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National Mission on Himalayan Studies (NMHS)

National Mission on Himalayan Studies (NMHS)

National Mission on Himalayan Studies (NMHS) HIMALAYAN RESEARCH FELLOWSHIP (PRO FORMA FOR THE ANNUAL PROGRESS REPORT) [Reporting Period: from June 2018 to April 2019]

Kindly fill the NMHS Fellowship Annual Progress Report segregated into the following 7 segments, as applicable to the NMHS Fellowship nature and outcomes.

1. Fellowship Grant Information and Other Details

2. Fellowship Description at Himalayan Research Associates (H-RAs) Level

3. Fellowship Description at Himalayan Junior Research Associates (H-JRFs) Level

4. Fellowship Description at Institutional/ University Level

5. Fellowship Concluding Remarks/ Annual Summary

6. Specific Research Question(s) Addressed with Succinct Answer(s)

7. Any other information

Please let us know in case of any query at: [email protected]

PRO FORMA NMHS-Fellowship Annual Progress Report (APR)

1. Fellowship Grant Information and Other Details

NMHS Fellowship Grant ID: GBPI / NMHS-2017-18 / HSF-02

Name of the Institution/ University: National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati

No. of Himalayan Research/Project 02 Associates:

No. of Himalayan Junior Research/Project 05 Fellows:

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2. Fellowship Description at H-RA Level

Himalayan Research Associates (H-RAs)

H-RAs Profile Description:

Date of Name of the PI S. No. Name of RA Research Title Qualification Joining and Designation

1. Dr. Nandana 4th June 2018 Database Dr. U.S.N. Murty, PhD, FRES Bhardwaj Development on Director (London) Himalayan Director Indigenous NIPER GUWAHATI Knowledge Systems (IKS) prevailing in NE hills.

2. Dr. Prakash 4th June 2018 Validation and Dr. U.S.N. Murty, PhD, FRES Kishore Hazam valuing of >5 Director (London) selected Director traditional, NIPER GUWAHATI ecological knowledge and practices

Progress Brief (to be filled for each H-RA in separate row):

RA Research Addressed Research/ Achievements No. Objective(s) Deliverables Experimental Work*

1. Database development  Database on the  In progress (Till date we Attached as Annexure 1 a on Himalayan Himalayan have listed over 400 medicinal and 1b Indigenous Knowledge indigenous of North East and Systems (IKS) prevailing Knowledge System a database preparation is in in NE hills. progress. The format and list  Digital Library on for the database has been the NE Himalayan shown in Annexure 1 IKS.  Silk as biomaterials used in NE India has been explored for the biomaterial application. 2 Validation and valuing Validation and  In progress (we have  Attached as Annexure 2- of >5 selected valuing of > 5 selected collected, dried and 5. traditional, ecological traditional, powdered noted medicinal knowledge and ecological knowledge plants used to treat various practices and practices ailments in North East India (Annexure 2)  Collection and IPR proceedings (Annexure 3a) have been initiated and we will file joint patents, if we get promising results after proper laboratory testing (Annexure 3 b).

 Approximately 75 traditional healers (Annexure 4) from , , , Tripura and attended the Traditional Healers Meet on 26th August 2018 at NIPER- NMHS Fellowship Grant Progress Page 2 of 122

G. A healthy discussion was conducted among the local healers and eminent scientists regarding therapeutic activity and rational use of natural products (Annexure 5). *Experimental work giving full details (in separate sheet, within 300 words) of experimental set up, methods adopted, data collected supported by necessary table, charts, diagrams & photographs. Note: Data, table and figures may be attached as separate source file (.docx, .xls, jpg, .jpeg, .png, .shp, etc. ).

3. Fellowship Description at H-JRF Level Himalayan Junior Research Project Fellows (H-JRFs)

H-JRFs Profile Description: S. Name of JRF Date of Joining Name of the PI Qualification No. 1. Chaudhari Vishal Sharad 30th July, 2018 Dr. Subham Banerjee M. Pharm. (JRF 04)

2. Datta Maroti Pawde 30th July, 2018 Dr. S. Tamilvanan M. Pharm. (JRF 03)

3. Aishwarya Jala 30th July, 2018 Dr. Alka Chaudhary M. Pharm. (JRF 05)

Siddhi Jain (JRF 01) 30th July, 2018 4. Dr. VGM Naidu M. Pharm. Shantanu PA (JRF 02) 30th July, 2018 5. Dr. VGM naidu M. Pharm.

Progress Brief (to be filled for each JRF in separate row): JRF Research Research/ Deliverable Achievements No. Objectives Experimental Work*

1. Pharmacological  Established the Selected selected Attached as Annexure 6 activities, efficacy, institutional capacity for medicinal plants

safety and determining safety and (NIP/18/12, NIP/18/13 efficacy, dosage testing and NIP/18/14) which dosage evaluation of and formulation of are endemic to NE bioactives compound phytomedicines. India and further in potential species.  Confirmed and tested tested for their efficacy record of 3 species of towards antioxidant, medicinal plants effective anticancer activities in for the treatment of few invitro and invivo human diseases and few models livestock diseases and determined their safety, From the studies it was efficacy and dosage levels. confirmed that  Strengthening the sector alcoholic extracts of through research and the above two plants development of traditional may be used as an drugs. adjuvant therapy along with the doxorubicin or chemotherapy to prevent dose dependent toxicity or side effects of the chemotherapy. Further preclinical toxicity studies and efficacy studies towards anticancer activities NMHS Fellowship Grant Progress Page 3 of 122

are in progress.

2. Pharmacological  Confirmed and tested Selected selected Attached as Annexure 7 activities, efficacy, record of 3 species of medicinal plants

safety and medicinal plants effective (NIP/18/01, NIP/18/11) for the treatment of few which are endemic to dosage evaluation of human diseases and few NE India and further bioactive compound. livestock diseases and tested for their efficacy determined their safety, towards antioxidant, efficacy and dosage levels. anticancer activities in  Facilitate the gradual invitro and invivo integration of traditional models drugs with modem medicine by giving due From the studies it was attention to traditional confirmed that practices and identifying alcoholic extracts of the beneficial and harmful the above two plants aspects through may be used as an investigation and research. adjuvant therapy along with the doxorubicin or chemotherapy to prevent dose dependent toxicity or side effects of the chemotherapy. Further preclinical toxicity studies and efficacy studies towards anticancer activities are in progress. 3. Sensitization and  Documentation of From the literature it Attached as Annexure 8. awareness about Phytochemical and was evident that medicinal awareness programmes Dhemaji district of Assam and Jayantia hills plant among in 10 villages for of Meghalaya Himalayan livelihood options. populations are mostly inhabitants for their  Database on high dependent on herbal rational use. value Medicinal plants and medicines for the their traditional use from treatment of various the selected villages of the diseases. We have region. conducted awareness programmes in three villages of two districts in Assam and Meghalaya states. Hereby, we are developing a novel herbal formulations for the treatment of rheumatoid arthritis from selected medicinal plant (NIP/18/22). 4. Identification,  Database on Two medicinal plants Attached as Annexure 9 collection and quantification of (NIP/18/08 and

quantification available resources for NIP/18/24) which are endengared have been of 5 most endangered selected species collected, extracted and plants with medicinal  Develop the NIP/18/08 was tested for potential in region. effective harvesting its efficacy towards guidelines to sustainably antimicrobial activity. improve the supply of and Hereby, we also access to, 5 important wild attempted to develop medicinal plants. novel formulations to increase bioavailability, efficacy and patient compliance for the bioactive compounds from medicinal plants of

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NE India having proven therapeutic efficacy for various diseases.

5. Extraction, isolation,  Database on From the Attached as Annexure 10 purification, quantification of available pharmacological efficacy characterization of resources for selected data it was found that bioactive compounds NIP/18/11 plants is species from 3 unexplored showing very good  species. Strengthen the efficacy and hence application of traditional further studies were drugs as certained to be performed for isolation safe and effective for and characterization of treatment. bioactive compounds from above plant. *Experimental work giving full details (in separate sheet, within 300 words) of experimental set up, methods adopted, data collected supported by necessary table, charts, diagrams & photographs. Note: Data, table and figures may be attached as separate source file (.docx, .xls, jpg, .jpeg, .png, .shp, etc. ).

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Annexure 1

List medicinal plants of found in North Easr India

Species Family

Abroma augusta L. Malvaceae Achyranthes asperaL. Amaranthaceae Aconitum heterophyllum Wall. Ex Royle Ranunculaceae Aconitum ferox Wall Ranunculaceae Actinodaphne angustifolia Nees Lauraceae Adhatoda zeylanicaL. Acanthaceae Adiantum capillus -venerisLinn. Pteridaceae Aegle marmelos (L.) Correa. Rutaceae Argemone mexicana L. Papaveraceae Allbizia odoratissima (Linn. f.) Benth Fabaceae Albizia lebbeck Benth. Fabaceae Alocasia cucullata (Lour.) Schott. Araceae Alocasia fornicata (Kunth) Schott Araceae Alpinia galanga (L.) Sw. Alpinia nigra (Gaertn.) Burtt Zingiberaceae Alpinia officinarumHance Zingiberaceae Anacardium occidentale Linn. Anacardiaceae Alstonia scholaris (Linn) R. Brown Apocynaceae Alternanthera sessilis (L.) R. Br. ex DC Amaranthaceae Amaranthus spinosusL. Amaranthaceae

Amaranthus viridis L. Amaranthaceae

Amorphophallus bulbifer (Roxb.) Blume Araceae Amorphophallus campanulatus (Roxb.) Blume Araceae Amorphophallus paeoniifolius (Dennst.) Nicolson Araceae Annona squamosa. L. Annonaceae Anisomeles indica Linn. Lamiaceae Neolamarckia cadamba (Roxb.) Bosser Rubiaceae Antidesma acidum Retz. Euphorbiaceae Antidesma bunius (L) Spreng., Euphorbiaceae Amomum subulatum Roxb. Zingiberaceae Aponogeton natans (Linn) Engler & K. Krause Aponogetonaceae Aporosa dioica (Roxb.) Müll.Arg. Euphorbiaceae Argyreia nervosa (Blume.) Boj. Convolvulaceae Arisaema tortuosum (Wall.) Schott Araceae Aristolochia clematitis L. Aristolochiaceae Aristolochia tagala Cham. Aristolochiaceae Artemisia vulgaris L. Asteraceae Artocarpus chama Buch.-Ham. ex Wall Moraceae Artocarpus gomezianus Wall. Ex.Trecul Moraceae Artocarpus lakoochaRoxb. Moraceae Aesculus indica (Colebr. ex Cambess.) Hook. Sapindaceae

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Aesculus assamica Griff. Sapindaceae Asparagus racemosus Willd. Liliaceae Averrhoa carambolaL. Averrhoaceae Azadirachta indica A. Juss. Meliaceae Baccaurea ramiflora Lour. Euphorbiaceae Bambusa balcooaRoxb. Poaceae Bambusa pallida Munro Poaceae Bambusa tulda Roxb. Poaceae Basella albaL. Basellaceae Bauhinia acuminata L. Caesalpiniaceae Bauhinia malabarica Roxb. Caesalpiniaceae Bauhinia purpurea L. Caesalpiniaceae Bauhinia variegata. L. Caesalpiniaceae Begonia hatacoa Buchanan-Hamilton ex D. Don Begoniaceae Begonia roxburghii A.DC. Begoniaceae Berberis aristata DC Berberidaceae Bergenia ciliata (Haw.) Sternb. Saxifragaceae Bidens pilosaL. Asteraceae Boerhavia diffusaL. Nyctaginaceae Bombax ceiba L. Bombacaceae Bridelia retusaSpreng. Euphorbiaceae Bridelia stipularis (L.) Blume Euphorbiaceae Brucea mollis Wall. ex Kurz. Simaroubaceae Buddleja asiatica Lour Scrophulariaceae Bursera serrata Wall.ex Colebr. Burseraceae Calamus latifoliaRoxb. Arecaceae Calamus tenuis Roxb. Arecaceae Callicarpa arborea Roxb. Verbenaceae Callicarpa longifolia Lam. Verbenaceae Callicarpa vestitaWall. Verbenaceae Calotropis procera (Willd.) R. Br. Apocynaceae Cannabis sativaL. Cannabaceae Capsicum frutescens Linn. Solanaceae Carallia brachiata( Lour) Merr. Rhizophoraceae Careya arborea Roxb. Lecythidaceae Carica papaya L. Caricaceae Caryota urensL. Arecaceae Cassia fistula Linn Caesalpiniaceae Cassia siameaLam. Caesalpiniaceae Cassia tora L. Caesalpiniaceae Castanopsis echinocarpa Miq. Fagaceae Celosia argentea L. Amaranthaceae Centella asiatica. (L.) Urban Apiaceae Cestrum nocturnum L. Solanaceae Chenopodium albumL. Chenopodiaceae

Chonemorpha fragrans (Moon) Alston Apocynaceae

Chrysophyllum lanceolatum(Bl.) D.C. Sapotaceae

Cinnamom camphora Nees Eberm. Lauraceae Cinnamomum tamala (Buch.-Ham.) T.Nees & C.H.Eberm. Lauraceae

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Cinnamomum bejolghota (Buch.-Ham.) Sweet Lauraceae Citrus limon (L.) Osbeck Rutaceae Citrus maxima (Burm.) Merrill. Rutaceae Citrus reticulatus Blanco Rutaceae Citrus sinensis (L.) Osbeck Rutaceae Clausena excavata Burm.f. Rutaceae Crecentia cujete L. Bignoniaceae Cissampelos pareira L. Menispermaceae Cissus repens Lam. Vitaceae Citrus medicaL. Rutaceae Clerodendrum colebrookianum Walp Verbenaceae Clerodendrum indicum (L.) Kuntze Verbenaceae Clerodendrum glandulosum Coleb. Verbenaceae Clerodendrum serratum (L.) Moon Verbenaceae Celastrus paniculatus Wild Celastraceae Coccinia grandis(L.) Voigt. Cucurbitaceae Combretum decundrumRoxb. Combretaceae Commelina benghalensis L. Commelinaceae Commelina paludosa Blume Commelinaceae Conyza stricta Wild. Asteraceae Coptis teeta Wall. Ranunculaceae Cordia dichotoma G. Forst. Boraginaceae Costus speciosus (J.Koenig) Sm. Costaceae Cirsium lipskyi Petr. Asteraceae Crataeva nurvala Buch.-Ham. Capparaceae Crotalaria albida Heyne ex Roth Fabaceae Crotolaria juncea L. Fabaceae Cucumis trigonus Roxb. Cucurbitaceae Curanga amara Juss. Scrophulariaceae Curcuma caesia Roxb. Zingiberaceae Curcuma amada Roxb. Zingiberaceae Curcuma aromatica Salisb. Zingiberaceae Curcuma zedoaria Rosc Zingiberaceae Cuscuta reflexa Roxb Cuscutaceae Cycas pectinata Buch.-Ham. Cycadaceae Emblica officinalis Gaertn. Euphorbiaceae Dalbergia pinnata (Lour.) Prain Fabaceae Dalbergia rimosa Roxb. Fabaceae Dalbergia stipulacea Roxb. Fabaceae Datura stramonium L. Fabaceae Deeringia amaranthoides (Lam.) Merrill Amaranthaceae Derris robusta (Roxb. Ex DC.) Benth. Fabaceae Dendrobium nobile Lindley Orchidaceae sinuata (Blume) Chew, Desmodium triquetrum (L.) DC. Fabaceae indicaL. Roxb. Dilleniaceae Dillenia scabrella Roxb. Dilleniaceae Dioscorea alataL. Dioscoreaceae Dioscorea bulbifera L. Dioscoreaceae

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Dioscorea hamiltonni Hook.f. Dioscoreaceae Dioscorea puber Blume Dioscoreaceae Dioscorea pentaphyllaL. Dioscoreaceae Dioscorea sativaL. Dioscoreaceae Diospyros kaki Thunberg Ebenaceae Diospyros lancaefoliaRoxb. Ebenaceae Diospyros malabarica(Desr.) Kostel. Ebenaceae Diplazium esculentum (Retz.) Sw Athyriaceae Drimycarpus racemosus (Roxb.) Hk. f. Anacardiaceae Drymaria cordata (Linn.) Willd Caryophyllaceae Duabanga grandiflora (Roxb.ex DC) Walp. Lythraceae Eclipta prostrata (L.) L. Asteraceae Ehretia acuminata (DC.) R. Br. Boraginaceae Elaeocarpus floribundus Blume Elaeocarpaceae Elaeocarpus sphaericus (Gaertn.) K.Schum. Elaeocarpaceae Elastostema platyphyllum Wedd. Urticaceae Enhydra fluctuans Lour, Asteraceae Equisetum debile Roxb. Equisetaceae Eryngium foetidumL. Apiaceae Eythrina arborescens (Roxb) Kuntze Fabaceae Euphorbia hirtaL. Euphorbiaceae Euryale feroxSalisb. Nymphaeaceae Ficus auriculataLour. Moraceae Ficus hirta Vahl Moraceae Ficus hispida L.f. Moraceae Ficus nervosa Heyne ex Roth Moraceae Ficus oligodon Miq. Moraceae Flacourtia jangomas (Lour.) Raeusch Flacourtiaceae Flemingia vestita Baker Fabaceae Firminia colorata (Roxb.)R. Br. Sterculiaceae Garcinia acuminutaPlanchon & Triana Clusiaceae Garcinia cowa Roxb. Clusiaceae Garcinia lanceaefoliavar. oxyphylla Clusiaceae Garcinia Morella(Gaetn.) Desr. Clusiaceae Garcinia pedunculataRoxb. ex Buch.-Ham. Clusiaceae Garcinia xanthochymus Hook. f. ex T. Anderson. Clusiaceae Glycosmis pentaphylla (Retz.) DC Rutaceae Gmelina arborea Roxb. Verbenaceae Grewia elastica Royle Tiliaceae

Grewia helicterifolia Wall. ex G.Don Tiliaceae Grewia hirsuta Vahl. Tiliaceae Grevillea robusta A. Cunn, Tiliaceae Grewia sapidaRoxb. Tiliaceae Grewia sclerophyllaRoxb. Tiliaceae Goniothalamus sesquipedalis (Wall.) Hook.F. & Thomson Annonaceae Gynocordia odorata R.Br. Flacourtiaceae Hedyotis corymbosa (L.) Lam. Rubiaceae Hedyotis diffusaWilld. Rubiaceae Helminthostachys zeylanica (L.) Hook. Helminthostachyaceae

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Hemidesmus indicus L. Apocynaceae Hibiscus rosa-sinensisL. Malvaceae Hibiscus sabdariffa L. Malvaceae Hypericum japonicum Thunb. ex Mur Hypericaceae Holarrhena antidysenterica (L.) Wall. ex A. DC. Apocynaceae Homalomena aromatica (spreng) Scott Araceae Houttuynia cordataThunb. Saururaceae Hyptis suaveolens (L.) Poit; Lamiaceae Hodgsonia macrocarpa (Blume) Cogn. Cucurbitaceae Hydrocotyle sibthorpioides Lam. Apiaceae Hydrolea zeylanica (L.) Vahl Hydrophyllaceae Hypericum japonicum Thunb. ex Murray Hypericaceae Ichnocarpus frutescens (L.) W. T. Aiton Apocynaceae Ipomoea aquatica Forssk. Convolvulaceae Ipomoea quamoclitL. Convolvulaceae Jatropha curcas L Euphorbiaceae, Jatropha gossypiifolia L. Euphorbiaceae, Jasminum sambac (L.) Aiton Oleaceae Justicia gendarussa Linn. Acanthaceae Kalanchoe pinnata (Lam.) Pers. Crassulaceae Kaempferia galanga L. Zingiberaceae Lagerstroemia parviflora Roxb. Lythraceae Lagerstroemia speciosa L. Pers. Lythraceae Lasia spinosa (L.) Thwaites Araceae Lawsonia inermis L. Lythraceae Leea macrophylla Roxb. ex Hornem. Leeaceae Leucas zeylanica (L.) W.T.Aiton. Lamiaceae Lippia javanica Burm F Spreng Verbenaceae Lippia geminate H.B. & K Verbenaceae Litchi chinensis Sonn. Sapindaceae Litsea cubeba L Lauraceae Litsea glutinosa (Lour.) C. B. Rob Lauraceae Litsea monopetala (Roxb.) Persoon Lauraceae Litsea salicifolia (Roxb. ex Nees) Hook.f. Lauraceae Lygodium japonium Thunb. Ex Murr. Lygodiaceae Lygodium microphyllum (Cav.) R.Br. Lygodiaceae Madhuca indicaJ.F.Gmel. Sapotaceae Maesa indica (Roxb.) A. DC. Myrsinaceae Macropanax undulatus (Wall ex D. Don) Seem Araliaceae Mallotus philippensis. (Lamk) Muell. Arg. Euphorbiaceae Mangifera andamanicaKing Anacardiaceae Mangifera indicaL. Anacardiaceae Mangifera sylvatica Roxburgh Anacardiaceae Manihot esculenta Crantz. Euphorbiaceae Marsilea minutaL. Marsileaceae Melastoma malabathricumL. Melastomataceae Melia azedarach. L. Meliaceae Meliosma simplicifolia (Roxb.) Walp. Sabiaceae Melocanna baccifera (Roxb.) Kurz. Poaceae Melodinus monogynus Roxb. Apocynaceae

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Meriandra strobilifera Benth Lamiaceae Merremia umbellata (L.) Hallier f. Convolvulaceae Mesua assamica (King & Prain) Kosterm. Calophyllaceae Mesua ferreaL. Calophyllaceae Meyna laxiflora Robyns Rubiaceae Meyna spinosa Roxb. ex Link Rubiaceae Mezoneuron cucullatum (Roxb.) Wight & Arn. Caesalpiniaceae Microcos paniculata L. Malvaceae Mimosops elengiL. Sapotaceae Mikania micrantha H. B. K. Asteraceae Mirabilis jalapaL. Nyctaginaceae Mollugo pentaphylla L. Molluginaceae Monochoria hastata (L.) Solms Pontederiaceae Monochoria vaginalis (Burm. f.) C. Presl ex Kunth Pontederiaceae Moringa oleifera Lam. Moringaceae Morus albaL. Moraceae Mucuna pruriens (L.) DC. Fabaceae Murraya koenigii (L.) Spreng Rutaceae Murraya paniculata (L.) Jack Rutaceae Mussaenda glabra Vahl Rubiaceae Nelumbo nucifera Gaertn. Nelumbonaceae Nerium indicum L. Apocynaceae Nicotiana Tabacum L Solanaceae Nicotiana rustica L. Solanaceae Nyctanthes arbor tristis Linn. Oleaceae Nymphaea nouchali Burm.f. Nymphaeaceae Nymphaea rubra Roxb. ex Andrews Nymphaeaceae Nymphoides cristata (Roxb.) Kuntze Menyanthaceae Ocimum basilicum L. Lamiaceae Ocimum sanctum L. Lamiaceae Oldenlandia corymbosa L. Rubiaceae (L.) Kurz, Bignoniaceae Osbeckia nepalensis J. D. Hooke Melastomataceae Ottelia alismoides. (L.) Pers Hydrocharitaceae Oxalis corniculataL. Oxalidaceae Oxalis debilis var. corymbosa (DC.) Lourteig Oxalidaceae Pandanus odoratissimus Linn. Pandanaceae Paederia foetidaL. Rubiaceae Parkia timoriana (A.D.C) Merrill Fabaceae. Passiflora foetidaL. Passifloraceae Pavetta subcapitata Hook.f. Rubiaceae Peperomia pellucida (L.) Kunth Piperaceae Piper longumL. Piperaceae Piper thomsonii (C. DC) Hooker, Fl. Piperaceae Pithecellobium monadelphum (Roxb.) Kosterm. Fabaceae Phlogacanthus curviflorus (Nees) Nees Acanthaceae Phlogacanthus thyrsiflorus Nees Acanthaceae Phlogacanthus tubiflorus Nees Acanthaceae Phyllanthus acidus. (L.) Skeels. Euphorbiaceae. Phyllanthus emblica L. Euphorbiaceae

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Phyllanthus fraternusG.L.Webster Euphorbiaceae Phyllanthuis urinariaL. Euphorbiaceae Physalis divaricata D. Don Solanaceae Plumbago indica. L. Plumbaginaceae Plumeria acuminata Ait. Apocynaceae Poikilospermum suaveolens (Blume) Merrill Moraceae Polygonum chinenseL. Polygonaceae Polygonum minus Huds. Polygonaceae Polygonum microcephalum D. Don Polygonaceae Polygonum orientale L. Polygonaceae Polygonum plebeium R.Br. Polygonaceae Pongamia pinnata (L.) Pierre Fabaceae Portulaca oleraceaL. Portulacaceae Pothos cathcartii Schott i Araceae Premna herbacea Roxb. Verbenaceae Premna latifolia Roxb Verbenaceae Prunella vulgaris L. Lamiaceae Prunus persica L. Rosaceae Pteris ensiformisBurm. f. Pteridaceae Psidium guajava L. Myrtaceae Pueraria phaseoloides (Roxb.) Benth. Fabaceae Rauvolfia serpentinaL. Benth. ex Kurz. Apocynaceae Rauvolfia tetraphylla L. Apocynaceae Rhynchotechum ellipticum (Wall. ex D. Dietr.) A. DC Gesneriaceae Ricinus communis L. Euphorbiaceae Rorippa indica (L.) Hiern Brassicaceae Rourea minor(Gaertn.) Alston. Connaraceae Rubus ellipticus f. denudatus Hook. Rosaceae Rumex maritimus L. Polygonaceae Rhus griffithii Hook. f. Anacardiaceae · Rhus chinensis Miller. (Rhus semialata Murr.) Anacardiaceae · Sabia lanceolataColebr Sabiaceae Saccharum spontaneumL. Poaceae Sambucus hookeri Rehder. Adoxaceae Saraca asoca (Roxb.), De. wild Caesalpiniaceae Sarcochlamys pulcherrima (Rpxb.) Goud. Urticaceae Semecarpus anacardium L.f. Anacardiaceae Scoparia dulcisL. Scrophulariaceae Schima wallichii (DC.) Korth. Theaceae. Sesbania grandiflora (L.) Pers. Fabaceae Setaria italica (L.) Beauv. Poaceae Sida acuta Burm. f. Malvaceae Sida cordifolia L. Malvaceae Sida rhombifolia L. Malvaceae Smilax aspericaulis Wallich ex A. DC. Smilacaceae Smilax ovalifolia Roxb. ex D.Don Smilacaceae Smilax sailenii Sarma et.al. Smilacaceae Smilax zeylanicaL. Smilacaceae Solanum anguivi Lam. Solanaceae Solanum feroxL. Solanaceae

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Solanum kurzii Brace ex Prain Solanaceae Solanum nigrum L. Solanaceae Solanum torvum Swartz Solanaceae Solanum spiraleRoxb. Solanaceae Solena amplexicaulis (Lamk) Gandhi Cucurbitaceae Spilanthes acmella Murr. Asteraceae Spilanthes paniculata Wall. Asteraceae Spondias pinnata Kurz Anacardiaceae Stellaria wallichiana Benth. ex Haines Caryophyllaceae Sterculia guttataRoxb. Sterculiaceae. Sterculia roxburghiiWall. Sterculiaceae. Sterculia villosa Roxb. Sterculiaceae Sterculia urensRoxburgh Sterculiaceae Streblus asper Lour Moraceae Symplocos racemosa Roxb. Symplocaceae Syzygium cumini (L.) Skeels Myrtaceae Syzygium formosum (Wall.) Masam. Myrtaceae Syzygium fruticosum DC. Myrtaceae Syzygium jambos (L.) Alston. Myrtaceae Syzygium cerasoides (Roxb.) Raizada. Myrtaceae Tamarindus indica L. Caesalpiniaceae Tagetes erectaLinn. Asteraceae Taxus wallichianaZucc. Taxaceae Tephrosia candida (Roxb.) DC. Fabaceae Tephrosia purpurea (L.) Pers. Fabaceae Terminalia arjuna (Roxb.) Wight & Arn. Combretaceae Terminalia bellirica (Gaertn.) Roxb. Combretaceae Terminalia chebula Retz. Combretaceae Terminalia citrina Roxb. ex Fleming Combretaceae Terminalia myriocarpaHeurck. And Muell. Combretaceae Tetrastigma lanceolarium (Roxb.) Planch. Vitaceae Tinospora cordifolia (Willd.) Miers Menispermaceae Thevetia peruviana (Pers.) K. Schum. Apocynaceae Thunbergia grandiflora (Roxb. ex Rottler) Roxb. Acanthaceae Trapa natans var. bispinosa (Roxb.) Makino Trapaceae Trevesia palmata (Roxburgh ex Lindley) Visiani Araliaceae Trichosanthes tricuspidata Lour. Cucurbitaceae Tylophora indica (Burm. f.) Merr. Apocynaceae Typhonium trilobatum (L.) Schott Araceae Uraria rufescens (DC.) Schindl. Fabaceae Uraria picta (Jacq.) DC. Fabaceae Vitex glabrata R. Br. Verbenaceae

Vitex negundo Linn. Verbenaceae Willoughbeia edulis Roxb. Apocynaceae Wrightia tomentosa Roem. & Schult Apocynaceae Xanthium strumarium L. Asteraceae Xanthium strumarium Linn Asteraceae Zanthoxylum armatumDC. Rutaceae Zanthoxylum budranga Wall. ex Hook. f. Rutaceae Zanthoxylum limonella(Dennst.)Alston Rutaceae

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Zanthoxylum nitidum (Roxb.) DC. Rutaceae Zanthoxylum oxphyllum Edgw Rutaceae

Zanthoxylum rhetsa(Roxb.) DC. Rutaceae montanum (J.Koenig) Link ex A.Dietrich Zingiberaceae

Zingiber officinale Roscoe Zingiberaceae Zingiber rubens Roxb. Zingiberaceae Zingiber zerumbet (L.) Roscoe ex Sm. Zingiberaceae Zingiber roseum (Roxb.) Roscoe Zingiberaceae Zingiber purpureum Roscoe Zingiberaceae Ziziphus jujuba (L.) Gaertn. Rhamnaceae

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Annexure 1b

Exploration of North-East natural resource silk for tissue repair and biomedical applications

The regions are physiographically categorized into the Eastern , Northeast hills (Patkai-Naga Hills and Lushai Hills) and the Brahmaputra and Barak Valley plains. At the confluence of the Indo-Malayan, Indo-Chinese and Indian biogeographical realms, the NE region is unique in providing a profusion of habitats, which features diverse biota (flora and fauna) with a high level of endemism. Among the natural resources available in North-East region, silk holds a very important place and sericulture represents a source of income generation for farmers in this area. There are different silk varieties such as muga silk, eri silk and mulberry silk widely available in North-East region being utilized in textile industry. In last two decades, utilization of silk fiber in non-textile applications such as tissue engineering and biomedical applications has been explored. The silk fibers are endowed with superlative material properties such as lustrous appearance, outstanding mechanical properties, tunable degradation profile, excellent biocompatibility, and large-scale production for various applications. Silkworm silk mainly comprised of two types of proteins, glue like hydrophilic, sericin and hydrophobic, fibroin protein. In this work, we intend to develop silk fibers based matrices, which are derived from North-East silk varieties for skin tissue engineering and wound healing. To attempt this, mulberry silk fibroin protein in combination with natural biopolymer, keratin was utilized and their potential for skin tissue engineering would be investigated. Silk fibroin and keratin was extracted following standard methods. The concentration of silk and keratin for fabrication of hydrogel was 2% W/V. The developed blended hydrogels were analyzed for physico-chemical, mechanical, thermal and degradation properties. The fabricated silk fibroin/keratin blended hydrogels showed porous structure with good interconnectivity and improved mechanical and degradation properties. Further studies would evaluate the cytocompatibility and in vivo evaluation of these matrices for wound healing applications. In conclusion, this silk based hydrogels would provide an economically viable skin substitute for wound healing. The outcome of this work open more avenues towards escalation of sericulture industry and in turn livelihood generation in farmers belongs to North-East region of India.

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Figure 1: Schematic representation showing extraction of silk from silk fibers.

Figure 2: Fabrication of hydrogels using silk fibroin from silk fibers and keratin.

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SF SF/KR (2:1) µm SF SF/KR (2:1) SF/KR (1:1) 161.63 28.72 SF/KR (1:2) 183.29 27.34

SF/KR (1:1) SF/KR (1:2)

Figure 3: Surface morphology of silk fibroin/keratin freeze gelled hydrogels pre- freezed at temperature -20 o C.

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Annexure 2

Till date we have collected the selected plants noted below, that have been used by local inhabitants in treatment of multiple ailments in North East India.

Details of collected plant materials

Table 1: List of plants collected for the NMHS project work

Code Sample picture Collection Area Parts

Auxiguri Barks and village, Baksa NIP/18/01 District

Guwahati, Lankeswar area, NIP/18/04 Whole plant Gauhati university

Leaves, stem, NIP/18/08 * National park

Bark, leaves and NIP/18/10 Lakhimpur district few seed

NIP/18/11 Guwahati

NIP/18/12 Plant Upper Assam

NIP/18/13 Plant Upper Assam

NIP/18/14 Plant Upper Assam

Fruit, leaves and NIP/18/22 Pathsala, Assam bark

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NIP/18/24* Leaves Borpeta, Assam

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Annexure 3a

MEMORANDUM OF UNDERSTANDING (MOU)

This non-binding, non-exclusive Memorandum of Understanding (“MoU”) is effective this…..day of….,2018, is entered into by …………… National Institute of Pharmaceutical Education and Research (NIPER)…………………………………………………….., India and ………Mr.……………………………………………………………………..NIPER, (“First Party”), which expression shall, unless repugnant to the context or meaning thereof, be deemed to include its successors and permitted assigns of the first part; AND: [Traditional healer] (“Second Party”), which expression shall unless repugnant to the context or meaning thereof, be deemed to include its successors and permitted assigns of the second part. WHEREAS First Party and Second Party (herein after individually referred to as “Party” and together as “Parties”), are desirous of working together with respect to carrying out research and development of traditional medicines acquired from the biological resource farm of the second party, who will also provide the knowhow of the concoction of the medicine that he prepares locally to assist in the development of the drug with value addition. To have a successful R & D and assistance from the second party (the Traditional Healer), the terms and conditions as set-forth below.

1.1 WHEREAS National Institute of Pharmaceutical Education and Research (NIPER) is the first national level institute in pharmaceutical sciences with an objective of becoming a centre of excellence for advanced studies and research in pharmaceutical sciences. NIPER will acquire raw materials from the Second party in an amount as agreed by both parties for Research & Development with value addition for new traditional drug. Data with regard to proprietary information, scientific, technical, clinical, experimental technology, results of clinical trials, indications, documents developments in Research & Development generated in Research &Development shall be confidential until the final product comes out. 1.2 Whereas Mr…… a traditional healer has agreed that he and his nominee will sell the raw materials from his biological resource farm in an amount in rupees as agreed by both the parties till the R&D work is going on and also share his traditional know how to prepare the medicine to facilitate the Research & Development. 1. DEFINITIONS In this MoU, except where the context or subject matter is inconsistent therewith, the following terms shall have the following meanings: 1.1“Traditional Healers” means a person in a society who uses long-established methods passed down from one healer to another to treat a person suffering from various illnesses, many of which have psychological underpinnings. Methods used by traditional healers include the use of roots, herbs and medicinal plants etc. 1.2“Traditional Knowledge” means knowledge, know how, skills and practices that are developed, sustained and passed on from generation to generation within a community, often forming part of its cultural or spiritual identity. 1.3 The biodiversity Act 2002 provides for the following exemptions-: a. Exemption to local people and communities of the area for free access to use biological resources within India b. Exemptions to growers and cultivators of biodiversity and to Vaids and Hakims to use biological resources.

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1.4 "biological resources" means plants, animals and micro-organisms or parts thereof, their genetic material and by-products (excluding value added products) with actual or potential use or value, but does not include human genetic material; 1.5 DATA” “Exclusive Data shall Include all confidential or proprietary information relating to scientific, technical, clinical, experimental technology, technical know-how, results of clinical trials, indications, documents, developments in Research & Development (R & D), other information in electronic or written form relating to product. 1.6 PATENT RIGHTS: “Patent Rights” means all rights, title and interests including the right to make, use, apply, store, import and manufacture, offer for sale, the products using the granted Indian patent of a product or a process pursuant thereto and all divisions, continuations, reissues substitutes and extensions thereof.

PURPOSE OF MOU 2.1 The Parties agree to work together in an endeavor to help enhancing the use of traditional knowledge and traditional medicines with value addition. 2.2. The second party will provide raw materials from his biologica resource farm for research & development to the first party. 2.3 The purpose of this MoU is to help summarize the principle terms of a mutual understanding between First and Second Party for the purpose of non-binding contractual basis, under which First Party shall buy raw materials (medicinal herb from biological resources) for R&D as & when required on a monthly basis, or otherwise from the second party with a cost as decided by both the parties amicably. The access to biological resources and associated knowledge will secure equitable sharing of benefits once Research &Development is completed. This will facilitate to start a business or accelerate their entrepreneurial endeavors in future. 2.4 Once the invention is developed, the first Party will initiate the filing of patent application as joint applicants with the second party and all rights title interest and benefit under patent rights will be shared equally. In case if the invention is licensed out later the consideration for the up-front monies and royalty etc. to be paid will be decided by both the parties mutually.

3. MUTUAL UNDERSTANDING

It has been mutually agreed by both the parties this MoU witnessed that in consideration of the premises and other good and valuable consideration, the Parties hereto agree as follows: IN WITNESS WHEREOF the parties hereto have hereunto set and subscribed their hands the day and year first hereinabove written

SIGNED AND DELIVERED by the within named

Dr. USN Murthy……..

In the presence of……………………………….

SIGNED AND DELIVERED by the within named

Mr…………

In the presence of……………………………….

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Annexure 3b

Table 10: A list of traditional drugs and formulations in their encoded form, which were collected from the traditional healers of North Eastern India by National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati. As per the claims, the traditional healers have treated the noted disorders mentioned in the list. The drugs have been collected and a series of laboratory testing regarding their safety/ toxicity and therapeutic activity will be conducted for the evaluation of the noted claims.

Traditional Healers Medicine Disease area (TMH) codes TMH/01 Cancer, Epilepsy, Diabetes, fatty liver

TMH/02

TMH/03 High Blood Pressure, Malaria

TMH/04 Jaundice

TMH/05 Diabetes

TMH/06 Diabetes, jaundice

TMH/07 Liver cirrhosis

TMH/08 Cancer, jaundice

TMH/11 Diabetes, malaria, high BP

TMH/13 Cancer, epilepsy, hypertension

TMH/17 Cancer, Hepato-protective

TMH/20 Cancer

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Annexure 4

Number of State traditional healers Details of Traditional healers per state Sri Amiya Choudhury, S/o Lt. DulalChoudhary, 61 yrs, Male Sri BitarayReang, S/o Sri KharendraReang, 31 yrs, Male Sri SurendraReang, S/o Lt. DodoniReang, 63 yrs, Male Sri Ratneshwar Chakma, S/o Lt. Singhanath Chakma, 45 Tripura 16 yrs, Male Sri Chandra ManikChakm, S/o Jubaraj Chakma, 70 yrs, Male

Sri KabendraReang, S/o Lt AmbikaReang, 41 yrs, Male

Sri Binoy Kumar , S/o Lt. Chaklaihareang, 52 yrs,

Male

Sri Gopal Chakma, S/o Lt Sukramoni Chakma, 66 yrs, Male Sri HarendraReang, S/o Lt. Khula Chandra Reang, 57 yrs, Male Sri Bikash Chandra Reang, S/o Lt. Suranjoy Reang,29 yrs, Male Rajesh Roy. Bishalgarh, Ph- 9862886286, 40 yrs, Male MithunDebnath. Sonamura, Ph-9436931768, 38 yrs, Male

GouraSingha. Dhalai, Ph- 94364477223, 52 yrs, Male SankarMajumder. Rastarmatha, Ph-9612123255, 54 yrs, Male Sanamani Chakma. South Tripura, Ph-8415939079, 55 yrs, Male Prabhat Chakma, South Tripura.Ph-8415939079, 55 yrs. Male Assam 40 Shri Jibeshwar Borah, President, 9854484840 Shri Debakantakoch, Secretary, 9954390900 Shri AjitBorbaruah, 9859355787 Shri MularamBorgohain, 9678283922

Shri LambodarBorpatroGohain, 9957885789

Shri JogenNeog, 7637932284 Shri Chandra Saikia, 7635894616 Shri KamleshwarSonwal, 7086315148

Shri Thagiram Borah, 9476501169

Shri Thitheswar Borah, 912379363 Shri Duleshwar Borah, 9435535512 Shri Atul Borah, 7086961963

Shri Hiranya Dev Goswami, 9957408755

Shri Kamakhya Chaudhary, 9435209004 Shri Amulyachaudhary, ---- Shri Anil Das, 8011396951

Shri JagdeepKakoti, 8011327106

Shri Atul Das, 8761956410 Shri GautamManikiyal, 7896427716 Shri Chao LikhamPumung, 81199844228

Shri Umesh Hazarika, 9508067838

Shri Narayan Dutta, 9101660701 Shri Bobhananda Dev Goswami, 8761855791 Shri Nirmal Hazarika, 7086902620

Shri Dibyajyoti Das, 8403997550

Shri AmbeshwarChetiya, 9678147380 Shri BhodiyaGogoi, 7035454336 Shri Atul Koch, 9957363005

Shri Ram Khanikar, 9954385946

Shri DibyaGogoi, ------Shri PunaramGogoi, 8720994946 NMHS Fellowship Grant Progress Page 23 of 122

Shri RajaniGohain, 9957112579 Shri JaykantaDoley, 9954378795

Shri MuhiswarDoley, 8876420015 Shri Deep SaikiaRajbor, ------Shri Santosh Rajbor, 9678506431 Shri SwapanSaroniya, 8724927652 Smti Shri LakhiDoley, 7578021906 SmtiPratibha Koch, 9869996047 SmtiJyotshna Borah,------Shri SingheswarShyam, Vill. Tipomia, P.O. Tipomia, Titabor, Jorhat Phone: 08486726324 Mr. SatyajitWakhet () Vill : VitarPawaigaon Assam 2 P.O. Margherita, Makumtila, Tinsukia

09435690269

Krishna Kumar Mesh, P.O. Margherita, Makumtila, Tinsukia

Mr. DwinBapung (Member JIMA) Meghalaya 6 Mr. Than Rasmut (Member JIMA)

Mrs. PanbiangKyndiah (Member JIMA)

Dr. V. Marak, Ph no: 7005753868

Mr. Meril M. Sangma, Phone no: 9862572871 (doubtful) Dr Carehome, Meghalaya Sikkim 5 Men Raj Dubba Vijay Bagdas Yamuna Prasad Bastola Krishna Prasad Pyakurel

Tika Prasad Sharma Mizoram 3 Dr. Zorin PuiiKhiangte Mr.Lalrikhuma David L. SiamtharaPakhuangte Meghalaya 2 Dr. RoiwandakaLaloo PanbiangKyndiah

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Annexure 5

We have organized Traditional Healers Meet on 26th August 2018 at NIPER Guwahati campus. An approximate number of 72 Traditional Healers participated from the states of , Assam, Meghalaya, Mizoram, Sikkim and Tripura. They actively participated in the display of their products openly without hiding any useful information which is the quite highlight of the workshop.

Figure: 1 Panel of scientific experts addressing the traditional healers from different states of North East India in “Traditional healers meets 2018” at NIPER Guwahati, Assam. From the left: Dr. USN Murthy (Director, NIPER Guwahati), Dr. S. Tamilvanan (Faculty, NIPER Guwahati), Dr. R.K. Sarma (Vice Chancellor, USTM), Dr. Pavan Kaushik (Regional Director CFLE), Dr. GN Qazi (Director General HIMSAR), Dr. PG Rao (Former Director CSIR NEIST Jorhat), Srinivas, Advisor (NIPER Guwahati).

Figure: 2 Panel of discussion with traditional healers from different states of North East India

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Figure: 3 A panel of experts having discussion with traditional healers from different states of North East India.

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Annexure 6

ABSTRACT:

Cancer is the second leading cause of mortality in human diseases worldwide. However, the available pharmacological options couldn’t increase the patient’s quality of life in most of the case. Therefore, there is a need to develop more effective therapeutic strategies to combat cancer, cancer induced bone loss and pain. In this context, it is noteworthy that some plants and plant based products seem to be effective against cancer, cancer-induced bone loss and neuropathy pain. In this study medicinal plant were studied to evaluate the anticancer, antioxidant and anti-inflammatory activity. Chloroform extract of NIP/18/13 and NIP/18/14 and Ethyl acetate extract of NIP/18/14 were shown to have anticancer activity when evaluated in MDA-MB-231 and FaDu cells. Medicinal plants were also proven to mask the cardiotoxicity induced by doxorubicin. Extracts of medicinal plants were also found to have antioxidant activity. Furthermore studies need to be conducted to evaluate their effects in other diseases also.

1. INTRODUCTION: Inflammatory diseases: Inflammatory diseases include a vast array of disorders and conditions that are characterized by inflammation. Examples include allergy, asthma, autoimmune diseases, celiac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, pre perfusion injury and transplant rejection. The inflammatory system is designed to recognize self from non-self and destroy nonself to protect the body. In diseases of the inflammatory system, the self-recognition can break down leading to conditions where the body destroys healthy tissue in a misguided attempt at protection. This can lead to rheumatoid arthritis, allergies, pain, COPD, asthma, , gout, graft rejection, and problems with chemotherapy. Non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and salicylates, leukotriene antagonists, and histamine receptor antagonists are used to treat inflammatory disorders.

Causative factors for inflammatory diseases:

I. Non-degradable pathogens that cause persistent inflammation. II. Viral infections. III. Immune system deregulation.

Oxidative stress and Inflammation and Interlink

Extensive research during the past 2 decades has revealed the mechanism by which continued oxidative stress can lead to chronic inflammation, which in turn could mediate most chronic diseases including cancer, diabetes, and cardiovascular, neurological, and pulmonary diseases. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes, including those for growth factors, inflammatory cytokines, chemokines, cell cycle regulatory molecules, and anti-inflammatory molecules.

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2.1 Epidemiology of inflammatory diseases:

2.1.1. Cancer

According to the National Cancer Registry Programme of the India Council of Medical Research (ICMR), more than 1300 Indians die every day due to cancer. Between 2012 and 2014, the mortality rate due to cancer increased by approximately 6%. In 2012, there are 478,180 deaths out of 2,934,314 cases reported. In 2013 there are 465,169 deaths out of 3,016,628 cases. In 2014, 491,598 people died in 2014 out of 2,820,179 cases. According to the Population Cancer Registry of Indian Council of Medical Research, the incidence and mortality of cancer is highest in the north-eastern region of the country. Breast cancer is the most common, and stomach cancer is the leading cause of death by cancer for the population as a whole. Breast cancer and lung cancer kill the most women and men respectively.

2.1.2. Diabetes

Until recently, India had more diabetics than any other country in the world, according to the International Diabetes Foundation, although the country has now been surpassed in the top spot by . Diabetes currently affects more than 62 million Indians, which is more than 7.1% of the adult population. The average age on onset is 42.5 years. Nearly 1 million Indians die due to diabetes every year.

According to the Indian Heart Association, India is projected to be home to 109 million individuals with diabetes by 2035. A study by the American Diabetes Association reports that India will see the greatest increase in people diagnosed with diabetes by 2030. The high incidence is attributed to a combination of genetic susceptibility plus adoption of a high-calorie, low-activity lifestyle by India's growing middle class.

2.2 Importance of indigenous medicinal plants:

The North-eastern part of India lies between lies between 21°34 ′N to 29°50 ′N latitude and 87°32 ′E to 97°52 ′E longitudes with an area of 262,230 km2 (101,250 sq mi) (1). It covers the states of Assam, Arunachal Pradesh, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura. Sikkim and Arunachal Pradesh falls under the Himalayan range whereas Manipur and Nagaland covers Naga hill. Meghalaya is occupied by Garo, Jayantiya and while Mizoram comes under Lusai hills (2).

The North-east India has the richest tank of plant diversity in India and is one of the „biodiversity hotspots‟ of the world (3). Almost all types of grassland, meadows, marshes, swamps, scrub forests, mixed deciduous forests, humid evergreen forests, temperate and alpine vegetation are found here. It is also considered as cradle of „angiosperms‟ as primitive plant families such as Magnoliaceae, Lauraceae, Hamamelidaceae, Degeneriaceae, Tetracentraceae and Lardizabalaceae are well found here (4).

Assam is one of the seven sister States of North Eastern India covering a total land area of 78,438 sq. kilometres with a population of about 31,205,576(5). The major ethnic groups are Ahom, Bodo, Borahi, Chutia, Deori, Dimasa, Garo, Kachari, Koch, Khamti, Karbi, Lalung, Mising, Moran, Mutuk, Naga, Rabha, Tai-Fake, Tiwa . Most of tribes still relay on their own traditional herbal remedies for common ailments as they are considered it to be effective, cheap, easily available NMHS Fellowship Grant Progress Page 28 of 122

and devoid of toxic side effects. Because of their proximity to nature and plants, they possess intimate knowledge about plant value and utilities developed through age-old trial and error method. The different ethnic communities considered it as hidden wealth of information about plants used for medicinal purposes (6).

2.3 Pharmacological Intervention:

2.3.1. NIP/18/12:

It belongs to Rutaceace family and is an important medicinal plant which is commonly known as Indian Prickly Ash, Pepper or Toothache tree. Local names of this plant are: Tejphal (), Tejowati (), Mukthrubi (Manipuri) and Timur (Nepal). It is widely distributed in India, from Kashmir to at altitudes up to 2,500 m, also occurs throughout North East India. It is also found throughout most of China, Taiwan, Nepal, Philippines, , Pakistan and Japan at altitudes of 1,300-1,500 m. Valleys and thickets in the mountains, wasteland and the under-storey of mixed forest are customary locations of the species (7).

Traditional uses

The bark, and seeds are extensively used in indigenous system of medicine as a carminative, stomachic and anti-helmintic. The fruit and seeds are employed as an aromatic tonic in fever and dyspepsia. An extract of the fruits is reported to be effective in expelling round worms. Because of their deodorant, disinfectant and antiseptic properties, the fruits are used in dental troubles, and their lotion for scabies.

Scientific works

Bark extract has shown anti-diabetic activity on STZ induced diabetes (8). Anti-oxidant and anti- inflammatory activities of ethanolic extract (9). Essential oils have anti nociceptive and anti- spasmodic activities (10). It has hepatoprotective activity in both CCl4 induced and paracetamol induced models. It enhances the therapeutic potential of cisplatin.

2.3.2. NIP/18/13:

It is an annual herb up to 50 cm tall, with strong lemony fragrance. Stems are erect, sometimes purple-red, densely covered with white velvet-hairy, much branched at base. -stalks are 2-5 mm, densely white velvet-hairy. Leaves are ovate to oblong, above white hairy, below velvethairy, yellowish glandular, margin sawtoothed . Tiny lilac or white are borne in spikes at branch- ends, which are cylindric, compact, 1-4.5 cm long, 0.8-1 cm wide. Axis is densely white hairy; bracts linear, up to 3.5 mm, densely white hairy. Flower-stalks are about 1 mm, densely white hairy. Calyx is tubular, up to 4 mm in fruit, tip recurved, densely gray woolly-hairy outside; teeth nearly equal, slightly closed in fruit. Flowers are funnel-shaped, about 3 mm, hairy, glandular outside, obscurely hairy annulate inside; upper lip oblong, notched, fringed with hairs; lateral lobes of lower lip less than half as wide as middle lobe. Stamens are 4, protruding out, hairless. Nutlets are 4, oblong, about 0.7 mm, sparsely brown hairy. It is used in seasoning food in NE India. It is found in disturbed forest or mountain valleys, at 400-600 m altitude, in NE India. It is also widely cultivated in NE India. Flowering: October-December.

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Medicinal uses

It is used as tonic, astringent, carminative and antiseptic. Decoction of leaves and flowers is given in tonsilitis, fever, cough, high blood pressure, nose bleeding, menstraual disorder, treatment of body itching.

2.3.3. NIP/18/14:

It is a semi-wild species of medicinal indigenous plant belonging to the Clusiaceae family. The fruit is commonly known as “Taikor” in and “Amlavetasa” in India. The mature fruit is greenish yellow and is consumed as a vegetable; Traditionally, the fruit has been using by the people of Assam as medicine to treat different types of stomach related diseases it is also used as an ingredient in cooking different types of meat and chicken. It is a rich source of secondary metabolites including xanthones, flavonoids, benzophenones, lactones, and phenolic acids with wide range of biological and pharmacological activities his fruit extract is reported to possess a variety of pharmacological benefits including antioxidant(11, 12) antimicrobial (13), antiinflammatory (14), hepatoprotective(15), and cardioprotective properties (16), antiplatlet and antioxidant (17).

Considering the preliminary data on anti-inflammatory activities the present study is been undertaken.

RESEARCH QUESTION/S:

 Which part of NIP/18/12, NIP/18/13 and NIP/18/14 crude extracts is biologically active?  Which solvent fraction shows maximum efficacy in various in-vitro screening models?  Which extract shows maximum efficacy against oxidative stress and cancer complications? 3. OBJECTIVE/S OF THE STUDY: 1. Pharmacological evaluation of crude extracts of NIP/18/12, NIP/18/13 and NIP/18/14 in the in vitro cancer cell lines. 2. Evaluation of anti-oxidant and cardio protective effect of extracts with potent antioxidant activity in Doxorubicin induced cardiotoxicity in Balb/c mice. 3. Preclinical toxicity studies and dosage evaluation for the potent medicinal plants.

4. STUDY AREA: Cancer is the second leading cause of mortality in human diseases worldwide. According to a national statistical report on the incidence and mortality in the USA, there were a total of

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1,529,560 new cancer cases and 569,490 deaths from cancer occurring in 2010 [18]. Although modern cancer therapies have been significantly increased patient survival rate in human medicine, the cancer patients are now facing new challenges resulting from severe chronic tumor induced bone loss and pain, affecting the social and behavioural aspects of humans. However, the available pharmacological options couldn’t increase the patient’s quality of life in most of the case [19, 20]. Therefore, there is a need to develop more effective therapeutic strategies to combat cancer, cancer induced bone loss and pain. India harbors a variety of flora and most of them have been noted to have ethno pharmaceutical importance, it is noteworthy that some plants and plant based products seem to be effective against cancer, cancer-induced bone loss and neuropathy pain.

5. METHODOLOGY ADOPTED: a. Objective 1: Pharmacological evaluation of crude extracts of NIP/18/12, NIP/18/13 and NIP/18/14 in the in vitro cancer cell lines. Extracts of different medicinal plants were tested in various cancer cell lines for their anticancer activity. Further evaluation of these plants in other human cancer

cells is in progress.

6.1.1. Cell viability assay by MTT method

Principle:

Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan crystals which are insoluble in aqueous solutions. The crystals are solubilized in Dimethyl sulfoxide (DMSO). The resulting purple solution is spectrophotometrically measured. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.

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Figure 1: Reduction of MTT to Colored Formazan Derivative (by viable cells)

Protocol:

 MTT assay was performed on the panel of human cancer cell lines such as MDA-MB-231, FaDu and HT-29 for evaluating the cytotoxicity of test compound.  Sub-confluent monolayer culture was trypsinised and collect cells in growth medium containing serum. Centrifuge the suspension (3000rpm, 5min) to pellet cells, resuspend in growth medium, and count cells.  Cells were plated at a density of 1×104 cells/well in a 96 well flat-bottomed microtiter tissue culture plates.  96 well plate was incubated overnight or for 24 hrs. After 24h test compound is added at different concentrations.

 Plates were incubated at 37˚C in CO2 incubator with 5% CO2 & 80% relative humidity for 48 hrs, and then the medium was replaced with fresh medium without FBS.  10μL of MTT solution (5 mg/ml in media or in PBS) is added to each well and reincubated for 4hrs at 37oC.  The supernatant culture medium was carefully aspirated and 200μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorbance was measured at 570 nm. Respective extracts of curcumin was used as standard anti-cancer activity drug.

6.2 Objective 2: Evaluation of anti-oxidant and cardio protective effect of extracts with potent antioxidant activity in Doxorubicin induced cardiotoxicity in Balb/c mice.

Extracts of medicinal plants were evaluated for free radicle scavenging and DPPH scavenging action to determine antioxidant activity. Further potent extract was assessed in Doxorubicin induced cardiotoxicity model.

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6.2.1. ABTS radical cation (ABTS.+) scavenging assay: ABTS radical cation scavenging activity of BO extracts was determined according to the method given by (Kraus, Pasieczny, Lariosa-Willingham, Turner, Jiang & Trauger, 2005) with some modification. ABTS radical cation was produced by reacting 7 mM ABTS with 2.4 mM potassium persulfate and kept in dark at room temperature for 12 -15 h. The ABTS.+ solution was diluted with phosphate buffer pH 7.4 to obtain the absorbance of 0.7 ± 0.2 units at 734 nm. Then 200 µl of diluted ABTS.+ solution was added to 20 µl of extract solutions at different concentrations (1.9- 1000 µg/ml). The ability of the extracts to neutralize ABTS.+ was measured after 20 min incubation in dark at room temperature by taking absorbance at 734 nm. Ascorbic acid, curcumin, BHT and trolox were employed as the standard. Results were expressed in terms of percentage free radical scavenging activity. % scavenging of ABTS = (A control - A extracts/A control) x 100

6.2.2. DPPH scavenging assay: The BO extracts were tested for their ability to scavenge free radicals by DPPH radical scavenging assay according to the procedure described by(Ak & Gulcin, 2008), with slight modification. Briefly, 20µl of the extract at each concentration (1.95 – 1000 µg/ml) was added to 200 µl of 0.2 mM DPPH in methanol and incubated in dark at room temperature for 20 min. Absorbance was read at 517 nm using a Spectra Max multi-mode detection reader (Molecular devices, CA, USA ). Ascorbic acid, curcumin, BHT and trolox were employed as the reference. Analyses were run in triplicates and percentage DPPH Radical Scavenging Activity (% DRSA) was calculated using the formula % scavenging of DPPH = (A control - A extracts/A control) x 100

6.2.3. In vivo-Cardioprotection study of NIP/18/12 HA extract on Doxorubicin induced cardiotoxicity:

Female Balb/c mice of 25-30gm of were used to test the cardio-protective activity of NIP/18/12 HA extract. All protocols were performed in accordance with guidelines of CPCSEA, India. The study was approved by the Institutional Animal Ethics Committee (IAEC), Guwahati, India. The animals were housed in IVC cages under standard conditions (temperature 23±1, light/dark cycle 12hrs). Acute oral toxicity study was performed for the maximum dose of 2000mg/kg for 7 days as per OECD guidelines. And choose 1/10th of the maximum as a dose of 100 mg/kg and 200 mg/kg.

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6.2.4. Study design:

Mice were divided into four groups. Group 1: normal control group 2: disease control: received single dose intraperitoneal injection of Doxo 15mg/kg on the 7th day of the study. Group 3: Given NIP/18/12 HA 100 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day. Group 4: Given NIP/18/12 HA 200 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day.

6.2.5. Electrocardiographic Recording and Analysis:

The ECG was performed on mice from 4 groups (n = 4 per group) to evaluate DOX cardiotoxicity at day 14th. Briefly, mice were anesthetized with 4% isoflurane and maintained anesthesia with 1-2% isoflurane followed by insertion of electrodes through a needle in the right hind limb, right front limb, left front limb. The data were collected by AD Instrument (Australia). T wave elevation and RR interval was analyzed using labchart 8 software (AD instrument). The animals underwent for imaging where animals were anesthetized with 4% isoflurane in an anesthesia chamber and placed on a heating pad and anesthesia was maintained using 1.5% isoflurane by a nasal mask throughput the experiment. Echocardiography was performed using a high-frequency (30MHz) small animal imaging system (Vevo 3100, FUJIFILM VisualSonics). Parasternal long-axis images and M-mode B-mode and Power Doppler (pw) images were obtained and analyzed for measuring left ventricle (LV) ejection fraction , cardiac output, fractional shortening (FS), systolic (LVIDs) and diastolic internal diameters (LVIDd).

6.2.6. Estimation of catalase (CAT) activity:

Catalase activity in tissue was determined by measuring the rate of decomposition of hydrogen peroxide at 240 nm. To 15 μl of sample, 2 mL of phosphate buffer (50 mM, pH 7) containing hydrogen peroxide was added (Aebi, 1984). The catalase activity was expressed as U/mg protein.

6.2.7. Estimation of Lactate dehydrogenase (LDH) activity:

The required amount of working solution(R1 and R2) was prewarmed at 37ºC , then serum sample of volume 0.02ml was mixed with 1ml working solution(0.8ml R1+0.2ml R2), mixed thoroughly and transferred the mixture to the thermostated cuvette and started the stop watch simultaneously. Recorded the first reading at 60th second and subsequently three more readings with 30 seconds interval at 340nm. The activity was expressed in mU/ml.

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The required amount of working solution (R1 and R2) was prewarmed at 37 ºC, then serum sample of volume 0.05ml was mixed with 1ml working solution(0.8ml R1+0.2ml R2), mixed and aspirated. After the initial delay of 300 seconds, recorded the absorbance of the test at an interval of 30 seconds for the next 90 seconds at 340nm. The mean change in absorbance is determined and calculated the test results. The activity was expressed in mU/ml.

6.2.9 Statistical analysis:

All the data was expressed as mean±SEM, n=3. Statistical analyses were done using one way ANOVA (analysis of variance) then evaluated with Tukey‟s test. Level of significance were taken as *P< 0.05, **P< 0.01 and ***P< 0.001.

7. RESULTS:

7.1 Objective 1: Pharmacological evaluation of crude extracts of NIP/18/12, NIP/18/13 and NIP/18/14 in the in vitro cancer cell lines.

MTT Results on Different Cell Lines:

MDA-MB-231 is a triple negative breast cancer cell line and Chloroform extract of NIP/18/13 (IC50 116.124) and NIP/18/14 (IC50 78.37) and Ethyl acetate extract of NIP/18/14 (IC50 90.44) had shown to have anti-cancer activity with respect to standard curcumin.

MTT Assay: Inhibitory concentrations of various extracts on MDA-MB-231 cell lines 1000 STD CURCUMIN *** NIP/18/12 C 800 *** NIP/18/13 C 600 NIP/18/14 C

(µg/ml) NIP/18/14 EA

50 400 NIP/18/14 EOH

IC 200

0

Extracts Fig 1: IC50 values from MTT Assay of different extracts in comparision with standard curcumin, when treatment given to MDA- MB-231 cell lines. All the data is performed in triplicate values with mean±SEM, n=3. ***P< NMHS Fellowship Grant Progress Page 35 of 122

0.001 NIP/18/12 C, NIP/18/14 ETOH Vs STD CURCUMIN. No significant difference was observed with remaining extracts.

FaDu is a oral cancer cell line and Chloroform extract of NIP/18/13 (IC50 66.99) and NIP/18/14 (IC50 48) and Ethyl acetate extract of NIP/18/14 (IC50 47) had shown to have anti-cancer activity with respect to standard curcumin.

MTT Assay: Inhibitory concentrations of various extracts on FaDu cell lines 400 *** STD CURCUMIN NIP/18/12 C 300 *** NIP/18/13 C NIP/18/14 C 200

(µg/ml) NIP/18/14 EA 50 NIP/18/14 ETOH IC 100

0

Extracts

Fig 2: IC50 values from MTT Assay of different extracts in comparision with standard curcumin, when treatment given to FADU cell lines. All the data is performed in triplicate values with mean±SEM, n=3. ***P< 0.001 NIP/18/12 C, NIP/18/14 ETOH Vs STD CURCUMIN. No significant difference was observed with remaining extracts.

MTT Assay: Inhibitory concentrations of various extracts on HT-29 cell lines 150 STD CURCUMIN *** NIP/18/13 C 100 NIP/18/14 C *** NIP/18/14 EA **

(µg/ml)

50 50

IC

0

Extracts Fig

3: IC50 values from MTT Assay of different extracts in comparision with standard curcumin, when treatment given to HT-29 cell lines. All the data is performed in triplicate values with mean±SEM, n=3. ***P< 0.001 NIP/18/12 C, NIP/18/14 ETOH Vs STD CURCUMIN. **P< 0.01 NIP/18/14 C Vs STD CURCUMIN.

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a. Objective 2: Evaluation of anti-oxidant and cardio protective effect of extracts with potent antioxidant activity in Doxorubicin induced cardiotoxicity in Balb/c mice.

Extracts of diverse medicinal plants were assessed for their free radicle scavenging activity and DPPH scavenging activity, further the potent extract was assessed in Doxorubicin induced cardiotoxicity model.

7.2.1. Evaluation of Antioxidant Activity by ABTS Assay

The ABTS assay measures the relative ability of antioxidants to scavenge the ABTS generated in aqueous phase, as compared with Ascorbic acid (water soluble vitamin C analogue) standard. The ABTS is generated by reacting with a strong oxidizing agent (eg, potassium permanganate or potassium persulfate) with the ABTS salt.

NIP/18/12 ETOH, NIP/18/12 HA, NIP/18/12 AQ, all extracts of NIP/18/13 and NIP/18/14 ETOH, NIP/18/14 HA, NIP/18/14 AQ were found have good free radicle scavenging activity.

ABTS Assay: Inhibitory concentrations of various extracts of NIP/18/12 500 Ascorbic acid **a 400 NIP/18/12 C NIP/18/12 EA 300 NIP/18/12 ETOH NIP/18/12 HA 50 (ug/ml) 50 200

IC NIP/18/12 AQ

100

0

Extracts

Fig 4: IC50 values from ABTS Assay of NIP/18/12 extracts compared to Ascorbic acid. All the data is performed in triplicate values with mean±SEM, n=3. **P< 0.01 EA vs Ascorbic acid. No significant difference is observed with remaining extracts.

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ABTS Assay: Inhibitory concentrations of various extracts of NIP/18/13 300 Ascorbic acid NIP/18/13 C 200 NIP/18/13 EA NIP/18/13 ETOH NIP/18/13 HA

50 (ug/ml) 50

IC 100 NIP/18/13 AQ

0

Extracts

Fig 5: IC50 values of NIP/18/13 extracts compared with standard Ascorbic acid .All the data is performed in triplicate values with mean±SEM, n=3. No significant difference was observed.

ABTS Assay: Inhibitory concentrations of various extracts of NIP/18/14 500 ***a ***a Ascorbic acid 400 NIP/18/14 C NIP/18/14 EA 300 NIP/18/14 ETOH NIP/18/14 HA 50 (ug/ml) 50 200

IC NIP/18/14 AQ

100

0

Extracts

Fig 6: IC50 values from ABTS Assay of NIP/18/14 extracts compared to Ascorbic acid. All the data is performed in triplicate values with mean±SEM, n=3. ***P< 0.001 C, EA, ET, AQ vs Ascorbic acid. No significant difference was observed with HA extract to ascorbic acid.

7.2.2. Evaluation of Antioxidant Activity by DPPH Assay

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical method is an antioxidant assay based on electron-transfer that produces a violet solution in ethanol. This free radical, stable at room temperature, is reduced in the presence of an antioxidant molecule, giving rise to colorless ethanol solution. NIP/18/12 C, NIP/18/12 EA, NIP/18/12 ETOH and NIP/18/13 EA were found have good DPPH scavenging activity

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DPPH Assay: Inhibitory concentrations of various extracts NIP/18/12 500 ***a Ascorbic acid 400 NIP/18/12 C NIP/18/12 EA 300 *a NIP/18/12 ETOH NIP/18/12 HA 50 (ug/ml) 50 200

IC NIP/18/12 AQ

100

0

Extracts

Fig 7: IC50 values from DPPH Assay of NIP/18/12 extracts compared to Ascorbic acid. . All the data is performed in triplicate values with mean±SEM, n=3. ***P< 0.001 AQ vs Ascorbic acid. *P< 0.05 HA vs Ascorbic acid. No significant difference is observed with remaining extracts.

DPPH Assay: Inhibitory concentrations of various extracts of NIP/18/13 1000 ***a Ascorbic acid 800 NIP/18/13 C NIP/18/13 EA 600 NIP/18/13 ETOH NIP/18/13 HA 50 (ug/ml) 50 400

IC ***a ***a NIP/18/13 AQ *a 200

0

Extracts

Fig 8: IC50 values from DPPH Assay of NIP/18/13 extracts compared to Ascorbic acid. All the data is performed in triplicate values with mean±SEM, n=3. *P< 0.05 ET vs AA, ***P< 0.001 C, HA, AQ vs Ascorbic acid. No significant difference was observed with EA extract to ascorbic acid.

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DPPH Assay: Inhibitory concentrations of various extracts of NIP/18/14 800 ***a ***a ***a Ascorbic acid ***a NIP/18/14 C 600 NIP/18/14 EA *a NIP/18/14 ETOH 400 NIP/18/14 HA

50 (ug/ml) 50

IC NIP/18/14 AQ 200

0

Extracts

Fig 9: IC50 values from DPPH Assay of NIP/18/14 extracts compared to Ascorbic acid. All the data is performed in triplicate values with mean±SEM, n=3. ***P< 0.001 C, EA, ET, AQ vs Ascorbic acid. *P< 0.05 HA vs Ascorbic acid.

Evaluation of cardio protective effect of extracts with potent antioxidant activity in Doxorubicin induced cardiotoxicity in Balb/c mice.

7.2.3. Effect on Body weights

Effect of NIP/18/12 HA extract on Body weights in Doxorubicin induced cardiotoxicity 40 Normal Doxorubicin control 30 NIP/18/12 HA 100mg/kg NIP/18/12 HA 200mg/kg 20

10

Body weight(gm) Body

0

GROUPS

Fig 10: Showing effect of NIP/18/12 HA extract on body weights in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. There was no significant difference observed.

7.2.4. Effect on Relative Heart Weights:

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Effect of NIP/18/12 HA extract on Relative Heart Weights in Doxorubicin induced cardiotoxicity 5 Normal 4 Doxorubicin control NIP/18/12 HA 100mg/kg 3 NIP/18/12 HA 200mg/kg

2

1

Relative Heart Weights Relative 0

GROUPS Fig 11: Showing effect of NIP/18/12 HA extract on Relative Heart weights in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. There was no significant difference observed.

7.2.5. Effect on cardiac catalase activity:

Effect of NIP/18/12 HA extract on cardiac catalase activity in Doxorubicin induced cardiotoxicity 150 Normal Doxorubicin control 100 NIP/18/12 HA 100mg/kg NIP/18/12 HA 200mg/kg

50

catalase activity (U/mg) activity catalase 0

GROUPS

Fig 12: Showing effect of NIP/18/12 HA extract on cardiac catalase activity in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. There was no significant difference observed.

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7.2.6. Effect on serum Lactate dehydrogenase activity:

Effect of NIP/18/12 HA extract on serum LDH activity in Doxorubicin induced cardiotoxicity 1000 *** Normal 800 Doxorubicin control @@@ NIP/18/12 HA 100mg/kg 600 @@@ NIP/18/12 HA 200mg/kg

400

200

LDH LDH Activity(mU/ml) 0

GROUPS

Fig 13: Showing effect of NIP/18/12 HA extract on serum lactate dehydrogenase activity in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. ***P< 0.001 compared to normal control (a) and Doxorubicin control (b).

7.2.7. Effect on serum CK-MB activity:

Effect of NIP/18/12 HA extract on serum CK-MB levels in Doxorubicin induced cardiotoxicity 150 Normal Doxorubicin control 100 *** NIP/18/12 HA 100mg/kg NIP/18/12 HA 200mg/kg @@@ @@@

MB mU/ml MB - 50

CK

0

GROUPS

Fig 14: Showing effect of NIP/18/12 HA extract on serum CK-MB activity in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. ***P< 0.001 compared to normal control (a) and Doxorubicin control (b).

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7.2.8. Effect on Heart rate:

Fig 15: Showing effect of NIP/18/12 HA extract on heart rate in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. No significant difference was observed.

7.2.9. Effect on Ejection Fraction:

Fig 16: Showing effect of NIP/18/12 HA extract on Ejection fraction in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. ***P< 0.001 compared to normal control (a) and Doxorubicin control (b) and **P< 0.01 compared to Doxorubicin control (b).

7.2.10. Effect on Stroke volume:

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Fig 17: Showing effect of NIP/18/12 HA extract on stroke volume in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. ***P< 0.001 compared to normal control (a) and Doxorubicin control (b) and *P< 0.05 compared to Doxorubicin control (b).

7.2.11. Effect on Cardiac output:

Fig 18: Showing effect of NIP/18/12 HA extract on Cardiac output in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. ***P< 0.001 compared to normal control (a) and Doxorubicin control (b) and **P< 0.01 compared to Doxorubicin control (b).

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7.2.12. Effect on Fractional Shortening:

Fig 19: showing effect of NIP/18/12 HA extract on ejection fraction in Doxorubicin induced cardiotoxicity. All the data is expressed as mean±SEM, n=3. **P< 0.001 compared to normal control and @P<0.01 to Doxorubicin control.

8. FUTURE PLAN: 1. To further collect and evaluate the medicinal plant species on various other human cancer cell line.

2. To assess the potential of extracts in Cisplatin induced nephrotoxicity and Streptozotocin induced diabetic complications.

3. Strengthening the sector through research and development of traditional drugs.

9. REFERENCES:

1. https://en.wikipedia.org/wiki/Northeast_India

2. Shankar R, Rawat MS. Conservation and cultivation of threatened and high valued medicinal plants in North East India. International Journal of Biodiversity and Conservation. 2013 Sep 30;5(9):584-91.

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3. Mao A A&Hynniewta T M, Floristic diversity of North East India, J Assam SciSoc, 41(4) (2000) 255-26.

4. Mao AA, Hynniewta TM, Sanjappa M. Plant wealth of Northeast India with reference to ethnobotany. Indian journal of traditional knowledge. 2009 Jan 1;8(1):96-103.

5. http://censusindia.gov.in/.

6. Sharma UK. PLANTS USED FOR ETHNOMEDICINE BY THE NEPALESE OF ASSAM, INDIA. InEthnobiology and conservation of cultural and biological diversity: proceedings of the Fifth International Congress of Ethnobiology, Nairobi, Kenya, September 2-6 1996 2002 (p. 83). National Museums of Kenya.

7. Karki H, Upadhayay K, Pal H, Singh R. Antidiabetic potential of Zanthoxylum armatum bark extract on streptozotocin-induced diabetic rats. International Journal of Green Pharmacy (IJGP). 2014;8(2).

8. Sati SC, Sati MD, Raturi R, Badoni P, Singh H. Anti-inflammatory and antioxidant activities of Zanthoxylum armatum stem bark. Global Journal of researches in engineering: J General Engineering. 2011 Jul;5:86.

9. Sati SC, Sati MD, Raturi R, Badoni P, Singh H. Anti-inflammatory and antioxidant activities of Zanthoxylum armatum stem bark. Global Journal of researches in engineering: J General Engineering. 2011 Jul;5:86.

10. Antinociceptive and anti-inflammatory activities of ethyl acetate fraction from Zanthoxylum armatum in mice. Fitoterapia. 2011 Apr 30;82(3):347-51.

11. Sharma, A., G.S. Joseph, and R.P. Singh, Antioxidant and antiplatlet aggregation properties of bark extracts of Garcinia pedunculata and Garcinia cowa. Journal of food science and technology. 51(8): p. 1626-1631.

12. Sarma, R., et al., Polyphenol rich extract of Garcinia pedunculata fruit attenuates the hyperlipidemia induced by high fat diet. Frontiers in pharmacology. 7.

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13. Negi, P.S., G.K. Jayaprakasha, and B.S. Jena, Antibacterial activity of the extracts from the fruit rinds of Garcinia cowa and Garcinia pedunculata against food borne pathogens and spoilage bacteria. LWT-Food Science and Technology, 2008. 41(10): p. 1857-1861.

14. Ruma, K., S. Kumar, and H.S. Prakash, Antioxidant, anti-inflammatory, antimicrobial and cytotoxic properties of fungal endophytes from Garcinia species. International Journal of Pharmacy and Pharmaceutical Sciences. 5(3): p. 889-897.

15. Mundugaru, R., M. Chakkravarthy, and R. Basavaiah, Hepatoprotective activity of fruit extract garcinia pedunculata. Bangladesh Journal of Pharmacology. 9(4): p. 483-487.

16. Mundugaru, R., et al., Cardioprotective activity of fruit of garcinia pedunculata on isoprenaline- induced myocardial infarction in rat. Bangladesh Journal of Pharmacology. 11(1): p. 231-235.

17. Sharma, P.B., P.J. Handique, and H.S. Devi, Antioxidant properties, physico-chemical characteristics and proximate composition of five wild fruits of Manipur, India. Journal of food science and technology. 52(2): p. 894-902.

18. Hullatti, K.K. & Rai, V.R. Antimicrobial activity of Memecylon malabaricum leaves. Fitoterapia 75, 409-411 (2004).

19. Aparna Areti, Dinesh Tummuri, ganesh Yerra, Asutosh Kumar, VGM Naidu*, Boswellia ovalifoliolata abrogates ROS mediated NF-kB activation, causes apoptosis and chemosensitization in Triple Negative Breast Cancer cells. Environmental Toxicology and Pharmacology. (Under review, Manuscript ID: ETAP-D-13-00447).

20. Ethanolic extract of Boswellia ovalifoliolata bark and leaf attenuates doxorubicin-induced cardiotoxicity in mice. Bandari Uma Mahesh, Shweta Shrivastava, Madhusudhana Kuncha, Bidya Dhar Sahu, Challa Veerabhadra Swamy, Rajeswara Rao Pragada, V.G.M. Naidu, Ramakrishna Sistla. Environmental Toxicology and Pharmacology, 2013 In Press.

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Annexure 7

COMPARATIVE PHARMACOLOGICAL EVALUATION OF NIP/18/11 AND NIP/18/01 EXTRACTS AGAINST ROS MEDIATED DISEASES, CANCER AND CHEMOTHERAPY INDUCED COMPLICATIONS

Abstract North-East India is blessed with rich bio-diversity giving and traditional know how both ideal and potent combination for discovering new chemical entities for treating various clinical pathologies. Through literature review and various interactions with local healer presently we discovered NIP/18/10 and NIP/18/01 has been used locally to treat various clinical pathologies like Antileukemia, antidiabetic, antimicrobial, cytotoxicity, traditional uses, nociceptive activity. But its efficacy against cancer and chemotherapy induced complications is still yet to be explored. Thus, aim of our study is to explore efficacy of NIP/18/10 and NIP/18/01 in various cancerous cell lines and in-vivo model of chemotherapy induced complications. In order to estimate the antioxidant activity, we performed ABTS, DPPH, HRA and Phosphomolybdenum assays. We found ethanolic, ethyl acetate and hydroalcoholic extracts of NIP/18/01 and NIP/18/11 were found to be significant reducing power compared to ascorbic acid. Further, to check its efficacy in doxorubicin induced cardiac toxicity model where we found that treatment of NIP/18/01 and NIP/18/11 was found to reverse the toxicity induced by doxorubicin. Further, we found both NIP/18/01 and NIP/18/11 significantly reduced cell viability of various colon cancer cell lines and FaDu cell line. We also screen the activity of bioactive compound Hispolon (Phellinus linteus) against RANKL induced osteoclastogenesis. We found Hisplon inhibited osteoclastogenesis by downregulating the expression of master transcriptional factors for osteoclast differentiation such as NFATc1 and c-FOS in the cells exposed at 2 and 4 μM concentration in presence of RANKL. In conclusion, further identification of pharmacologically active plant and phytoconstituent and its validation is needed to identify the drugs against cancer and chemotherapy induced complication

1. Introduction Cancer is the leading cause of death worldwide: around 14 million new cases were diagnosed worldwide accounting for 8.2 million deaths in 2012 [1]. Cancer is an abnormal growth of cells caused by multiple changes in gene expression leading to dysregulated balance of cell proliferation and cell death and ultimately evolving into a population of cells that can invade tissues and metastasize to distant sites, causing significant morbidity and, if untreated, death of the host. Cancer development is a multistep process in which a cell acquires essential alterations that dictate the progressive transformation of normal cells into cancer cells. These cellular alterations include

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evading apoptosis, self-sufficiency in growth signals, limitless replicating potential, escaping growth suppressors, sustained angiogenesis, and tissue invasion and metastasis. Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries [2]. The burden of cancer is increasing in economically developing countries as a result of population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and ‘‘westernized’’ diets. It is estimated that if the current trend continues, there will be 22 million new cases and around 13.2 million deaths from cancer worldwide occurring each year by 2030 [3]. Chemotherapeutic techniques have a range of side effects that depend on the type of medications used. The most common medications mainly affect the fast dividing cells of the body, such as blood cells and the cells lining the mouth, stomach, and intestines. Damage to specific organs may occur, with resultant symptoms like cardiotoxicity, hepatotoxicity, nephrotoxicity, ototoxicity and encephalopathy. Depression of the immune system, which can result in potentially fatal infections, is also a common side effect of cancer chemotherapy. Hence, there is urgent need for the development of the novel anticancer molecule to combat both cancer and chemotherapy induced complication 2. Literature Review (max. 1000 words) Antioxidants have become one of the most important topics of today’s world because of its health benefits. These are a diverse group of chemicals which protect the body from oxidative damage induced by free radicals and reactive oxygen species by suppressing their formation [5]. Plants are the basis of life on earth and are central to people's livelihoods. Tribal people are the ecosystem people who live in harmony with the nature and maintain a close link between man and environment. Indian subcontinent is being inhabited by over 53.8 million tribal people in 5000 forest dominated villages of tribal community and comprising 15% of the total geographical area of Indian landmasses, representing one of the greatest emporia of ethnobotanical wealth [6].The tribal people have a deep belief in their native folklore medicine for remedies and they rely exclusively on their own herbal cure. Recently significant attention has been paid towards the use of natural health products as complementary or alternative approaches and efforts have been made for the development of anti-cancer activity molecules from natural sources. 2.1. NIP/18/11 [7] It possesses some of the pharmacological activities which are given. Antileukemia [8], antidiabetic [9], antimicrobial [10], cytotoxicity [12] traditional uses [13], nociceptive activity[14] 2.2. NIP/18/01

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The plant is having ethanomedicinal anticancer activity which is used by the North-East people of India [11]. It possesses the antioxidant and cytotoxic activity against Dalton’s Lymphoma cancer [15] Rationale of the study: Hypothesis of present study is, NIP/18/11 and NIP/18/01 have ethnomedicinal anticancer activity used in north-east India (sharma et.,al, 2001). Till now there is no scientific data proved for this activity. This study completely focuses on elucidation of antioxidant and anticancer activity of different extracts of both the plants and characterization of the compound involved for the activity.

(Background study and the study of research context) S. No. Reference Year Major objective Methodology adopted Findings

1 Kumar, D., et al., 2010 Development of An aliquot of 20 µl of each Betulinic acid, was Anti-leukemic HPLC method sample was injected onto isolated from the activity of the HPLC column ethyl acetate Dillenia indica L. Isolation of (XTerraTM RP C18 fraction by silica fruit extract and Pharmacologically column, 4.6_250mm, 5 gel column quantification of active compounds mm particle size) and chromatography betulinic acid by elution was carried out and was identified Screening in HPLC. with acetonitrile: water and characterized. human leukemic Phytomedicine. (9:1) pH3.0 (with cell lines U937, 17(6): p. 431-435. phosphoric acid) ataflow- HL60 and K562 rateof1ml/min and the eluate was monitored at 210nm

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2 Kumar, S., V. 2011 To investigate Blood glucose levels were Daily oral Kumar, and O. antidiabetic, measured using blood administration Prakash, hypolipidemic glucose test strips with DIME (250 and Antidiabetic, histopathological elegance glucometer on 500 mg/kg body hypolipidemic analysis of Dillenia weekly intervals till the weight) and and indica (D. indica) end of study (i.e. 3 weeks). glibenclamide (10 histopathological methanolic leaves Other parameters e.g. liver mg/kg) showed analysis of (DIME) extract in profile, renal profile and beneficial effects Dillenia indica alloxan induced total lipid levels were on blood glucose (L.) leaves extract diabetic rat by determined in normal and level (P < 0.001) as on alloxan administering oral alloxan induced diabetic well as improving induced diabetic doses (250 and 500 rats after oral kidney, liver rats. Asian mg/kg body administration of the functions and Pacific journal of weight). extract for 21 days. hyperlipidaemia tropical medicine. Histopathological changes due to diabetes. The 4(5): p. 347-352. in diabetic rat organs extract treatment (pancreas, liver and also showed to kidney) were also enhanced serum observed after extract insulin level and treatment body weight of diabetic rats as compared to diabetic control group. Furthermore, the extract has a favorable effect on the histopathological changes of the pancreas, liver and kidney in alloxan induced diabetes.

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3. Apu, A.S., et al., 2010 Different solvent  Disc diffusion The study Antimicrobial fraction of Dillenia method. confirms the Activity and Brine indica Linn. was  Brine shrimp moderate Shrimp Lethality collected and (Artemia salina) antimicrobial and Bioassay of the efficacy was lethality bioassay. potent cytotoxic Leaves Extract of evaluated by disc activities Dillenia indica diffusion of Dillenia Linn. Journal of method. Further, Young indica leaves the fractions Pharmacists. 2(1): extract and were screened for p. 50-53. therefore cytotoxic activity demands the using brine isolation of active shrimp (Artemia principles and salina) lethality thorough bioassay. bioassay.

4. Alam, M.B., et 2012 to evaluate the In- Antioxidant activity was Rats were dosed at al., vivo analgesic and measured by DPPH 200 and 400 mg/kg Antinociceptive antinociceptive and 400 mg/kg was and antioxidant properties of The methanolic extracts found to be activities of the methanolic extract Dillenia indica bark was significantly Dillenia indica of Dillenia indica evaluated against acetic increase pain bark. international bark. acid induced writing test, threshold and found Journal of formalin induce pain and to be anlagesic Pharmacology. hot plate method 8(4): p. 243-251.

3. Research Question/s  Which part of NIP/18/11 and NIP/18/01 crude extracts is biologically active?  Which solvent fraction shows maximum efficacy in various in-vitro screening models?  Which extract shows maximum efficacy against cancer and cancer complications?

4. Objective/s of the Study

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 Collection, extraction and determination of anti-oxidant properties of medicinal plants of NE India  To determine organoprotective activities for the extracts which showed potent anti-oxidant activity  To determine anti-cancer activity of the selected medical plants of NE India  Pre-clinical toxicity studies for the extracts having good efficacy

5. Study Area Head and neck cancer is emerging as a major socio-economic burden in India due to its varying degrees of structural and functional deformities depending on site, size, and metastatic patterns compromising the self-esteem well-being. Asian countries account for 57% of all global head and neck cancer cases all around the world especially India. Head and neck cancers in India accounted for 30% in males except in Dibrugarh, Assam where it accounted for 49.6%. Females to constitute 11-16% cases in all sites in most of the prevalence studies exploring the Indian scenario. Worse, over 2,00,000 cases of oral cancer which occur each year, only 80,000 cases are successfully diagnosed in India. Aizawl is becoming the world capital for lower pharyngeal and tongue cancer, due to the highest incidence rate in the. With over a century of research anti- neoplasm, oral cancer research effective standardized therapy based on etiology is not established. Oral cancer is triggered by etiologic factors like tobacco, betel quid (pan), Alcohol and in combination with tobacco smoking, Human Papilloma Virus (HPV) like HPV 16 and HPV 22, oral cavity lichen planus, and prolonged immunosuppressive treatments. Assam sits at the heart of North-East India rich in natural flora and fauna and home to various unexplored variety of herbal species. Hence our aim is to identify and isolated pharmacologically active anti-cancer molecules form plants native to Assam region

6. Methodology Adopted Antioxidant Assays FRAP Assay The reducing power of the extracts was determined by the method reported by [15] with slight modification. Briefly, 0.2 ml of each extract was mixed with 0.2 ml of phosphate buffer solution (0.2M, pH 6.6) and 0.2 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min and the reaction was stopped by addition of 0.2 ml of 10% trichloroacetic acid , followed by addition of 0.5 ml of distilled water and 0.2 ml of 0.1% ferric chloride, and the absorbance was measured at 700 nm spectrophotometrically against blanks. Ascorbic acid was employed as the reference. Results were expressed in terms of their reducing capacity.

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Phosphomolybdenum method: Total antioxidant capacity can be calculated by the method described by [16]. 0.1 mL of sample (100µg/ml) solution is combined with 1 mL of reagent (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tube is capped and incubated in a boiling water bath at 950C for90 min. After cooling the sample to room temperature, the absorbance of the aqueous solution is measured at 695 nm against blank. A typical blank solution contained 1 mL of reagent solution and the appropriate volume of the same solvent used for the sample and it is incubated under same conditions as rest of the sample. 4.3.3 Hydroxyl Radical Scavenging Activity: Scavenging activity of hydroxyl radicals was determined by the method reported by [17]. Briefly an amount of 100 μl of phenanthroline (1.85 mM), 100 μl of Ferrous sulphate (1.85 mM) and 100 μl of H2O2 (0.05%) were added into 100 μl of the extract dissolved in potassium phosphate buffer (0.76 M, pH 7.4), and the mixture was incubation at 40°C for 1 h. The absorbance was measured by spectrophotometer at 532 nm. Ascorbic acid was used as the reference standard. Results were expressed in terms of percentage free radical scavenging activity. % scavenging of hydroxyl radical = (A sample- A negative cntrl/A blank - A negative cntrl) x 100 Cell viability assay MTT assay [18] Principle: Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan crystals which are insoluble in aqueous solutions. The crystals are solubilized in Dimethyl sulfoxide (DMSO). The resulting purple solution is spectrophotometrically measured. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.

Figure 1: Reduction of MTT to Colored Formazan Derivative (by viable cells) Protocol MTT assay is performed on the panel of human cancer cell lines such as HCT-15, HCT-116 and HT-29 for evaluating the cytotoxicity of test compound.

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Trypsinize a sub-confluent monolayer culture and collect cells in growth medium containing serum. Centrifuge the suspension (3500rpm, 2min) to pellet cells, resuspend in growth medium, and count cells. Plate the cells at a density of 4×103 cells/well in a 96 well flat-bottomed microtiter tissue culture plates. Incubate the 96 well plate overnight or for 24 hrs. After 14hrs test compound is added at different concentrations. Plates are incubated at 370C in CO2 incubator with 5% CO2 & 80% relative humidity for 48 hrs, and then the medium is replaced with fresh medium without FBS. 100μL of MTT solution (5 mg/ml in media ) is added to each well and re-incubated for 4hrs at 37oC. The supernatant culture medium is carefully aspirated and 200μL of dimethyl sulfoxide (DMSO) is added to each well to dissolve the formazan crystals, and the absorbance is measured at 570 nm. Curcumin is used as standard anti-cancer activity drug. Cardio-protective activity of NIP/18/11 and NIP/18/01 Based on the previous antioxidant assays it was proven that hydroalcoholic extract of NIP/18/11 and NIP/18/01 was having good antioxidant property. Hence, for this two extracts cardioprotective activity was done on doxorubicin induced cardiotoxicity on Balb/c mice. Experimental design: 25-30 gms Balb/c femal mice was taken were grouped into six groups consisting of four mice in each group. Group 1: normal control group 2: disease control: received single dose intraperitoneal injection of Doxo 15mg/kg on the 7th day of the study. Group 3: Given NIP/18/11 HA dose of 100 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day. Group 4: Given NIP/18/11 HA dose of 200 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day. Group 5: Given NIP/18/01 HA dose of 100 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day. Group 6: Given NIP/18/01 HA dose of 200 mg/kg orally from day one to day fourteen and an IP injection of Doxo on 7th day. Electrocardiographic Recording and Analysis: The ECG was performed on mice from 6 groups (n = 4 per group) to evaluate DOX cardiotoxicity at day 14th. Briefly, mice were anesthetized with 4% isoflurane and maintained anesthesia with 1-2% isoflurane followed by insertion of electrodes through a needle in the right hind limb, right front limb, left front limb. The data were collected by AD Instrument (Australia). T wave elevation and RR interval was analyzed using labchart 8 software (AD instrument).

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The animals underwent for imaging where animals were anesthetized with 4% isoflurane in an anesthesia chamber and placed on a heating pad and anesthesia was maintained using 1.5% isoflurane by a nasal mask throughput the experiment. Echocardiography was performed using a high-frequency (30MHz) small animal imaging system (Vevo 3100, FUJIFILM VisualSonics). Parasternal long-axis images and M-mode B-mode and Power Doppler (pw) images were obtained and analyzed for measuring left ventricle (LV) ejection fraction , cardiac output, fractional shortening (FS), systolic (LVIDs) and diastolic internal diameters (LVIDd). Biochemical parameters: On 14th day, body weights of mice were recorded and blood samples were collected from intra cardiac puncture. Serum was separated by centrifugation at 8000 × g for 10 min and used to determine LDH from Sigma & CKMB using Accurex kit Mumbai. Heart was dissected and minced into small pieces and homogenized in ice-cold phosphate buffer saline (PBS) (0.05 M, pH 7) to obtain 1:9 (w/v) whole homogenate and was centrifuged at 17,000g for 60 min at 4 0C, and supernatant was used for the assay of Catalase.

Hispolon ameliorates osteoporosis by inhibiting RANKL induced osteoclastogenesis: An in vitro study Cell proliferation assay We employed a method as described by “Naidu et al” [9]. RAW 264.7 cells were seeded in 96- well plate at the density of 1 x 103 cells. After 12 h, HISP treatment was given at varying concentrations (1, 2, 4 and 8µM) for 48 h then cell viability was determined by MTT [3-(4, 5 - dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method. At the end of the experiment, the plate was treated with media containing MTT (500μg/mL) for 3 h at 37 °C. Cells were washed with PBS and solubilized by the addition of 200μL DMSO. The resulting intracellular purple formazan was quantified with multi-detection plate reader (Spectramax M4, Molecular Devices, USA) at 570nm. In vitro osteoclastogenesis assay This assay was performed using RAW 264.7 cells to evaluate the effect of Hispolon on RANKL-mediated osteoclastogenesis [3]. Cells were seeded (10 × 103 cells/well) into 24 well cell culture plate and incubated overnight. Then, the cells were stimulated with RANKL (100ng/mL) and treated with various concentration of HISP (1, 2 and 4 μM). The exhausted medium was replaced with fresh one, every second day. After 5 days incubation, cells were fixed with 4 % paraformaldehyde and washed with PBS twice. Further, cells were stained for tartrate-resistant acid phosphatase (TRAP) with leucocyte acid phosphatase kit from Sigma (St. Louis, MO, USA) activity to identify formation and abundance of osteoclasts using TRAP staining kit. TRAP-positive multinucleated giant cells with >3 nuclei were scored as osteoclasts and counted using phase-contrast microscope at 100× magnification. NMHS Fellowship Grant Progress Page 56 of 122

Actin cytoskeleton staining To investigate the RANKL-induced cytoskeletal changes, the formation of F-actin ring formation was studied in RAW 264.7 cells. RAW 264.7 cells (10 × 103) were incubated with RANKL 100 ng/ml along with Hispolon (4μM) for five days. After five days, cells are fixed with 4% paraformaldehyde. Fixed cells were stained with FITC-Phalloidin. Nuclei were visualized with 1μg/ml DAPI (4′, 6-diamidino-2-phenylindole; Sigma). Cells were visualized under 100× original. The experiment was performed in duplicate on two independent occasions in a 24-well plate.

Western blotting analysis for protein expression analysis RAW 264.7 cells were pre-treated with varying concentrations of Hispolon (1, 2 and 4µM). After 6h, they were stimulated with RANKL (100ng/mL) for 30 min. Then, the cells were washed with ice-cold PBS and lysed in RIPA buffer containing phosphate and protease inhibitor cocktails. Protein concentration was estimated by the Bradford protein assay. For western blotting analysis, protein samples were separated on an 8-12% SDS-PAGE and then transferred to a nitrocellulose membrane (Amersham Bioscience). Blocking of non-specific proteins was done with the usage of 5% bovine serum albumin for 2h. Primary antibodies were given to probe membranes for 12h at 4 °C. Then, the membranes were washed and incubated with respective secondary antibodies for 2 h. The antigen-antibody complex was visualized with the aid of ECL detection kit (Amersham Bioscience). For immune blot analysis, quantifications were done by densitometry scanning with NIH Image J software. Statistical Analysis: Data presented were representative results from a triplicate set of three independent experiments or the mean ± SEM of those experiments. One-way ANOVA was used to test statistical significance between groups using GraphPad Software Prism 5. A P-value of <0.05 was considered to be statistically significant.

7. Research Findings

Objective (s) Deliverables Methodology adopted Achievements so far

(As per sanctioned (As per sanctioned letter) letter)

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Overall Objective  Established the  Collected and  Plant materials from two institutional capacity authenticate (Botanist medicinal plant species for determining Pharmacological safety and efficacy, from Guwahati of NE were collected, activities, efficacy, dosage testing and University) the two dried and extracted with formulation of safety and dosage medicinal plants solvents of increasing phytomedicines. evaluation of  Confirmed and (NIP/18/11 and polarity. Extracts were bioactive compounds tested record of 3 weighed and percentage species of medicinal NIP/18/01) which in potential species of yield was calculated. plants effective for are endemic to NE the treatment of few  We have collected 7 Himalayan regions Objective 1 human diseases and solvent extracts from 2 few livestock  Solvent extraction with different plants which Collection, diseases and increasing polarity and determined their are endemic to NE India. extraction and safety, efficacy and distillation  Determined antioxidant dosage levels.  Preparation of crude determination of  Strengthening the sector assays for the 7 extracts extracts by percolation anti-oxidant through research and from 2 medicinal plants method development of and found that ethyl properties of  Antioxidant activities traditional drugs. acetate and alcoholic medicinal plants of for the crude extracts extracts has been NE India are determined by showed potent anti- DPPH, ABTS, FRAP oxidant activity assays compared to .Ascorbic acid

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Objective 2 To find the efficacy of Chemotherapeutic drugs Determined the efficacy of extracts from the used for the treatment of selected 2 medicinal plants To determine organ selected medicinal cancer are mostly causes for their use as adjuvant protective activities plants dose dependent orange therapy along with for the extracts toxicity. For example chemotherapeutic drugs like doxorubicin which is a doxorubicin to prevent dose which showed good chemotherapeutic dependent cardiotoxicity. potent anti-oxidant agents causes activity cardiotoxicity. To prevent doxorubicin induced cardiotoxicity, plants extracts which showed potent antioxidant activities are tested for their effect on cardio protective effect in mouse models.

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Objective 3  To screen and evaluate  The efficacy of  From the results, it was the anti-cancer activity different extracts was found that ethylacetate To screen and evaluate on different cancer cell screened on different extracts of two medical the anti-cancer activity lines. cancer cells lines such plants has showed potent on different cancer cell  Research article as Colon-rectal cancer cytotoxicity on colon lines. communicated (HCT116, HT15 HT- cancer cells as well as Hispolon ameliorates 29) and oral cancer cell head and neck cancer

osteoporosis by lines (Fadu) cells. Further studies are inhibiting RANKL in progress to determine induced selective cytotoxicity osteoclastogenesis: An towards cancer cells. Hispolon is a bioactive in vitro study compound from Phellinus (Bioscience Reports linteus. Several reports has Publisher:MDPI been reported that Hispolon  Hispolon showed Portland Press I.F 2.9) is having anti-inflammatory significant inhibition of and anti-oxidant activities. formation of However there is no studies osteoclastogenesis at on the effect of Hispolon on 4µM concentrations in bone resorption. Hence in RANKL induced the current study we studied osteoclastogenesis the effect of Hispolon on models confirmed by TRAP staining &  RANKL induced organised F-actin osteoclastogenesis and formation and further further LPS induced inhibiting the NFKB bone lysis in mouse translocation which is models. key regulatory protein for upregulation of

inflammatory cytokines.

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RESULTS: 1. Effect of different NIP/18/11 and NIP/18/01 solvent extracts on antioxidant activity Figure 1

A B

C D FRAP Assay

E. Phosphomolybdenum Assay NIP/18/11 F Phosphomolybdenum Assay NIP/18/01

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Figure 1 : Antioxidant assays (A. ABTS, B. DPPH, C. FRAP assay D. Hydroxylradical scavenging assay, and Phosphomolybdenum assay of NIP/18/11 E. and NIP/18/01 F) were performed. Results were analysed by using Graph Pad Prism mean ± SEM for a triplicate experiment.

Hydroalcoholic extracts shows a good antioxidant activity in all in-vitro antioxidant assays:

In order to estimate the antioxidant properties of the extracts we isolated few NIP/18/11 and NIP/18/01 in different solvent fractions and performed ABTS, DPPH, FRAP, HRA and Phosphomolybdenum Assay in ABTS and DPPH assay ethyl acetate and aqueous fraction of NIP/18/11 and NIP18/01 were found to be significant in scavenging free radicals compared to ascorbic acid. In Phosphomolybdenum Assay hydroalcoholic fraction of both NIP/18/11 and 01 was having significant antioxidant activity. The order of activity was found to be NIP/18/01 HA > NIP/18/11 HA > NIP/18/11 ET-OH > NIP/18/01 ET-OH > NIP/18/11 EA > NIP/18/11 CH > NIP/18/11AQ. Similar results were observed in hydroxyl radical scavenging assay where hydroalcoholic fractions were showing maximum efficacy the order of scavenging is as follows Ascorbic acid > NIP/18/11 HA > NIP/18/11 ET-OH > NIP/18/11 EA > NIP/18/11 CH. In ethanolic extracts were found to be more potent and hydroalcoholic fraction the order is as follows NIP/18/11 ET-OH >NIP/18/01 ET-OH> NIP/18/01 HA > NIP/18/11 HA > NIP/18/11 EA > NIP/18/01 AQ > NIP/18/EA

2. Effect of extracts on body weight, heart index and biochemical parameters in cardioprotective activity (heart weight/body weight)

Body weight Heart index B CK-MB A C

E Catalase D LDH Effect of HA extract of NIP/18/11 and NIP/18/01

on biochemical parameters. The data was analysed by One Way ANOVA using

Graph Pad Prism where n= 4. *P <0.05 represents Normal control vs Disease # control. P<0.05 represents Disease control vs treatment groups. Figure 2 : Effect of HA extract of NIP/18/11 and NIP/18/01 on biochemical parameters. The data was analysed by One Way Anova using Graph Pad Prism where n= 4. **P <0.01 represents Normal control vs Disease control. #P<0.05 represents Disease control vs treatment groups

Table 3: Effect of extracts on cardiac parameters by AD instrument.

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PARAMETERS Normal Disease NIP/18/11 NIP/18/11 NIP/18/01 NIP/18/01 HA 100 HA 200 HA 100 HA 200 % T wave elevation 2.38 46.08 41.58 18.67 14.57 4.18 compared with respective pretreatment (mV) RR Interval (s) Mean 0.12 ± 0.14 ± 0.13 ± 0.02 0.14 ± 0.13 ± 0.01 0.14±0.02 ± SD 0.02 0.01 0.01 P Amplitude (mV) 0.09 ± 0.06 ± 0.08 ± 0.09 ± 0.07 ± 0.10 ± Mean ± SD 0.022 0.027 0.008 0.012 0.040 0.023

ST Height (mV) 0.006598 0.10152 0.050228 ± 0.038723 0.08694 ± 0.10715 ± Mean ± SD ± ± 0.102 ± 0.036 0.111 0.017 0.017 0.102

ECG Images by AD

Instrument

P wave P wave Elevated Normal T Normal T wave wave Disease control control

NIP/18/11/

HA 100 NIP/18/01/ HA 200

NIP/18/01/ NIP/18/01/ HA 100 HA 200 Figure 3 Shows various ECG of various animals. Doxorubicin treatment induced significant T- wave changes in T wave abnormalities. SD rat were treated with 100 and 200 mg/kg. treatment with NIP/18/11 and 01 extract was able to reverse the doxorubicin induced changes in ECG

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Effect of HA extrac of NIP/18/11 and NIP/18/01 on cardiac parameters by PSLAX Vevo Lazer X 3100. The data was analysed by One Way ANOVA using Graph Pad Prism where n= 4. ***P <0.001 represents Normal # ## control vs Disease control. P<0.05, P < 0.01 represents Disease control vs treatment groups.

5.5 Effect of extracts on biochemical parameters

Figure 4: Effect of hydroalcoholic extracts of NIP/18/11 and 01 on cardiac output parameters. the results were obtained by PSLAX Vevo Lazer X 3100. The data was analysed by One Way ANOVA using Graph Pad Prism where n= 4. ***P <0.001 represents Normal control vs Disease control. #P<0.05, ##P < 0.01 represents Disease control vs treatment groups.

Thickness of Left ventricular posterior wall

Normal control Disease control

NIP/18/11/HA 100 NIP/18/11/HA 200

.

NIP/18/01/HA 100 NIP/18/01/HA 200

Figure 5: Left ventricular posterior wall thickness measured in Vevo Lazer X 3100.

Hydroalcoholic extracts of NIP/18/01 and 11 was able to reverse Doxorubicin induced cardiac toxicity

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Hydroalcoholic extracts of both the plants was showing good antioxidant activity in the in-vitro assays thus, was chosen to explore its efficacy against doxorubicin induced cardiac toxicity. Bab/c mice of weight 20-25g weight were chosen and was randomly divided in to normal, disease control, hydroalcoholic extract of NIP/18/11 (100mg/kg), hydroalcoholic extract of NIP/18/11 (200 mg/kg), hydroalcoholic extract of NIP/18/01 (100mg/kg), ), hydroalcoholic extract of NIP/18/01 (200 mg/kg). Doxorubicin treatment induced significant physiological and morphological changes as evident in the results of biochemical (figure 2), ECG (figure 3), altered cardiac output parameters (figure 4) and left ventricular posterior wall thickness. Hydroalcoholic extracts of both NIP/18/11 and NIP/18/01 at 200 mg/kg was significant in reversing the doxorubicin induced changes in cardiac hypertrophy model.

5.6 Effect of extracts on different cell lines of colon cancer on HCT 115 and HCT 116

Colon cancer

IC 50 values of NIP/18/11 and NIP/18/01 compared with standard curcumin on HCT 15 and HCT 116 cell lines. All the data were expressed as mean ± SEM for a triplicate experiment by One Way ANOVA. **P < 0.01, ***P < 0.001 represents ## @ NIP18/11 vs NIP/18/01, P < 0.01 represents NIP/18/11 vs Curcumin, P < 0.05 represents NIP/18/01 vs Curcumin.. IC 50 values of NIP/18/11 and NIP/18/01 on HT-29 cell line. All the data was expressed for a single experiment.

Figure 14 and 15: IC 50 values of NIP/18/11 and NIP/18/01 compared with standard curcumin. All the data were expressed as mean ± SEM for a triplicate experiment. **P < 0.01, ***P < 0.001 represents NIP18/11 vs NIP/18/01, ##P < 0.01 represents NIP/18/11 vs Curcumin, @P < 0.05 represents NIP/18/01 vs Curcumin.

Figure 16

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Figure 16: IC 50 values of NIP/18/11 and NIP/18/01. All the data were expressed as mean ± SEM for a duplicate experiment.

Hispolon ameliorates RANKL induced osteoclastogenesis an in-vitro model of bone resorption

Figure 1: Effect of Hispolon on cell viability of RAW 264.7 cells. RAW 264.7 cells were incubated with different concentrations of Hispolon concentration (1, 2, 4 and 8 µM) for 2 days. We observed Hispolon remains non-toxic in all concentration. However, at the 8µM sight but insignificant reduction in the cell viability was noticed.

Effect of Hispolon on cell viability

Cytotoxicity assessment of Hispolon on RAW 264.7 cell was performed by MTT assay. We

observed that incubation of RAW 264.7 cells with various concentration (1,2, 4 and 8µM) of

Hispolon for 2 days did not alter cell viability. However, at 8 µM concentration there was a slight

but insignificant reduction in the cell viability was observed (figure 1).

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Figure 2: HISP attenuates RANKL-mediated osteoclastogenesis in RAW264.7 cells. (A) RAW 264.7 cells (10×103 per well) were incubated with 100 ng/ml RANKL, 4 μM of HISP or both RANKL and HISP (1, 2 and 4μM) for 5 days and then stained for TRAP. Magnification, 100× original. (B) Bar graphs showing the quantification of multinucleated osteoclasts. Data expressed as mean ± SEM (n = 3). **P < 0.01 and ***P < 0.001 significant vs. RANKL control.

Effect of Hispolon on RANKL induced osteoclastogenesis

To demonstrate the anti-osteoclastogenic activity of Hispolon, we performed an in vitro dose-

dependent osteoclastogenesis assay. In 24 well plate RAW 264.7 cells were seeded at a density

1×104 per well to see the formation of multinucleated giant cells induced by RANKL treatment

(100 ng/mL) for 5 days. TRAP staining was performed in order to visualize the formation of

osteoclasts. We found that HISP treatment has significantly reduced the osteoclasts number in in-

vitro osteoclastogenesis assay at 2 and 4 μM concentration (figure 2).

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Figure 3: HISP blocks F-actin ring formation induced by RANKL stimulation. RAW264.7 cells were cultured with RANKL 100ng/mL along with HISP (4μM) for five days and stained with FITC-phalloidin for F-actin rings. Nuclei were visualized with DAPI. F-actin containing patches are commonly found in cells treated with RANKL only (red arrows), which unlike, HISP treated (4μM) cells fail to develop an organized actin ring.

HISP blocks the occurrence of RANKL-mediated F-Actin ring formation

To test the effect of HISP on the formation of F-Actin ring mediated by RANKL stimulation, after

attachment, 4μM of HISP along with RANKL (100ng/mL) was added to the culture for 5 days to

initiate osteoclastogenesis. Along with osteoclastogenesis, the formation of F-actin ring was

observed in the disease control group. To stain the F-actin ring, the procedure is followed described

in Methods and Materials. HISP treatment blocked the occurrence of F-acting ring formation as

compared to the control group (figure 3). This data suggest that Hispolon abrogates RANKL-

mediated F-actin ring formation in osteoclast precursor cells. Nuclei were visualized with DAPI. F-

actin containing patches are commonly found in cells treated with RANKL only (red arrows),

which unlike, HISP treated (4μM) cells fail to develop an organized actin ring.

Figure 4: HISP suppresses RANKL-mediated activation of NF-ĸB. (A) RAW264.7 osteoclast precursors were pre-treated for 6 h with increasing doses of Hispolon (0–4μM), and then stimulated with RANKL (100ng/mL) for 30 min. Total protein extracts were analysed by western blotting using antibodies against phospho-NF-κB (p65), total NF-κB, phospho IKKα/β(176/180), total IKKα/β and β-actin. (B) Graphical depiction of western blotting analysis of phospho-NF-κB and phospho IKKα/β (176/180). Data expressed as mean ± SEM (n=3), **P<0.01, ***P<0.001 significant vs. RANKL control.

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HISP suppresses the osteoclastogenesis by RANKL-mediated phosphorylation of NF-κB

NF-κB pathway plays a crucial role in RANKL- mediated osteoclast differentiation and function.

Thus, we investigated whether HISP has any modulatory effects on NF-κB activation in RAW264.7

cells. RAW264.7 cells were seeded on 6-well plate and pre-treated with varying concentration of

HISP. After 6h, cells were stimulated with RANKL (100ng/mL) for 30 min. As shown in (figure

4)., Hispolon significantly reduced the RANKL-mediated phosphorylation subunit of IKKα/β as

well as the phosphorylation of NF-κB/p65 in a concentration-dependent pattern. These results

clearly demonstrate that the anti-osteoclastogenic effect of HISP is associated with inactivation of

NF-κB signaling.

Figure 5: HISP inhibits RANKL-mediated activation of ERK/MAPK. (A) RAW264.7 cells were incubated for 6 h with increasing doses of HISP (0–4μM), and then stimulated with RANKL (100ng/mL) for 30 min. Phosphorylation of p38 and ERK was determined on indicated times by western blotting. (B) and (C) the relative protein expression levels of p38 and ERK were quantified using Image-Pro Plus 6.0 software and normalized to β-actin. Data expressed as mean±SEM (n = 3), ***P < 0.001 significant vs. RANKL control.

HISP inhibits the ERK/MAPK signalling

MAPK signaling is another crucial mechanism that is responsible for the formation and function of

an osteoclast. We next investigated the effect of Hispolon on MAPK signaling cascade involved in

RANKL-mediated osteoclastogenesis. RAW264.7 cells were exposed with HISP in different

concentrations along with stimulation of RANKL (100ng/mL) for 30 min. With immunoblotting

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analysis given in figure 5, HISP markedly inhibited the phosphorylation of p38 and ERK1/2. These

results indicate the inhibitory action of HISP on ERK/MAPK signaling activation.

Figure 6: HISP suppressed the RANKL-induced expression of osteoclast-specific like NFATc1, c-Fos and Cathepsin-K protein expression. (A) Effect of HISP on protein expression of c-FOS, NFATc1 & Cathepsin-K. (B) (C) & (D) Graphical representation of fold changes in the c-FOS, NFATc1 and Cathepsin- K respectively. Protein expression normalized to β-actin. Data expressed as mean±SEM (n = 3). **P < 0.01, ***P < 0.001 significant vs. RANKL control.

HISP attenuates the RANKL-mediated downregulation of NFATc1 and c-FOS

NFATc1 and c-Fos are master transcriptional factors in RANKL mediated osteoclastogenesis. In

our study we found that, RANKL stimulation significantly increased the expression of NFATc1 and

c-Fos whereas, HISP significantly abolished the RANKL induced expression of NFATc1 and c-

Fos. We also observed that Hispolon inhibited the RANKL-induced expression of Cathepsin-K

(figure 6). These results clearly showed that Hispolon inhibits the RANKL induced

osteoclastogenesis by downregulating the NFATc1, c-Fos and Cathepsin-K.

8. Future Plan (including monthly plan)

To further collect and test medicinal plants for treatment of few human disease and life style disease and determine their safety efficacy and doasage level. Secondly strengthening the sector through research and development of traditional drugs.

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Further we will teat the effect of these extract in different human cancer cell lines and also check the efficacy in different chemotherapy and diabetes induced complications.

9. Reference/s Stewart, B. and C.P. Wild, World cancer report (2014). World. Parkin, D.M., et al., Estimating the world cancer burden: Globocan (2000). International journal of cancer, 2001. 94.2: (153-156). Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians (2015); 61.2:( 69-90). Huang, E.S. Internal medicine: handbook for clinicians, resident survival guide. Arlington, VA: Scrub Hill Press (2000); p.130. Salvayre, A.N, Dousset, N., Ferretti, G., Bacchetti, T., Curatola, G. and Salvayre, R. Antioxidant and cytoprotective properties of high-density lipoproteins in vascular cells. Free Radic Biol Med (2006); 41.7: 1031-1040. Chowdhuri SK: From Ethnobotany. In Studies in Botany Volume 2. 7th edition. Edited by: Mitra D, Guha J. Chowdhuri SK, Kolkata: Manasi Press; 2000:(855-867) Kumar, D., et al., Anti-leukemic activity of Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC. Phytomedicine (2009) 17(6): p. 431-435 Kumar, S., V. Kumar, and O. Prakash, Antidiabetic, hypolipidemic and histopathological analysis of Dillenia indica (L.) leaves extract on alloxan induced diabetic rats. Asian Pacific journal of tropical medicine. 4(5): p. 347-352. Apu, A.S., et al., Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn. Journal of Young Pharmacists. 2(1): p. 50-53. Rai, P.K. and H. Lalramnghinglova, Ethnomedicinal plant resources of Mizoram, India: Implication of traditional knowledge in health care system. Ethnobotanical Leaflets. 2010(3): p. 6. Akter, R., et al., Cytotoxic activity screening of Bangladeshi medicinal plant extracts. Journal of natural medicines. 68(1): p. 246-252. Saiful Yazan, L. and N. Armania, Dillenia species: A review of the traditional uses, active constituents and pharmacological properties from pre-clinical studies. Pharmaceutical biology. 52(7): p. 890-897. Alam, M.B., et al., Antinociceptive and antioxidant activities of the Dillenia indica bark. international Journal of Pharmacology. 8(4): p. 243-251 Ashish KS, antibacterial, anti-alpha glucosidase and antioxidant properties of Dillenia pentagyna Asian journal of pharmaceutical & clinical Research (2013) Barreira, J. o., Ferreira, I. C. F. R., Oliveira, M., & Pereira, J. A. (2008). Antioxidant activities of the extracts from chestnut flower, leaf, skins and fruit. Food Chemistry, 107(3), 1106-1113

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Marwah, R. G., Fatope, M. O., Mahrooqi, R. A., Varma, G. B., Abadi, H. A., & Al-Burtamani, S. K. S. (2007). Antioxidant capacity of some edible and wound healing plants in Oman. Food Chemistry, 101(2), 465-470. Olabinri, B. M., Odedire, O. O., Olaleye, M. T., Adekunle, A. S., Ehigie, L. O., & Olabinri, P. F. (2010). In vitro evaluation of hydroxyl and nitric oxide radical scavenging activities of Artemether. Research Journal of Biological Sciences, 5(1), 102-105. Riccardi et al. Analysis of apoptosis by propidium iodide staining and flow cytometry.Nat protoc (2006);1:(1458–1461)

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Annexure 8

Sensitization and awareness about medicinal plant among Himalayan inhabitants for their rational use

Abstract (200-250 words) Herbal drugs and plant products have been used to treat multiple ailments in NE India. However, there is a concern about their rational use as many of the plant drugs have been reported to have noted toxicity and side effects. Therefore we had organised awareness camps for the awareness and rational use of herbal drugs in the states of Meghalaya and Assam. Other than that, NE India is considered to be biodiversity hotspot as a large number of plants with therapeutic activity is indigenously found here. Therefore we explored the possibilities of developing potential formulations for rheumatoid arthritis. It has been observed that native extracts have lower bioavailability resulting in lower therapeutic activity. Hence, we attempt to develop a formulation with better bioavailability and improved medicinal activity. In one such attempt to improve the therapeutic efficacy of NIP/18/22 in the treatment of rheumatoid arthritis (RA) we incorporating drug into the janus nanosized emulsion. The janus nanosized emulsion were synthesized using high shear homogenization method and their size and were evaluated by dynamic light scattering and morphology will be evaluated by scanning electron microscopy (SEM). The solid-state behaviour of the nanoparticles will be also characterized operating X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopy (FTIR). Furthermore, the therapeutic efficacy of the prepared nanosized emulsion on the adjuvant induced arthritic rats will be assessed. The severity of arthritis will be determined by measuring the arthritic score on alternate days until mean arthritic score of 4 will be observed. The NO and PO levels will be also analyzed in serum samples. NF-κB and iNOS expression levels will be determined in spleen tissue samples by real time RT-PCR.

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a) Dhemaji (Assam) tour for awareness of herbal drug usage and conservation of the plants.

An awareness campaign on the usage and safety of herbal drugs was conducted in Dhemaji District of Assam. The president and secretary of All Assam Herbal Healers Association, Shri. Jibeshwar Borpatra Gohain and Shri. Debakanta Koch have discussed the usage if herbal products in treatment of multiple ailments. They have treated

Figure: A poster on awareness of herbal drugs for the traditional healers of Dhemaji (Assam)

Figure: Adressing the local peoples regarding safety usage of herbal drugs by Traditional healers of Dhemaji and NIPER Guwahati researchers and a discussion among NIPER Guwahati researchers and traditional healers of Assam

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b) Meghalaya tour for awareness and conservation of herbal drug usage.

We conducted an awareness campaign regarding the usage of herbal drugs and their conservation in few places in Meghalaya. A group of local inhabitant and traditional healers have attended the meeting and given their inputs regarding the same.

Figure: A poster on awareness campaign regarding the usage of herbal drugs

Figure: A poster on awareness campaign regarding the toxicity of herbal drugs

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Figure: Visit to Meghalaya herbal garden conservation site maintained by Dr. H C Carehome (President of Meghalaya Herbal Healers Association) in association with NIPER Guwahati.

Figure: Adressing the local peoples regarding safety usage of herbal drugs by Traditional healers of Meghalaya and NIPER Guwahati researchers

Rheumatoid arthritis

Introduction (max. 500 words) Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by infiltration of inflammatory cells and proliferation of synovial fibroblasts, which causes destruction of the synovial membrane, progressive bone destruction and joint deformity. The treatment principle of RA is to slow down the development of the disease, reduce the rate of NMHS Fellowship Grant Progress Page 76 of 122

disability, and improve the quality of life of RA patients. NIP/18/22 is documented in the traditional literature for rheumatoid arthritis. NIP/18/22 possess the anti-inflammatory and immunomodulatory activity. NIP/18/22 does not inhibit cyclo-oxygenase enzyme and is therefore devoid of gastric irritation at the therapeutic doses. It is a novel compound having both anti-inflammatory and immunomodulatory properties fused in a single molecule with a mechanism of action clearly distinct from classical NSAID and immunomodulatory. Indomethacin (Indo) is a nonsteroidal anti-inflammatory drug that is effective to inflammatory pain and commonly used to relieve RA. However, long-term oral administration can cause toxicity. This study was to develop and optimize a delivery system based on Janus nanosized emulsion loaded with NIP/18/22 and Indomethacin. The Janus nanosized emulsion were prepared by high shear homogenization method and optimized by the Box–Behnken design (BBD). The in vitro characteristics nanosized emulsion were investigated. The delivery mechanism of nanosized emulsion will be elucidated using confocal laser scanning microscope (CLSM).

2. Literature Review (max. 1000 words) (Background study and the study of research context) S. Reference Year Major Methodology Findings No. objective adopted 1. Qian Kang 2018 Transdermal In vivo anti Cel-Indo-NLCs et al delivery system rheumatic activity showed of nanostructured assessment. prominent lipid carriers effect of loaded with Histological Celastrol and decreasing paw Indomethacin: analysis oedema, optimization, characterization inhibiting and efficacy inflammation evaluation for rheumatoid and pain by arthritis regulating the levels of IL-1b, TNF-a, b- endorphin and Substance P.

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2 Anum Gul 2018 Rutin and Real-time reverse The present et al. rutin- transcriptase PCR study conjugated gold (RT-PCR) analysis demonstrated

nanoparticles the preclinical

ameliorate characterization collagen- of rutin and R- induced AuNPs that arthritis in rats showed through prominent anti- inhibition of arthritic effect NF-κB and in CIA rats by iNOS activation acting in a pleiotropic manner by improving the arthritic scoreas well as by downregulating the oxidative 3. Amarinder 2019 Rohitukine Western blotting stresssuppressive markers . Singh et al. inhibits NF-κB effect of RHK Spontaneous activation on LPS-induced motor activity induced by LPS production of and other NO was inflammatory mediated at the agents transcriptional level.

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3. Research Questions These research questions were raised while reviewing literature as follows: 1. What is the need for the sensitization and awareness about the use of medicinal plant in local communities of NE India? 2. What measures should be taken to enforce the rational use of medicinal plants? 3. What are the current scenarios of treatment strategies? What are the reasons behind failure of current therapy? 4. Why there is need of active plant constituent’s loaded formulations? What is the role of two active plant constituent’s selected to make rational combination? 5. Will janus nanosized emulsion of these active plant constituents solve the problem? What studies to be required to do make it successful?

4. Objectives of the Study

Objectives:

1. Awareness programme for the the sensitization and awareness about the use of medicinal plant in local communities of NE India. 2. Strategies to enforce the rational use of herbal products and medicinal plants in NE India. 3. Analytical method development and validation of HPLC method for determination of NIP/18/22 4. Conduction of pilot scale formulation development works using the plant-derived solid powder and the selected nanoparticulate drug carrier systems 5. In-vitro characterisation of drugs loaded janus nanosized emulsion and In-vivo pharmacokinetic studies and Pharmacodyanamic studies

5. Study Area (max. 150 words) The prevalence of RA is seen more in women than in man. There is a striking imbalance between the sexes, with females representing the majority of autoimmune disease cases. Thus, women are more affected than men; it is also true for RA, where the sex ratio is typically around 3:1. The mortality rate of RA patients is twice more than compared to that of the normal population. The estimate of the worldwide prevalence of RA was published as part of the Global Burden of Disease 2010 study. The global prevalence of RA in patients, from 5 to 100 years of age, in the year 2010 was estimated to be 0.24% with 95% CI (0.23%– 0.25%).

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6. Methodology Adopted a) Preparation of Janus nanosized emulsion The previously used method as described in the patent document (Indian Patent No. 201711034483, published on 23-11-2018) was followed with slight modification to prepare Janus nanosized emulsions. Olive oil and silicone oil were mixed with slight heating of 50- 55º C. Tween 80 was mixed with water at the same temperature condition in a separate beaker. Then the oil phase was slowly added to the water phase and stirred for 15 min using a magnetic stirrer. The formed crude emulsion mixture was homogenized for 10 min at 15000 rpm using a polytron high-speed mixer. b) Photomicrograph of Janus particles Using an optical microscope fitted with internal software to view images on the desktop display, the droplets of emulsion were visualized (Fig 1). The presence of double-faced head or bicompartmental structures in the dispersed oil droplets of the emulsion indicated the formation of Janus nanosized emulsion.

c) Analytical method development and validation

The Thermo Scientific UHPLC system consisted of a quaternary pump, auto sampler and UV detector. The resolution and better peak shape of analytes were achieved on a hypersil C18 column (4.6 × 250 mm, particle size 5 µm). A 20 µL aliquot of each sample was injected into the HPLC system. The system was analysed in isocratic mode with a mobile phase consisting of 10 mM sodium acetate buffer (pH 5.5) : methanol (62: 38, v/v) at a flow rate of 1.0 mL/min. Mobile phase was filtered through 0.22 µm membrane filter and degassed

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ultrasonically for 15 minutes prior to use. The absorption wavelength was set to 257 nm for NIP/18/22 (Chhonker et al 2015).

Currently the HPLC development work is going on in our laboratory.

7. Research Findings

Objectives Objective/s Deliverables Methodolog Achievement so y adopted far

1 Sensitization and  Documentatio Awareness We have conducted awareness about n of Phytochemical programmes for awareness medicinal plant among and awareness the rational use workshops in Himalayan inhabitants programmes in 10 of medicinal selected places in NE for their rational use. villages for livelihood plants have been India. These type of options. conducted awareness  Database on progrmmes will be high value Medicinal further continued for plants and their the benefits of local traditional use from the inhabitants of NE selected villages of the India. region.

2 Analytical method Analytical RP- work is going on in development and HPLC method our laboratory validation of HPLC method for determination of NIP/18/22

3 Preparation of Janus Prototype formed high shear Accomplished nanosized emulsion homogenization method

4 In-vitro Particle size along characterisation Janus with zeta potential

nanosized emulsion Dynamic light determination is accomplished. scattering Microscopy method accomplished.

8. Future Plan Development of database on high value medicinal plants and their traditional use from the various villages of the region and conducting awareness programmes for the rational usage of medicinal plants in NE India. 9. References 1. Kumar, Vikas, Santosh K. Guru, Shreyans K. Jain, Prashant Joshi, Sumit G. Gandhi, Sandip B. Bharate, Shashi Bhushan, Sonali S. Bharate, and Ram A. Vishwakarma. "A NMHS Fellowship Grant Progress Page 81 of 122

chromatography-free isolation of rohitukine from leaves of Dysoxylum binectariferum: Evaluation for in vitro cytotoxicity, CDK inhibition and physicochemical properties." Bioorganic & medicinal chemistry letters 26, no. 15 (2016): 3457-3463. 2. Lakdawala, A. D., M. V. Shirole, S. S. Mandrekar, and A. N. Dohadwalla. "Immunopharmacological potential of rohitukine-a novel compound isolated from the plant Dysoxylum-binectariferum." Asia Pacific Journal of Pharmacology 3, no. 2 (1988): 91-98. 3. Chhonker, Yashpal S., Hardik Chandasana, Ashok Kumar, Deepak Kumar, Tulsankar Sachin Laxman, Sunil Kr Mishra, Vishal M. Balaramnavar, Shishir Srivastava, Anil K. Saxena, and Rabi S. Bhatta. "Pharmacokinetics, tissue distribution and plasma protein binding studies of rohitukine: a potent anti-hyperlipidemic agent." Drug research 65, no. 07 (2015): 380-387.

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Annexure 9

IDENTIFICATION AND COLLECTION AND QUANTIFICATION OF 5 MOST ENDANGERED PLANTS WITH MEDICINAL POTENTIAL IN REGION

Abstract North east region of India is one of major biodiversity hotspot, rich in several plant species with known medicinal activities. The ethnic communities of North East region are rich in traditional knowledge. This helps these people to cure many diseases as so many remote areas are not able to get facilities. However, repeated uses of these plants have caused their existance in threat and few of them have been categorized in endangered category. To reveive and explore their medicinal properties, we have collected NIP/18/08 and NIP/18/24 for research purpose. One of the plant falls under the genous Clerodendrum, which is also one of the medicinally important plant genus found in the NE India. Plants of these species have been reported to possess several medicinal activities. A variety of constituents have been isolated and characterized from leaves, roots, and stems of the clerodendrum species used as folk and traditional medicine to treat many kinds of diseases, such as cold, hyperpyrexia, asthma, furunculosis, hypertension, rheumatism, , mammitis, toothache, anorexia, leucoderma, leprosy, arthrophlogosis, and other inflammatory disease in various parts of the world such as India, China, Kore Japan, , and Africa. Hence, this project aims at the antimicrobial activities of the selected medicinal plant of the genus Clerodendrum found in North east India. Extraction was performed with various solvents and testing was performed for the preliminary antimicrobial activity of the plant against E.coli, S aureus and Gentamicin resistant MRSA bacterial species.

Introduction India is one of the mega biodiversity hotspots of the world. Presently, it has a rich vegetation of more than 45,000 plant species, of which 15,000-20,000 plants have known medicinal activity. Within the country, a major portion of these plants are found in North-East India. NE India comprised of eight states namely Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Tripura and Sikkim and supports 50% of India’s biodiversity. The ethnic communities of North East region are rich in traditional knowledge. These peoples are still practicing traditional medical approaches to cure diseases with the use of natural products found in these parts of the country. Medicinal uses like antipyretic, anti-inflammatory, anti-helminthic, antiviral, antibacterial, antidiarrheal, antioxidant have been some of the prominent activities found in the natural products used by the ethnic tribes of NE India. Clerodendrum is also one of the important plant genus found in the NE India. Plants of these species have been reported to possess activities such as anti-inflammatory, anti-nociceptive, anti-oxidant, anti-hypertensive, anticancer, antimicrobial, anti-diarrheal, hepatoprotective, hypoglycemic, hypolipidemic, memory enhancing, neuroprotective, and other activities as well. Therefore, we have selected one of the newly reported plant species of this genus for our study.

Literature review Clerodendrum is a genus of flowering plants belonging to the family Lamiaceae (Verbenaceae)1. It is native to tropical and warm temperate regions of the world, with most of the species occurring in tropical Africa and southern Asia, but with a few in the tropical America and northern Australia, and a few extending north into the temperate zone in eastern Asia2.

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Plants belonging to genus Clerodendrum are well known for their pesticidal properties, and various Clerodendrum species like C. indicum, C. phlomidis, C. serratum var. amplexifolium, C. trichotomum, C. chinense, C. petasites, etc. have been historically used as folk and traditional medicine to treat many kinds of diseases, such as cold, hyperpyrexia, asthma, furunculosis, hypertension, rheumatism, dysentery, mammitis, toothache, anorexia, leucoderma, leprosy, arthrophlogosis, and other inflammatory disease in various parts of the world such as India, China, Kore Japan, Thailand, and Africa3. A variety of constituents have been isolated and characterized from leaves, roots, and stems of the clerodendrum species and they are monoterpene and its derivatives, sesquiterpenes, diterpenoids, triterpenoids4,5, flavonoid and flavonoid glycosides6, phenylethanoid glycosides7, steroids and steroid glycosides8, cyclohexylethanoids, anthraquinones, cyanogenic glycosides, and others. Some of these constituents have been evaluated with a number of biological properties, mainly including anti-inflammatory and anti-nociceptive, anti-oxidant9, anti-hypertensive, anticancer, antimicrobial, anti-diarrheal, hepatoprotective, hypoglycemic and hypolipidemic, memory enhancing and neuroprotective, and other activities.

Research questions

  Which part of the selected medicinal plant of the genus Clerodendrum is biologically active?  Which solvent fraction shows maximum efficacy in various in-vitro screening models?  Which extract shows maximum efficacy as antimicrobial agent?

Aim & objectives

Aim: To test the antimicrobial activities of the selected medicinal plant of the genus Clerodendrum found in North east India.

Objectives:

1. To develop database on quantification of available resources for selected species. 2. To develop harvesting guidelines for sustainable supply of medicinal plants. 3. To evaluate the anti-microbial activity of prepared plant extracts. 4. To collect, authenticate and process for extraction of the selected medicinal plant of the genus Clerodendrum found in North east India.

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Experimental design

Anti-microbial activities

Work done so far:

Objective 1: To collect, authenticate and attain the extracts from medicinal plants.

NIP/18/08 was collected and it was washed and dried under shade for 15days. The dried plant materials were ground to coarse powder with the help of MF grinder drive. The powder is collected and it is used for further successive solvent extraction based upon polarity. The extraction process is done by hexane, chloroform, ethyl acetate, methanol, hydro alcohol (1:1). Later solvent recovery is done by Rota vapour followed by lyophilization. The yielded crude dry extracts were stored in 2oC - 8oC refrigerator and used for further studies.

Table 2: Percentage yield of NIP/18/08 extracts

Sr.no Extract name Percentage yield of Percentage yield NIP/18/08 Leaves NIP/18/08 Flowering Parts 1 Chloroform extract 0.9% 2.3% 2 Ethyl acetate extract 1.4% 2.8% 3 Alcoholic extract 2.74% 3.7% 4 Hydro alcoholic extract 4.2% 5%

Objective 2: To test the preliminary antimicrobial activity of the plant against E.coli, S aureus and Gentamicin resistant MRSA bacterial species.

Principle: The methodology of antibacterial activity studied in this work is based on the Zone of Inhibition. Cups cut out of the agar are filled with appropriate standard and test solutions. Then, the diffusion of the solutions takes place into agar. So that after incubation, cups are found to be

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surrounded by circular zones of inhibition in which the organism has failed to grow owing to the antibacterial action of the standard and test solutions.

Methodology: Agar plates were prepared by the autoclaved nutrient agar medium and the plates were incubated overnight. Mid log phase bacterial culture of Escherichia coli was diluted to a bacterial count of 3 × 107 CFU/mL) in phosphate buffer saline solution. 100 µL of bacterial suspension was spreaded over agar plates and 4 holes are cut in the agar by means of a sharp 10mm cork borer. 50µL of plant extracts and standard solution were added into the cups and incubated for 16 hours at 37°C.

Results

a) Table 1: Evaluation of Antimicrobial assay for endangered plant (NIP/18/08) extracts against Escherichia coli, Staphylococcus aureus and Gentamicin resistant MRSA bacterial species.

S. Drug/Extract Conc. Zone of Inhibition in cm no (µg/ml) E S aureus Gentamicin coli resistant MRSA 1 Streptomycin 10 1.5 1.7 1.4

2 NIP/18/08/Leaf/Chloroform 1000 NO NO NO

3 NIP/18/08/Leaf/ Ethylacetate 1000 NO NO NO

4 NIP/18/08/Leaf/Ethanol 1000 NO NO NO

5 NIP/18/08/Leaf/Hydro- 1000 NO NO NO alcohol

6 NIP/18/08/Flower/Chlorofor 1000 NO NO NO m

7 NIP/18/08/Flower/Ethyl 1000 NO NO NO acetate

8 NIP/18/08/Flower/Ethanol 1000 NO NO NO

9 NIP/18/08/Flower/Hydro- 1000 NO NO NO alcohol

* We have collected and described in details about 2 endangered medicinal plants as noted in Annexure 2

REFERENCE

1. Kubitzki, K., Rohwer, J. G. & Bittrich, V. Date of publication: 28.7.1993 Volume III Flowering Plants. : Lilianae (except Orchidaceae) Edited by K. Kubitzki (1998) Date of publication: 27.8.1998 Volume IV Flowering Plants. Monocotyledons: Alismatanae and Commelinanae (except Gramineae. (1993). 2. Mabberley, D. J. Mabberley’s Plant-Book: A Portable Dictionary of Plants, their NMHS Fellowship Grant Progress Page 86 of 122

Classification and Uses: Third Edition. 3. Baker, J. T. et al. Natural product drug discovery and development: new perspectives on international collaboration. J. Nat. Prod. 58, 1325–57 (1995). 4. Xu, R.-L., Jiang, H.-L., Wang, R., Shi, Y.-P. & Assoc Huan-Yang Qi, by. DIVERSE TERPENOIDS FROM THE LEAVES OF Clerodendrum trichotomum. Chem. Nat. Compd. 51, (2015). 5. Pandey, R., Verma, R. K. & Gupta, M. M. Neo-clerodane diterpenoids from Clerodendrum inerme. doi:10.1016/j.phytochem.2004.11.007 6. Sinha, N., Seth, K., Pandey, V., Dasgupta, B. & Shah, A. Flavonoids from the Flowers of Clerodendron infortunatum. Planta Med. 42, 296–298 (1981). 7. Chae, S., Kang, K. A., Kim, J. S., Hyun, J. W. & Kang, S. S. Trichotomoside: A New Antioxidative Phenylpropanoid Glycoside fromClerodendron trichotomum. Chem. Biodivers. 3, 41–48 (2006). 8. Kanchanapoom, T., Kasai, R., Chumsri, P., Hiraga, Y. & Yamasaki, K. Megastigmane and iridoid glucosides from Clerodendrum inerme. 9. Ali, M. et al. Selected hepatoprotective herbal medicines: Evidence from ethnomedicinal applications, animal models, and possible mechanism of actions. Phyther. Res. 32, 199–215 (2018)

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ACTIVE PLANT CONSTITUENTS ENRICHED NANOSTRUCTURED LIPID CARRIERS AGAINST ORAL CANCER MITIGATION IN ASSAM

Abstract

Oral squamous cell carcinoma (OSCC) is one of the most leading cancers observed in the north-eastern region (NER) of India. Quercetin and piperine, (an active plant constituents) were selected to mitigate oral cancer burden in NER specially Assam. Initially, a modern analytical method for the simultaneous detection of these two active plant constituent mixtures were successfully developed and validated through RP-HPLC method as per regulatory ICH criterion. The retention time of both quercetin and piperine were found to be at 2.82 and 10.98 min, respectively. After successful validation, we further developed nanostructured lipid carrier (NLCs) by solvent evaporation mediated cold high pressure homogenization method followed by ultrafiltration technique. Compritol 888 ATO as solid lipid and squalene as liquid lipid were selected herein to solubilise the active natural constituent’s in order to formulate well designed NLCs. Four major formulations namely, blank NLCs, Piperine-loaded NLCs, Quercetin-loaded NLCs and combinational quercetin and piperine loaded NLCs were prepared with the earlier adopted method preparation the design of NLCs. Particle size for all NLCs were found within nano- range (<200±20nm) with a negative zeta potential value which reflects the stability of the prepared NLCs dispersion. Entrapment efficiency and other characterization are in progress. Further in vitro cellular evaluation and in vivo animal experimentation will be done in a due course of project tenure.

1. Introduction

Cancer is one of the most lives threatening disease in the world. Being a multifactorial type of disease, oral cancer is ranked 6th in that list of cancers. It is classified under the class of head and neck cancer (HNC)[1]. It has been observed that continuous and chronic use of tobacco, areca nut and betel quid leads to cause precancerous lesion in oral cavity. Most affected areas are oral squamous epithelial cells, tongue, gingiva, jaw bone and lips. The oral squamous cell carcinoma (OSCC) is the cancer of epithelial tissue of oral mucosal linings of oral cavity. It accounts for 90% of all head and neck cancer cases. Early stage symptoms are not able to detect is the main prognostic factor for in this case.

Herbal compounds have been used widely for treatment of various diseases either alone or in combination of two or more drugs. These drugs have their own properties with potential to cure so many diseases with lesser side effects. Like synthetic drugs, herbal medicines have been used through various route of administration like dermal, oral, inhalational etc. Combinations of drugs have been used to treat a disease for synergistic effect or to counteract the side effect of one drug.

Quercetin, naturally occurring potent anti-oxidant obtained from various edible sources such as red onion, apple, tomato, red grapes, green and black tea, and green leafy vegetables. Quercetin showed dose dependant cell cycle arrest with inhibition at G1 and S phase along with decreased expression of cyclin D1 in SCC 25 cell lines. In SAS cell lines, quercetin showed to inhibit PIK3 and MAPK pathways with reduction of expression of metalloproteinases[2]. Second drug, piperine is obtained from fruits of black pepper and long pepper. It comes from the category of spices. Studies on anti-cancer activity of piperine in oral cancer KB cell lines had shown to modulate cellular content of DNA with the G2/M phase arrest of cell cycle, along with generation of reactive oxygen species (ROS)[3]. Drugs, quercetin and piperine have been explored widely in cancer cell line studies. These drugs had proved their anti-cancer potential especially in oral cancer. They have NMHS Fellowship Grant Progress Page 88 of 122

shown to inhibit cell proliferation with their cytotoxic potential at very less concentration. Quercetin causes dysfunction mitochondria in cancer cell while piperine inhibits cell cycle at nuclear level. This shows quercetin acting in cytoplasm and piperine inside the nucleus. Both drugs act on different pathways in prognosis of oral squamous cell carcinoma, which may give rise to new combination drugs from natural origin as a treatment. This gives rationality to this combination in treating oral cancer.

2. Literature review

Table 1 Literature review

Sr. Reference Year Major objective Methodology Findings No. adopted

1. Yi-Shih 2018 Quercetin induced Cell morphology 40 μM of quercetin Ma et apoptosis of human and viability for 0, 12, 24 and 48 al[2] oral cancer SAS cells assays. h causes changes in through mitochondria cell morphology and endoplasmic Measurement of and decrease cell reticulum mediated reactive oxygen viability signaling pathways species (ROS), Quercetin increases Western blotting the activities of analysis. caspase-3, caspase-8 and caspase-9 in SAS cells along with mitochondrial dysfunction

2. Mitali 2017 Epidemiological Survey 3rd most cancer Dandekar survey of head and burden in India et al[4] neck cancer in India along with factors causing it

3. ICMR 2017 A Report on Cancer Case Report A complete report report Burden in North on population Eastern States of India suffering from OSCC along with statewise distribution in North eastern India

4. Sahabjada 2017 Piperine Triggers MTT Assay for Significant Siddiqui Apoptosis of Human Cell Viability, reduction in et al[3] Oral Squamous viability of KB cell Carcinoma Through lines at lower conc, Cell Cycle Arrest and Piperine Induces Mitochondrial piperine Nuclear demonstrated NMHS Fellowship Grant Progress Page 89 of 122

Oxidative Stress Condensation apoptotic cell death

in KB cells in a dose-dependent Assessment of manner as Mitochondrial evidenced Membrane Potential (DC) by the increased permeability of cells to DAPI showing

nuclear apoptotic bodies and condensed chromatin.

5. Jiaping 2014 Mechanisms of Research Thorough study Xue et Cancer Induction by regarding induction al[5] Tobacco-Specific of oral cancer by nitosamines NNK and NNN

6. Guo 2012 Development of a Emulsion Increased retention Chen-yu Quercetin-loaded of Quercetin NLCs et al[6] nanostructured lipid evaporation– in the skin, carrier formulation for solidification at suppression of low temperature edema, retention of topical delivery radical scavenging activity after encapsulation

7. Sunil 2008 Study of Review Effects of tobacco, Kumar et carcinogenesis areca nut, betel quid al[7] induction due to and other ingredients of Paan ingredients in Masala and other causing oral sub factors mucuoasal fibrosis and leukoplakia

3. Research Questions

Several things to be taken into consideration regarding the selection of the disease, its treatment, population affected, area to be focused, strategies to be made etc. These research questions were raised while reviewing literature as follows:

1. What are the diseases is of more concern in north east region of India? What is its prevalence and pathophysiological condition? 2. What are the current scenarios of treatment strategies? What are the reasons behind failure of chemotherapeutics?

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3. Why there is need of active plant constituent’s enriched formulations? 4. What is the role of two active plant constituent’s selected to make rational combination? 5. Will NLCs of these active plant constituent’s solve the problem? 6. What studies to be required to do make it successful?

4. Objectives of the Study

As north east region of India is very diverse in plant species and rich in herbal medicines which had been used by native population since so long time. Using potential of natural herbal medicines in treatment of dangerous death causing diseases like cancer is a new research area in pharma industry. In order to overcome side effects of chemotherapeutic drugs used in treatment of oral squamous cell carcinoma (OSCC) along with replacing them by is a principle behind this research. Developing a nanoformulation loaded gel of combination of herbal drugs having activity against oral squamous cell carcinoma along with its characterization is main objective of the project.

Objectives:

1. Analytical method development and validation of HPLC method for determination of quercetin and piperine 2. Formulation & optimization of combination of piperine & quercetin loaded nanostructured lipid carriers 3. In-vitro characterisation drugs loaded NLCs formulation 4. In-vitro evaluation of NLCs in cell lines 5. Transformation of NLCs into NLCs loaded gel formulation for topical application 6. Characterisation of NLCs loaded gel formulation 7. In-vitro release studies along with ex-vivo permeation studies 8. In-vivo pharmacokinetic studies 9. IVIVC study

5. Study area

Oral squamous cell carcinoma (OSCC) comes under the class of head and neck cancer. It accounts for about 90% of all head and neck cancer cases. As per 2014 report from World Health Organization (WHO), 53842 cases in males and 23161 cases in females were found in India. This also showed mortality rates of 18.3% in males and 6.8% in females caused because of mouth and oropharynx cancer. According to the recent survey of Indian Council of Medical Research (ICMR), Bengaluru, more number of cases of oral cancer was observed in North east states of India as compared with rest of other states. This comparison showed that five year cumulative survival rate in early stages of HNC are 73.6 % and 40.5% and in locally advanced stages it drops to 44.5% and 16.9% in rest of India and north east India respectively. The detection of oral cancer in later stages III or IV is the significant reason for very less survival rate in case of OSCC. Many districts in north east of India showed large number of Population Based Cancer Registries (PBCRs) are Kamrup urban district, Cacchar district, East Khasi Hills district, Dibrugarh district (Assam state), Meghalaya state, Papumpare and Pasighat district (Arunachal Pradesh state), Aizwal district (Mizoram state) etc.

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6. Methodology Adopted

A) Analytical method

Selection of analytical wavelength

The ultraviolet spectrum for both quercetin and piperine was obtained individually for drugs in methanol, as these drugs are more soluble in it. Primary standard solution was prepared with pure drug dissolved in methanol (1000µg/mL) and it was used for further dilutions to prepare secondary stock solutions. Aliquots of these drugs having concentration 10µg/mL were taken in 1 mL cuvette made up of quartz and were scanned for spectrum showing wavelength with maximum absorption (λmax) in the range190-800 nm of UV-Visible light. Methanol was used as blank solution as drugs were dissolved in it in a double beam UV spectrophotometer. Finally, the overlay of UV absorption spectrum for both drugs was obtained to determine the isobestic wavelength for maximum absorption of both drugs.

Fig.1 UV-Vis absorption spectrum of Quercetin and piperine

Instrumentation

An analytical ultrahigh performance liquid chromatography (uHPLC) with a pump model 3000 and UV/Visible detector (Thermo-scientific technologies limited) was used. The system with Chromeleon software was used for controlling the instrument parameters. A C18 column (Hypersil, 150mm×4.6mm, 5µm particle size) was used throughout the analysis procedures. Column temperature controlled with oven which can accommodate column inside the chamber.

Chromatographic conditions

A reverse phase chromatographic technique with isocratic elution mode was used for analytical study. The mobile phase constitutes of Acetonitrile and 2% acetic acid in water with pH adjusted to 2.6, in the ratio of 40:60, v/v. Before use, mobile phase was filtered through membrane filter 0.45µm range equipped with vacuum filtration assembly and then sonicated in bath sonicator for 20 min to remove soluble gases which may cause problems in analysis process. The flow rate of mobile phase in defined ratio was kept constant at 1mL/min with injection volume of 20µL. The

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column temperature was maintained at 35± 0.2 oC with column oven .After purging, equilibration was performed with mobile phase for stable baseline, and then sample was injected from glass vial. Run time for the single elution was kept at 15 min. The absorbance measurement was performed at maximum wavelength of 346nm obtained as isobestic from overlay spectrum of both drugs.

Sample preparation

Primary stock solution of 1000µg/mL for both quercetin and piperine were prepared in methanol. These solutions were covered with aluminium foil and stored in refrigerator at 4oC. Secondary standards were prepared with proper dilution of primary standard solutions.

Assay validation

According to International on Harmonization Conference (ICH) guidelines for validation of analytical procedures, method validation was performed for simultaneous estimation of quercetin and piperine with HPLC. Validation of assay was performed with respect to linearity, range, precision, accuracy/recovery, selectivity/specificity, sensitivity, system suitability parameters etc. Analysis of variance (ANOVA) test was applied in order to verify the validity of this method.

Linearity and range

The linearity study determines concentrations of sample are in a range, so that in this range the analytes response is linearly proportional to its concentration. Calibration curves with 7 different drug concentrations were obtained with specified HPLC method conditions and parameters. Accurately weighed 10mg of quercetin and piperine was dissolved in 10mL of methanol separately to prepare the primary stock solution of 1000µg/mL. After diluting the above solutions, working standard solutions were prepared. Working standard solutions were prepared as 2, 4, 8, 16, 32, 64, 128 µg/mL for quercetin and 1, 2, 4, 8, 16, 32, 64 µg/mL for piperine. Solutions were made in triplicate and then injected. Calibration curve was plotted as concentration (µg/mL) against AUC (mAU*min). Linearity was evaluated by linear regression using ANOVA.

Precision

The precision is the degree of closeness of experimental results between a series of measurements obtained from multiple analysis of the same homogenous sample under the prescribed similar conditions. Precision of the method was performed with determination of instrumental precision, repeatability (intra-day precision), intermediate precision (inter-day precision) and was expressed as %RSD (relative standard deviation). Instrumental precision was performed in 6 replicate injections of sample of 50 µg/mL of quercetin and piperine each. Standard deviation along with relative standard deviation were calculated, which should be around 2% considered as acceptable. In repeatability studies, 3 concentration levels were evaluated for analysis which includes low, medium and high level quality control samples. Low level 2, medium level 16 and high level 128µg/mL for quercetin and low level 1, medium level 8 and high level 64µg/mL for piperine were injected in triplicates on the same day for intraday precision and three different samples of these concentrations on three consecutive days for interday precision under the similar experimental conditions. For these samples, standard deviation and relative standard deviation were calculated.

Accuracy/recovery

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Accuracy of an analytical method is the degree of closeness of measured values to the true values. In order to determine the accuracy of this analytical method, percentage recovery of the known amounts of quercetin and piperine is used as a key determinant. Samples of known concentrations of low, medium and high quality control levels i.e. 2, 16 and 128 µg/mL for quercetin and 1, 8, 64µg/mL for piperine respectively. These samples were prepared in triplicates. Accuracy was determined by comparing the results of these samples with nominal concentrations.

Sensitivity

Limit of detection (LOD) and limit of quantitation (LOQ) were used for determination of sensitivity of the analytical method. These parameters were determined according to method described by ICH guidelines. Slope of the calibration curve (S) and standard deviation of the intercept of calibration curve (σ), using equation LOD=3.3× (σ/S). Limit of quantitation was calculated from equation LOQ=10× (σ/S). Both LOD and LOQ parameters were calculated with the help of calibration curves of quercetin and piperine.

System suitability

In accordance with Ep, the system suitability test was performed which confirms proper working of the equipment to perform analysis. The test was performed with injections of standard solution of 50µg/mL of quercetin and piperine in the replicates of six, which were analysed for their peak area, retention time for drugs (tR), tailing factor (T), peak resolution (R), theoretical plate (N) along with capacity factor (K) etc.

Application of the method

The validated HPLC method was further used for simultaneous estimation of quercetin and piperine from both drugs loaded nanostructured lipid carrier formulations. The drug release studies were performed for NLC formulation, and as samples were prepared according to the method and then analysed.

B) Preparation of Nanostructured lipid carriers (NLCs)

Nanostructured lipid carriers were prepared by solvent evaporation emulsification method at room temperature with high shear homogeniser (HSH). Initially weighed quantity of solid lipid, Compritol 888 ATO was dissolved in certain amount of Chloroform till solution becomes clear. Liquid lipid i.e. squalene was added to the mixture along with surfactant Span 80 and piperine to make lipid drug mixture. Second drug, Quercetin was solubilized in acetone and then slowly transferred to previous mixture with vortexing to get clear yellow coloured solution. Separately cold water with tween 80 was mixed and kept for homogenization. Drug lipid mixture was added dropwise to aqueous phase under high shear homogeniser at 15000 rpm till complete evaporation of chloroform in order to get piperine and quercetin loaded NLCs. NLCs dispersion was filtered through 0.2µm filter under cooling centrifuge at 4o C with 3500 rpm for 30 min. Then final NLC dispersion was obtained with cut off particle size of 200nm. It was further characterized for several properties.

Individually blank NLCs, Piperine loaded NLCs, Quercetin loaded NLCs and both quercetin and piperine in combination loaded NLCs were prepared with this optimized method of preparation for NLCs.

Formulations Solid Liquid Quercetin Piperine Surfactant Solvents Water

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Lipid(mg) Lipid(µL) (mg) (mg) % w/v mL

Blank NLCs 400 300 - - 0.1 Chloroform 20

Piperine loaded 400 300 - 20 0.1 Chloroform 20 NLCs

Quercetin 400 300 10 - 0.1 Chloroform, 20 loaded NLCs Acetone

Quercetin and 400 300 10 20 0.1 Chloroform 20 Piperine loaded NLCs

Table 2 Components of NLCs formulation

C) Characterization of NLCs

Particle size, PDI and zeta potential

The mean diameter of prepared NLCs was measured with the help of photon correlation spectroscopy using particle size analyser (Zetasizer; Malvern instruments Ltd.) at a fixed angle of 90o with He-Ne laser of 633nm at ambient room temperature. Evaluation of particle size was done with volume distribution. The zeta potential was determined with the help of microscopic electrophoresis system at room temperature.

Differential scanning calorimetry

In order to determine the presence of any crystal form of active plant constituents in lipid matrix, Differential Scanning Calorimetry (DSC) studies was performed with DSC instrument of METTLER TOLEDO. Aluminium pans were used for recording of thermograms. An empty pan was used as a reference. Around 3-5mg of sample was placed in sample pan and crimped with crimping machine for tight sealing of pan. Heating rate was kept constant at10o/min within the range of room temperature (25oC) to 500oC. Nitrogen purging was performed at 20mL/min flow rate to maintain stable inert atmosphere.

Results and discussion

Method Validation

Linearity and range

The standard calibration curve was found to be linear over the concentration range of 2-128 µg/mL and 1-64 µg/mL for quercetin and piperine respectively. Correlation coefficients (R2) obtained from linear regression analysis was 0.9982 for quercetin and 0.9996 for piperine. Equation of the calibration curve based mean peak and concentration (µg/mL) for quercetin and piperine were y = 0.6218x - 0.7915 and y = 1.9524x - 1.1863 respectively.

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Fig.2 Calibration curve for quercetin Fig.3 Calibration curve for piperine

Precision

Precision studies for analytical method were performed under the same instrumental conditions includes instrumental, intra-day and inter-day precision and were expressed as their corresponding percent relative standard deviation (% RSD). For precision, relative standard deviation less than 2% was considered as acceptable. Lower values of % RSD showed low variability between the sample concentrations.

Table 3 Intraday precision values for proposed method

Analytes Nominal Intraday concentration precision level (µg/mL) (Repeatability) %R.S.D.

Quercetin 2 1.15

16 1.48

128 0.50

Piperine 1 0.87

8 1.81

64 0.96

Table 4 Interday precision values for proposed method

Analytes Nominal % R.S.D. of Interday concentration precision (Intermediate)

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(µg/mL) Day-1 Day-2 Day- 3

Quercetin 2 1.15 4.84 2.50

16 1.06 1.48 1.24

128 0.50 0.59 2.83

Piperine 1 1.23 2.10 0.87

8 1.35 1.06 1.81

64 2.35 2.77 0.96

Accuracy/recovery

Results for accuracy of proposed method are shown in table 3. Recovery ranged between 102.04 – 146.42% for quercetin and 97.88 – 139.71% for piperine with R.S.D. less than 3%. This showed that proposed method is accurate for the simultaneous estimation of both drugs.

Table 5 Accuracy of proposed method determined according to ICH Q2

Analytes Nominal Found % Mean ±SD(n=3) % concentration concentration Recovery Accuracy R.S.D. (µg/mL) (µg/mL) (n=3)

Quercetin 2 2.9 145.55

2 2.97 148.36 146.42 1.69 1.15

2 2.91 145.34

16 18.13 113.32

16 17.82 111.35 111.57 1.65 1.48

16 17.61 110.05

128 128.20 100.15

128 130.22 101.73 102.55 2.90 2.83

128 135.39 105.78

Piperine 1 1.41 141.00

1 1.39 139.53 139.71 1.21 0.87

1 1.40 139.53

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8 7.99 99.87

8 7.78 97.30 97.88 1.78 1.81

8 7.72 96.47

64 70.71 110.49

64 69.58 108.72 109.93 1.05 0.96

64 70.78 110.59

Sensitivity

According to the formula, limit of detection (LOD) was found to be 90ng/mL and 65ng/mL for quercetin and piperine respectively. In similar way, the limit of quantitation (LOQ) values was obtained as 300ng/mL and 200ng/mL for quercetin and piperine respectively. These values are adequate for accurate and precise detection and quantitation of quercetin and piperine.

System suitability

Various system suitability parameters like retention time (Rt), peak resolution (R), theoretical plate (N), effective plate (E), capacity factor (K) along with tailing factor (T) were determined for both drugs quercetin and piperine. The sample of 50(µg/mL) concentration of each drug was injected for determination of system suitability parameters. Their values were compared with required limits shown in the table. And finally proposed method showed to fulfil requirement being in accepted limits.

Table 6 System suitability parameters

Analytes Conc. % R.S.D. Peak Theoretical Capacity Tailing (µg/mL) resolution plate (N) factor factor Retention Peak (R) (K) (T) time area

(Rt-min)

Quercetin 50 2.82 30.508 4.94 11050 0.88 1.20

Piperine 50 10.73 84.250 2.13 13649 6.15 1.02

Required R>2 N>2000 1-10 T<1.5 limits

Application of method

In order to estimate two drugs quercetin and piperine simultaneously from drugs loaded into nanostructured lipid formulations. A chromatogram of drugs analysed from NLC formulation is presented. Chromatogram shows both peaks are well resolved at a single wavelength of 346nm.

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Quercetin itself showed maximum absorption at 369nm while piperine at 329nm. Method allowed determining both drugs at a single wavelength with maximum absorption with UV detection system. Entrapment efficiency was calculated for both drugs in the nanostructured lipid carriers and are represented in table.

Fig. 4 Chromatogram showing peak for standard drug solution at 346nm, 1) Quercetin 2) Piperine

Formulation development

Nanostructured lipid carriers were prepared with solvent evaporation high shear homogenization method for combination of quercetin and piperine drugs along with blank and individual drugs. The formulation consists of following components:

Table 7 Components of NLC formulation

Sr.No. Components Blank Piperine Quercetin Piperine and NLCs NLCs NLCs Quercetin NLCs

1. Compritol 888 ATO (Solid 400 400 400 400 lipid), mg

2. Squalene (Liquid lipid), mg 250 250 250 250

3. Piperine, mg - 20 - 20

4. Quercetin, mg - - 10 10

5. Surfactants, drops 1 1 1 1

6. Water, mL 25 25 25 25

Values of particle size and zeta potential measure with Zetasizer instrument for each formulation have mentioned in Table 8 in triplicates. The size was found to be well below 200nm with PDI values less than 0.3 i.e. in well accepted limits.

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Sr. No. Formulation Particle Avg. SD PDI Zeta potential size (nm) (nm) (mV)

1 Blank NLC 1 127.8 129.7 1.76 0.194 -0.47

2 Blank NLC 2 129.9 0.183 -0.686

3 Blank NLC 3 131.3 0.161 -2.53

4 Piperine NLC 1 141.5 153.3 14.96 0.182 -1.21

5 Piperine NLC 2 148.2 0.25 -0.889

6 Piperine NLC 3 170.1 0.23 0.329

7 Quercetin NLC 1 148.1 157 8.71 0.14 -3.22

8 Quercetin NLC 2 157.4 0.128 -1.03

9 Quercetin NLC 3 165.5 0.061 1.89

10 Quer + Pip NLC 196.8 179.0 15.49 0.186 0.159 1

11 Quer + Pip NLC 171.9 0.096 -2.43 2

12 Quer + Pip NLC 168.4 0.141 0.195 3

DSC results

Differential scanning calorimetry study was performed with METLER TOLEDO instrument. Thermogram for crushed Compritol 888 ATO showed sharp endotherm at melting temperature of 73-74oC. Thermograms for APIs showed sharp endotherm of melting of drugs while in case of lyophilised NLCs the sharp endotherm at melting temperature of APIs is absent which showed that drug is completely encapsulated inside the matrix in amorphous form. No endotherm or exotherm was observed in blank NLCs as the lipid has already left its crystalline nature in formulation.

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Fig. 5 Themogram of Crushed Compritol 888 ATO

Fig. 6 Thermogram of Piperine API

Fig. 7 Thermogram of Quercetin API

Fig. 8 Thermogram of Blank lyophilised NLCs

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Fig. 9 Thermogram of Blank lyophilised NLCs

Research Findings Table 9 Research findings of the project Sr. Objective/s Deliverables Methodology Achievement so far No. adopted

1. Analytical method Thermo Accomplished development and validation Scientific of HPLC method for Analytical determination of quercetin HPLC and piperine instrument

2. Formulation & optimization Solvent Accomplished of combination of piperine evaporation & quercetin loaded high shear nanostructured lipid carriers homogenization method

3. In-vitro characterisation Particle size Particle size along with drugs loaded NLCs analyser zeta potential formulation determination is (Zetasizer) accomplished.

Morphological study and entrapment efficiency determination is remained.

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Future plan

Sr. Objective August December July January July 2020 January

No. 2018 2018 2019 2020 To 2021

To To To To December To

December June December June 2020 June

2018 2019 2019 2020 2021

1. Analytical method development

2. Formulation development and optimization

3. Characterization of prepared NLCs

4. In-vitro evaluation of NLCs in cell lines

5. Formulation of NLCs loaded gel

6. Characterisation of NLCs loaded gel formulation

7. In-vitro release studies along with ex-vivo permeation studies

8. In-vivo pharmacokinetic studies

9. IVIVC study

Reference

1. Niaz, K., et al., Smokeless tobacco (paan and gutkha) consumption, prevalence, and contribution to oral cancer. Epidemiology and health, 2017. 39. 2. Ma, Y.S., et al., Quercetin induced apoptosis of human oral cancer SAS cells through mitochondria and endoplasmic reticulum mediated signaling pathways. Oncology letters, 2018. 15(6): p. 9663- 9672.

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3. Siddiqui, S., et al., Piperine triggers apoptosis of human oral squamous carcinoma through cell cycle arrest and mitochondrial oxidative stress. Nutrition and cancer, 2017. 69(5): p. 791-799. 4. Dandekar, M., et al., Head and neck cancers in India. Journal of surgical oncology, 2017. 115(5): p. 555-563. 5. Xue, J., S. Yang, and S. Seng, Mechanisms of cancer induction by tobacco-specific NNK and NNN. Cancers, 2014. 6(2): p. 1138-1156. 6. Chen-yu, G., et al., Development of a quercetin-loaded nanostructured lipid carrier formulation for topical delivery. International journal of pharmaceutics, 2012. 430(1-2): p. 292-298. 7. Kumar, S., Panmasala chewing induces deterioration in oral health and its implications in carcinogenesis. Toxicology mechanisms and methods, 2008. 18(9): p. 665-677.

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Annexure 10

Title of Research: Extraction, isolation, purification and characterization of bioactive compounds from 3 unexplored species

Abstract (200-250 words) North-east part of India is rich in plant biodiversity whose healing power has been realized and documented since ages. Most of the populations rely on traditional medicines hence, it is important to characterize the medicinal importance of each plant. In this study, we have identified chemical constituents (37 compounds in chloroform, 77 compounds in ethyl acetate and 90 compounds in ethanol, 73 compounds in hydro-alcoholic extract) from bioactive extracts of NIPER/18/11. Further purifications are under progress. The chromatographic analysis of the dried extracts was performed using QTOF-MS/MS coupled with Agilent 1290 infinity II UHPLC series. Several secondary plant metabolites were identified in each extract of bark and leaves.

1. Introduction (max. 500 words) Medicinal plants have been known to be an important potential source of therapeutics or curative aids. This involves the use of medicinal plants not only for the treatment of diseases but also as a potential material for maintaining good health and conditions. Himalayan topography is the prime source of diversity of plants that are used by the local inhabitants for preparing various kinds of medicines. Tribal people of various ethnic groups in the Himalayan region are dependent on these medicinal plants for cure and management of various diseases. However not much information is available on the biological activities as well as chemical composition of these plants. In our interests to explore flora of Himalayan region we have shortlisted few plants to identify their biological activities and chemistry.

2. Literature Review (max. 1000 words) Literature pertaining to the ethno medical plants from North Est India was retrieved. Several plants were selected based on their availability and medicinal value.

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S. Reference Year Major objective Methodology Findings No. adopted 1 Phytochemical analyses 2015 Identification of Review Description and activity of herbal ethnomedicinal value of article about anti- medicinal plants of plants from NE region diabetic, anti- North-East India for anti- cancer and diabetic, anti-cancer and anti- anti-tuberculosis and tuberculosis their docking studies

2 Evaluation of phenolic 2016 Screening of plants from NE Review Description content variability along region for said biological article about with antioxidant, activities antioxidant, antimicrobial, and antimicrobial, cytotoxic potential of and cytotoxic selected traditional plants medicinal plants from India

3 Review on 2015 Identifying anticancer plants Review Anticancer ethnomedicinal, from NE region article plants from phytochemical and Himalayan pharmacological region evidence of Himalayan anticancer plants

3. Research Question/s  What are the chemical components present in the bioactive extracts?  Which components are responsible for the particular activity?  How to isolate and characterize the bioactive components from the crude extract?

4. Objective/s of the Study  To find out the various components present in bioactive extracts using UPLC-QTOF- MS/MS.  Isolation and characterization of bioactive components by chromatographic procedures and spectroscopic methods.  Quantitative analysis of biomarkers in the crude extracts

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5. Study Area (max. 150 words) Plants from North East region of India are being widely used by local people for treatment for various diseases as well as a part of their regular diet. As there is lack of any scientific reports on the biological as well as chemical properties of these plants, we undertook this opportunity to identify potential bioactivities and compounds responsible for particular activity. Three plants (NIPER/18/1, NIPER/18/10 and NIPER/18/11) based on the in-house in vitro testing (anticancer and antioxidant) were selected for phytochemical investigations. To develop chemical fingerprints of bioactive extracts LC-MS approach has been selected for rapid identification and de-replication of components. Moreover, with the application of advanced chromatographic methods purification of pure compounds can be achieved. 6. Methodology Adopted OBJECTIVE-1 Identification of various components present in bioactive extract The chromatographic analysis of the dried bioactive extracts from plant NIPER/18/11 was performed using QTOF-MS/MS coupled with Agilent 1290 infinity II UHPLC series. The dried samples were dissolved in methanol, vortexed, spinned, diluted and filtered through 0.22 µ nylon syringe filter. The diluted sample solution was used for LCMS analysis in both positive and negative ESI ionization modes. The LCMS conditions were optimized for suitable separation of compounds and MS source and acquisition parameters. The separation of compounds was achieved on an Eclipse Plus C18 column (2.1X100mm, 1.8um) with with gradient mode. Both mobile phase i.e mobile phase A ( 95:5::Water : Methanol with 0.1% Formic acid and 5mM Ammonium formate) and mobile phase B (95:5::Methanol: Water with 0.1% Formic acid and

5mM Ammonium Formate) were set as follows: Tmin/% proportion of solvent (B): 0/3, 0-3/3, 3-

17/95, 17-22/95, 22-22.1/3, 22.1-25/3.The flow rate of the mobile phase is 0.4 ml/min. Mass spectrometric detection was carried out using QTOF mass analyser equipped with atmospheric pressure electrospray ionization (ESI) source.

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7. Research Findings Objective (s) Deliverables Methodology Achievements so far adopted (As per (As per sanctioned letter) Objectivesanctioned 1 Identification of Qualitative LC- NIPER/18/11 has been investigated letter) chemical constituents in MS (UPLC- for 37 compounds in chloroform, 77 bioactive extracts QToF) compounds in ethyl acetate and 90 compounds in ethanol, 73 compounds in hydro-alcoholic extract. Objective 2 Purification of pure Flash Fractionation of extract is under progress compounds chromatography and Semi- Objective 3 Quantitative analysis preparativeUPLC-MS (TQD)HPLC Will be uptaken after fulfilling objective 1 and 2

Figure 1 Total ion chromatogram of chloroform extract of NIPER/18/11

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Figure 2 Total ion chromatogram of ethylacetate extract of NIPER/18/11

Figure 3 Total ion chromatogram of ethanolic extract of NIPER/18/11

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Figure 4 Total ion chromatogram of hydroalcoholic extract of NIPER/18/11

Table 1. Component list of chloroform extract of NIPER/18/11

Name Formula m/z Mass RT Area Relative area (%)

Apigenin C15 H8 O5 286.07 268.036 14.42 51019669 59.43577

Ulopterol C15 H18 O5 301.104 278.115 14.48 7071818 8.238371

Peonidin C16 H13 O6 300.063 301.07 14.18 3593739 4.186555

Phloretin C15 H14 O5 275.092 274.084 12.03 2220142 2.586372

Malvidin C17 H15 O7 330.074 331.081 13.44 1974583 2.300306

Apigravin C15 H16 O4 261.113 260.106 15.85 1354491 1.577925

Scopoletin-7-glucoside C16 H18 O9 353.089 354.096 12.73 1336540 1.557013

Celerin C15 H16 O4 261.113 260.105 16.93 1170332 1.363388

Angelicin C11 H6 O3 187.038 186.031 8.273 721572 0.840601

Ferulic acid 4-O-glucoside C16 H20 O9 357.118 356.111 15.85 659748 0.768579

Xanthoxyletin C15 H14 O4 259.098 258.09 16.41 481152 0.560522

Apigenin 7-O-glucoside C21 H20 O10 450.14 432.107 11.86 451856 0.526393

Protocatechuic aldehyde C7 H6 O3 137.026 138.033 3.609 390803 0.455269

Fraxoside; Fraxin C16 H18 O10 393.08 370.093 18.82 354393 0.412853

6-Hydroxyluteolin 7-O-rhamnoside C21 H20 O11 466.133 448.098 12.13 254283 0.296229

Theaflavin C29 H24 O12 587.118 564.131 21.34 245717 0.28625

Alloxanthoxyletin C15 H14 O4 259.097 258.09 15.29 242996 0.28308

Isoferulic acid C10 H10 O4 193.053 194.06 9.396 218425 0.254456

Mellein C10 H10 O3 179.069 178.062 5.08 189418 0.220664

Vanillic acid C8 H8 O4 169.048 168.041 16 120442 0.14031

Benzoic Acid C7 H6 O2 121.031 122.038 5.416 120209 0.140038

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Herniarin C10 H8 O3 177.054 176.046 9.577 116014 0.135151

Coumaric acid C9 H8 O3 163.042 164.049 7.389 106116 0.123621

Marmesin C14 H14 O4 247.098 246.091 15.38 101135 0.117818

Rrutaretin C14 H14 O5 263.092 262.085 13.6 99026 0.115361

Paeonol C9 H10 O3 165.057 166.064 8.884 94535 0.110129

Syringic acid C9 H10 O5 199.061 198.054 13.68 80822 0.094154

Sinapoylquinic acid C18 H22 O10 399.13 398.122 17.71 68302 0.079569

Resveratrol 3-O-glucoside C20 H22 O8 389.125 390.133 8.259 64418 0.075044

Cyanidin 3-O-arabinoside C20 H19 O10 464.096 419.097 10.46 38241 0.044549

Auraptene C19 H22 O3 343.158 298.16 18.17 37976 0.04424

Urolithin A 3,8-O-diglucuronide C25 H24 O16 603.094 580.105 9.056 30215 0.035199

Cyanidin 3-O-galactoside C21 H21 O11 448.104 449.111 21.54 28005 0.032625

Homovanillic acid C9 H10 O4 181.052 182.059 7.549 26724 0.031132

Kaempferol 7-O-glucoside C21 H19 O11 446.088 447.095 20.03 25262 0.029429

Bergaptol C11 H6 O4 203.033 202.027 11.2 23691 0.027599

Daphnoretin C19 H12 O7 397.057 352.059 13.44 8584 0.01

Table 2. Component list of ethyl acetate extract of NIPER/18/11

Name Formula m/z Mass RT Area Relative area (%)

Auraptenol C18 H20 N2 O6 361.14 360.13 15.91 22022718 22.90216098

Angelicin C11 H6 O3 187.037 186.03 7.974 11859417 12.33300437

Malvidin C17 H15 O7 330.074 331.081 13.42 11022266 11.46242304

Sinapoylquinic acid C18 H22 O10 399.13 398.123 17.7 9004570 9.364153494

Umbelliferone C9 H6 O3 185.022 162.033 4.813 3117819 3.242324251

Petunidin 3-O-rhamnoside C22 H23 O10 492.128 447.129 11.17 1756939 1.827099626

Catechol C6 H6 O2 109.031 110.038 2.156 1009155 1.049454035

Protocatechuic aldehyde C7 H6 O3 137.026 138.033 4.06 967108 1.005727953

Celerin C15 H16 O4 261.112 260.105 15.67 946939 0.984753536

Quercetin-3-rutinoside C27 H30 O16 655.151 610.153 16.27 824874 0.85781406

Herniarin C10 H8 O3 199.035 176.046 8.023 513542 0.534049501

Kaempferol 3-O-rhamnoside C21 H19 O10 476.096 431.098 11.67 485132 0.504504992

Catechin (+) C15 H14 O6 291.086 290.078 12.95 458592 0.476905158

Quercetin 3,4'-O-diglucoside C27 H30 O17 625.141 626.148 16.85 457243 0.475502288

Theaflavin C29 H24 O12 587.118 564.131 21.32 402595 0.418672005

Scopoletin-7-glucoside C16 H18 O9 353.088 354.095 12.63 401729 0.417771423

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Eriodictyol C15 H12 O6 287.059 288.066 10.19 398928 0.414858569

Cinnamoyl glucose C15 H18 O7 311.112 310.105 11.35 389228 0.404771215

Ellagic acid arabinoside C19 H14 O12 435.053 434.046 2.647 385842 0.40125

Isoferulic acid C10 H10 O4 193.053 194.06 9.348 383744 0.39906822

Celereoside C20 H24 O10 442.172 424.139 19.19 371614 0.386453827

Dihydroxyflavone C15 H10 O4 299.058 254.06 16.07 353804 0.367932612

Scopoletin C10 H8 O4 191.037 192.044 9.826 348032 0.361930116

Quercetin 3-O-xyloside C20 H18 O11 457.074 434.084 10.94 341698 0.355343178

Urolithin A 3,8-O-diglucuronide C25 H24 O16 598.141 580.107 9.279 330886 0.344099418

Kaempferol 7-O-glucoside C21 H19 O11 492.091 447.094 10.86 321277 0.334106697

Resveratrol C14 H12 O3 246.114 228.08 15.73 304580 0.316742928

Paeonol C9 H10 O3 165.057 166.064 5.86 263882 0.274419717

Pelargonidin C15 H11 O5 316.058 271.059 11.49 257638 0.267926373

Rrutaretin C14 H14 O5 263.093 262.085 13.94 246382 0.256220882

Dihydrocaffeic acid C9 H10 O4 181.052 182.06 7.979 241636 0.251285358

Petunidin C16 H13 O7 362.063 317.065 11.62 239571 0.249137895

Cyanidin C15 H11 O6 332.053 287.054 11.57 221300 0.230137271

Hydroxycaffeic acid C9 H8 O5 195.032 196.039 7.288 218711 0.227444884

Methoxynobiletin C22 H24 O9 450.175 432.141 9.478 199365 0.207326331

Delphinidin C15 H11 O7 348.048 303.049 10.63 190362 0.19796381

Benzoic Acid C7 H6 O2 121.03 122.038 5.397 181923 0.189187812

Vanillin C8 H8 O3 151.041 152.049 5.494 171607 0.178459859

Homovanillic acid C9 H10 O4 181.052 182.059 6.837 169314 0.176075291

Syringic acid C9 H10 O5 199.062 198.054 14.8 149426 0.155393095

Malonylgenistin C24 H22 O13 563.107 518.109 8.407 133020 0.138331947

Naringenin C15 H12 O5 317.069 272.071 10.07 125497 0.130508527

Hispidulin C16 H12 O6 345.065 300.066 13.13 123019 0.127931572

Peonidin C16 H13 O6 346.068 301.07 9.45 118535 0.123268511

epi-Catechin-3-gallate (ECG) C22 H18 O10 443.097 442.09 13.94 118526 0.123259151

Hesperetin C16 H14 O6 301.074 302.082 10.86 117995 0.122706947

Eriodictyol 7-O-glucoside C21 H22 O11 495.115 450.12 9.753 99953 0.103944468

epi-Gallocatechin-3-gallate (EGCG) C22 H18 O11 476.118 458.083 11.69 96471 0.100323419

Vanillin 4-sulfate C8 H8 O6 S 233.012 232.004 10.06 93879 0.097627912

Gallic acid C7 H6 O5 169.016 170.023 8.309 91007 0.094641223

Hesperetin C16 H14 O6 325.068 302.077 15.32 90893 0.094522671

Hydroxyphenylacetic acid C8 H8 O3 151.041 152.049 6.3 83071 0.086388311

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Cyanidin 3-O-arabinoside C20 H19 O10 464.095 419.097 9.348 79174 0.082335691

Cyanidin 3-O-galactoside C21 H21 O11 448.1 449.108 10.59 73716 0.076659734

Osthol C15 H16 O3 289.11 244.112 15.81 70115 0.072914933

Bergenin C14 H16 O9 373.081 328.083 6.915 67668 0.070370216

Rhamnazin C17 H14 O7 329.07 330.077 11.62 58973 0.061327995

Biochanin A C16 H12 O5 329.07 284.071 11.62 58614 0.060954659

Phloretin C15 H14 O5 319.082 274.084 6.633 55651 0.057873336

Dihydroquercetin C15 H12 O7 303.054 304.061 8.492 49856 0.051846922

Ellagic acid acetyl-arabinoside C21 H16 O13 475.054 476.061 11.62 45521 0.04733881

3-Feruloylquinic acid C17 H20 O9 367.104 368.112 9.959 42893 0.044605865

Methoxynobiletin C22 H24 O9 431.135 432.143 14.89 42522 0.04422005

Acetylglycitin C24 H24 O11 533.134 488.136 11.62 42319 0.044008943

Malonylgenistin C24 H22 O13 519.114 518.107 13.58 38416 0.039950083

Quercetin C15 H10 O7 301.035 302.042 0.606 38331 0.039861689

2,3-Dihydroxybenzoic acid C7 H6 O4 153.02 154.028 10.63 34650 0.036033694

Myricetin C15 H10 O8 336.071 318.037 16.01 32743 0.034050541

Caffeoylquinic acid C16 H18 O9 353.088 354.096 7.578 30240 0.031447587

Gardenin B C19 H18 O7 357.101 358.108 15.45 29835 0.031026414

Naringenin C15 H12 O5 273.075 272.068 14.33 24991 0.025988977

Daphnetin C9 H6 O4 177.021 178.028 7.036 24622 0.025605241

Sinapic acid C11 H12 O5 269.067 224.069 7.249 24032 0.024991681

Isoxanthohumol C21 H22 O5 353.14 354.148 9.45 23839 0.024790973

Naringenin 7-O-glucoside C21 H22 O10 479.122 434.123 10.67 22902 0.023816556

Daphnoretin C19 H12 O7 397.057 352.059 13.42 19320 0.020091514

Caffeic acid C9 H8 O4 179.036 180.044 17.41 9616 0.01

Table 3. Component list of ethanolic extract of NIPER/18/11

Name Formula m/z Mass RT Area Relative area (%)

Malvidin C17 H15 O7 330.074 331.081 13.42 12121308 13.30988

6-Hydroxyluteolin 7-O-rhamnoside C21 H20 O11 493.098 448.105 10.83 9778235 10.73705

Pelargonidin C15 H11 O5 316.058 271.059 12.35 5949447 6.532829

Petunidin C16 H13 O7 316.058 317.065 12.35 5929302 6.510708

Medicagol C16 H8 O6 319.022 296.033 2.097 4517314 4.960266

6-Hydroxyluteolin 7-O-rhamnoside C21 H20 O11 449.108 448.099 7.778 3772036 4.141908

Petunidin 3-O-rhamnoside C22 H23 O10 492.128 447.13 11.18 3120941 3.426969

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2,3-Dihydroxybenzoic acid C7 H6 O4 153.021 154.028 2.336 2070594 2.273629

Apigenin 7-O-glucuronide C21 H18 O11 469.072 446.084 7.479 2011726 2.208989

Cyanidin 3-O-galactoside C21 H21 O11 448.101 449.108 10.83 1990132 2.185277

Quercetin-3-rutinoside C27 H30 O16 655.152 610.154 16.27 1912951 2.100528

Rhoifolin C27 H30 O14 601.156 578.164 16.83 1715133 1.883313

Quercetin 3,4'-O-diglucoside C27 H30 O17 625.141 626.148 16.84 1659342 1.822051

Sinapoylquinic acid C18 H22 O10 399.13 398.123 17.72 1390365 1.526699

Kaempferol C15 H10 O6 309.038 286.049 1.918 1340710 1.472175

Dehydrodiferulic acid C20 H18 O8 387.107 386.098 15.31 1230285 1.350922

p-Coumaroyl malic acid C13 H12 O7 281.063 280.056 11.73 991020 1.088196

Luteolin C15 H10 O6 309.038 286.049 1.615 893871 0.981521

Cyanidin C15 H11 O6 286.047 287.054 12.05 853312 0.936985

Celerin C15 H16 O4 261.112 260.105 15.66 637514 0.700026

4-Hydroxybenzoic acid 4-O-glucoside C13 H16 O8 345.083 300.086 2.369 559705 0.614588

Delphinidin C15 H11 O7 302.042 303.049 10.06 518668 0.569527

1-Acetoxypinoresinol C22 H24 O8 434.182 416.149 11.88 472036 0.518322

Dihydrocaffeic acid C9 H10 O4 181.052 182.06 9.346 461025 0.506231

Apigravin C15 H16 O4 261.112 260.105 15.55 394791 0.433503

Catechol C6 H6 O2 109.03 110.038 2.336 311590 0.342143

Homovanillic acid C9 H10 O4 181.052 182.06 7.973 273114 0.299895

Marmin C19 H24 O5 355.153 332.165 18.15 271714 0.298357

Malonylgenistin C24 H22 O13 563.108 518.109 10.76 262437 0.288171

Methoxynobiletin C22 H24 O9 431.136 432.143 14.87 256567 0.281725

Galangin C15 H10 O5 269.048 270.055 15.83 254840 0.279829

Nordentatin C19 H20 O4 330.169 312.135 20.58 249496 0.273961

Hispidulin C16 H12 O6 299.059 300.066 12.43 244693 0.268687

Ellagic acid acetyl-arabinoside C21 H16 O13 477.068 476.061 0.612 239734 0.263241

Geraldone C16 H12 O5 329.07 284.071 14.02 229742 0.25227

Fraxoside; Fraxin C16 H18 O10 393.083 370.093 20.54 228226 0.250605

Formononetin C16 H12 O4 291.061 268.072 15.76 209794 0.230366

Dihydroxyphenylacetic acid C8 H8 O4 167.036 168.044 6.641 194108 0.213142

Matairesinol C20 H22 O6 359.151 358.143 17.49 181644 0.199455

Myricetin C15 H10 O8 336.072 318.038 16.03 178615 0.196129

Acetylgenistin C23 H22 O11 519.118 474.12 11.63 168351 0.184859

Alloxanthoxyletin C15 H14 O4 259.096 258.089 14.61 162052 0.177942

Osthol C15 H16 O3 245.117 244.11 6.924 160182 0.175889

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Vanillic acid C8 H8 O4 167.036 168.043 5.912 156193 0.171509

Benzoic Acid C7 H6 O2 167.036 122.038 5.912 154340 0.169474

Dihydroxyflavone C15 H10 O4 253.053 254.06 11.35 145475 0.15974

Xanthoxyletin C15 H14 O4 259.096 258.089 12.16 116656 0.128095

Heraclenin C16 H14 O5 309.072 286.083 15.15 115562 0.126894

Naringenin C15 H12 O5 317.069 272.071 9.895 103056 0.113161

Apigenin 7-O-glucoside C21 H20 O10 477.103 432.108 9.951 99737 0.109517

Quercetin C15 H10 O7 320.079 302.045 14.97 98613 0.108283

Gallic acid C7 H6 O5 169.016 170.023 8.33 97314 0.106856

Daphnetin C9 H6 O4 177.021 178.028 5.862 93931 0.103142

epi-Catechin-3-gallate (ECG) C22 H18 O10 487.088 442.09 8.416 91803 0.100805

Naringenin 7-O-glucoside C21 H22 O10 479.118 434.12 6.302 91705 0.100697

Peonidin C16 H13 O6 346.068 301.07 9.485 88747 0.097449

Kaempferol 3-O-rhamnoside C21 H19 O10 476.096 431.097 11.69 77387 0.084975

Apigenin C15 H10 O5 315.054 270.055 11.12 75904 0.083347

Quercetin C15 H10 O7 301.038 302.045 7.702 75107 0.082472

Kaempferol 7-O-glucoside C21 H19 O11 492.091 447.092 10.22 70607 0.07753

Rhamnetin C16 H12 O7 315.054 316.061 11.12 68605 0.075332

Catechin (+) C15 H14 O6 291.088 290.08 10.38 68343 0.075044

Urolithin B 3-O-glucuronide C19 H16 O9 433.081 388.083 10.49 66742 0.073286

Quercetin 3-O-xyloside C20 H18 O11 433.081 434.088 10.49 66742 0.073286

Osthenol C14 H14 O3 231.103 230.096 7.379 65710 0.072153

Scopoletin C10 H8 O4 191.036 192.044 9.854 63858 0.07012

Myricetin C15 H10 O8 317.033 318.04 6.474 62987 0.069163

Rrutaretin C14 H14 O5 263.093 262.086 13.44 58284 0.063999

Quercetin 3-O-xyloside C20 H18 O11 435.091 434.084 7.252 53603 0.058859

Cyanidin 3-O-arabinoside C20 H19 O10 464.095 419.097 8.236 51574 0.056631

Acetyldaidzin C23 H22 O10 459.131 458.124 8.051 48643 0.053413

Acetylglycitin C24 H24 O11 533.134 488.136 12.02 48110 0.052827

Isorhamnetin-3-glucoside C22 H22 O12 477.103 478.111 10.03 45807 0.050299

Pinocembrin C15 H12 O4 257.081 256.073 15.59 44014 0.04833

Paeonol C9 H10 O3 165.057 166.064 5.912 37092 0.040729

Biochanin A C16 H12 O5 329.07 284.071 12.47 35517 0.039

Rhamnazin C17 H14 O7 329.07 330.077 12.47 35483 0.038962

Protocatechuic aldehyde C7 H6 O3 137.025 138.033 7.865 34873 0.038293

Auraptene C19 H22 O3 343.157 298.159 17.69 34465 0.037845

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Pabulenol C16 H14 O5 287.09 286.082 17.51 34210 0.037565

Cinnamic acid C9 H8 O2 193.052 148.054 9.383 33859 0.037179

Daphnoretin C19 H12 O7 397.057 352.059 13.44 31042 0.034086

Caffeic acid C9 H8 O4 179.037 180.044 5.912 30945 0.033979

Hydroxyphenylacetic acid C8 H8 O3 151.041 152.048 5.51 28931 0.031768

Chicoric acid C22 H18 O12 473.074 474.081 6.8 28149 0.030909

Resveratrol 3-O-glucoside C20 H22 O8 435.133 390.135 8.887 27001 0.029649

epi-Gallocatechin-3-gallate (EGCG) C22 H18 O11 457.079 458.086 12.79 19382 0.021283

Isoferulic acid C10 H10 O4 193.052 194.059 9.383 19300 0.021192

Malonyldaidzin C24 H22 O12 547.11 502.112 9.258 17010 0.018678

4-Ethylphenol C8 H10 O 167.073 122.074 10.55 9107 0.01

Table 4. Component list of hydroalcoholic extract of NIPER/18/11

Name Formula m/z Mass RT Area Relative area (%)

Malvidin C17 H15 O7 330.074 331.081 13.42 6657002 18.24336

Peonidin C16 H13 O6 300.063 301.07 14.18 6472615 17.73805

Petunidin 3-O-rhamnoside C22 H23 O10 492.129 447.13 11.2 3004680 8.234256

Pelargonidin C15 H11 O5 316.058 271.06 12.36 2977183 8.158901

Petunidin C16 H13 O7 316.058 317.065 12.36 2946501 8.074818

Sinapoylquinic acid C18 H22 O10 399.13 398.123 17.72 2170715 5.948794

Apigenin C15 H8 O5 286.07 268.036 14.42 1707838 4.68029

Rhoifolin C27 H30 O14 601.155 578.167 17.74 843729 2.31222

Kaempferol 7-O-glucoside C21 H19 O11 448.101 447.094 5.737 807106 2.211855

Apigenin C15 H10 O5 288.086 270.052 14.26 773112 2.118696

Xanthoxyletin C15 H14 O4 259.095 258.087 6.169 553784 1.517632

Kaempferol 3-O-rhamnoside C21 H19 O10 476.097 431.099 11.69 515348 1.412299

Auraptenol C18 H20 N2 O6 361.14 360.132 17.19 468257 1.283247

Osthenol C14 H14 O3 231.104 230.096 4.339 462497 1.267462

4-Hydroxybenzoic acid 4-O-glucoside C13 H16 O8 345.084 300.086 2.391 444276 1.217528

Catechol C6 H6 O2 109.031 110.038 2.326 376884 1.032842

Episesaminol C20 H18 O7 371.113 370.107 9.842 358055 0.981241

Matairesinol C20 H22 O6 376.175 358.141 13.86 327863 0.898501

Cyanidin C15 H11 O6 286.047 287.054 12.04 272185 0.745917

Medicagol C16 H8 O6 314.066 296.031 1.73 257366 0.705306

Cyanidin 3-O-arabinoside C20 H19 O10 464.097 419.099 9.373 244315 0.66954

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Homovanillic acid C9 H10 O4 181.052 182.06 7.983 229009 0.627594

Fraxoside; Fraxin C16 H18 O10 393.08 370.094 16.2 222017 0.608432

6-Hydroxyluteolin 7-O-rhamnoside C21 H20 O11 466.133 448.099 12.12 218481 0.598742

Dihydroquercetin C15 H12 O7 303.053 304.06 1.111 182615 0.500452

Liqcoumarin C12 H10 O4 217.053 218.06 6.448 174000 0.476843

Syringaresinol C22 H26 O8 436.197 418.164 11.98 163969 0.449353

Ooreoselone C14 H12 O4 245.081 244.073 3.878 154503 0.423412

Kaempferol 7-O-glucoside C21 H19 O11 492.091 447.093 10.21 154367 0.423039

2,3-Dihydroxybenzoic acid C7 H6 O4 153.021 154.028 6.02 145953 0.399981

Cyanidin 3,5-O-diglucoside C27 H31 O16 610.155 611.162 9.373 138002 0.378191

Delphinidin C15 H11 O7 302.042 303.049 10.04 136713 0.374659

Protocatechuic aldehyde C7 H6 O3 137.025 138.033 4.052 129590 0.355138

Daphnetin C9 H6 O4 177.021 178.028 8.289 123896 0.339534

Celerin C15 H16 O4 261.112 260.105 15.55 122976 0.337013

Vanillin 4-sulfate C8 H8 O6 S 233.012 232.004 10.08 122698 0.336251

6-Geranylnaringenin C25 H28 O5 409.202 408.195 17.13 112446 0.308156

Dihydroxyphenylacetic acid C8 H8 O4 167.036 168.044 7.293 111654 0.305985

Apigenin C15 H10 O5 269.048 270.055 15.82 108959 0.2986

1-Acetoxypinoresinol C22 H24 O8 439.137 416.148 10.2 105516 0.289164

p-Coumaroyl malic acid C13 H12 O7 281.064 280.057 8.774 103920 0.28479

Methoxynobiletin C22 H24 O9 455.131 432.142 13.86 102269 0.280266

Malonylgenistin C24 H22 O13 563.107 518.109 8.401 92853 0.254461

Rhamnetin C16 H12 O7 339.048 316.057 0.898 84805 0.232406

Urolithin A 3,8-O-diglucuronide C25 H24 O16 579.102 580.109 5.529 77703 0.212943

Episesamin C20 H18 O6 355.118 354.111 17.21 71627 0.196292

Vanillic acid C8 H8 O4 167.036 168.044 7.983 70870 0.194218

Chicoric acid C22 H18 O12 475.088 474.078 9.482 59936 0.164253

Quercetin C15 H10 O7 325.035 302.044 0.613 59132 0.16205

Alloxanthoxyletin C15 H14 O4 259.098 258.091 16.13 58370 0.159962

Oxypeucedanin C16 H14 O5 309.072 286.082 15.16 57589 0.157821

6-Geranylnaringenin C25 H28 O5 407.188 408.195 10.47 50574 0.138597

Rosmarinic acid C18 H16 O8 405.086 360.088 7.117 49258 0.13499

Pinocembrin C15 H12 O4 257.081 256.072 12.75 47794 0.130978

Hydroxyphenylacetic acid C8 H8 O3 151.041 152.049 5.529 45887 0.125752

Apigenin 7-O-glucuronide C21 H18 O11 447.093 446.085 7.991 44111 0.120885

Apigravin C15 H16 O4 261.11 260.103 0.613 43604 0.119496

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Celereoside C20 H24 O10 469.135 424.136 6.398 41899 0.114823

epi-Gallocatechin-3-gallate (EGCG) C22 H18 O11 476.12 458.085 11.08 39973 0.109545

Cyanidin 3-O-galactoside C21 H21 O11 448.101 449.108 10.59 38813 0.106366

epi-Gallocatechin-3-gallate (EGCG) C22 H18 O11 457.08 458.087 12.79 38749 0.106191

Methoxynobiletin C22 H24 O9 477.141 432.142 1.255 37477 0.102705

Rrutaretin C14 H14 O5 263.093 262.086 13.44 36034 0.09875

Cinnamic acid C9 H8 O2 147.046 148.053 7.33 33320 0.091313

5,6-Dihydroxy-7,8,3',4'- C19 H18 O8 373.094 374.102 1.087 29021 tetramethoxyflavone 0.079531

Isoimperatorin C16 H14 O4 271.095 270.088 15.81 27293 0.074796

Hesperetin C16 H14 O6 303.089 302.08 2.9 26348 0.072206

Rosmarinic acid C18 H16 O8 383.075 360.087 11.36 25110 0.068813

Herniarin C10 H8 O3 177.054 176.047 17 21267 0.058282

Didymin C28 H34 O14 639.197 594.198 7.894 18841 0.051633

Isoferulic acid C10 H10 O4 193.052 194.06 8.115 18618 0.051022

Heraclenin C16 H14 O5 287.09 286.083 17.5 18530 0.050781

Naringin 4'-O-glucoside C33 H42 O19 760.271 742.237 17.36 13111 0.03593

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Figure 5: Major chemical compounds identified in NIPER/18/11

8. Future Plan (including monthly plan) 1. Bioassay guided isolation of chemical compounds from the extracts NIPER/18/11. 2. Identification of compounds in bioactive extracts of NIPER/18/10 and NIPER/18/1 3. Characterization of pure compounds by UV, IR, Mass and NMR techniques 4. Quantitative analysis of biomarkers

9. Reference/s 1. Suhitha, Sivasubramanian, Seenivasan K. Devi, Krishnasamy Gunasekaran, H. Carehome Pakyntein, Atanu Bhattacharjee, and Devadasan Velmurugan. "Phytochemical analyses and activity of herbal medicinal plants of North-

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East India for anti-diabetic, anti-cancer and anti-tuberculosis and their docking studies." Current topics in medicinal chemistry 15, no. 1 (2015): 21-36. 2. Singh, Garima, Ajit K. Passsari, Vincent V. Leo, Vineet K. Mishra, Sarathbabu Subbarayan, Bhim P. Singh, Brijesh Kumar et al. "Evaluation of phenolic content variability along with antioxidant, antimicrobial, and cytotoxic potential of selected traditional medicinal plants from India." Frontiers in plant science 7 (2016): 407. 3. Tariq, Akash, Sakina Mussarat, and Muhammad Adnan. "Review on ethnomedicinal, phytochemical and pharmacological evidence of Himalayan anticancer plants." Journal of ethnopharmacology 164 (2015): 96-119.

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Annexure 11

Conferences and workshops

1. Scientific writing and publication “From bench to paper” at National Workshop organized by Himalayan Pharmacy Institute, Sikkim, Sikkim Central university, India – 737136 on October 6th 2018.

2. “Herbal medicines in modern therapy” at Continuous Education Programme organized by Association of Registered Pharmacist Assam, sponsored by Pharmacy Council of India at Central Government Health Scheme – Guwahati (India) – 781016 on 6th January 2019.

3. DBT-NER Training Program on Preclinical Imaging 2019 January 7th, 2019 KS 3rd Floor Seminar Room (9.30am -1.00pm).

4. Pharma Nanotech – 2018.SERB – DST Sponsored National Seminar entitled “Emerging global trends in pharmaceutical nanotechnology” :: November 23 – 24, 2018 DEPARTMENT OF PHARMACEUTICAL SCIENCES, Dibrugarh University.

5. International Training Workshop of “Quality by Design (QbD)” – Practical Implementation of tools & tactics of Quality by Design (QbD) & Lean Six Sigma (L6σ) in Pharmaceutical Product Development, on 12th-13th April, 2019 at Hotel Kamat Lingapur, Begumpet, Hyderabad; organized by QbD-Expert

6. Travel grant for Asian Association of School of Pharmacy (AASP) Conference on 3-5 th July, Ajou University (Suwon), Republic of Korea.

7. Society for the study of xenobiotics (SSX). “Merging in vitro DMPK to translational and quantitative DMPK” JN Tata Auditorium, Indian Institute of Science (IISc), Bangalore, India-560012 October 9th to 13th 20018

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4. Fellowship Description at Institutional/ University Level

Annual Deliverables/ Outputs (during the reporting year)

S. Deliverables/ Parameters No. Description No. 1. No. of Research Publications (monograph/ Nil NA articles/ peer-reviewed articles): 2. No. of Data Sets generated: NA NA 3. No. of Conferences/ Workshops attended: 07 Annexure 11 4. No. of Sites/ Study Area Covered: 02 Annexure 8 5. No. of Best Practices suitable for IHR: NA NA 6. New Observations/ Innovations NA NA

5. Fellowship Concluding Remarks/ Annual Summary

Conclusions summarizing the achievements and indication of remaining work (within 300 words): The North-east India has the richest tank of plant diversity in India and is one of the ‘biodiversity hotspots’ of the world. Assam is one of the seven sister States of North Eastern India covering a total land area of 78,438 sq. kilometres with a population of about 31,205,576. Most of tribes still relay on their own traditional herbal remedies for common ailments as they are considered it to be effective, cheap, easily available and devoid of toxic side effects. Because of their proximity to nature and plants, they possess intimate knowledge about plant value and utilities developed through age-old trial and error method. The different ethnic communities considered it as hidden wealth of information about plants used for medicinal purposes. Exploration of medicinal plants in the Himalayan regions of North east India is a matter of attraction to the scientists, traders as well as pharmaceutical industries. Fellowships offered to JRFs at NIPER Guwahati helped to perform research under drug discovery & development by exploring the efficacy, safety for the use of medicinal plants for the treatment of various diseases. The research work performed by the JRFs supported by NMHS were broadly categorised in to 3 categories

A) Pharmacological evaluation (biological efficacy, safety and dosage) of unexplored medicinal plants of Himalayan regions of NE states (JRF 01 & JRF 02) B) Development of novel formualtions to improve efficacy, safety and patient compliance for the bioactive compunds having proven biological activity. (JRF 03 & JRF 04) C) Extraction, isolation and biological charecterisation of bioactive compounds from the medicinal plants of NE India. (JRF 05)

In addition to above studies, we are also conducting awareness progammes for the safe use along with development of data base for the medicinal plants grown in himalayan region of NE states.

The data generated at the end of this project will give the value addition to the medicinal plants which inturn give insight to the farmers for the farming in large quantities of the respective medicinal plants.

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