In vitro and in vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album & Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats

Accepted Manuscript Full Length Article In vitro and in vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album & Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats Syed Murthuza, B.K. Manjunatha PII: DOI: Reference:

S2314-8535(18)30264-6 https://doi.org/10.1016/j.bjbas.2018.10.001 BJBAS 309

To appear in:

Beni-Suef University Journal of Basic and Applied Sciences

Received Date: Revised Date: Accepted Date:

2 June 2018 14 September 2018 25 October 2018

Please cite this article as: S. Murthuza, B.K. Manjunatha, In vitro and in vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album & Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats, Beni-Suef University Journal of Basic and Applied Sciences (2018), doi: https://doi.org/ 10.1016/j.bjbas.2018.10.001

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“In vitro and in vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album & Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats”

1. Mr. Syed Murthuza Research Scholar Department of Biotechnology, The Oxford College of Engineering Bommanahalli Bengaluru-560068 [email protected] 2. Dr. B.K. Manjunatha Professor and Dean Research Department of Biotechnology, The Oxford College of Engineering Bommanahalli Bengaluru-560068 [email protected] Acknowledgements: The authors are greatly thankful to Director of Minorities, Government of Karnataka and Greatly thankful to Department of Biotechnology, The Oxford College of Engineering, Bangalore. We greatly acknowledge the constant support of RB & HSD, Bhabha Atomic Research Center, Mumbai, and Board of Research in Nuclear Science, Government of India (Sanction No. 2012/34/21/BRNS).

In-vitro and In-vivo evaluation of anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album and Anthocephalus cadamba in murine macrophages raw 264.7 cell lines and Wistar albino rats. ABSTRACT:

The present study reports the in-vitro and in-vivo anti-inflammatory potency of Mesua ferrea, Saraca asoca, Viscum album and Anthocephalus cadamba, potent medicinal plants of Western Ghats of Karnataka India. Lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cell line was used in in-vitro assay. Pretreatment with methanolic extracts at non toxic concentrations abridged the LPS-induced protein levels. Among the four plants tested, methanolic extract of Viscum album (70.20±1.094) and Mesua ferrea (68.29±2.862) showed potent anti-inflammatory activity at 100 µg concentration with an IC50 value of 57.23±1.922 and 63.36±3.791 µg/ml. In vivo anti-inflammatory activity was evaluated using carrageenan-induced paw edema in Wistar albino rats, Viscum album (0.6167±0.01667) and Mesua ferrea (0.6833±0.01667) showed potent anti-inflammatory activity (at 100 mg/Kg.bw). The obtained results indicate the potency of active constituents of Mesua ferrea and Viscum album in the development of effective anti-inflammatory drugs. KEYWORD: Antiinflammatory; Lipopolysaccharide; Paw edema; Medicinal plants.

1.

INTRODUCTION:

Inflammation, the first line of defence against pathogens can contribute to all phases of tumorigenesis including tumour initiation, promotion and metastasis. Inside this scaffold, the Toll-like receptor (TLR) pathway plays a vital role (Maiorov et al., 2013). Inflammation is a critical component of tumor progression. Many cancers arise from sites of infection, chronic irritation and inflammation (Coussens and Werb 2002). Inflammatory cells can furthermore secrete reactive oxygen species (ROS) that encourage mutations, lead to failure of DNA repair, activation of oncogenes and eventually cancer. ROS promote activation of inflammatory genes which is regulated via c-MYC, KRas and Wnt signal pathways, which takes part in tumorigenesis (Trinchieri; 2012 and Chang 2010). Chronic inflammation has come up as one of the trademark of cancer; in addition it play a key role in modulating sensitivity of tumors which leads to opening of several transcription factors, modulating the expression of many mediators in tumor cells and cells of the microenvironment promoting cancer progress (Hanahan and Weinberg 2011; Das et al., 2016 and Reuter 2010) The inflammation might direct the origination of cancer, considering that chronic inflammation is measured by infiltration of mononuclear immune cells, tissue damage, fibrosis, and amplified angiogenesis (Nambiar et.al, 2011). Further, augmented genomic break, increased DNA synthesis, cellular proliferation, pauses in DNA repair pathways, inhibition of apoptosis and promotion of angiogenesis are linked with inflammation (Hofseth and Ying 2006). Each of this process has been drawn in initiation and progression of cancers. Throughout chronic inflammation, pro-inflammatory molecules, such as cytokines, ROS, and NFkB are upregulated (Sarkar and Fisher 2006). Collectively, these processes provide an encouraging microenvironment for the exponential development of malignant cells. Thus, inflammation may offer equally the key mutations and favourable condition for tumour growth.

Ethnomedicinal plants contain diverse range of bioactive phytoconstituents which exhibits antiinflammatory property (Abraham et al., 1993 and Kim et al., 2003). Future development of antiinflammatory drug may en-routes for evaluation of more herbal products that either stimulates the production of anti-inflammatory cytokines or regulates it (Hebbar et al., 2002; Romay et al., 1998 and Sidhu et al., 1999). In this context, an attempt is made herewith to screen the ethnomedicinal plants for their anti-inflammatory activity. Further work viz., isolation, characterization and mechanism of action study is under process.

2.

MATERIALS AND METHODS.

2.1. Collection and Identification of Plant Material: The whole plant of Viscum album was collected from Shivamogga, Karnataka. The stem bark of Mesua ferrea, Saraca asoca, and Anthocephalus cadamba were collected from Shetihalli reserve forest, Western Ghats of Shivamogga, Karnataka. Collected plants were taxonomically authenticated and deposited with voucher number OX/BT-239, OX/BT-240, OX/BT-241 and OX/BT-242 respectively in the Department of Biotechnology, The Oxford College of Engineering, Bengaluru. 2.2. Extraction of Plant Material: Stem bark was washed under running tap water to remove adhered dirt, followed by rinsing with distilled water, shade dried and pulverized in a mechanical grinder to obtain coarse powder. The powdered material was stored in an airtight container until use. 250 g of each plant material was subjected to soxhlation using methanol for about 48 hours. The extracts were filtered and concentrated to dryness in vacuum under reduced pressure using rotary flash evaporator (Ika, Germany) (Manjunatha et al., 2007). 2.3. Invitro Antiinflammatory Activity. 2.3.1. The HRBC Membrane Stabilization: Human blood mixed with equal volume of Alsevers solution (2% Dextrose, 0.8% Sodium citrate, 0.5% Citric acid and 0.42% NaCl) and centrifuged at 3000 rpm for 10 min. The packed cell was washed with isosaline and 10% suspension was made. Crude drug was prepared to a concentration of 50 mg/ml using distilled water. 1 ml of each extracts was mixed with 1 ml of 2 mM phosphate buffer, 2 ml of hyposaline and 0.5 ml of HRBC suspension. This mixture was incubated at 37 0C for 30 min and centrifuged at 3000 rpm for 20 min. The supernatant was collected and measured spectrophotometrically at 560 nm. Diclofenac (50 mg/ml) was used as a reference standard and a control was prepared by omitting the extracts (James et al., 2009).

2.3.2. Heat Induced Hemolysis:

Heat-induced hemolysis assay was performed by following the method of Shinde et al., 1999. The reaction mixture (2 ml) consisted of 1 ml of test drug (50 mg/ml) and 1 ml of HRBCs suspension. Reaction mixture with 1 ml saline was used as control and Diclofenac (50 mg/ml) as the standard drug. The reaction mixtures were incubated in a water bath at 56 0C for 30 min, cooled and centrifuged at 2500 rpm for 5 min. The absorbance of the supernatants was measured at 560 nm. 2.3.3.

Proteinase Inhibitor Assay:

The test was performed according to the modified method of Oyedepo and Femurewa 1995. The reaction mixture (2 ml) contained 0.06 mg/ml trypsin, 1 ml 20 mM Tris-HCl buffer (pH 7.4) and 1 ml test sample. The mixture was incubated at 37 0C for 5 min and added with 1 ml of 0.8% (w/v) Casein. The mixture was again incubated for 20 min, 2 ml of 70% per-chloric acid was added to terminate the reaction. Obtained cloudy suspension was centrifuged at 3000 rpm for 5 min and the absorbance of supernatant was measured at 210 nm. 2.3.4. Inhibition of Albumin Denaturation Assay The extracts were screened for their anti-inflammatory activity following the method of Mizushima and Kobayashi 1968, with slight modification. The standard drug Diclofenac (50 mg/ml) and test sample (50 mg/ml) was dissolved in 1 ml of 2.5% Dimethyl formamide (DMF) and diluted with 0.2 M phosphate buffer (pH 7.4). Test solution (1 ml) containing different concentration of drug was mixed with 1 ml of 1 mM albumin solution in 0.2 M phosphate buffer and incubated at 27 0C in water bath for 10 min. The turbidity was measured at 660 nm spectrophotometrically. Percentage inhibition of denaturation was calculated. Percentage Inhibition = Abs Control - Abs sample × 100 Abs Control 2.4. Antiiflammatory Assay Using RAW 264.7 Cells: 2.4.1.

Cell Culture

The murine monocytic macrophage RAW 264.7 cell line (ATCC, Manassas, VA, USA) was cultured in DMEM (2 mM L-glutamine, 45 g/L glucose, 1 mM sodium pyruvate) with 10% FBS. The cultured cells were incubated at 37 °C with 5% CO2.

2.4.2. Inhibition of Nitric Oxide (NO) Production The cells (1×106 cells/ml) were seeded in 96-well plate and incubated for 24 h at 37 °C with 5% CO2. Then, 100 μl of extract (1 mg/ml) in DMSO was serially diluted to give a two-fold concentration variation and then added onto cells. Cells were then stimulated with and 10 μg/ml E.coli LPS and incubated at 37 °C for another 17 h. Then, 100 μL of supernatant from each well was mixed with an equal volume of Griess reagent and incubated for 10 min. The absorbance of the resultant solution

was measured with a microtitre plate reader (Tecan device) at 550 nm. The concentrations of nitrite were calculated from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated (Kiemer and Vollmar 2001). 2.5.

Invivo Antiinflammatory Activity.

2.5.1. Dosage fixation and Grouping of Animals: For oral administration, suspensions of extracts were prepared in 1% gum tragacanth following the LD50 value of the extract (1/10th of LD50). The animals were divided into six groups of six animals each; the body weight of each animal was recorded. Group 1 was treated as control, group 2 received Diclofenac 10 mg/kg.bw. Animals in groups 3 were treated with M. ferrea 250 mg/kg.bw, group 4 with S.asoca 120 mg/Kg.bw, group 5 with V.album 150 mg/Kg.bw, group 6 with A. cadamba 200 mg/Kg.bw. 2.5.2. Carrageenan Induced Paw Edema in Rats: Antiinflammatory activity was determined in albino rats of either sex. Test drugs were administered orally to the respective groups one hour before Carrageenan injection. Acute inflammation was induced by injecting Carrageenan (0.1 ml of 1% w/v suspension) in the right hind paw of the rats under the plantar aponeurosis. The inflammation was quantitated in terms of ml i.e. displacement of water by edema using a digital plethysmometer immediately before and after Carrageenan injection at +1,+2, +3, +4, +5 and +6 h. The percentage inhibition of edema was calculated for each group with a vehicle-treated control group (Winter et al., 1962). The difference in the initial ‘0’ h and volume at +1 h indicate paw edema at 1st h following Carrageenan administration. Accordingly, paw edema at +2, +3, +4, +5 and +6 h were calculated. Then percentage inhibition of paw edema was calculated. Percentage inhibition of paw edema =

(1– average inflammation of the drug treated Average inflammation of the control group) × 100

2.6. Statistical Analysis The values expressed are means of three replicate determinations ± standard deviation. The statistical analysis was carried out by analysis of variance (ANOVA).

3.

RESULTS:

3.1. Invitro Antiinflammatory activity: A study on the ability of plant extracts in the stabilization of RBCs membrane indicates that, methanolic extracts of M. ferrea (71.78±0.9180) and V. album (67.06±2.763) has highest potential followed by S. asoca (54.80±0.9771) and A. cadamba (45.56±4.181) compared with standard drug Diclofenac (75.16±1.973) as shown in Table-I.

The plant methanolic extracts were effective in inhibiting the heat induced haemolysis of the erythrocyte membrane. V. album (48.69±2.526) exhibited significant inhibition of hemolysis followed by M. ferrea (46.69±1.833), S. asoca (35.52±0.855) and A. cadamba (33.4±1.775) respectively when compared with Diclofenac 50 μg/ml (57.71±0.5279) as shown in Table-I. Denaturation of proteins is a well documented cause of inflammation. As part of the investigation on the mechanism of the in-vitro antiinflammatory activity, the ability of plant methanolic extracts to prevent protein denaturation was studied. The plant extracts were effective in inhibiting heat induced albumin denaturation. Maximum inhibition of 66.58±2.095 was observed in M. ferrea methanol extract followed by V. album (64.85±2.236), S. asoca (48.88±4.0210) and A. cadamba (39.45±2.458) compared with standard Diclofenac 74.42±0.4964 as shown in Table-I. Among the 4 plants studied, V. album exhibited significant anti-proteinase activity. The maximum inhibition was observed in V. album methanol extract (59.91±1.909) followed by M. ferrea (48.83±5.277), S. asoca (44.48±3.850) and A. cadamba (42.12±4.324). Whereas, the standard drug Diclofenac showed the maximum proteinase inhibitor activity (62.95±0.8139) compared with extracts (Table-I). 3.2. In-vitro antiinflammatory activity of plant extracts in murine macrophage RAW 264.7 cells. The study results showed that V. album (70.20±1.094) possessed better activity followed by M. ferrea (68.29±2.862), S. asoca (41.12±1.852), A. cadamba (56.13±1.596) and M. ferrea: S. asoca combination (66.59±1.631) at the concentration of 100 µM respectively. The drug inhibited NO release with the IC50 value 57.23±1.922 µg/ml of V. album, M. ferrea (63.36±3.791 µg/ml), S. asoca, (110±5.993 µg/ml), A. cadamba (77.58±3.549 µg/ml) and M. ferrea: S asoca combination (63.07±1.957 µg/ml). Further, NO inhibitory activity of the extracts were in a dose-dependent manner as depicted in Table-II and Graph I and II. 3.3. Effect of plant extracts on paw volume and percentage inhibition in carrageenan-induced paw edema. The study revealed that, the percent reduction in paw edema on 5th hour recorded highest in V. album (0.6167±0.01667) at 25 mg/Kg b.w followed by S. asoca (0.5833±0.01667) at 100 mg/Kg b.w, A. cadamba (0.7667±0.0833) at 100 mg/Kg b.w and M. ferrea (0.6833±0.01667) 50 mg/Kg b.w, in a dose-dependent manner as shown in Table-III.

4.

DISCUSSION.

The erythrocyte membrane is analogous to the lysosomal membrane which is utilized to study antiinflammatory activity. Stabilization of lysosomal membrane is fundamental in restraining the inflammatory response by preventing the release of lysosomal constituents of activated neutrophils, such as bacterial proteases, which causes auxiliary tissue inflammation and extracellular release

(Manjunatha et al., 2013 and Nanumala 2012). In our study all the methanolic extracts significantly stabilized HRBC membrane and effectively inhibited the heat induced haemolysis indicating the antiinflammatory ability of the phytoconstituents present in the extracts. Our investigation showed that, all methanolic extracts possess significant anti-proteinase activity and it is logical to predict that, the anti-inflammatory activity of the tested drug may be due to the anti proteinase activity of the extracts (Sakat et al., 2010). Generally biological proteins lose their role when denatured; denaturation of proteins is a well accepted case of inflammation (Leelaprakash and Das 2011), so any phytoconstituent which has ability to defend denaturation of protein could contribute significantly in the course of inflammation. In our investigation it was evident that, the extracts possess ability to inhibit heat induced albumin denaturation. Macrophages play a decisive role in inflammatory responses through the generation of various cytokines. Activated macrophages act in response to pathogen invasion by releasing various proinflammatory cytokines and inflammatory mediators such as nitric oxide (NO). Our present study constantly demonstrated that, the crude extract inhibited the inflammatory response in LPS-stimulated RAW 264.7 macrophages without cytotoxicity. Among four plants, Viscum album and Mesua ferrea methanol extracts exhibited significant reduction of NO level. The results reveal that the extract may work as efficient anti-inflammatory agents by inhibiting the production of NO. The inhibitory action of NO production in macrophages possibly due to the presence of higher level of phenolic content, prior studies have also reported that phenolic compounds act as excellent anti-inflammatory agents and they play a key role in oxidative stress and inflammation (Alvarez-Suarez et al., 2017 and Yen 2008). NO is a sturdy vasodilator and is also concerned in carrageenan-induced edema, which may be linked to its capacity to increase vascular permeability and edema through changes in local blood flow (Salvemini et al., 1996). Inhibition of NO would diminish the vasodilation and in-turns, decrease in edema which is evident in our study as depicted in table-III. The percent inhibition of inflammation of all extracts was found to be dose dependent. Carrageenan-induced paw edema as an in-vivo model of inflammation has frequently used to assess the anti-edematous effect of the drug candidate. In the early hyperemia, 0-2 h following carrageenan injection, there is a discharge of histamine, serotonin and bradykinin on vascular permeability. The inflammatory edema reaches its utmost level at early phase and after that, it starts deteriorating (Arulmozhi et al., 2005).

5. CONCLUSION:

This research provides the scientific groundwork for the traditional usage of Mesua ferrea, Viscum album, Saraca asoca and Anthocephalus cadamba for various inflammatory diseases. However, further studies on isolation of bioactive compounds, establishment of SAR and analysis of the molecular mechanisms responsible for its anti-inflammatory potential will help in considering these plant genetic resources for anti-inflammatory treatment. Conflicts of Interest: The authors do not have any potential conflicts of interest. Abbreviation: LPS- Lipopolysaccharide ROS-Reactive Oxygen Species DMEM - Dulbecco’s Modified Eagle Media FBS- Fetal Bovine Serum DMSO- Dimethyl Sulfoxide DMF- Dimethyl Formamide NO- Nitric Oxide HIH- Heat Induced Hemolysis HRBC- Human Red Blood Cell

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A n t i i n f la m m a t o r y a c t i v i t y o f M . f e r r e a , V . a l b u m , A . C a d a m b a , S . a s o c a a n d M .f e r r e a : S .a s o c a c o m b i n a t i o n m e t h a n o l i c e x t r a c t s i n m u r i n e m a c r o p h a g e s R A W 2 6 4 . 7 c e l l i n e .

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Graph-I: Effect of M. ferrea, V. album, S. asoca, A. cadamba and M .ferrea: S. asoca combination on inhibition of NO production in murine macrophages RAW 264.7 cell line was Statistical significant at p<0.0001 means ±SE of 3 trials for each concentration. Antiinflammatory studies using murine macrophage RAW 264.7 cells of plant extracts IC50 Values 125

Concentration of plant M

100

75

50

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MfM

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IC50 Values in g.

Graph –II: IC50 value of M. ferrea, V. album, S. asoca, A. cadamba and M .ferrea: S. asoca combination on inhibition of NO production in murine macrophages RAW 264.7 cell line was Statistical significant at p<0.0001.

Sl No.

Name of the (methanol extract)

Invitro Antiinflammatory Mean ± SEM

plant

1.

Diclofenac Standard

HRBC membrane stabilization 75.16±1.973

Heat Induced hemolysis 57.71±0.5279

Albumin denaturation assay 74.42±0.4964

Proteinase inhibition assay 62.95±0.8139

2.

M. ferrea

71.78±0.9180*

46.69±1.833

66.58±2.095*

48.83±5.277

3.

V. album

67.06±2.763

48.69±2.526*

64.85±2.236

59.91±1.909*

4.

S. asoca

54.80±0.9771

35.52±0.855

48.88±4.021

44.48±3.850

5.

A. cadamba

45.56±4.181

33.4±1.775

39.45±2.458

42.12±4.324

Table-I: Effect of M. ferrea, S. asoca, V. album and A. cadamba on HRBC membrane stabilization activity, heat-induced hemolysis, and Albumin denaturation assay was statistically significant at p<0.0001 and p<0.0073 for proteinase inhibition assay, means ±SEM of 3 trials for each concentration. Sl No.

Plant Name

Antiinflammatory activity at 100 µg.

IC50 Value in µg

1.

V. album

70.20±1.094

57.23±1.922

2.

M. ferrea

68.29±2.862

63.36±3.791

3.

S. asoca

41.12±1.852

110±5.993

4.

A. cadamba

56.13±1.596

77.58±3.549

5.

M. ferrea,: S. asoca

66.59±1.631

63.07±1.957

Table-II: IC50 value of M. ferrea, V. album, S. asoca, A. cadamba and M .ferrea: S. asoca combination on inhibition of NO production in murine macrophages RAW 264.7 cell line was Statistical significant at p<0.0001. Name of the Group & Dose/kg bw.

Mean paw volume ± SEM (mL) (% Inhibition) 1st

2nd

3rd

4th

5th

Vehicle Control

1.31 ±0.017

1.36 ±0.045

1.34 ±0.066

1.32 ±0.084

1.34 ±0.071

Std. (diclofenac 10 mg)

1.917±0.08333

1.767±0.04410

1.683±0.03333

1.633±0.01667

1.567±0.03333

M. ferrea (250 mg)

1.700±0.1155

1.583±0.1093

1.233±0.08819

1.000±0.05774

0.6833±0.01667

S. asoca (120 mg)

1.700±0.1528

1.377±0.06227

1.100±0.05774

0.8333±0.03333

0.5833±0.01667

V. album (150 mg)

1.867±0.01667

1.467±0.06667

1.167±0.03333

0.9333±0.03333

0.6167±0.01667

A.cadamba (200 mg)

1.333±0.1202

1.167±0.08819

0.9333±0.06667

0.8333±0.06667

0.7667±0.08333

Table-III: Effect of M. ferrea, S. asoca, V. album and A. cadamba on paw volume reduction and percentage inhibition in carrageenan-induced paw edema for was statistically significant at p<0.0001

Highlights of the Study:

1. In the present research work we aimed to investigate the antiinflammatory efficacy of four ethnomedicinal plants from Western Ghats of Karnataka, India. by Invivo by in vivo in carrageenan-induced paw edema in Wistar albino rats and in vitro by LPSactivated murine macrophage RAW 264.7 model, HRBC membrane stabilization, Heat-induced hemolysis, Albumin denaturation activity & Proteinase inhibitory activity. 2. This research provides the scientific groundwork for the traditional usage of Mesua ferrea, Viscum album, Saraca asoca and Anthocephalus cadamba for various inflammatory diseases. However, further studies are needed for isolation of bioactive compounds for analysis of the molecular mechanisms responsible for its antiinflammatory potential. All necessary files have been uploaded: Manuscript: Abstract Include keywords All figures (include relevant captions) All tables (including titles, description, footnotes) Ensure all figure and table citations in the text match the files provided Indicate clearly if color should be used for any figures in print Graphical Abstracts Further: Manuscript has been 'spell checked' and 'grammar checked All references mentioned in the Reference List are cited in the text, and vice versa Permission has been obtained for use of copyrighted material from other sources (including the Internet) Relevant declarations of interest have been made.