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1. TO DETERMINE ANALGESIC, ANTI-INFLAMMATORY AND ANTI-OXIDANT ACTIVITIES OF CAPPARIS CARTILAGINEA DECNE USING IN VITRO AND IN VIVO MODELS M.Phil. Scholar Ashok Kumar Supervisor Dr. Geeta Kumari Assistant Professor Department of Pharmacology Faculty of Pharmacy University of Sindh, Jamshoro 2 1

2. Pain

3. Market available drugs as analgesic & anti-inflammatory Non steroidal Anti-inflammatory drugs (NSAIDs) Glucocorticosteroids (GCs) Naproxen Nimesulide Diclofenac sodium Ibuprofen Celecoxib Prednisolone Hydrocortisone Betamethasone Dexamethasone 3

4. Capparis cartilaginea Decne Common n ame Caper Local name Kabar, Khawarg Family Capparidaceae (Capparaceae) PHARMACOLOGICAL USES TRADITIONAL USES Anti-inflammatory and anti-oxidant, anticancer and antimicrobial Headache, earache, rheumatic fever, childbirth associated pain A. R. Hamed eta al, 2007 4

5. Pharmacological activity Reference Anti-inflammatory and Anti-oxidant B. A. Moharram et. al, Rheumatic fever B. Alsharif et. al, Eyes infection and skin diseases B. Alsharif et. al, Headache, earache B. Alsharif et. al, Healing of bruises B. Alsharif et. al, Childbirth associated pain B. Alsharif et. al, Comforting colic B. Alsharif et. al, Cure peptic ulcer A. R. Hamed et. al, Anti-microbial A. R. Hamed et. al, 5 Literature survey

6. Capparis cartilaginea Decne collected from Dadu Leaves shade dried and coarsely powdered Maceration in aqueous, hydroethanol, ethanol for 72 hr 6 Filtration Drying Dried Extracts Method of collection and preparation of plant extracts

7. OBJECTIVES To prepare total extracts of the plant leaves by using aqueous, ethanol and hydroethanol solvents. To determine in vitro anti-inflammatory activity by protein denaturation assay. To determine in vitro anti-oxidant activity by ABTS assay. To determine acute oral toxicity of potent plant extract. To determine in vivo anti-inflammatory and analgesics activities in various animal models 7

8. INVIVO ASSAYS INVITRO ASSAYS PROJECT OUTLINE Acute oral toxicity test Formalin-induced nociception assay Acetic acid-induced writhing assay TPA-induced dermatitis model Protein denaturation assay ABTS assay 8

9. 9 Sonicator CFEC-ROHS, China Hot air oven LDO-o6oE Labtech, Korea Beakers (100 mL- 500 mL) Bomex Spectrophotometer R.M marketing, England Centrifuged machine Noke, China pH meter Comen, China Thermostat Water bath HH-4, China Test tubes Pyrex, England Vortex Mixer Noke, China Magnetic Stirrer   Bottles Pyrex, England Electronic Balance Shimadzu, Japan Rotavap RO1, Buchi, Switzerland Micro pipettes (2 μ L-100 μ L) Biohit, Finland Experimental Apparatus and Materials

10. 10 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) Sigma Disodium hydrogen phosphate Scharlab S.L, Spain Hydrochloric acid BDH, England Potassium dihydrogen phosphate Sigma Aldrich, Germany Sodium chloride BDH Laboratory Suppliers Ethanol, methanol BDH Laboratory Suppliers Diclofenac sodium Sigma Aldrich, USA Phosphate buffer saline (pH 6.4)   Egg albumin/egg albumin powder   Normal saline   Experimental Chemicals

11. Denatured proteins lose their 3D structure and therefore cannot function To determine anti-inflammatory activity Spectrophotometric method Protein Denaturation Assay Heat 11

12. Mixture of Egg albumin & PBS (pH6.4) 2.8 ml (albumin+PBS) and 0.2 ml of sample Heated at 70°C for 5 mins Allowed to stand at room temperature Protein Denaturation Method Incubated at 37°C for 15 mins Absorbance (660 nm) H. D. Tharindu et. al, 2023 12

13. ABTS ( 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ABTS will be used to determine the scavenging activity of ABTS + radical cation Spectrophotometric method 13 ABTS ABTS* Free radical cation K 2 S 2 O 8   B. Alsharif et. al, 2023 ABTS free radical scavenging Activity

14. Weigh Extract Dif : Conc : of extract (12.5, 25, 50, 100, 250, 500 µg/ml) ABTS (7 mM ) + K 2 S 2 O 8 (2.45 mM ) (1:1 v/v) Dilute until get absorbance (735 nm) of 0.700 ± 0.05 2.7 mL of ABTS + 0.3 mL of varying conc. Of sample and incubate at room temp: for 15 min Absorbance (734 nm) 14 B. Alsharif et. al, 2023 ABTS Method

15. Experimental Animals The handling and care of animals will be carried out according to National Institute of Health (NIH) Guide for the care and use of laboratory animals (NIH, 2011) 12 hours light and dark cycle Temperature 25°C (±2°C ) Relative humidity 45% (±15%) 15

16. Acute Oral Toxicity Test Group 01 Control (only vehicle) Group 02 100 ug/ml Group 03 250 ug/ml Group 04 500 ug /ml Day 01 Day 07 Acute signs of toxicity: Continuously for 4 hours on Day 1, then at least once in a day for 14 days Collection and weighing of vital organ Hematological analysis Oral Administration of potent extract M. Kifayatullah et. al, 2015 Day 14 Any behavioral change 16

17. Formalin-induced nociception assay Each group consist of 6 mice Group 01 Control Group 02 Formalin sol. Group 03 O nly vehicle Group 04 Formalin sol. + Saline Group 05 Formalin sol. + Potent extract Group 06 Indomethacin early phase at 0-10 minutes late phase at 10-30 minutes Paw flicks, licks, bites or shakes after injection Moharram Al- mahbashi et. al, 2018 Biorender.com Intra-plantar Administration of potent extract 17

18. Acetic acid-induced writhing assay 18 Each group consist of 6 mice Before 30 minutes of Acetic acid Group 01 Diclofenac sodium (Standard) Acetic acid 0.6% Group 02 O nly vehicle Acetic acid 0.6% Group 03 Potent extract Acetic acid 0.6% From 5 minutes up to 20 minutes Abdominal contraction (writhes) will be counted A. K. Gupta et. al, 2015 Intra-peritoneal injection to mice Writhes Writhes

19. TPA-induced dermatitis model Each group consist of 5 mice Group 01 Control Group 02 TPA Group 03 Low dose of potent extract Group 04 High dose potent extract Group 05 Indomethacin Group 06 Only potent extract (High dose) Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is a protein kinase C activator Its application to the skin induces inflammation and epidermal hyperplasia Wu, Chen et al., 2020 19

20. TPA-induced dermatitis model 4 hours later Difference in the thickness or weight Mice will be sacrificed and 6 mm plugs of ears will be collected Extract, Indomethacin or vehicle will topically be applied on right ear before TPA application Grouping of mice Extent of Ear edema 20 N. Hirasawa et. al, 2009

21. Time Frame 21 To conduct l iterature review 1 month Collection, dry and making total extract of leaves 2 months For in vitro anti-inflammatory and anti-oxidant activities of extracts 2 months For in vivo anti-inflammatory and analgesics activates of most potent 4 months Analyze data and write thesis 3 months

22. POSSIBLE OUT COMES ANTI-INFLAMMATORY ANALGESICS ANTI-OXIDANT A new Analgesics and Anti-inflammatory agent from natural source 22

23. References 23 B. A. Moharram, H. M. AL- Mahbashi , T. Al- Maqtari , R. Saif -Ali, I. A. Al- Hakami , and W. T. Alhaj , "Leaves of Capparis cartilaginea Exhibit a Promising Antidiabetic Activity in Alloxan -Induced Diabetic Rats," vol. 12, pp. 65-75, 2023 B. Alsharif, G. A. Babington, N. Radulović , and F. Boylan , "Volatiles of Capparis cartilaginea Decne. from Saudi Arabia," Plants, vol. 11, p. 2518, 2022. B. Alsharif, N. Hante , B. Govoni , H. Verli , W. Kukula -Koch, M. Jose Santos-Martinez, and F. Boylan , "Capparis cartilaginea decne (capparaceae): isolation of flavonoids by high-speed countercurrent chromatography and their anti-inflammatory evaluation," Front Pharmacol , vol. 14, 2023. A. R. Hamed, K. A. Abdel- Shafeek , N. S. Abdel- Azim , S. I. Ismail, and F. M. Hammouda , "Chemical Investigation of Some Capparis Species Growing in Egypt and their Antioxidant Activity," Evid Based Complement Alternat Med, vol. 4, pp. 25-8, 2007. B. Alsharif, N. Hante , B. Govoni , H. Verli , W. Kukula -Koch, M. Jose Santos-Martinez, and F. Boylan , "Capparis cartilaginea decne (capparaceae): isolation of flavonoids by high-speed countercurrent chromatography and their anti-inflammatory evaluation," Frontiers in Pharmacology, vol. 14, 2023

24. 24 H. D. Tharindu Madhuranga and N. Samarakoon , "In vitro Anti-Inflammatory Egg Albumin Denaturation Assay: An Enhanced Approach," Journal of Natural & Ayurvedic Medicine, vol. 7, p. 000411, 2023. M. Kifayatullah , M. S. Mustafa, P. Sengupta , M. M. R. Sarker , A. Das, and S. K. Das, "Evaluation of the acute and sub-acute toxicity of the ethanolic extract of Pericampylus glaucus (Lam.) Merr . in BALB/c mice," Journal of Acute Disease, vol. 4, pp. 309-315, 2015. B. Moharram, H. Al- mahbashi , R. Saif -Ali, and F. Aqlan , "PHYTOCHEMICAL, ANTIINFLAMMATORY, ANTIOXIDANT, CYTOTOXIC AND ANTIBACTERIAL STUDY OF CAPPARIS CARTILAGINEA DECNEFROM YEMEN," International Journal of Pharmacy and Pharmaceutical Sciences, vol. 10, p. 38, 2018. Wu, J.-Y., Y.-J. Chen, et al. (2020). " Chrysoeriol ameliorates TPA-induced acute skin inflammation in mice and inhibits NF- κB and STAT3 pathways." Phytomedicine 68 : 153173. N. Hirasawa , Y. Ohsawa , K. Ishihara, T. Seyama , J. Hong, and K. Ohuchi, "Analysis of the Mechanism for the Development of Allergic Skin Inflammation and the Application for Its Treatment: Establishment of a Modified Allergic Dermatitis Model in Mouse Ear Lobes by Application of 12-O-Tetradecanoyl Phorbol 13-Acetate: Putative Involvement of Thymic Stromal Lymphopoietin and Roles of Histamine," Journal of Pharmacological Sciences, vol. 110, pp. 245- 250, 2009

25. 25 A. K. Gupta, D. Parasar , A. Sagar , V. Choudhary , B. S. Chopra, R. Garg , Ashish , and N. Khatri , "Analgesic and Anti-Inflammatory Properties of Gelsolin in Acetic Acid Induced Writhing, Tail Immersion and Carrageenan Induced Paw Edema in Mice," PLOS ONE, vol. 10, p. e0135558, 2015.

26. QUESTIONS REMARKS SUGGESTIONS THANK U COMMENTS 26