STUDY REPORT Preclinical evaluation and development of herbal

STUDY REPORT Preclinical evaluation and development of herbal product Zindrol therapeutic benefit in management of cancer Confidential 1 Providing Integrated Research Solutions in Preclinical

INTRODUCTION Confidential 2 Providing Integrated Research Solutions in Preclinical

Introduction • Ginger (Zingiber officinale) is a flowering plant whose rhizome, ginger root or ginger, is widely used as a spice and a folk medicine. It is a herbaceous perennial plant which reaches heights of 1 -3 feet and grows annual pseudo stems (false stems made of the rolled bases of leaves) • It belongs to : Family Genus Species : : : Zingiberaceae Zingiber Zinginber officinale • It is Native to India and Malaysia, Commercially grown mainly in India, followed by Jamaica, Africa and China • The parts which are widely used is the Rhizomes. It is most beneficial part of the plant. It is a branch with thick, thumb-like protrusions. Individual divisions of the rhizome are called hands. The fleshy, yellow interior of the root is the most widely used portion 3 Providing Integrated Research Solutions in Preclinical

Chemical components & Uses S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Bioactive Components in ginger 6 -gingerol 6 -shogaol α-curcumene 6 -prandol 8 -gingerol 10 -gingerol Zingiberene ß-sesquiphellandrene 1 -dehydrogingerdione 6 -gingerdione 10 -gingerdione 4 -gingerdiol 6 -gingerdiol 8 -gingerdiol 10 -gingerdiol diarylheptanoids Camphene Bornyl methyl ether ß-Bisabolene Zingerone S. No Uses 1 Digestive aid 2 Curbing motion sickness 3 Anti-inflammatory 4 Circulatory stimulatory 5 Expectorant for Treatment of asthma 6 Antipyretic 7 Analgesic 8 Anti-viral 9 Anti-tumor 4 Providing Integrated Research Solutions in Preclinical

Shogaols • Shogaols are one of the bioactive component of ginger. • It is a pungent constituent of ginger similar in chemical structure to gingerol. The most common of the group is (6)-shogaol. it is produced when ginger is dried or cooked. • (4)-Shogaol, (8)-shogaol, (10)-shogaol, and (12)-shogaol (all found in ginger) together constitute the group shogaols. • Multiple therapeutic effects have been reported for Shogaol such as anticancer, neuroprotective, antidiabetic, hepatoprotective, cardioprotective, antioxidant, anti-inflammatory, immunostimulatory etc. • Shogaol has demonstrated potent anticancer activity in various types of cancers such as breast, pancreas, prostate, leukemia, colon, lung, ovary, kidney etc. Hence, based on the multiple therapeutic benefits of shogaol, the aim of the project is to enrich the ginger extract with high shogaol content 5 Providing Integrated Research Solutions in Preclinical

Therapeutic Benefits of Shogaol 6 Providing Integrated Research Solutions in Preclinical

OBJECTIVE AND PROJECT STRATEGY Confidential 7 Providing Integrated Research Solutions in Preclinical

Product development strategy- for Shogaol enriched ginger extract Extraction and characterization Collection of Ginger Rhizome Pre-formulation Primary Efficacy tests Pre-formulations DNA fingerprinting Extraction: Water/alcoholic In vitro efficacy and selectivity tests In vitro selectivity tests Enrichment process Formulation development Oral product Efficacy Safety In vivo Efficacy tests Chemical characterization Stability Formulation 8 Providing Integrated Research Solutions in Preclinical

EXPERIMENTAL DATA Confidential 9 Providing Integrated Research Solutions in Preclinical

STATUS OF STUDIES S. No. Studies 1 2 3 4 5 6 7 Details Standardization & Preparation of shogaol enriched ginger extract Extraction, Characterization and Preformulation Efficacy and mechanistic studies for anticancer potential in vitro Efficacy and mechanistic studies for anticancer potential in vivo Safety studies Other indications 4 a. Chemical characterization DNA fingerprinting In vitro cytotoxicity in 10 cancer cell lines 4 b. Selectivity index determination in 2 normal cells 4 c. Apoptosis assays 4 d. Anti-angiogenesis assay: Inhibition of proliferation of human endothelial cells 4 e. Anti-angiogenesis assay: Inhibition of VEGF 5 a. Anticancer potential in vivo based on cancer cell line selected from 4 a 6 a. Acute toxicity in rodents 6 b. Dose range finding studies: Pilot study for determination of suitable dose for continuous administration in rodents 7 a. Antioxidant potential by free radical scavenging assay 7 b. Immunity & Inflammation assay by modulation of cytokine release 7 c. Protective ability against DNA damage 7 d. Cytoprotection Study: Protection of cells against damage 10 Providing Integrated Research Solutions in Preclinical

1. CHARACTERIZATION Providing Integrated Research Solutions in Preclinical

Extraction and enrichment. . 1 Blank; Ethanol 95% Crude ginger in Et. OH 95%, normal p. H 6 -Shogaol standard Crude ginger in Et. OH 95%, p. H 1 12 Providing Integrated Research Solutions in Preclinical

Extraction and enrichment. . 2 Crude ginger extract (Et. OH 95%, Normal p. H) heat at 150°C for 3 h Ginger extract processed to 20% Gingerol in Et. OH 95% normal p. H Crude ginger extract (Et. OH 95%, p. H 1) heat at 150°C for 3 h Ginger extract processed to 20% Gingerol in Et. OH 13 95% p. H 1 Providing Integrated Research Solutions in Preclinical

Extraction and enrichment. . 3 Ginger extract processed to 20% Gingerol in Et. OH 95% normal p. H subjected to heat at 150°C For 3 hrs Ginger extract processed to 20% Gingerol in Et. OH 95% p. H 1 subjected to heat at 150°C For 3 hrs 3. 05 Ginger extract processed to 20% Gingerol subjected to heat at 150°C For 3 hrs at normal p. H Ginger extract processed to 20% Gingerol subjected to heat 14 at 150°C For 3 hrs at p. H 1 Providing Integrated Research Solutions in Preclinical

2. FORMULATION Confidential 15 Providing Integrated Research Solutions in Preclinical

Formulation development Statement of Ingredients and Additives according to FSSAI Formula of Ginger S Capsule Providing Integrated Research Solutions in Preclinical

Technical specification: Co. A Confidential 17 Providing Integrated Research Solutions in Preclinical

Certificate of Analysis

3. SAFETY STUDIES Confidential 19 Providing Integrated Research Solutions in Preclinical

List of Safety studies S. No. Studies 1 Acute Oral Toxicity 2 Dose Range Finding (DRF) or Maximum Tolerable Dose (MTD) Determination Study Confidential 20 Providing Integrated Research Solutions in Preclinical

Acute oral Toxicity Confidential 21 Providing Integrated Research Solutions in Preclinical

In vivo safety studies S. No. Model Objective 1 Acute Oral To determine toxic and safe Toxicity dose level Design STUDY Single dose administration of Shogaol ginger extract Species Balb/c mice (Female) ROA Oral No. of animals/Set 3 No. of Set 2 Observation Two weeks Single dose of Ginger Extract with high Shogaol content (GEHS) at 2000 mg/kg (In two separate sets) was orally administered to the female BALB/c mice (n=3) and observed for two weeks. (As per OECD Guideline 423) 22 Providing Integrated Research Solutions in Preclinical

In vivo safety studies : Acute Oral Toxicity v Writhing was observed till 1 hour post dosing and recovered later. v Body weight gain was observed during the experimental period. v No mortality was observed at 2000 mg/kg in any of the set. v Hence, as per OECD 423 guideline, LD 50 is under the category 5. LD 50 : >2000 - 5000 mg/kg The highest dose for MTD study is 2000 mg/kg 23 Providing Integrated Research Solutions in Preclinical

Dose Range Finding (DRF) or Maximum Tolerable Dose (MTD) Determination Study Providing Integrated Research Solutions in Preclinical

In vivo safety studies S. No. 2 Model Objective Design Dose To determine the maximum STUDY 14 days daily dose administration Range tolerable dose & dose at Species BALB/c mice (Female) Finding which efficacy study can be ROA Oral conducted Dose 3 different doses; 2000, 1000 and 500 mg/kg (Based on Acute Oral Tox Study) Observation § § § Body weight Feed consumption Clinical sign Haematology & Biochemistry Gross pathology v 32 Female BALB/c mice were grouped into four groups each consisting of 8 animals. v Animals from group G 1 received 0. 5% CMC in distilled water (Normal Control). v Selected Doses : 2000 mg/kg (G 2), 1000 mg/kg (G 3) and 500 mg/kg (G 4). 25 Providing Integrated Research Solutions in Preclinical

In vivo safety studies : Dose Range Finding Study v Body weight gain was observed throughout the experimental period. v Writhing was observed till one-hour daily post dosing in all the animals at 2000 mg/kg and in some animals at 1000 mg/kg. Writhing was not observed in any of the animal at 500 mg/kg. v No change in feed consumption and no mortality was observed at all tested doses v At 2000 mg/kg, changes in certain hematological and biochemical parameters were observed v MTD of Ginger Extract with high Shogaol content (GEHS) found to be more than 2000 mg/kg v Highest efficacy dose selected for in-vivo efficacy study is 1000 mg/kg 26 Providing Integrated Research Solutions in Preclinical

4. EFFICACY STUDIES Confidential 27 Providing Integrated Research Solutions in Preclinical

List of efficacy studies S. No. 1 Study Anticancer Potential: In vitro cell based assay i In vitro cytotoxicity ii Apoptosis iii Angiogenesis 2 Anticancer Potential: In vivo Assays 3 Additional efficacy studies i Antioxidant potential by free radical scavenging assay ii Immunity & Inflammation assay by modulation of cytokine release iii Protective ability against DNA damage iv Cytoprotection Study: Protection of cells against damage Confidential 28 Providing Integrated Research Solutions in Preclinical

Confidential 29 Providing Integrated Research Solutions in Preclinical

IN VITRO ASSAY (Screening, Efficacy and Mechanism) Providing Integrated Research Solutions in Preclinical

ANTICANCER POTENTIAL : IN VITRO CELL BASED ASSAYS Confidential 31 Providing Integrated Research Solutions in Preclinical

List of Studies S. No. Study Title 1 In vitro cytotoxicity- Killing of cancer cells 2 In vitro Apoptosis -How the cancer cells are being killed 3 In vitro Anti-Angiogenesis: How the microvasculature in cancer is being targeted Confidential 32 Providing Integrated Research Solutions in Preclinical

ANTICANCER POTENTIAL In vitro cytotoxicity- Killing of cancer cells 33 Providing Integrated Research Solutions in Preclinical

Study design: In vitro cytotoxicity TEST SYSTEM: Human Cancer cell lines TEST ITEMS: • 95% Ethanolic extracts were diluted in Serum Free Medium to obtain concentration range of 0. 001%v/v – 0. 1 %v/v for treatment of cells. STUDY DESIGN: • Plating of cells in 96 -well culture plates for 24 h. • Treatment of cells with Test Items for 72 h. • Determination of cytotoxic effect of Test Item by MTT assay. INTERPRETATION: • Determination of % inhibition of cell viability/ % cytotoxicity. • Determination of IC 50 values (concentration resulting in 50% cell death). • Determination of selectivity index (IC 50 in normal cells/IC 50 in cancer cells). S. No. Type Name of Cell lines 1 B 16 -F 10 (Mouse Skin Melanoma) 2 PA-1 (Human Ovarian Cancer) 3 MDA-MB-231 (Human Breast Cancer) 4 Cancer Cell Lines A 549 (Human Lung Carcinoma) 5 HT 29 (Human Colon Cancer) 6 Hep. G 2 (Human Hepatocarcinoma) 7 MG-63 (Human Osteosarcoma) 8 THP-1 (Human Leukemia) 9 Normal Cells HEK 293 (Human Embryonic Kidney Cells) 34 Providing Integrated Research Solutions in Preclinical

Results: In vitro cytotoxicity and IC 50 values Cytotoxic effect of various Ginger Extracts on a panel of Cancer Cell Lines Enriched Ginger extracts show better cytotoxic effects than crude extract in different Cancers as: 35 Skin > Breast > Bone > Leukemia> Colon> Lung Providing Integrated Research Solutions in Preclinical

Results: IC 50 Values IC 50 values of different Ginger Extracts in panel of Cancer Cell Lines Lower IC 50, better activity than crude extract 36 Enriched Ginger extracts show better cytotoxic effects in different Cancers as: Skin > Breast > Bone > Leukemia> Colon> Lung Providing Integrated Research Solutions in Preclinical

Results: Selectivity index in normal cells Selectivity index of various Ginger Extracts in normal cells (wrt HEK 293) Highest Selectivity • Selectivity index <1 = Non selective, • Selectivity index 1 -3 = Moderate selectivity • Selectivity index>3 = Good selectivity Enriched (20% Gingerol and 20% Ginger) extracts show good selectivity in normal cells 37 Providing Integrated Research Solutions in Preclinical

Interpretations from cytotoxicity data Based on the IC 50 and selectivity index obtained in 8 cancer cell lines and 1 normal cells, following can be concluded: 1. Out of 8 cancer cell lines, best activity of the extracts has been observed in B 16 F 10 (Mouse Skin cancer), MDAMB-231 (Human Breast Cancer), MG-63 (Human Bone cancer), THP-1 (Human Leukemia), HT-29 (human colon cancer) and A 549 (Human Lung Cancer). 2. Of the 3 extracts tested, 20% Ginger Extract showed most cytotoxic effects with high selectivity index. 3. Based on IC 50 values obtained in cancer cells and selectivity index in normal cells, following is the order of activity: Skin > Breast > Bone > Leukemia> Colon> Lung 4. For apoptosis following cell lines are selected: Skin cancer, Breat cancer 38 Providing Integrated Research Solutions in Preclinical

ANTICANCER POTENTIAL In vitro Apoptosis -How the cancer cells are being killed 39 Providing Integrated Research Solutions in Preclinical

Study design: Mitochondrial depolarization by JC-1 staining method TEST SYSTEM: • B 16 F 10 (Mouse Skin cancer) • MDA-MB 231 (Human breast cancer) TEST ITEMS: • 95% Ethanolic extracts were diluted in Serum Free Medium to obtain concentration range of 0. 001%v/v – 1 %v/v for treatment of cells. STUDY DESIGN: • Plating of cells in 96 -well culture plates for 24 h. • Treatment of cells with Test Items for 48 h. • Determination of Mitochondrial Membrane Potential (MMP) by JC-1 staining based fluorescence assay. INTERPRETATION: • Healthy cells show red fluorescence and apoptotic cells green fluorescence. • A decrease in mitochondrial membrane potential (MMP) indicates depolarization and pro-apoptotic potential. • % decrease in mitochondrial membrane potential was determined. Healthy cells Apoptotic cells 40 Providing Integrated Research Solutions in Preclinical

Results – Mitochondrial Depolarization in Skin cancer (B 16 F 10 cell line) • Apoptotic potential of extracts was determined in B 16 F 10 (Skin cancer) cells by determining their effect on Mitochondrial Membrane Potential by JC-1 assay. • Ginger extracts demonstrated a decrease in mitochondrial membrane potential resulting in mitochondrial depolarization. Enriched Ginger extracts show potent apoptotic effect by enhancing mitochondrial depolarization in Skin cancer 41 Providing Integrated Research Solutions in Preclinical cells

Results – Mitochondrial depolarization in breast cancer (MDA-MB-231 cell line) • Apoptotic potential of extracts was determined in MDA-MB-231 (Breast cancer) cells by determining their effect on Mitochondrial Membrane Potential by JC-1 assay. • Ginger extracts demonstrated a decrease in mitochondrial membrane potential resulting in mitochondrial depolarization. Enriched Ginger extracts show potent apoptotic effect by enhancing mitochondrial depolarization in Breast 42 cancer Providing Integrated Research Solutions in Preclinical cells

ANTICANCER POTENTIAL In vitro Anti-Angiogenesis: How the microvasculature in cancer is being targeted 43 Providing Integrated Research Solutions in Preclinical

Study design - Angiogenesis a) Inhibition of endothelial cells proliferation § Human endothelial cells (EA. hy 926) were treated with extracts and effect on cell proliferation was determined by MTT assay after 72 h. § A decrease in cell number/proliferation indicates anti-angiogenic effect. § The concentrations of test item that inhibited endothelial cell proliferation (inhibition of test item treated cells < Inhibition of SFM treated cells) were considered as non-cytotoxic doses demonstrating anti-angiogenic effects. b) Inhibition of VEGF secretion • Human endothelial cells (EA. hy 926) were treated with extracts at noncytotoxic concentrations. • Levels of secreted VEGF in culture supernatants after 72 h of treatments were estimated by ELISA. 44 Providing Integrated Research Solutions in Preclinical

Results: Anti-Angiogenic effect by inhibition of cell proliferation • Inhibitory effect of Ginger Crude and enriched extracts on proliferation of endothelial cells (Ea. hy 926) was determined by MTT assay. • Ginger Crude and enriched extracts demonstrated antiproliferative effect on endothelial cells. • Enriched extracts showed better inhibition of proliferation than crude extract. Antiproliferative doses Enriched Ginger extracts show better anti-angiogenic effect than crude extract by inhibition of endothelial cells 45 proliferation Providing Integrated Research Solutions in Preclinical

Results: Anti-Angiogenic effect by inhibition of VEGF secretion § Inhibition of VEGF secretion suggests anti-angiogenic potential. § Ginger extracts demonstrated inhibition of VEGF secretion in endothelial cells as compared to untreated levels. Enriched Ginger extracts show better anti-angiogenic effect than crude extract 46 Providing Integrated Research Solutions in Preclinical

ANTICANCER POTENTIAL : IN VIVO ASSAYS Confidential 47 Providing Integrated Research Solutions in Preclinical

List of Studies S. No. 1 Study Title In-vivo Anti-Tumor Activity Syngeneic solid tumor model in immunocompetent mice Confidential 48 Providing Integrated Research Solutions in Preclinical

In-vivo Anti-Tumor Activity Syngeneic solid tumor model in immunocompetent mice Providing Integrated Research Solutions in Preclinical

In vivo efficacy studies S. No. 3 Model Objective In-vivo syngeneic solid To determine the in- tumor vivo anti-cancer activity Design STUDY 28 days daily dose administration to bearing mice Species Balb/c mice immunocompetent Ro. A Oral mice Dose 3 different doses (Based on study MTD study) Observation q q q model in solid tumor Body weight Tumor volume % T/C Tumor Growth inhibition and Delay % Anti-cancer activity v. Animals of group G 1 received 0. 5% CMC(Normal Control). v. Animals of group G 2, G 3 and G 4 received orally at 1000 , 400, and 160 mg/kg of Ginger Extract with high Shogaol content (GEHS) respectively once daily for 21 days v. Animals of group G 5 received 12 mg/kg of 5 -FU, i. p. as a reference standard 50 Providing Integrated Research Solutions in Preclinical

Result v 21 Days daily oral administration of Ginger Extract with high Shogaol content (GEHS) at a dose of 1000 mg/kg showed Shogaol content (GEHS) showed dose dependent inhibition in tumor significant (p<0. 001) reduction in tumor size when compared to growth vehicle control group v 1000 mg/kg, 400 mg/kg, 160 mg/kg showed tumor growth inhibition of 41. 73%, 21. 75% and 18. 56% respectively 51 Providing Integrated Research Solutions in Preclinical

EFFICACY STUDIES IN MULTIPLE THERAPEUTIC AREAS Confidential 52 Providing Integrated Research Solutions in Preclinical

Antioxidant potential 53 Providing Integrated Research Solutions in Preclinical

Study design: Antioxidant assay TEST SYSTEM: Cell free assay TEST ITEMS: • The ethanolic extracts were diluted in 80% methanol to obtain concentration range of 0. 05%-1% v/v METHOD: • Free radical scavenging activity of test items were evaluated spectrophotometrically by DPPH assay in vitro INTERPREATTAION: • Free radical scavenging activity of TI determined by : 1. Decrease in absorbance values in presence of TI 2. % inhibition in absorbance Free radicals produced during oxidative stress are responsible for aging. Scavenging of free radicals generated indicates antioxidant potential. 54 Providing Integrated Research Solutions in Preclinical

Results: Antioxidant potential Fresh Ginger Crude Ginger in 95% Et. OH; Conc (%) in 95% Et. OH; in 95% Et. OH p. H 1 heat at p. H 1 150 °C 20 % Gingerol extract in 95% Et. OH 20 % Gingerol extract in powder 95% Et. OH; heat at 150 p. H 1 heat at °C 150 °C 0 0 0 0. 05 2 3 1 40 38 43 0. 1 2 1 3 51 52 67 0. 25 3 2 9 72 70 73 0. 5 8 9 14 74 74 74 0. 75 12 13 19 75 74 75 1 13 16 24 76 75 75 IC 50 value 6% 3. 5% 1. 59% 0. 052% 0. 053% 0. 044% IC 50 value of positive control 13. 95 µg/m. L Lowest IC 50 • Out of 6 extracts the best activity was observed in 20 % Ginger extract in 95% Et. OH; p. H 1 heat at 150 °C with IC 50 value 0. 044% • Based on the IC 50 value the order of antioxidant potential is as follows: 20 % Ginger extract in 95% Et. OH; p. H 1 heat at 150 °C > 20 % Gingerol extract in 95% Et. OH = 20 % Gingerol powder heat at 150 °C > Fresh Ginger in 95% Et. OH; p. H 1 heat at 150 °C > Crude Ginger in 95% Et. OH; p. H 1> Crude Ginger in 95% Et. OH 55 Enriched Ginger extracts show better antioxidant potential than crude extract Providing Integrated Research Solutions in Preclinical

ANTI-INFLAMMATORY POTENTIAL by estimation of TNF-α levels against LPS stimulation Confidential 56 Providing Integrated Research Solutions in Preclinical

Study design: Anti-inflammatory potential TEST SYSTEM: • Mouse macrophage cell line (RAW 264. 7) TEST ITEMS: • 95% Ethanolic extracts of the test item were used for further dilution in Serum Free Medium for treatment of cells • STUDY DESIGN: § Non-cytotoxic concentrations (<30% cell death) were identified by MTT assay. § For anti-inflammatory activity, cells were Co-treated with extracts (at non-cytotoxic concentrations) and inflammatory stimulus (Lipopolysaccharide from E. coli; LPS) for 24 h. § Cell free supernatants were collected. § Levels of cytokine (TNF-α) were determined by ELISA. INTERPRETATION: § % Inhibition in TNF-α levels was determined as compared to LPS stimulated inflammatory levels. Confidential 57 Providing Integrated Research Solutions in Preclinical

Results – Anti-inflammatory potential § LPS treatment resulted in stimulation of key immunomodulatory cytokine (TNF-α) as compared to untreated levels. § Test items demonstrated good anti-inflammatory potential by inhibition of TNF-α as compared to LPS stimulated levels. Enriched Ginger extracts show better Anti-inflammatory effect than crude extract by inhibition of TNF-α secretion against LPS 58 stimulated levels Providing Integrated Research Solutions in Preclinical

HEPATOPROTECTION • Cytoprotection in liver cells against oxidative stress Confidential 59 Providing Integrated Research Solutions in Preclinical

Study design - Hepatoprotective potential TEST SYSTEM: § Human liver cell line (Hep. G 2) was used to determine cytoprotective potential of extracts. TEST ITEMS: • 95% Ethanolic extracts of the test item were used for further dilution in Serum Free Medium for treatment of cells STUDY DESIGN: § Cells were pretreated with extracts for 24 h. § Cells were then exposed to oxidative damage (t-BHP) for 2. 5 h. § Cell viability was assessed by MTT assay. INTERPRETATION: § Restoration of cell viability against t-BHP induced cytotoxicity reflects cytoprotective ability. Confidential 60 Providing Integrated Research Solutions in Preclinical

In vitro Cytoprotection in liver cells – Results § Liver cells (Hep. G 2) when subjected to oxidative stress (t-BHP) resulted in loss of cell viability. § A restoration of cell viability against oxidative stress induced cell death suggests hepatoprotective ability. Enriched Ginger extracts show better hepatoprotective potential than crude extract by restoration of cell viability against Confidentialthan 61 oxidative stress Providing Integrated Research Solutions in Preclinical

DNA PROTECTION ABILITY Protection against oxidative stress 62 Providing Integrated Research Solutions in Preclinical

Study design: DNA protection assay TEST SYSTEM: Plasmid DNA TEST ITEMS: • 95% Ethanolic extracts of the test item were used for further dilution STUDY DESIGN: • Incubation of Plasmid DNA with different conc. of extracts and Trolox (positive control) for 2 h at 37 C. • Oxidative damage with 30 % H 2 O 2 and UVB irradiation for 30 min at 37 C. • After irradiation samples were run on 1 % agarose gel electrophoresis. • Segregated form of plasmid DNA bands were analyzed and captured using BIORAD Gel documentation System. • Modulation in the plasmid DNA pattern of test herb(s) treated DNA, as compared to control untreated DNA was observed. INTERPRETATION: Restoration of Native DNA against oxidative damage 63 Providing Integrated Research Solutions in Preclinical

Results: DNA protection ability against oxidative stress • Effect of ginger extracts on restoration of Native DNA against oxidative damage was studied. • Ginger extracts demonstrated good DNA protection ability as suggested by restoration of the supercoiled form of DNA. Enriched Ginger extracts show better DNA protection ability than crude extract by restoration of supercoiled DNA 64 against oxidative stress Providing Integrated Research Solutions in Preclinical

Ginger Extract with high Shogaol content – Overall Summary Confidential 65 Providing Integrated Research Solutions in Preclinical

Ginger Extract with high Shogaol content – Overall Summary • Strong Anticancer activity - Ginger Extract with high Shogaol content demonstrated better anticancer activity in vitro then crude extract in cancer cell lines, with good safety index in normal cells: Skin > Breast > Bone > Leukemia> Colon> Lung - 21 Days daily oral administration of Ginger Extract with high Shogaol content (GEHS) showed dose dependent inhibition in tumor growth - Enriched Ginger extracts show potent pro-apoptotic effect by enhancing mitochondrial depolarization in Skin cancer and breast cancer cells. - Enriched ginger extracts demonstrate better antiangiogenic potential in vitro then crude extract by inhibition of endothelial cells proliferation. • Enriched ginger extracts demonstrate better antioxidant potential then crude extract. • Enriched ginger extracts demonstrate better anti-inflammatory activity in vitro than crude extract by cytokine inhibition in immune cells. • Enriched ginger extracts demonstrate better hepatoprotective activity in vitro than crude extract by restoration of cell viability in liver cells against oxidative damage. • Enriched Ginger extracts show better DNA protection ability than crude extract by restoration of supercoiled DNA against oxidative stress. Confidential 66 Providing Integrated Research Solutions in Preclinical

Efficacy assessment of Zindrol Capsule using in vitro assay Confidential 67 Providing Integrated Research Solutions in Preclinical

In vitro anticancer potential TEST SYSTEM: Results Human Breast Cancer cell line (MDA-MB-231) TEST ITEMS: • • Contents of Zinderol capsule were dissolved in DMSO to prepare stock solution. This stock solution was further diluted in serum free medium for treatment of cells. STUDY DESIGN: • • • Cells were plated for 24 h in 96 -well plates. Cells were treated with Test Item for 72 h. The cytotoxic effect of Test Item was determined by MTT assay. Confidential Zinderol demonstrated in vitro anticancer potential by inducing cytotoxic effect in breast cancer cells 68 Providing Integrated Research Solutions in Preclinical

Antioxidant potential Results TEST SYSTEM: Cell free assay TEST ITEMS: • Zinderol was dissolved in 80% methanol to obtain stock concentration of 100 mg/ml. • Stock solution was diluted using 80% Methanol to achieve final concentrations ranging from 10 µg/m. L- 500 µg/m. L. STUDY DESIGN: • Free radical scavenging activity of Zinderol was evaluated spectrophotometrically by DPPH assay in vitro. • Free radical scavenging activity of TI was determined by : 1. Decrease in absorbance values in presence of TI 2. % inhibition in absorbance Zinderol demonstrated antioxidant Confidential potential by free radical scavenging effect 69 Providing Integrated Research Solutions in Preclinical

Anti-inflammatory potential Results TEST SYSTEM: Mouse macrophage cell line (RAW 264. 7) TEST ITEMS: • • Contents of Zinderol capsule were dissolved in DMSO to prepare stock solution. This stock solution was further diluted in serum free medium for treatment of cells. STUDY DESIGN: • • • Cells were plated for 24 h in 24 -well plates. Cells were treated with Test Item and stimulated with LPS (50 ng/ml). Supernatants were collected and levels of TNF-alpha were determined by ELISA. Confidential Zinderol demonstrated anti-inflammatory potential by inhibition of TNF-α secretion 70 Providing Integrated Research Solutions in Preclinical

Anti-fatigue potential TEST SYSTEM: Plasmid DNA TEST ITEM: • Contents of Zinderol capsule were dissolved in DMSO to prepare stock solution. • This stock solution was further diluted in serum free medium for treatment of cells. Results STUDY DESIGN: • Plasmid DNA was incubated with different conc. of Test Item for 2 h at 37 C. • Oxidative damage was induced with 30 % H 2 O 2 and UVB irradiation for 30 min at 37 C. • After irradiation samples were run on 1 % agarose gel electrophoresis. • Segregated form of plasmid DNA bands were analyzed and captured using BIORAD Gel documentation System. • Restoration of Native DNA against oxidative damage was determined. Zinderol demonstrated anti-fatigue. Confidential potential by restoration of DNA damage 71 Providing Integrated Research Solutions in Preclinical

Zindrol : Key Outcomes Higher Shogaol content Anticancer effects in vitro & in vivo Good antioxidant potential Potent DNA protection ability Potent immunostimulatory & antiinflammatory action Potent cytoprotective Good safety profile Confidential 72 Providing Integrated Research Solutions in Preclinical

Thanks Confidential 73 Providing Integrated Research Solutions in Preclinical