Biological activity of Simarouba amara (Aubl.) bark

 

Hegde Veena¹, Thippeswamy N. B²*, K. G Sandesh³

^1,2 Department of P.G Studies and Research in Microbiology, Kuvempu University,

Jnana Sahyadri, Shankaraghatta-577451, Shivamogga, Karnataka, India.

³Director, Niranthara Scientific Solutions Pvt Ltd, Bengaluru-560060, Karnataka, India.

 

ABSTRACT:

Simarouba amara (Aubl.) bark belongs to Simaroubaceae family and used to treat diarrhoea. S. amara (Aubl.) aqueous bark extract (SAAE) was qualitatively, quantitatively analyzed for phytochemicals and presence of terpenoids, phenol and flavonoids spectrophotometrically. SAAE was further evaluated for acute toxicity (1000-5000mg/kg b.w), anti-inflammatory and immunostimulatory activity in female BALB/c mice. Cell migration inhibition in peritoneal exudates was assessed in carrageenan induced peritonitis. Further, studies focused on immunostimulation in cyclophospahmide (CP) induced immuno-suppressed mice. Cell mediated (macrophages, T- cell, T_H cells) and humoral immune (immunoglobulins) responses were evaluated after treating with SAAE in groups of mice. Pre-treatment of SAAE (100, 200, and 300mg/kg, b.w, p.o.) showed antiinflammatory activity by reducing the accumulation of polymorpho neuclear leukocytes (PMNLs) and nitrite (NO^-₂) in dose dependent manner. At 300mg/kg b.w SAAE reduced the PMNLs count (2.2×10⁶cells/mL) and NO^-₂ (5.3µM±1.5) significantly (P<0.001) with half of inhibitory concentration (IC₅₀) at 194.95mg/mL and 197.64mg/mL respectively. SAAE was successful in stimulating the immune system in immune suppressed mice (increased the differential, total WBC, splenocytes, RBC, platelets and haemoglobin level) significantly (P < 0.01) compared to CP induced group and control. SAAE at the dose 300mg/kg b.w improved the macrophage activity to engulf the carbon particles in circulation system in carbon clearance test. Whereas SAAE increased the T-lymphocytes, rosettes formation and increased T_H cells against SRBC. Significant results in T-cell population test and delayed hypersensitivity test indicates the cell mediate response and activation of adaptive immunity. Moreover dose dependent increase in antibody titer test exhibited the humoral immune resonse of SAAE. In addition, SAAE displayed nontoxic nature even at the highest dose 5000mg/kg b.w in an acute toxicity study. It could be due to the presence of phytoconstituents like proteins, carbohydrates, cardiac glycosides, tannins, diterpenoids along with terpenoids (260µg/g), flavonoid (100µg/g), and phenol (144µg/g). The synergetic activity of secondary metabolites of SAAE was responsible for compelling outcome in anti-inflammatory, immunostimulatory, cell mediated and humoral immune response.

 

 

INTRODUCTION:

Practise of non-steroidal anti-inflammatory (NSAIDs) and glucocorticoids drugs in inhibiting inflammation is common. According to statistics, NSAIDs with greater risk are extensively used in worldwide^[1]. Drugs express severe side effects and patient display weak anti-inflammatory responses after a long run and susceptible to infections. The downside of the drug made treatment for inflammation to strenuous. This lacuna can be dissolved by using efficient immunomodulators. Approach of immunomodulators/ immunotherapy in treatment would be a new hope for preventing the disease as well as promotion of health by strengthening tissues so that they can withstand exogenous endogenous stress^[2,3]. Unfortunately, these have not been meaningfully interpreted in modern scientific language nor have they been subjected to verification by rigorous experimental or clinical methodology. About 83% of herbal supplements to treat dysfunction or diseases are available in market (morphine from Papaver somniferum, Aswagha from Withania somnifera, Ephedrine from Ephedra vulgaris, curcumin from Curcuma longa) and their need is suspecting to reach 95% in upcoming years^[4]. Therefore overpowering immunoceutical agent development is in entail. As such Simarouba amara (Aubl.) bark aqueous extract has been reported for wide array of medicinal property viz, amoebicide, analgesic, anthelmintic, antidysenteric, antileukemic, antimalarial, antimicrobial, antitumorous, astringent, febrifuge, skin hydrator with active component quassinoids has a long history in herbal medicine in many other countries. Withal water extracts of Simarouba is the subject of one U.S. patent for increasing skin keratinocyte differentiation, skin hydration and moisturization till to date^[5,6]. In addition, bark inhibited the S. aureus, S. typhi, K. pneumonia, P. auregenosa and Enterotoxigenic E. coli growth effectively and antidiarrhoeal property was recorded in our previous study. Stock-still no scientific validated information is available to confirm its pharmacological effect. Thus, present study was to configure the stated activity of S. amara (Aubl.) bark aqueous extract (SAAE) in respective experiments.

 

MATERIALS AND METHOD:

Experimental animals:

Female BALB/c mice (22-25g b.w) were used for the experimental studies. Animals were housed under temperature (22-25°C), relative humidity (50- 65%) 12 h light/dark cycle. They were allowed for free access of food and water ad libitum. The animals were handled according to Institutional Animal Ethics Committee (IAEC) standard guidelines in experiments (NGSMIPS/IAEC/MARCH-2018/103).

 

Antigen preparation:

Fresh sheep blood (SRBC) was collected in sterile Alsever’s solution (1:1) washed three times with phosphate buffered saline (pH 7.2) by centrifugation at 3000 ×g for 10 min. Then it was stored in PBS at 4ºC for use in the immunization.

 

Plant extraction and phytochemical screening:

S. amara (Aubl.) bark powder was extracted with distilled water (10:100 w/v) as solvent by boiling for 15 min. Filtered extract was concentrated in lyophilizer and stored in vials at 4ºC. Extract was subjected to qualitative phytochemical analysis^[7] and quantitatively total phenolic content, terpenoids^[8], flavonoids^[9] were determined by using spectrophotometer.

 

Acute toxicity Study:

A toxicity study was performed by managing five groups of six mice each. SAAE was administered in doses (1000-5000mg/kg b.w) orally at once. The mortality and behaviour changes were observed for one to seven days^[10].

 

SAAE on carragenan induced peritonitis:

Overnight fasted mice were divided into five groups of five mice each. Group I (control) received saline 1mL/100g b.w, group II (positive control) dexamethasone 2mg/kg b.w, p.o., group III, IV, and V received SAAE (100, 200, and 300mg/kg b.w, p.o.) respectively. Mice were injected with carrageenan (0.25mL) (1% carrageenan in 0.9% saline) via i.p, after one hour of pre-treatment with SAAE. Four hours later, mice from each group were sacrificed by cervical dislocation and abdomen was opened longitudinally. The peritoneal cavity was washed with 2mL of saline containing 3mM EDTA. Exudates were determined for PMNLs count (Neubauer chamber after dilution with Turk solution; 0.01% crystal violet in 3% acetic acid) accumulated NO^-₂ (nitrite assay according to colorimetric assay based on Griess reaction)^[11].

 

SAAE on cyclophosphamide (cp) induced immunosuppression:

The experiment was performed according to the method for 15 days^[12]. Briefly, 30 mice were fasted for 18 h and divided into six groups of five mice each. Group I received saline (1mL/100g b.w, p.o.), cyclophosphamide (30mg/kg b.w, i.p/day) was subjected to group II, III, IV, V, and VI for initial three days. These immunosuppressed groups (III-VI) were dosed with positive control; levamisole (50mg/kg b.w, p.o./day), IV, V, and VI with SAAE (100, 200  and 300mg/kg b.w, p.o./day) respectively and dosed from day four to 15. Then blood samples were collected on 16^th day in an anticoagulant containing vial for determination of immune cells; total WBC, differential WBC, RBC cells, platelets haemoglobin concentration and splenocytes from spleen.

 

SAAE on phagocytic activity (carbon clearance test):

The experiment was performed according to the method with modification^[13]. Parameter was studied for seven days in groups of mice. Group I received saline (1mL/100g b.w, p.o.), group II dosed with positive control; levamisole (50mg/kg b.w, p.o./day). Group III, IV and V with SAAE 100, 200 and 300mg/kg b.w, p.o. respectively. On 9^th day, blood was collected to anticoagulant containing vial from retro-orbital plexuses or tail cut method immediately from groups of all mice. Then, Indian ink [(Indian ink (3mL): saline (4mL): 3% gelatin (4mL)] was injected through intravenous route (tail vein) and blood was collected after 15 min of injection. After, 25-50µL of blood sample was mixed with 2 to 4mL of 0.1% sodium carbonate solution to lyses the erythrocyte. Further absorbance was observed at 660nm. Then carbon clearance was calculated using the equation:  K= (Log_e OD₁-Log_eOD₂)/15

 

OD₁ and OD₂ represents optical density of blood sample at 0 and 15 min,

 

Phagocytic Index (α) = (³√ K) × Weight of Animal/ Weight of liver + Weight of Spleen

 

SAAE on T-cell population (erythrocyte rosette assay):

Animals were dosed studied for 10 days. Group I received saline (1mL/100g b.w, p.o.), Group II dosed with positive control; levamisole (50mg/kg b.w, p.o./day). Group III, IV and V administered with SAAE (100, 200 and 300mg/kg b.w, p.o.). On 11^th day, plasma was separated from mice in each group. Equal volume of (0.25mL) plasma and 0.5% SRBC were mixed, incubated for 5 min at 37°C. Mixed suspension was centrifuged at 1000rpm for 5 min and supernatant was cooled at 4°C for 2 h. Then lymphocytes were counted (Neubauer chamber) and lymphocyte binding with three or more erythrocytes considered as a rosette^[4].

 

SAAE on Antibody titer:

Animals were dosed studied for 14 days. Group I received saline (1mL/100g b.w, p.o.), group II dosed with positive control; levamisole (50mg/kg b.w, p.o./day). Group III, IV and V with SAAE (100, 200 and 300mg/kg b.w, p.o.). Mice were immunized with 0.1mL SRBC (1×10⁸) i.p on day seven. After day 14, blood was withdrawn from mice in each group and serum separated was used for antibody titer. Result obtained was expressed in log₁₀ 2 of the serum, and antibody titer obtained was recorded as primary antibody response^[14].

 

SAAE on delayed type of hypersensitivity (DTH):

The experiment was studied for seven days^[15] . Group I received saline (1mL/100g b.w, p.o.), group II dosed with positive control; levamisole (50mg/kg b.w, p.o./day). Group III, IV and V administered with SAAE (100, 200 and 300mg/kg b.w, p.o.). The animals were immunized with 0.1mL of SRBc  containing 1×10⁸ cells, i.p on day 0 and challenged on 7^th day with 0.05mL of 2 x 10⁸ SRBC on the right hind paw. Meanwhile, the contra lateral paw received equal volume of saline. The thickness of the foot pad was measured after post challenge (24 h and 48 h) using screw gauge. The difference in the thickness of the right hind foot paw was used as a measure of DTH reaction left hind paw was considered as control.

Statistical Analysis:

Difference between group means was analyzed with one way ANOVA followed by Dunnett and Turkey’s test. P < 0.05, P < 0.01and P < 0.001 were considered as statistically significant (Graph Pad Prism version 5.0; Graph Pad Software, La Jolla, CA).

 

RESULTS AND DISCUSSION:

Phytochemical screening:

A qualitatiative phytochemical screening showed meticulous presence of cardiac glycosides, phenols, tannins, flavonoids, terpenoids, and diterpenes. SAAE has total phenol (144µg/g), followed by total flavonoid (100µg/g), terpenoids in higher amount (260µg/g) quantitatively. Withal, chromatogram of crude aqueous extract showed more water soluble components in the extract (data not shown). Precisely, glycolysis pentose phosphate pathway are the producer of complex secondary metabolites in cytosol, endoplasmic reticulum plastids. Flavonoids, monoterpens, diterpenoids, plastoquinone, gibberlins, tochopherol etc have beneficial role in medicine. Higher amount of terpenoids in SAAE confirms biological activity. Since, terpenoids are important molecule as antibiotics, hormones, antifeedant become part of treatment. For instance sesquiterpens (C₁₅) has been used as antimalarial drug.

 

Acute toxicity study:

Oral treatment with SAAE in single oral dose of 1000-5000mg/kg b.w did not produce any clinical sign of toxicity (piloerection, altered locomotion activity, sign of diarrhoea, lethargic, sedative effect) or mortality in a group of mice after 24h. Withal, food, water intakes and body weight gain of the animals were normal as like in control group during the a week of observation (Fig.1). The LD₅₀ was not observed in the study and result suggested the low or nil toxicity of SAAE. Blend of secondary phytochemicals and metal ions like Mg²⁺, Zn^2+, Ca²⁺ and Fe²⁺( ICP-OES method, previously published data) in aqueous extract might be reason for non toxic nature even at the highest dose.

 

Figure. 1 Acute toxicity study of SAAE; weight of the mice before and after treatment.

 

SAAE on carrageenan induced peritonitis:

Carrageenan induces inflammation and increased accumulation of PMNLs and nitrite in peritoneal exudates. The valuses found to be highly significant (P < 0.001) compared to control. Besides, SAAE reduced the infiltration of PMNLs and NO^-₂ accumulation in dose dependent manner. Dexamethasone is glucocorticoids used to treat inflammation and inhibits the PMNLs in our experiment and significant (P < 0.001) compared carrageenan induced group. SAAE at 300mg/kg b.w significantly (P  < 0.001) reduced the PMNLs against carrageenan group (Fig. 2a). Whereas, measured nitrite level is an indicator of nitric oxide. Higher dose in our experiment works well in reducing the nitric oxide accumulation significantly (P < 0.001) against carrageenan induced group (Fig. 2b). Moreover, half of the inhibitory concentration (IC₅₀) of SAAE in PMNLs and NO^-₂ was found to be 194.95 and 197.6mg/mL. Among the available phlogistic agent (dextran, bradykinin, β glucan) well studied carrageenan initiates an inflammation within three to four hours. Peritonium contains lesser quantity PMNLs (neutrophils; first line defender, basophils and eosinophils), macrophages and lymphocytes. Up on stimulation, de-granulation event releases enzymes, reactive oxygen species (ROS; nitric oxide), cytokines (TNF-α, IL-1β, IL-6, IL-4, IL-10, and IL-12), histamine, lipid mediators (leukotrienes), platelet activating factors, in addition with recruiting other immune cells via signals (ecosanoids; cylcooxygenase pathway, PGE₂, cell signalling; NFƙB) and initiate inflammation^[16]. Fundamentally, corticosteroids are released by adrenal gland, have key role in homeostasis of heart, immune, nervous and endocrine system. Majority of drugs works by inhibiting PMNLs and associated signals. Altered work efficiency takes system into imbalance. Various findings on molecular pharmacology of components in plants (curcummin, epigallocatechin-3-gallate, zingerone) on inflammation exhibited triggering inflammatory markers (JAK-STAT pathway, blocking iNOS expression, and cytokine mediated NFƙB initiation) at various levels and speedy recovery without affecting the synchronization in functioning^{[9,12,16,17,18]}. S. amara (Aubl.) bark has triterpenoid; quassinoid, alkaloid; 5-hydroxycanthin-6-one, β-sitosterol along with other metabolites^[19]. Notable reduction in cell migration (PMNLs) and ROS in SAAE pre-treatment might with its constituents and agreed with previous likely findings. As per document inhibition of leukocytes anti-inflammatory molecule in treatment is an inappropriate which destroy the defense^[20]. Thus necessity of searching a novel compound/agent which functions on cell migration is to be concerned. SAAE showed anti-inflammatory action and might become one of promising agent for treatment.

 

Figure 2 a and b: Inhibitory ability of SAAE on cell migration (PMNLs and nitrite) in peritoneal cavity. Effective in dose dependent manner. Data were expressed as Mean ± SE (n = 5) and analysed with one-way ANOVA followed with Tukey’s test. The asterisk stands for * P < 0.05,  ** P < 0.01 & ***P < 0.001 compared to model control (carrageenan). Dex: Dexamethasone.

 

SAAE on cyclophosphamide induced immunosupression:

SAAE stimulate the haematopoiesis, splenocyte production efficiently at higher dose significantly (P < 0.01) (300mg/kg b.w) in immuno-suppressed mice (Table 1). Cyclophosphamide is a strong immunosuppressive agent used during transplantation, autoimmune disorders and used as immunosuppressant in our study. Cyclophosphamide affects the precursor in bone marrow and limits haematopoiesis^[21]. Data on Beetroot juice and involvement of its phytoconstituents, amino acids, carbohydrates, vitamin-C, B₅, B6 (12%), B12, calcium, phosphorous, zinc, magnesium (56%), selenium, cobalt, iron copper have been found critical for high quality production and active haematopoiesis^[2]. Majorly, polyphenols has the regulation capacity over protein synthesis, transportation across the membrane, cell growth, exclusively viable activity of CD4⁺ T-cells which maintains the cellular homeostasis by reducing the oxidative stress on the RBC and increases the GSH (glutathione) level^{[10,22,23,24]}. In contrast, levamisole; a heterocyclic compound (immunostimulating agent) which mimic the thymic hormone thymopoietin; contain imidazole ring, which stimulates the lymphocyte proliferation, regulatory T-cells, neutrophils, macrophages and reduced oxidative stress and boost the immune system^[25]. Likely mechanism observed could be the reason for obtained results in our experiment.

Table 1. Immunostimulaatory effect of SAAE on cyclophosphamide (CP) induced immunosuppression

Test	Control	CP (30mg/kg)	CP + LM (50mg/kg)	CP+ SAAE (100mg/kg)	CP+ SAAE (200mg/kg)	CP+ SAAE (300mg/kg)
Erythrocytes (106/mm3)	7.45±0	6.3±0.1	8.9±0.3	7.7±0.6	8.25±0.2	8.9±0.3
Hemoglobin (g/dL)	9.9±0	6.5±0.2	11±0.1	10.7±0.5	10.8±0.4	12.2±0.8
Leukocytes (103/mm3)	4.43±0.8	3±0.2	11.0± 0	8.52±0.5	14.05±1.4	25.90±1.3**
Neutrophils (%)	67±1.4	56±0.9	84± 0.3	78.5±0.4	84.5±4.4	87±2.9**
Lymphocytes (%)	10.5±0.4	7.5±0.4	14.5±0.5	12.5±3.4	14.5±0.5	20±0**
Monocytes (%)	0.5±0.4	0±0	1±0	0.5±0.4	0.5±0.4	0.5±0.4
Eosinophils (%)	1±0	1±0	0.5±0.4	0±0	1±0	0±0
Basophils (%)	0±0	0±0	1±0	0±0	0.5±0.4	0±0
Platelets (109/L)	7±0	3±0.2	8.8±0	7.35±0	8.3±0.5	8.85±1.0
Spleenocytes (108cells/mL)	1.15±0.1	0.9±0.1	2.35±0	1.7±0.5	2.69±1.1	3.1±0.6**

CP was induced for  initial three days for immunosupression and treated with respective treatment in mice groups. LM: Levamisole (positive control). (SAAE) administration increases the leukocytes and splenocytes in dose dependent manner. Data were expressed as Mean±SEM (n = 5) and analysed with one-way ANOVA followed with Dunnett test. The asterisk** stands for P < 0.01 compared with CP induced group and control.

 

SAAE on phagocytic activity (carbon clearance test):

Clearance of foreign particles is related to mononuclear phagocytic system. An enhanced activity of macrophages in processing of colloidal carbon particles in circulating system was observed in study with SAAE. This was absolutely associated with dose. SAAE increased the rate of carbon clearance at 300mg/kg b.w efficiently (P < 0.05) (Table 2). Chiefly, macrophages carries radical dependent (ROS; super oxide anion, H₂O_2, hydroxyl) respiratory burst to process an antigen ^[17]. For an optimum antigen mopping, reactive species needs vitamins, minerals and nutrients. A document clearly stated that Zn²⁺/Cu²⁺ are essential for anti-oxidant enzyme; super oxide dismutase (SOD) effective ROS. Withal, presence of hydroxyl (-OH) groups in polyphenols helps them to chelate the metal ion and successful scavenging activity. Elements like zinc (12%), vitamin A (44%), E, C (31%), D, selenium, together with flavonoids and terpenoids helps macrophages to remain active in antigen processing via producing dependent cytokines (IL-1, IL-6 and TNF-α) by mononuclear cells. Moreover, macrophages and dendritic cells alarm the tissue for invading infections. Thus, they are potential enough to switching adaptive immunity from an innate immunity. Comparebly, pre-treatment of SAAE up to a week might enhance the macrophages activity through functioning of varied molecule in it^[26].

 

Table 2. Effect of SAAE on carbon clearance

Group	Rate of carbon clearance (K)	Phagocytic Index (α)
Control	0.008±0.002	1.0 ± 0.01
Levamisole 50mg/kg	0.029±0.03	3.62 ± 0.07
SAAE 100mg/kg	0.0123±0.01	2.46 ± 0.32
SAAE 200mg/kg	0.0205±0.01	2.56 ± 0.14
SAAE 300mg/kg	0.044±0.04*	5.5 ± 0.16*

SAAE increases activity of macrophages and enhanced phagocytosis in dose dependent manner. Data were expressed as Mean ± SEM (n = 5) and analysed with one-way ANOVA followed with Dunnett test. The asterisk* stands for P < 0.05 compared with control.

 

SAAE on T-cell population (erythrocyte rosette assay):

Treatment with SAAE up to 10 days increased the T cell number against SRBC in all doses of SAAE. Mean while efficient erythrocyte rosette formation were observed in 300mg/kg b.w (P < 0.01) followed by 200mg/kg b.w. Whereas, SAAE at 100mg/kg b.w was not effective in showing avidity with SRBC, hence failed to form rosettes (Fig. 3). Primarily, T-cell maturation in thymus and their rosette formation against specific antigen become down side in immunosuppressed, malnutrition and patient with alcoholic liver disease. As such, measurement of rosettes provides the most sensitive information on T- cell population. In likely manner, a finding in our experiment is in agreement with previous studies^[27]. Pronounced effect of SAAE in present study has limiting to draw an inference in involvement of avid T-cell subpopulation in immuno-competency and it needs an established study.

 

Figure 3: SAAE increased T-lymphocytes. Data were expressed as Mean ± SEM (n = 5) and analysis was performed with one-way ANOVA followed with Dunnett test. Asterisk** stands for P < 0.01 & ***P < 0.001 compared with control.

 

SAAE on humoral immune response (antibody titer):

Measure of antibodies reveals level of antibodies raised after SRBC stimulation. SAAE might be involved in maturation and activation of B-cells via α-globulin antibodies. SAAE had shown maximum antibody titer value at 300mg/kg b.w significantly as compared to control (Fig. 4). This is the evidence for humoral response and adaptive immunity. Fundamentally, immunoglobulins are glycoprotein; its function depends on enzyme cerruloplasmin. For their maturation and activity demand involvement of fatty acids, vitamin C, B₆, B₁₂, zinc, manganese, selenium in bone marrow ^[28]. Withal, SAAE has shown positive result for pentose sugar, amino acid (data not shown) with the other active secondary molecules and suspect active antibody production against SRBC.

 

Figure 4: SAAE on humoral immune response. SAAE increases the immunoglobulins and antibody titer against SRBC in dose dependent manner. Data were expressed as Mean ± SE (n = 5) and analysis was performed with One-Way ANOVA followed with Dunnett test.  Asterisk stands for * P < 0.05, **P < 0.01 & ***P < 0.001 compared with control.

 

SAAE on delayed hypersensitivity test (DTH):

DTH response is declined with age and other ailments and has correlation with disease burden^[29]. Oral administration of SAAE interestingly enhances the cell mediated immune protection at moderate dose 200mg/kg but was diminished after 24 h. In contrast, higher dose 300mg/kg b.w increases the number of Th1cells gradually and protective response was observed after 48 h significantly (P < 0.01) (Fig. 5). In fact, cytotoxic T-cells are depleted with increase in memory T cell. The decreasing diverse T-cell receptors (TCR) are the reason for not being available for protection. SAAE might evoke a classical CD4⁺ T cell-mediated DTH within 24 h against antigen or the primary immune component is elicited by B cells which activated rapidly after immunization or the faster production of serum IgM antibody, complement activation at early and post antigen challenge. ^[30].

 

Figure 5. Effect of SAAE on DTH. SAAE stimulate the T_H cells and increases delayed hypersensitivity. Data were expressed as Mean ± SEM (n = 5) and analysis was performed with one-way ANOVA followed with Dunnett test and asterisk** stands for P < 0.01 compared with control.

 

CONCLUSION:

Taken collectively, the experimental result showed that requirement of dose for optimal immune response is greater than the amount required for its generation and maturation. In addition, SAAE exhibit the similar mechanism like as levamisole in restoring the immunosupression. More water soluble phytonutrients in SAAE stimulated the immune system through the gut-associated lymphoid tissues (GALT) and are integrating with different elements of the immune system at varied time point without disturbing balanced function. Moreover, a first-hand study on anti-inflammatory and immunostimulatory property needs an advanced study for better understanding of its elaborative mechanism. Nevertheless, our primitive study suggested that SAAE has merit to become a promising immunostimulating anti-inflammatory agent.

 

ACKNOWLEDGMENT:

The authors are thanks to Mr. Sandesh K. Gowda Director, Niranthara Scientific Solutions Pvt Ltd., Bengaluru, Karnataka for adequately providing the plant sample. I thank to Vasanth Kumar for helping in lyophilisation. Thanks to Navya Nagaraj for procuring the animal to laboratory and Mr. Santhosh K.N and Prashanth J for their assistance in the animal experiment. Add on authors are grateful to Department of P.G Studies Research in Microbiology, Kuvempu University, Shankaraghatta for providing the laboratory facility.

 

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Accepted on 21.01.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(9):4165-4171.