTH response i.e. increase in footpad thickness using vernier calipers. The observation in table 1 indicate that mice treated with dose i.e 200mg/kg (Group-III) and dose 370mg/kg (Group-IV) increase response in footpad edema was found to be statistically significant (*p < 0.05), (**p < 0.01), when compared to control (Group-I) and with (Group-II) Cyclophohphmide.

Table 1 Delayed type hypersensitivity of methanolic extract of Pavonia odorata roots and Isolated compound.

Groups	Treatment	DTH Response (mm) 24 h mean SEM	DTH Response (mm) 48 h mean SEM
I	Control	0.2670±0.0109	0.2670 ±0.01097
II	CP 30mg/kg	0.5537±0.5537*	0.5160±0.01658
III	MEPO (200mg/kg)	1.328±0.0248*	1.172±0.0113*
IV	ICPO (370mg/kg)	1.617±0.0607**	1.544±0.0117**

Values are expressed as mean, SEM, (n=6). All the groups were compared with control and standard groups using ANOVA followed by Dunnett's -test. Significant values are expressed as (*=p<0.05), (**p<0.01).

Results of Pavonia odorata methanolic extract and Isolated compound on Hematological Parameters:

The results obtained on Hematological Parameters are given in table 2 Administration of methanolic extract of Pavonia odorata and isolated compound at both levels showed statistically significant (*p < 0.05) increase in RBC count and WBC count, when compared to cyclophosphamide (20mg/kg) treated and control-treated mice

Table 2 Effect of methanolic extract of Pavonia odorata and isolated compound on WBCs and Platelet count in DTH Model

S. No	Group	Hematological Parameters
S. No	Group	WBC count (Thousand/mm3)	Platelet count (Thousand/mm3)
I	Control	6.180 + 0.6198	430.8 + 75.52
II	CP 30 mg/kg	5.513 + 0.3469	406.0 + 56.12
III	MEPO 200mg/kg	8.338 + 0.6343*	664.7 + 67.20*
IV	ICPO 370mg/kg	9.77 + 0.4190*	748.5 + 25.00*

Data were expressed as a Mean + SEM, n=6; using one-way analysis variance (ANOVA) followed by Dunnett s test. *P<0.05 was considered as statistically significant.

Effect of Pavonia odorata methanolic extract and Isolated compound on carbon clearance test for phagocytic activity in mice:

The result obtained in the carbon clearance test for phagocytic index given in table 3 The results obtained in the Carbon clearance test for the phagocytic index are given in table 3 and figure 2. The rate of clearance (phagocytic Index) is generally measured by the rate of removal of carbon particles from the bloodstream. The phagocytic index of an extract of Pavonia odorata and isolated compound increase (1.250 and 1.452) and was found to be statistically significant (p < 0.05) when compared to vehicle-treated control group-I (0.8499) and also with Cyclophosphamide Group-II (0.7128).

Table 3 Effect of Pavonia odorata methanolic extract and Isolated compound carbon clearance test for phagocytic index

Sr. No	Groups	Phagocytic Index
I	CONTROL	0.8499 ±0.007027
II	CP (20 mg/kg)	0.7128±0.024
III	MEPO (200 mg/kg)	1.250±0.008*
IV	ICPO (370 mg/kg)	1.453±0.044***

Data were expressed as a Mean + SEM, n=6; using one-way analysis variance (ANOVA) followed by Dunnett's test. *P<0.05 was considered as statistically significant compare with vehicle and standard.

Effect of Pavonia odorata methanolic extract and Isolated compound Cyclophosphamide induced immunosuppression in mice:

The results obtained in the cyclophosphamide-induced immunosuppression are given in table 4 and 5. Cyclophosphamide at the dose of 30mg/kg i.p. (Group-II) caused a significant reduction in total DLC, as compared to control Group-I (table 4), methanolic extract of Pavonia odorata and isolated compound showed highly significant (P < 0.001) increase DLC when compared with cyclophosphamide group-II. However the increased hematological parameters were more with the methanolic root extract of P odorata and isolated compound as compared to the standard group at the mentioned dose, but both doses showed similar levels of significance.

Effect of Pavonia odorata methanolic extract and Isolated compound of Nitroblue Tetrazolium (NBT) Dye Test:

The results obtanied in the NBT test are given in table 6. The results indicate that the methanolic extracts of Pavonia odorata and isolated compounds have stimulated phagocytosis of NBT dye by the neutrophils at concentrations of 5ug/m1,10ug/m1, 25ug/m1, and 100 ug/m1. The methanolic extract of Pavonia odorata and isolated compound showed significant (P<0.01) activity compared to the normal control the activity of both the extracts was comparable at all concentrations.

Table 4 Effect of methanolic extracts of Pavonia odorata and isolated compound on differential leukocyte counts in a cyclophosphamide-induced immunosuppression

Group No	Neutrophils (%)	Lymphocytes (%)	Eosinophils (%)	Basophils (%)	Monocytes (%)
Control	19.84±0.58	69.83±1.37	2.60±0.08	1.65±0.08	1.63±0.07
CP 30mg/kg	10.94±0.77	59.17±1.17	0.65±0.07	0.34±0.04	0.31±0.16
MEPO 200mg/kg	12.67±0.84	63.83±0.54*	1.30±0.01***	0.32±0.03	0.33±0.07
ICPO 370 mg/kg	15.17±0.60***	65.00±1.00***	1.30±0.01***	0.32±0.03	0.40±0.08

Data were expressed as a Mean + SEM, n=6; using one-way analysis variance (ANOVA) followed by Dunnett's test. *P<0.05 was considered as statistically significant compare with vehicle and standard.

Table 5 Effect of methanolic extract of Pavonia odorata and isolated compound on Haematological Parameters by cyclophosphamide-induced immunosuppressant

S. No	Group No	RBC (106/mm3	Hb (g%)	Platelets (105/mm3)	WBC (103/mm3)
I	Control	6.43±0.06	9.62±0.08	6.57±0.07	4.52
II	CP 30mg/kg	5.02±0.09	8.12±0.12	4.73±0.05	2.28
III	MEPO 200mg/kg	5.77±0.13*	9.13±0.06*	5.70±0.14**	3.14**
IV	ICPO 370 mg/kg	5.74±0.13*	9.20±0.09*	5.74±0.13**	3.67**

Values are expressed as mean±SEM *P < 0.05, **P < 0.01 as compared to Group II (Cyclophosphamide) standard.

Table. 6 Nitroblue Tetrazolium (NBT) qualitative test on methanolic extract of Pavonia odorata and isolated compound

Concentration In µg/ml	Normal Control	MEPO	ICPO
5	16.00±0.057	15.00±0.57	18.42±0.57
10	16.00±0.057	16.00±0.57	32.33±0.33***
25	17.67±0.88	17.33±0.33	36.00±0.57***
50	18.33±0.33	20.67±0.33*	43.00±0.57***
100	21.00±0.57	23.00±0.57**	51.67±13.84***

Values are expressed as Mean ± SEM., **P < 0.01, ***P,0.001 (n=3) by ANOVA followed by Dunnett's test.

DISCUSSION:

In the present study, Pavonia odorata and isolated compound showed an overall stimulatory effect on the system in mice Stimulatory effect was observed on both in vivo and in vitro models. within the DTH test, the DTH response, which directly correlates with cell-mediated immunity (CMI), was found to be the best at the isolated compound of P odorata 370mg/kg. The mechanism behind this elevated DTH during the CMI may be too sensitized T lymphocytes when challenged by SRBC antigen and secrets a range of molecules including proinflammatory lymphokines, attracting more scavenger cells to the location of reaction.²⁴ Increases in DTH response indicate that the P odorata Extract and Isolated compound has stimulatory and accessory cell types required for the expression of reaction.²⁵ The effect of methanolic extract of Pavonia odorata and isolated compound on the footpad thickness and hematological data like WBC and Total Platelet counts of antigenically challenged mice in (tables 1 and 2) showed, the (P<0.05) significant decrease in the footpad thickness, WBC and Total Platelet counts in cyclophosphamide group as compared to control group. While in extract-treated group animals showed (P<0.05) significant increase. General characteristics of DTH are an invasion of immune cells at the site of injection and induction became apparent within 24 to 48 hrs. An increase in Paw edema after 24 hrs of the challenge was observed in all extract-treated group animals showed (P<0.05) significant increase. General characteristics of DTH are an invasion of immune cells at the site of injection and induction became apparent within 24 to 48 hrs. An increase in paw edema after 24 hrs of the challenge was observed in all extract-treated groups when compared to control. An increase in DTH response of animals revealed the stimulatory effect of MEPO on T 1ymphocytes i.e. cell-mediated immunity or specific immunity. The result indicates that there was a significant difference in the foot paw thickness at doses of 200mg/kg bd.wt (P<0.01), and 370mg/kg bd.wt (p<0.01) methanolic extract of P odrata and isolated compound treated group when compared against normal control and standard. Because of the pivotal role played by the macrophages in coordinating the processing and presentation of antigen to B-cells. Phagocytic index (Table 3) was (*P<0.05) and ***P<0.001 significantly increased as compared to control and Cyclophosphamide group. Hence, the increased clearance rate of carbon particles from circulation in animals reflects the enhancement of the phagocytic function of mononuclear macrophage and non-specific immunity. Phagocytosis by macrophages is important against pathogenic microorganisms and its effectiveness is markedly enhanced by the opsonization of parasite with antibodies and complement C3b leading to more rapid clearance of parasite from the blood. The modulation of immune response using plant products as a possible therapeutic measure has become a subject of active scientific investigation.²⁶ Based on obtained results of methanolic extract of Pavonia odorata and the isolated compound was found to have an immunostimulant potential.

Immunomodulatory activity of methanolic extract of Pavonia odorata and the isolated compound was investigated by evaluating their effects on cyclophosphamide-induced myelosuppression (table 4 and 5) in mice at 2 dose levels of 200mg/kg and 370 mg/kg p.o. The results revealed the effect of the extracts in counteracting the myelosuppression induced by cyclophosphamide as indicated by increase RBC, total WBC, platelet counts, Hb%, and DLC in the extract-treated groups (Group III, IV,) when compared to cyclophosphamide-treated group (Group II). The isolated compound was found to be more effective than the methanolic extract at the mentioned doses, but both showed a similar level of significance.

Immunomodulatory activity of methanolic extract P odorata and isolated compound. was also evaluated on human neutrophils using the parameters, viz., NBT assay (table 6). From these parameters, the process of immunomodulation (immunostimulation) of the methanolic extract P odorata and isolated compound at various concentration range can be assessed by observing stimulation (opsonization) of neutrophils (NBT assay), Both the methanolic extracts and isolated compound demonstrated potent in vitro immunomodulatory activity the isolated compound showing more activity than the methanolic extracts.

CONCLUSION:

It is concluded from the studies of in vivo and in vitro models that the methanolic extracts and isolated compound demonstrated potent in vitro immunomodulatory activity the isolated compound showing more activity than the methanolic extracts. The significant increase response in immunomodulatory potential of isolated compound (α- Pinene) roots of Pavonia odorata which modulate and potentiate humeral as well as cellular immunity. This emphasizes the future scope of this study.

ACKNOWLEDGEMENT:

The author is thankful to B N College of Pharmacy, Bhupal Nobles University, providing good facilities and infrastructure to carry out the project.

CONFLICT OF INTERESTS:

The authors hereby declare that they have no conflicts of interest either to disclose

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Received on 07.06.2020 Modified on 31.07.2020

Research J. Pharm. and Tech. 2021; 14(7):3489-3494.

DOI: 10.52711/0974-360X.2021.00605