INTRODUCTION:

In view inflammation cause of development in some chronic diseases like rheumatoid arthritis, obesity, diabetes, asthma, inflammatory bowel disease,Parkinson disease, depression of CNS diseases¹. An increasing in outcome of inflammatory pathways resulting in some chronic diseases.

The inhibition of some inflammatory mediators in plays important role in treatment of inflammatory diseases. And the pathogenesis of inflammation is a complex process among cells in the immune system, which is regulated by cytokine networks, and many proinflammatory genes. One of the important classes of immune cells is macrophages, which play a central role in inflammatory response and act as defense against invading agents (bacteria, virus, and fungi). Macrophages respond to pathogen attack by releases various cellular signaling molecules and antimicrobial agents². However, an exaggerated production of these inflammatory mediators by macrophages defensive systems can cause damage to the host³. In detection of pathogenic substances by its pattern–recognition receptors as which used in a wide range of soluble pro-inflammatory mediators⁴. The biological factors released by macrophages that are involved in the inflammation response, are as reactive oxygen species (ROS), nitric oxide (NO), many pathways have been described to explain the effect of anti-inflammatory compounds on macrophages. The importance of these inflammation factors is known and elucidated from many researches. Thus, the effects on them might be used to explain the anti-inflammatory effort of many drugs in cellular level. In order to elucidate the anti-inflammatory properties of drugs, macrophages cell line provides with an excellent in-vitro model. RAW 264.7 macrophages provide us with an excellent model for anti-inflammatory drug screening and Further the key inflammation cytokines and mediators can be studied in LPS-stimulated RAW264.7.the lip polysaccharide Ipswich is gram negative bacteria and a pathogenic factor in septic shock. Which also an inflammatory stimulus of cytokines through transcription factor activation including pro inflammatory gene⁵.

Continuing advances in our understanding of action mechanism of anti-rheumatic agents will benefit the future development of RA regimens with greater efficacy and less toxicity. There are a number of currently available anti-rheumatic drugs, although none of them seems to cure RA or can be used preventively to reduce the risk of contracting the disease⁶. In some study shown that Indian medicinal plants has great effect in cure and prevention of Arthritis⁷. Unani system of medicine has rich treasure of therapeutically active products and extensive beneficial clinical experience for various diseases like skin diseases, neurological disorders, on many inflammatory and non-communicable diseases including rheumatoid arthritis and many more⁸ Unani system of medicine, many poly herbal formulations are being used for the treatment of arthritis and the few UNIM formulation like UNIM-301 and UNIM-302 important poly herbal proven clinical efficacy ⁹. Therefore, Unani system of medicine used to treatment of arthritis diseases by much Poly herbal formulation. Thus, there is considerable research interest in the identification of other anti-inflammatory agents especially those which are being used in traditional medicine since centuries. The present study to look the “Effect of Anti-inflammatory Activity of Aqueous, Hydro-ethanol and Methanol extracts of two Unani formulations”.

MATERIALS AND METHODS:

Chemicals required:

DMSO from Hi media, Bismuth sub nitrate were from Hi media; Phosphate Buffer Saline (PBS)Sigma, Bismuth sub nitrate were from Hi media, Phosphate Buffer Saline (PBS), Antimyotic-Antibiotic were from Gibco-life technologies, Fetal bovine serum (FBS), ; DMEM Media were from sigma, MTT -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Griess reagent, LPS(lip polysaccharide).

Name of the UNIM formulations:

UNIM-301: contains is Operculina turpenthum, Alpinia galanga, Withania somnifera, Colchicum luteum and Zingier officinale based poly herbal Unani formulation.

UNIM-302: contains Matricaria chamomilla and Butea monosperma based poly-herb Unani formulation.

Extraction of UNIM formulation:

UNIM formulations UNIM 301 and UNIM 302 powder form was taken the GMP pharmacy of NRIUM-SD Hyderabad .prepared aqueous, methanol, Hydroethanol (50:50) extraction by dissolving 10g of powder of two Unani formulation in 100ml of solvent shaking incubator at 25°C stirring at 50rpm for overnight .mixture filtered through what man No.1 filter paper and mixture was lyophilized by rotary vacuum evaporator. The obtained extract were kept in desiccators and used for different assay.

DPPH Scavenging Activity:

The DPPH (2, 2-diphenyl-1-picryl-hydrazyl-hydrate) scavenging activity was assessed according to the method¹⁰ Briefly an aliquot of 80µl of 0.1mM DPPH in methanol was mixed with 20µl of test extracts with different concentrations (0.1–1000µg/ml) in a micro test plate. The reaction mixture was allowed to stand in dark and the absorbance was determined after 30 min at 517 nm with a multimode reader (Infinite pro M 200; Te can, Austria). Positive Control as Ascorbic acid.

Cell viability and cytotoxicity Assay:

RAW cells were from NCCS Pune. these RAW cells were grown in medium containing DMEM, FBS, 100 U/ml L-glutamine, FBS,L-glutamine, streptomycin at 5% CO2 incubator with 37ºC. macrophages were treated with both UNIM formulation with various concentration (0.1-1000ug) different extracts of Aqueous(AQ), Hydroethanol (HE), Methanol (ME). then after incubation cells were washed with PBS and MTT (0.5mg/ml PBS).were added to each well and incubated for 3hrs at 37ºC. the formazan crystals in cells were dissolved in DMSO measured absorbance at 570nm using a Multimode plate Reader (Infinite pro M 200; Tecan, Austria).

Determination of Nitric oxide (NO):

NO production (NO2+NO3 level) in the supernatants of the cultured RAW 267 cells was determined by Griess reagent. Briefly, the macrophages were plated in a96-well plate (1*10⁵ cells/well) for16 h. were treated with UNIM 301 and UNIM 302 of all extract (AQ, HE, ME) various concentration (0.1-1000µg) along with LPS (1ug/ml). After 24hrs treatment, 100μl of supernatants were mixed with equal volume of Griess reagent which contain (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride) and incubated at room temperature for10 min. Next, the absorbance was measured at 550nm by Multimode plate Reader (Infinite pro M 200; Tecan, Austria)

Determination of Reactive oxygen species (ROS):

The intracellular ROS were determination by a fluorometric assay using dichlorofluorescein-diacetate (DCFH-DA) as the probe. The prepared RAW264.7 macrophages(1*10⁵ cells/well in 96-well plate and incubated for16 h) were treated with 0.1-1000ug/ml (AQ, HE,ME) of extracts of UNIM 301,UNIM302 along with Lbs (1ug/ml) and incubated for 24 h; subsequent the samples were again incubated with 10 nm of DCFHDA for 30 min in the dark. After washing twice with PBS, fluorescence was measured in a Multimode plate Reader (Infinite pro M 200; Tecan, Austria) with excitation and emission wavelengths of 485 nm, and 535 nm, respectively.

RESULTS:

Extraction:

The yield of extract of both the Unani formulations of UNIM 301 and UNIM 302 solvent of extract shown in percentage. The result extraction yield of the formulation UNIM 301Aqueous, Hydroethanol (50:50), Methanol is 15.11, 11.01,17.41 and UNIM 302 Aqueous, Hydroethanol (50:50), Methanol is 11.21, 06.99, 14.26 respectively. The. highest extraction yield was from Methanolic extract of UNIM-301 and least extraction Yield from hydro-ethanol extract of UNIM-302 in the different solvent system the extraction yield varied from one formulation to other formulation.

DPPH Radical Scavenging activity:

Antioxidant activity in UNIM formulation is evaluated by DPPH scavenging activity. The results shown Hydro ethanol extract of UNIM 302 exhibited the highest scavenging activity With IC 50 value of 229µg/ml followed by aqueous extract of UNIM 301 shown lowest scavenging activity with IC 50 value of 412µg/ml. however the result of the study shown (Table 1) that UNIM 302 shown better antioxidant properties in compared with UNIM 301 in comparison with positive control standard of ascorbic acid.

Table 1: Antioxidant activities measured by DPPH of UNIM 301and UNIM 302 formulations of different extracts.

Name of Unani formulation extract	*IC₅₀ (DPPH)**
UNIM-301Aq	412
UNIM-301HE	336
UNIM-301ME	303
UNIM-302 Aq	264
UNIM- 302 HE	229
UNIM-302 ME	319
Ascorbic Acid	14.11

Effect of Unim formulation on LPS-induced cytotoxicity in RAW 264.7macrophages:

To evaluate possible toxicity of UNIM formulation on RAW 264.7 cells MTT assay was performed. The cytotoxicity activity of UNIM formulation of UNIM 301 and UNIM 302 observed in dose dependent manner. Pre tremens of cells with UNIM formulation of UNIM 301 and UNIM 302 showed a significant increase in cell viability. The UNIM 301 Aqueous extract IC ₅₀ 680, and Hydro ethanol extract IC ₅₀ 350, Methanol extract IC ₅₀ 552.followed by UNIM 302 Aqueous extract IC₅₀ 529, Hydro ethanol extract IC ₅₀300, Methanol extract IC ₅₀ 253.

(Fig1 (A) (B)) Keeping in this view the results showed that no cytotoxicity effect of these both formulation against macrophage.

Figure 1 (A): Cytotoxic effect of UNIM 301 AQ, HE, ME.

The % inhibition was evaluated on RAW264.7 cells by using different concentration (0.1 to 1000 μg ml-1)of aqueous , hydro-ethanol and methanol extracts UNIM-301. Data represent the mean ± SD from 3 independent experiments. *P < 0.05 along with control.

Figure 1 (B): Cytotoxic effect of UNIM 302 AQ, HE, ME.

The % inhibition was evaluated on RAW264.7 cells by using different concentration (0.1 to 1000 μg ml-1)of aqueous , hydro-ethanol and methanol extracts UNIM-302. Data represent the mean ± SD from 3 independent experiments. *P < 0.05 along with control.

Effect of Unani formulation on LPS-induced NO production in RAW264.7 macrophages:

In these study macrophages cells were activated by LPS and NO production was measured as nitrite concentration in the culture medium. In compared to the untreated control. these aqueous, methanol, and hydro ethanol extract of UNIM 301,and UNIM 302 significantly inhibits (P<0.05) the nitrite accumulation in LPS stimulated RAW 264.7, (Table2), (Fig 2(A)(B)). among these the aqueous extract of UNIM 302 (96.1% at 0.1 µg) shown the highest inhibition and least inhibition at Hydro ethanol extract of UNIM 301 is (15.1% at 1000µg ).and we observed that treatment of macrophages with all extract of UNIM 301 and UNIM 302 causes as modest decreases in the endogenous NO levels in RAW 264.7 cells.

Table 2: Effect of UNIM 301 and UNIM302 on LPS-induced NO production in RAW264.7 macrophages

Concentration µg/ml	UNIM301 Aqueous	UNIM301 Hydroethanol	UNIM301 Methanol	UNIM302 Aqueous	UNIM302 Hydroethanol	UNIM302 Methanol
0.1	94.15±2.90	75.88±1.24	91.50±0.99	96.55±0.85	83.80±2.52	76.51±1.56
1	81.01±1.74	59.26±0.81	62.45±1.99	90.63±2.45	73.69±1.71	52.48±0.29
10	75.46±0.61	38.01±1.30	50.59±0.31	80.52±0.69	65.70±1.49	50.28±1.04
100	68.09±1.12	25.46±0.95	46.48±2.35	60.15±4.68	31.34±2.15	33.17±1.23
1000	27.62±1.59	21.97±1.39	31.71±1.48	35.95±3.70	15.11±1.46	18.90±0.81

Data expressed Mean ± SD

Table3: Intracellular ROS levels of UNIM 301 and UNIM 302 in LPS-induced RAW264.7 macrophages.

Concentration µg/ml	UNIM301 Aqueous	UNIM301 Hydroethanol	UNIM301 Methanol	UNIM302 Aqueous	UNIM302 Hydroethanol	UNIM302 Methanol
0.1	811.6±1.69	594±13.06	640.3±43.6	743±16.3	566.1±26	932 ±12.1
1	544±29.5	476.6±49.2	543.6±23.4	530.3±36.0	525±3.75	828.6±14.8
10	436.3±10.65	453.3±27.2	519.6±12.3	451.3±64.0	502±2.96	799 ± 34.32
100	326.6±4.02	430.6±7.6	317.3±11.2	372.3±11.4	486±5.71	321.3±5.79
1000	249.6±4.49	213.1±9.3	77.36±2.16	269.3±1.2	199±36.3	138.3±13.1

Data expressed Mean ± SD

Figure 2 (A): Inhibitory effect of UNIM 301on LPS-induced NO production in RAW264.7 macrophages.

Effect of UNIM formulation on NO production in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with UNIM 301 in different concentrations (0.1to1000ug) .Data represent the mean ± SD p < 0.05from three independent experiments. Indicate significant differences compared to the LPS-treated group.

Effect of Unani formulations on LPS-induced ROS level in RAW 264.7macrophages:

The DCFH-DA assay was employed to investigate the inter cellular ROS level in LPS–stimulated macrophages. Exposure of cells with LPS caused a significant increase in ROS generation as measured by DCF-DA in the culture medium, which converts to highly fluorescent dichlorofluroscein in the presence of intracellular ROS. Pre trement of cells shown significant (P<0.001) decreasing trends of ROS production in UNIM 301, UNIM 302 in LPS stimulated RAW264.7 cells. The methanol extract of UNIM302 shown 138% at 1000 µg and UNIM 301 methanol extract shown 77% 1000µg resented in respectively (Table3 (Fig 3(A) (B)).

Figure 2 (B): Inhibitory effect of UNIM 302on LPS-induced NO production in RAW264.7 macrophages

Effect of UNIM formulation on NO production in LPS-stimulated RAW 264.7 cells. Cells were pre-treated with UNIM 302 in different concentrations (0.1to1000ug).Data represent the mean ± SD p < 0.05from three independent experiments. Indicate significant differences compared to the LPS-treated group.

Figure 3(A): Effect of UNIM 301 (AQ, HE, ME) on intercellular ROS levels in LPS-stimulated RAW 264.7 cells.

RAW cells were pretreated with UNIM 301concentrations (0.1to 1000ug) along with LPS for 24 hrs. The data shown represent the mean ± SD of three independent experiments. p<0.05. the control group with LPS treated group.

Figure 3 (B): Effect of UNIM 302 (AQ, HE, ME) on intercellular ROS levels in LPS-stimulated RAW 264.7 cells.

RAW cells were pretreated with UNIM 302 concentrations (0.1to1000ug) along with LPS for 24 hrs. The data shown represent the mean ± SD of three independent experiments. p<0.05. the control group with LPS treated groups

DISCUSSION:

Inflammation is found to be a physiological process that eliminates noxious or infectious stimuli, and also which initiate the repair and healing process of the damage tissue ¹¹. The raw macrophages found to play the role of maintaining ghomeostastic function and in production of inflammatory mediators and proinflammatory cytokines ¹². As chronic inflammation causes abnormal increase of pro-inflammatory cytokines which are responsible for increase in nitric oxide respectively. The various inflammatory diseases such as parkinsons, Alzhemiers, and multiple Sclerosis, Arthritis¹³. The exact mechanism of chronic inflammatory diseases not understood The natural sources of antioxidant could be the alternative to synthetic antioxidants in counteracting oxidative stress associated diseases. The naturally occurring substances have been recognized to have antioxidant abilities. and where as various in vitro methods have been used to assess their free radical scavenging and antioxidant activity. the free radical scavenging activity by its synthetic antioxidant, their cause of adverse side effect leading to carcinogenicity and effective natural antioxidant become crucial as¹⁴so far natural antioxidant are believed to be safer and bioactive. A.¹⁵. in the present Antioxidant activity determined by using 1,1-diphenyl-2-picrylhyrazyl (DPPH) radical method. Assay based on the measurement of the loss of DPPH color absorbance at 517nm. In this free radical of purple color and in the presence of an antioxidant color changes to yellow based on the efficiency of the antioxidant. DPPH assay antioxidant activity of Ascorbic Acid¹⁶Results shown that all the extracts of AQ, HE, ME of UNIM 302 showed better antioxidant compared to UNIM 301 the activity increased with increase in concentration. In some studies reported that plant flavonoids that show antioxidant activity in vitro also functions as antioxidants in vivo¹⁷. and it has also been reported that these plant derived polyphenolic flavonoids exhibit their antioxidant potential through various mechanisms including scavenging of ROS, inhibiting lipid per oxidation as well as chelating metal ions¹⁸. As such some of natural antioxidants from food supplements and traditional medicines including the Unani system of medicine which is being practiced from centuries and these in use formulations can be the excellent source for the natural antioxidants and will help in combating various diseases with fewer side effects no side effects.

In some studies shown the plant extract of Withania somnifera has a potential source of Natural antioxidant ¹⁹ in related to biological activities posses by within somnifera such as Anti-inflammatory property, infertity Activity, Hepatoprotective Activity and Anti-convulsant activity²⁰. Methonolic extract of Withania somnifera response in Antibacterial activity²¹.

In study Butea monosperma the plant used as Anti-inflammatory agent, immunomodulatory Agent, Protein Synthesis inducer, Anti-cancerous Agent²². An In-vivo Animal study shown that ethanolic extract of Butea monosperma posses’ anti-inflammatory activity a review study²³.

The current study MTT Assay was employed to test the potential toxicity of Unani formulation .more specifically these UNIM 301, 302 of both formulation of all the extracts didn’t effect the RAW 264.7 cell viability did not show the cytotoxic effect. Thus these Unani formulation protective effects to macrophages against LPS induced. And result were in corresponding to earlier studies by²⁴. reported that these extract from few plants used in Unani system of medicine have no cytotoxicity effect against U937 cell lines. This was correlated to long history used in Unani medicine. Where NO is known to act as an intracellular messenger that, in many cases, is involved in promoting inflammatory responses²⁵. Over-production of NO by macrophages may lead to various pathological disorders such as inflammation²⁶. Data of the present study indicates that these both Unani formulation 301, 302 of all three extracts exhibited improved inhibitory effect on NO production in LPS induced RAW macrophages. Chamomile inhibits NO production in macrophages as reported in earlier literature²⁷.

ROS Level maintenance of cellular hemostasis, and over production of ROS level to be harmful in inflammatory disease and removal of pathogens. These ROS acts in triggering various biological responses in exposure to stress full environment. And in excessive ROS production and oxidative stress for treatment of inflammatory disease disorders²⁸ some studies shown that ROS impacts in signaling pathways, including signaling pathway MAPKs, AKT, NF-Kb^.29. Some clinical studies shown Unani treatment was found to be effective in reducing knee pain effect the osteoarthritis patients. in support to Maticaria chamomilla and Asarum eurpeaum has nerve periarticular muscle wasting join stable. the Matricaria chamomilla have anti-inflammatory, antioxidant and analgesic properties.. in vitro studies shown that Matricaria chamomilla extract inhibited both cyclo-oxygenase and lipo-oxygenase, and consequently prostaglandins and leukotrienes³⁰. as Unani system of medicine, many poly herbal formulations are being used for the treatment of arthritis .Consistently our study showed that treatment of RAW Cells with UNIM formulation UNIM301, UNIM 302 decrease in NO and ROS level in concentration dependent manner .and these UNIM formulations might contribute to the inhibition of inflammatory reaction in LPS stimulated RAW cells. Also observed that UNIM 302 elicited better activity in relation to UNIM 301.

CONCLUSION:

In Conclusion study demonstrated extracts of aqueous methanol hydro-ethanol both Unani formulations inhibits NO and ROS production in LPS induced RAW macrophages. DPPH activity in all different extracts UNIM 302 exhibited better antioxidant compared to UNIM 301. thus our finding suggested that these UNIM formulation that extracts potential anti-inflammatory properties against LPS activated Raw cells. Hence, these unim formulation extracts can be used as natural sources of antioxidants potent anti inflammatory agent and exhibits inflammatory preventive properties.

ACKNOWLEDGEMENT:

We would like to acknowledge Department of Science & Technology, National Research Institute of Unani Medicine for Skin Disorders. and Director of NRIUM-SD for providing necessary facility.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Yu et al., The pivotal role TBK1 in inflammatory responses mediated by macrophages. Mediators of Inflammation. 2012; 979105.

2. Blazovics et al., Cytokines, prostaglandins, nutritive and non-Nutritive factors in inflammatory bowel diseases. Orvosi Hetilap. 2004; 145, 2523–2529.

3. Huo et al., Anti-inflammatory effects of linalool in RAW264.7 macrophages and lip polysaccharide-induced lung injury model. Journal of Surgical Research. 2012; 180, e47–e54.

4. Vargas et al., Ragweed pollen extract intensifies lip polysaccharide-induced priming of NLRP3 inflam-masome in human macrophages. Immunology. 2013; 138,392–401.

5. Liu and Mali. NF-kB activation as a pathological mechanism of septic shock and inflammation. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 290, L622–L645.

6. Jue et al., Nuclear factor κB (NF-κB) pathway as a therapeutic target in rheumatoid arthritis. J Korean Med Sci. 1999; 14(3):231-238.

7. Nabarun Mukhopadhyay, Sampath. V, Sameer Pai, U. V. Babu, Richard Lobo. Antiarthritic Medicinal Plants: A Review. Research J. Pharm. and Tech. 2019; 12(1): 375-381. doi: 10.5958/0974-360X.2019.00068.4

8. AYUSH Department Unani system of medicine the science of health and healing. Ministry of Health & FW. (2013); Govt of India.

9. Siddiqui KM, Parveen S, Jamil SS. Multicentric observational studies of polyherbalUnani oral and local formulations in cases of rheumatoid arthritis. Planta Med. 2011; 77:149.

10. Youwei Z, Jinlian Z, Yonghong P .A comparative study on the free radical scavengingactivities of some fresh flowers in southern China. LWT- Food Science and Technology.2008;41:1586-1591.

11. Mariathasan S, Monack DM. Inflammasome adaptors andsensors: intracellular regulators of infection and inflammation. Nat Rev Immunol. 2007; 7: 31–40.

12. Duffield JS. The inflammatory macrophage: a story of Jekylland Hyde. Clin Sci. 2003;104: 27–38.

13. Heiss E, Herhaus C, Klimo K, Bartsch H, Gerhauser C. Nuclearfactor kappa B is a molecular target for sulforaphane mediated anti-inflammatory mechanisms. J Biol Chem. 2001; 276:32008–32015.

14. M. A. Ebrahimzadeh, S. M. Nabavi, S. F. Nabavi, F. Bahramian, and A. R. Bekhradnia. Antioxidant and free radical scavengingactivity of H. officinalis L. var. angustifolius, V. odorata, B.hyrcana and C. speciosum. Pakistan Journal of Pharmaceutical Sciences. 2010; vol. 23, no. 1, pp. 29–34.

15. A. A. Adedapo, F. O. Jimoh, S. Koduru, P. J. Masika, and A.J. Afolayan, “Assessment of the medicinal potentials of themethanol extracts of the leaves and stems of Buddleja saligna, ”BMC Complementary and Alternative Medicine. 2009. Vol. 9, Article 21.

16. Geetha S, Sai-Ram M, Mongia SS, Singh V, Ilavazhagan G, et al Evaluation ofantioxidant activity of leaf extract of Sea buckthorn (Hippophae rhamnoides L.) on chromium (VI) induced oxidative stress in albino rats. J Ethnopharmacol. 2003; 87:247–251.

17. Ch. Madhu, J. Swapna, K. Neelima, Monic V. Shah. A Comparative Evaluation of the Antioxidant Activity of Some Medicinal Plants Popularly Used in India. Asian J. Res. Pharm. Sci. 2(3): July-Sept. 2012; Page 98-100

18. Swahili F Natural antioxidants: An overview. In Natural Antioxidants, Chemistry, Health Effects and Applications. Shahidi F (ed) Champaign: 1997; AOCS Press; 1-11.

19. Kalim MD, Bhattacharyya D, Banerjee A, Chattopadhyay S Oxidative DNA damagepreventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complement Altern Med. 2010; 10:77 .

20. Munhoz CD, Garcia-Bueno B, Madrigal JLM, Lepsch LB,Scavone C, Leza JC.. Stress-induced neuroinflammation: mechanisms and new pharmacological targets. Bruz J Med Biol Res. 2008; 41: 1037-1046.

21. Patel D.S., Shah P. B., Managoli N. B.. Evaluation of In-vitro Anti-oxidant and Free Radical Scavenging activities of Withania somnifera and Aloe vera. Asian J. Pharm. Tech. 2(4): Oct. - Dec. 2012; Page 143-147.

22. Sudhanshu Kumar Meher, Banmali Das, Purnendu Panda, GC Bhuyan, MM Rao. Uses of Withania somnifera (Linn) Dunal (Ashwagandha) in Ayurveda and its Pharmacological Evidences. Research Journal of Pharmacology and Pharmacodynamics. 2016; 8(1): January-March, 23-29. doi: 10.5958/2321-5836.2016.00006.9.

23. T. Jeyanthi, P. Subramanian, P. Kumaravel. A Comparative Analysis of Antibacterial Activity of Withania somnifera Root Extract with Commercial Antibiotics. Asian J. Pharm. Res. 3(2): April- June 2013; Page 98-102.

24. Shweta Singh Chauhan, Pramod Kumar Mahish. Flavonoids of the flame of forest- Butea monosperma. Research J. Pharm. and Tech. 2020; 13(11):5647-5653. doi: 10.5958/0974-360X.2020.00984.1

25. Nilesh Gupta, U.K Jain, Ankit Jain, Goutam Lovanshi, Nitin Mathan, Vipin Tiwari. Review of Some Important Medicinal Plants Possesses Anti-Inflammatory Activity. Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1506-1512.

26. Choi MS, Lee SH, Cho HS, et al. Inhibitory effect of obovatol on nitric oxide production and activationof NF-kappaB/MAP kinases in lipopolysaccharide-treated RAW 264.7cells. Eur J Pharmacol. 2007; 556:181–189.

27. Natarajan Bhaskaran, Sanjeev Shukla, Janmejai K Srivastava, and Sanjay Gupta Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking Rel A/p65 activity. Int J Mol Med. 2010; December ; 26(6): 935–940.

28. Paola Rosanna D, Salvatore C. Reactive oxygen species, inflammation, and lung diseases. Curr Pharm Des. 2012; 18(26):3889±900.

29. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, et al. ROS and ROS-Mediated Cellular Signaling. OxidMed Cell Longev. 2016; 2016:4350965. PMCID: PMC4779832.

30. Hörmann HP, Korting HC. Evidence for the efficacy and safety of topical herbal drugs in dermatology: Part I: Anti-inflammatory agents. Phytomedicine. 1994; 1:161–71.

Received on 09.12.2020 Modified on 27.04.2021

Research J. Pharm.and Tech 2022; 15(4):1560-1566.

DOI: 10.52711/0974-360X.2022.00260