Evaluation of microbial activity, standardization, characterization of total extract and active phytoconstituents

of Roylea elegans

A Synopsis

Submitted in

Partial Fulfilment for the Degree

Of

Doctor of Philosophy

(Pharmacy)

Supervised by Submitted by Dr. Anurekha Jain Neeru

Department of Pharmaceutical SciencesJayotiVidyapeeth Women’s University

Jaipur (Rajasthan), IndiaJanuary, 2017

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Table of Content

Outline Page No.

Introduction………………………….......................3-7

Objective of the Study……………………………….8

Review of Literature…………………………………9-13

Research Methodology…………………………….14-16

Activity Plan……………………………………….17

Bibliography…………………………………………18

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INTRODUCTION

Herbal medicine is defined as a plant part like leaf, seed, stem, roots, rhizomes flower and whole

plant and has an active principle which have any pharmacological activity or capable to treat any

disease. Plants are the natural source of herbal medicine and also natural sources of drugs. Plants

have been used in the treatment of various diseases from time immemorial. The traditional Indian

system of medicine, Ayurveda, Siddha, Unani systems, is based on the use of plants and other

natural substances. There are 2000,000 to 250,000 species of flowering plants growing on earth

which belong to 10,500 genera and about 300 families.

ADVANTAGES OF HERBAL MEDICINE

Herbal medicine have long history of use and better patient tolerance as well as

acceptance.

Medicinal plants have a renewable source, which is our only hope for sustainable supplies

of cheaper medicine for the world growing populations.

Availability of medicinal plants is not a problem especially in developing countries like

India having rich agro-climatic, cultural, and ethnic biodiversity.

The cultivation and processing of medicinal herbs and herbal products is environmental

friendly.

EXTRACTION- Extraction is defined as separation of mixture of constituents from crude drug

with the help of suitable solvents. In this method solvents are used according to polarity.

Extraction, as the term is used pharmaceutically, involves the separation of medicinally active

portions of plant or animal tissues from the inactive or inert components by using selective

solvents in standard extraction procedures. The purposes of standardized extraction procedures for

crude drugs are to attain the therapeutically desired portion and to eliminate the inert material by

treatment with a selective solvent known as menstruum.

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The extract thus obtained may be ready for use as a medicinal agent in the form oftinctures and

fluid extracts, it may be further processed to be incorporated in any dosage form such as tablets

or capsules, or it may be fractionated to isolate individual chemical entities such as ajmalicine,

hyoscine and vincristine, which are modern drugs. Thus, standardization of extraction

procedures contributes significantly to the final quality of the herbal drug.

HOT CONTINUOUS EXTRACTION (SOXHLET)-In this method, the finely ground crude

drug is placed in a porous bag or “thimble” made of strong filter paper, which is placed in

chamber E of the Soxhlet apparatus. The extracting solvent in flask A is heated, and its vapors

condense in condenser D. The condensed extractant drips into the thimble containing the crude

drug, and extracts it by contact. When the level of liquid in chamber E rises to the top of siphon

tube C, the liquid contents of chamber E siphon into flask A. This process is continuous and is

carried out until a drop of solvent from the siphon tube does not leave residue when

evaporated. The advantage of this method, compared to previously described methods, is that

large amounts of drug can be extracted with a much smaller quantity of solvent. This effects

tremendous economy in terms of time, energy and consequently financial inputs. At small

scale, it is employed as a batch process only, but it becomes much more economical and viable

when converted into a continuous extraction procedure on medium or large scale.

STANDARDIZATION- Standardization is defined as the process in which crude drug or its

part is standardizing by different parameters to check the quality, safety and efficacy of

particular plant. Accounting toWHO it is the process involving the physicochemical evaluation

crude drug covering the aspects,as selection and handling of crude material,safety,efficacy and

stability assessment of finished product,documentation of safety and risk based on

experience,provision of product information to consumer and product promotion.

IMPORTANCE OF STANDARDIZATION- In olden times,vaidyas used to treat patients on

individual basis,and prepare drug according to the requirement of the patient. In almost all the

traditional system of medicine, the quality control aspect has been considered from its inspection

of itself Rishis,Vaidyas and Hakims. Unlike in olden times where traditional practitioners

prepared and tested the qualities of herbal medicines, the problem faced today are these of

economics of industrial scale production ,shelf life and distribution to long distances. These have

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necessitated development of modern and objective standards for evaluating the safety, quality and

efficacy of these medicines.

It is the cardinal responsibility of the regulatory authorities to ensure that the consumers

get the medication, which guarantee Purity, safety, potency and efficacy. Herbal product has been

enjoying renaissance among the customers through out the world. However,one of the

impedimentsin the acceptance of the Ayurvedic formulations the lack of standard quality control

profile. The quality of herbal medicine i.e. the profile of the constituents in the final product has

implication in efficacy and safety.

The task of lying down standards for quality control of herbal crude and their formulation

involves biological evaluation for a particular disease area, chemical profiling of the material and

lying down specification for the finished product. Therefore, incase of herbal drugs and product,

the word"Standardization"should encompass entire field of study from cultivation of medicinal

plant to its clinical application.

PARAMETERS OF STANDARDIZATION- The bioactive extract should be standardized on the

basis of active principles or major compounds along with the chromatographic fingerprints

(TLC,HPTLC, HPLC and GC). The standardization of crude drug materials includes the following

steps:

1. Authentication(Stage of collection, parts of the plant collected, regional status, botanical

identity like phytomorphology, microscopical and histological analysis, taxonomical identity,

etc.)

2. Foreign matter(herbs collected should be free from soil, insect parts or animal excreta,

etc.)

3. Organoleptic evaluation(sensory characters–taste, appearance,odor, feel of the drug,etc.)

4. Tissues of diagnostic importance present in the drug powder.

5. Ash values and extractive values.

6. Volatile matter

7. Moisture content determination

8. Chromatographic and spectroscopic evaluation. TLC, HPTLC, HPLC methods will

provide qualitative and semiquantitative information about the main active constituents present

in the crude drug as chemical markers in the TLC fingerprint evaluation of herbals(FEH).

9. Determination of heavy metals–e.g.cadmium,lead,arsenic,etc.

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10. Pesticide residue–WHO and FAO (Food and Agricultural Organization) setlimits of

pesticides, which are usually present in the herbs.These pesticides are mixed with the herbs

during the time of cultivation. Mainly pesticides like DDT, BHC, toxaphene, aldrin causes

eriousside- effects in human beings if the crude drugs are mixed with these agents.

11. Microbial contamination–usually medicinal plants containing bacteria and molds are

coming from soil and atmosphere. Analysis of the limits of E.coli and molds clearly throws

light towards the harvesting and production practices.The substance known as afflatoxins will

produces eriousside-effects if consumed along with the crude drugs. Afflatoxins should be

completely removed or should not be present.

12. Radioactive contamination–Microbial growth in herbals are usually avoided by

irradiation. This process may sterilize the plant material but the radio activity hazard should be

taken into account. The radioactivity of the plant samples should be checked accordingly to the

guideline of International Atomic Energy(IAE) in Vienna and that of WHO.

ISOLATION- Isolation of single phytoconstituents from mixture of phytoconstituents with the

help of suitable solvent. Solvents are used according to polarity.

IDENTIFICATION AND CHARACTERIZATION- Due to the fact that plant extracts usually

occur as a combination of various type of bioactive compounds or phytochemicals with different

polarities, their separation still remains a big challenge for the process of identification

andcharacterization of bioactive compounds. It is a common practice in isolation of these

bioactive compounds that a number ofdifferent separation techniques such as TLC, column

chromatography, flash chromatography, Sephadex chromatography and HPLC, should be used to

obtain pure compounds. The pure compounds are then used for the determination of structure and

biological activity. Besides that, non-chromatographic techniques such as immunoassay, which

use monoclonal antibodies (MAbs), phytochemical screening assay, Fourier-transform infrared

spectroscopy (FTIR), can also be used to obtain and facilitate the identification of the bioactive

compounds.

FLAVONOID- The term “flavonoid” is generally used to describe a broad collection of natural

products that include a C6-C3-C6 carbon framework, or more specifically a

phenylbenzopyranfunctionality. Depending on the position of the linkage of the aromatic ring

to the benzopyrano (chromano) moiety, this group of natural products may be divided into

three classes: the flavonoids (2-phenylbenzopyrans) 1, isoflavonoids (3-benzopyrans) 2, and

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the neoflavonoids (4-benzopyrans) 3. These groups usually share a common chalcone

precursor, and therefore are biogenetically and structurally related.

2-PHENYLBENZOPYRANS (C6-C3-C6 BACKBONE)-Based on the degree of oxidation and

saturation present in the heterocyclic C-ring, the flavonoids may be divided into the different

groups.

ISOFLAVONOIDS- The isoflavonoids are a distinctive subclass of the flavonoids. These

compounds possess a 3-phenylchroman skeleton that is biogenetically derived by 1,2-aryl

migration in a 2-phenylchroman precursor. Despite their limited distribution in the plant

kingdom, isoflavonoids are remarkably diverse as far as structural variations are concerned.

This arises not only from the number and complexity of substituents on the basic 3-

phenylchroman system, but also from the different oxidation levels and presence of additional

heterocyclic rings. Isoflavonoids are subdivided into the different groups.

NEOFLAVONOIDS- The neoflavonoids are structurally and biogenetically closely related to

the flavonoids and the isoflavonoids and comprise the 4-arylcoumarins (4-aryl-2H-1-

benzopyran-2-ones), 3,4-dihydro-4-arylcoumarins, and neoflavenes.

MINOR FLAVONOIDS- Natural products such as chalcones and aurones also contain a C6-

C3-C6 backbone and are considered to be minor flavonoids. These groups of compounds

include the 2′-hydroxychalcones, 2′-OH-dihydrochalcones, 2′-OH-retro-chalcone, aurones (2-

benzylidenecoumaranone), and auronols.

ANTIMICROBIAL AGENTS- Antimicrobial agents have the abilitythat a drugkills or suppresses

the growth of microorganisms.

MECHANISMS OF ANTIMICROBIAL AGENTS

A. Inhibition of cell wall synthesis

B. Inhibition of functions of cellular membrane

C. Inhibition of protein synthesis

D. Inhibition of nucleic acid synthesis

E. Inhibition of folic acid synthesis

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F. Antiviral agents

PLANTS USED AS ANTIMICROBIAL AGENTS- Natural antimicrobials are secondary

metabolites that can be found in plants, animals, and microorganisms.Plants,especially herbs and

spices, are been given more attention in natural antimicrobial research, however; this chapter

focuseson the newly discoveries in fruits and vegetables.Plants are the largest biochemical and

pharmaceuticalstores ever known on our planet. These living stores are able togenerate endless

biochemical compounds. In their living, humanand animals are using only a small portion (1

to10%) of plantsavailable on Earth (250,000 to 500,000 species). Although, plants have many

other defense mechanismsagainst micro and macro organisms, but in this review we focusonly

on their chemical products of important pharmaceuticalcharacteristics (secondary metabolites).

Medicinal plants are rich ina numerous variety of secondary metabolites of

antimicrobialproperties such as saponines, tannins, alkaloids, alkenyl phenols,glycoalkaloids,

flavonoids, sesquiterpenes lactones, terpenoids andphorbol esters. Plants are able to develop

new, faster and naturalantimicrobials and then man-made remedies, and that is explaining why

plants succeed in its fightingagainst microbes since millions of years while human failed.

1. Garciniamangostana

2. Caesalpiniacoriaria

3. Psidiumguajava

4. Commiphoramolmol

5. Boswelliapapyrifera

6. Centratherumpunctatum

7. Pelargoniumsidoides

8. Thonningiasanguinea

9. Acacia nilotica,

10. Cinnamomumzeylanicum

11. Syzygiumaromaticum

12. Cinnamomumcassia

13. Prosopisjuliflora

ANTIBACTERIAL AGENTS- Anything that destroys bacteria or suppresses their growth or

their ability to reproduce. Heat, chemicals such as chlorine, and antibiotic drugs all have

antibacterial properties.

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MECHANISM OF ACTION- Antibacterial agents act against bacterial infection either by killing

the bacterium or by arresting its growth. They do this by targeting bacterial DNA and its

associated processes, attacking bacterial metabolic processes including protein synthesis, or

interfering with bacterial cell wall synthesis and function.

PLANTS USED AS ANTIBACTERIAL AGENTS

Calendula (Calendula officinalis) Cinnamon Stick (Cinnamomumzeylanicum) Dried Clove Bud (Syzygiumaromaticum) Garlic (Allium sativum) Echinacea (Echinacea angustifolia) Oregon Grape Root (Mahoniaaquifolium) Marshmallow Root (Althaea officinalis L.) Usnea (Usneabarbata

ANTIFUNGAL AGENTS- An antifungal agent is a drug that selectively eliminates fungal

pathogens from a host with minimal toxicity to the host.

MECHANISM OF ACTION- The three major groups of antifungal agents in clinical use, azoles,

polyenes, and allylamine/thiocarbamates, all owe their antifungal activities to inhibition of

synthesis of or direct interaction with ergosterol. Ergosterol is the predominant componentof the

fungal cell membrane.

PLANTS USED AS ANTIFUNGAL AGENTS

1. Lyciumchinense

2. Baseonemaacuminatum

3. Erythrinaburtii

4. Aquilegia vulgaris

5. Helichrysumaureonitens

6. Alpiniaofficinarum

7. Adina cordifolia

8. Malussylvestris

9. Inulaviscosa

10. Swartziapolyphylla

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OBJECTIVES OF THE STUDY

To collect and authenticate the plant.

To extract out various phytoconstituents by soxhlet method.

To standardize total extract.

To perform phytochemical screening for flavonoids.

To characterise flavonoid by various chromatography techniques.

To elucidate structure of different flavonoid through spectroscopy methods.

To study plants used in antimicrobial activity.

To evaluate antimicrobial activity.

To evaluate antibacterial activity.

To evaluate antifungal activity.

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REVIEW OF LITERATURE

Upadhyay, G. et al; Evaluate hepatoprotective activity of R. elegans (Aerial parts).Roylea

elegans (Aerial parts) are widely used against liver disorders in various tribal areas. Therefore,

the present investigation shows the ethno pharmacological claims of the drug. Following

method used the hydro alcoholic extract of R. elegans (100, 200 & 400 mg/kg, p.o.) was

evaluated for its hepatoprotective activity against CCl4 (1:1 ratio of CCl4 and olive oil, s.c.)

and PCM (3 mg/kg, p.o.) induced hepatotoxicity in Wistar rats. The effect was evaluated with

control and standard drug silymarin (50 mg/kg, p.o.) treated animals and compared it. The

hepatoprotective effect was evaluated by estimating following parameters the serum levels of

SGPT, SGOT, ALP and total billirubin, tissue parameters (TBARS and reduced Glutathione),

and by histological studies. Hydro-alcoholic extract showed a dose dependent hepatoprotective

effect against both the toxicants. R. elegans extract (200 mg/kg and 400 mg/kg) showed

significant (P<0.05) hepatoprotective activity in comparison to the control and toxicant treated

animals. Following research conclude that the present investigations clearly indicate the

hepatoprotective effect of RE against CCL4 and PCM induced hepatotoxicity and validating

its traditional use for liver disorders.

Ahmad, N. et al; (2005), five new anamorphic powdery mildews from India.Mycelium is

defined as it is present on leaves, amphigenoususually epiphyllous, effuse, covering entire

surface of leaves,white, persistent. Present study shows five new anamorphic powdery mildews

viz., Oidiumcassiae-Ieschenaultianae, O. cassiae-torae, O.crotalariae,

O.heliotropiistrigosumand O. roylaecollected on Cassia leschenaultianaL., Cassia

toraeL.,Crotalaria junceaL., Heliotropiumstrigosum, and Roylea cinerea (D. Don)

Baill.,respectively, from NorthernIndia. This study evaluated that Roylea cinerea (D. Don)

Baill, [=R. elegans Wall.], (Lamiaceae) Pithoragarh, no Powdery mildew has so far been

reportedon R. cinerea (=R. elegans). It, therefore, is given a new specific name on the basis of

its occurrenceon the present host.

Majumdar, P. L. et al; (1979), Structure of Moronic Acid.Recent studies showed that isolation

of two furanoid diterpenes isomers in Roylea eleganswhich are royeleganin and royelegafuran.

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Further investigation of this plant showed that yielded a new triterpene which is moronic acid.

This study was done by C13 NMR (Nuclear magnetic resonance) and it evaluated structure of

naturally occurring substance.

Upadhyay, G. et al; (2011), Pharmacognostical studies and evaluation of quality parameters of

Roylea elegans Wall. (Aerial parts).Roylea elgans belonging to family Lamiaceae is commonly

a shrub of monotypic genus. The aerial parts of the plant are widely used fortreatment of

various liver disorders, especially, jaundice and liver debility. Present investigations shows

that,morphological, microscopical (qualitative and quantitative), physic-chemical and

phytochemical characters of the plant. This study is helpful for identity and characteristics of

plant.

Prakash, V., et al; (2010), Traditional uses of ethnomedicinal plants of lower foot hills of

himachal Pradesh-I.This study evaluated the uses of Roylea Cinereacommonly known as kodu

belonging to family Lamiaceae. Shoots of this plant are crushed and eaten with salt to

strengthen the liver by local villagers and young shoots are used as insect repellent for cattle

during rainy season. In skin infection shoot extraction is used. Flowers used in snuffing

purpose during cold season by some people of himachal Pradesh.

Prashad, D., et al; (2014), Antifungal activity of essential oils and plant extracts against

Corticiumsalmonicolor, the incitant of pink canker in apple.Apple botanical name is Malus x

domestica Borkh. It is one of the most important fruit crops which are grown in the temperate

region of the world. In apple, pink canker caused by CorticiumsalmonicolorBerk. Oils of some

plants likeBrassica junceavar. cunefoliahave maximum wound recovery (81.27%) when

applied it as paint against C. salmonicolorunder field conditions for two consecutive years

then followed by Oleaeuropea(75.83 %) with more than 10 mm callus formation respectively.

There is combination of cow urine + Melia azedarach + Vitex negundo +

Artimisiaroxburghiana + Juglans regia + Roylea elegans (1:1:1:1:1:1) which shows maximum

wound recovery (58.54 %) followed by another combination of cow urine + Melia azedarach

+ Vitex negundo + Artimisia roxburghiana + Juglans regia (1:1:1:1:1) (55.44%) which is

useful for callus formation of 5-10 mm respectively.

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Rawat, R., et al; (2013), Roylea cinerea (D. Don) Baillon: A Traditional Curative of

Diabetes, its Cultivation Prospects in Srinagar Valley of Uttarakhand.Roylea cinerea (D. Don)

Baillon, belonging to family lamiaceae and is synonym of R. elegans.This plant is traditionally

used for the cure of ailments such as fever, jaundice, skin disease, malaria and most

prominently in diabetes. In this studyprovides us information about this plant like on the

herbal uses, phytochemical composition, and propagation behaviour of this plant. This plant is

propagated both by seeds and vegetativly. In sandy soil, highest seed germination was

observed, whereas the maximum vegetative propagation is done by stem cutting with proper

farmyard compost in spring season.

Mathur, A., et al; (2010), phytochemical investigation and in vitro antioxidant activities of

some plants of uttarakhand.In this study, it showed that traditional Solvent Extraction (TSE) were

utilized to determine the content of antioxidants in Menthapiperita(root), Syzigumcuminii(seeds),

Murrayakoenigii(leaves), Roylea cinerea(bark) and Leucascephalotes(leaves) extracts. This

extraction method was performed at 60ºC by employing four solvents, methanol, aqueous, hexane

and petroleum ether. Antioxidant activity is done by DPPH (2, 2-Diphenyl-1-picrylhydrazyl) Assay

and Superoxide Anion Radical Scavenging Activity. Methanol extract showed maximum

antioxidant capacity extracted using the TSE, and then followed by aqueous extracts in all the

plants. The antioxidant capacity within the specific extract of the plant, correlated with the Total

Phenolic Content (TPC). Conventional method is used for phytochemical investigation of active

constituents of plants extracts. By this study,it is possible that to formulate the formulation of some

agents which are responsible for antioxidant activity.

Rawat, D. S., et al; (2013), studies on traditional herbal pediatrics practices in jaisinghpur,

district kangra (himachalpradesh, india).Some peoples used crushed Leaves of “Kouru” Roylea

cinerea with the help of a clean pastel and mortar. After crushing, Juice is filtered with a fine

clean cloth piece. Take 1–2 drops of juice is used, twice as nasal drops for three consecutive

days. It is considered that is good for cough.

Raj, H., (2014),Integration of soil solarization with soil amendments, botanicals and microbial

pesticides for the management of soil-borne diseases of strawberry.In this study fourteen native

plants were evaluated against threeimportant soil-borne pathogens like Rhizoctoniasolani,

Sclerotiumrolfsii, Fusariumoxysporumf.sp. fragariaeof strawberry to know their efficacy by

poison food technique in potato dextrose agar medium. Fresh leaves which are 45 to 60 days

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old of Aloe barbadensis, Bougainvillea glabra, Eucalyptus globulus, Lantana camara,

Menthalongifolia , Ocimum sanctum, Roylea elegans, Cryptolepsisbuchanani, Menthapiperita ,

Thujachinensis, Urticadivocia, Vitex negundo, Pogostemonebenghalensis and seeds of Melia

azedarach were taken and 100 % formulations were made on weight and volume (w/ v) basis

in the water (Raj and Tomar, 2013). Out of these five plants like, M. azedarach, R. elegans, B.

glabra, O. sanctum, C. buchanani) most effective were taken to make a soil amendment by

adding equal parts of leaves of all except for M. azedarach where mature seeds (light yellowish

colour) were taken.

Chandel, S., (2014), Botanicals, biofumigants and antagonists application in managing stem rot

disease caused by Rhizoctoniasolani Kuhn in carnation.In this study hot water extracts of nine

plants species namely Melia azedarach, (Darek- seed extract) and leaf extracts of Eucalyptus

globules (Safeda), Roylea elegance (Karu), Vitex negundo (Banna), Adhatodavasica (Basuti),

Tageteserecta (Marigold), Murrayakoenigii (Currypatta), Menthaarvensis (Pudina), Cannabis

sativa (Bhang) and two commercial formulations for neem were evaluated at 3 and 5 % in vitro

against Rhizoctoniasolani. In this study evaluated that R. elegans inhibit percentmycelial

growth at concentration 35.10.

Gautam, R. H., et al; (2011), bioformulation of different plant species show effective against

five major post-harvest rots in apple.In this study evaluation of bioformulation of different

plant species show effective against five major post-harvest rots in apple viz., blue mould rot

(Penicilliumexpansum), bitter rot (Glomerellacingulata), brown rot (Moniliniafructigena),

pink mould rot (Tichotheciumroseum) and whisker’s rot (Rhizopussiolonifer).

Dua, V. K., et al; (2011),Antiprotozoal activities of traditional medicinal plants from the

Garhwal region of North West Himalaya, India. In this study, new plant-derived biologically

active compounds against protozoan parasites, an ethnopharmacological study was carried out

to evaluate extracts from selected 17 traditional medicinal plants which were used by healers

from the Garhwal region of North West Himalaya for the treatment of protozoal infections.

These researches evaluate the investigation of components of traditional medicines as potential

new antiprotozoal agents. On the other hand since herbalism has become the main stream

throughout the world, investigation demonstrates that these non-polar plants extracts of the

plants like Artemisia roxburghiana, Roylea cinerea, Leucascephalotes, Nepetahindostanaand

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Viola canescens examined in this study could play an important role in herbal formulations for

the treatment of vector borne protozoal diseases.

Neeru; et al; (2016), Pharmacognostic Standardization Parameters of Roylea elegans Wall

(Aerial Parts).The qualitative and quantitative microscopy, physicochemical evaluation,

phytochemical screening and fluorescence analysis of the plant were done by the standard

procedure recommended in the WHO guidelines. Macroscopic study shows that leaves were dark

green with lemon like odor and bitter taste, 2-8 cm length and 1-8 cm wide, shape: ovate, hairy

upper and lower surface, apex: acute and having reticulate veination, Stems: were light green

Microscopic evaluation of the leaves powder shows the presence of trichomes (unicellular covering

and glandular), upper epidermis, vessels, xylem fibres, wavy trichomes. The transverse section of

the leaf shows the presence of epidermis layer followed by cuticle layer, lignified vascular bundles,

trichomes, collenchyma, and palisade cells. Various pharmacognostical parameters help to evaluate

the identification and standardization of Roylea elegans (aerial part).

Neeru; et al; (2016), Determination Of Total Phenolic Content, Total Flavonoid Content And

Total Antioxidant Capacity Of Different Extracts Of Roylea Elegans Wall (Aerial

Parts).Petroleum ether, chloroform, ethyl acetate, methanol and water extracts of Roylea elegans

(aerial parts) was screened for assessing bioactive phytochemical constituents. The preliminary

phytochemical screening using the standard phytochemical tests detected the presence of alkaloids,

steroids, flavonoids, terpenoids and phenols in different solvent extracts. Total phenolic and

flavonoid contents along with total antioxidant capacity of the ethyl acetate extract of Roylea

elegans (aerial parts) was evaluated to explore the reliable and potential sources of novel natural

antioxidants. The ethyl acetate extract showed high flavonoid content. Due to high flavonoid

content it showed better antioxidant activity.

Bhatt U. P., et al; (2015), Evaluation of In Vivo and In Vitro Anti-diabetic Activity of Roylea

cinerea.In the present study, we investigated In vivo &In vitro anti-diabetic activity of different

fractions (petroleum ether, ethyl acetate and methanolic) of aerial part of Roylea cinerea. In

vivo method Glibenclamide, an oral hypoglycemic agent was used as a positive control against

alloxan-induced diabetic rats and In vitro method all three fractions of Roylea cinerea were

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evaluated for antidiabetic activity by determining their inhibitory effect on alpha amylase and

alpha-glucosidase enzyme. The methanolic extract of aerial part of Roylea cinerea was found

to exhibit potent antidiabetic activity In vitro as well as In vivo. The methanolic extract showed

maximum reduction (41.20% In vivo and 80% In vitro) in blood glucose levels.

Almeida R. N., (2001), Plants with central analgesic activity.A systematic analysis study was

carried out through a recent computer search of data on Natural Products Alert (NAPRALERT

– SM) and information from several journal articles, mainly. The plant selection was based on

the effects presented in specific animal models for evaluation of analgesic activity, involving

mechanisms controlled by the central nervous system, such as: a) tail flick response to radiant

heat, b) tail flick reaction time test, c) tail immersion test, d) hot water induced analgesia, e)

naloxon reversed analgesic effect, f) inhibited substance P- induced nociceptive response, g)

hot plate method, h) hot wire method, i) tail pinch method, j) acetic acid-induced writhing, k)

tail clip mehtod and l) tail pressure method. Roylea elegans Lamiaceae Dried leaf India i. p.

350 and 500 mg.

Peter K. V., (2006), herbs and plants used as anticancer.Herbal therapies for the treatment of

cancer are of different types. It may be an alternative or blood purifier, which destroy toxins.

These herbs are used fresh along with a detoxifying diet. This category of herbs is claimed to

cure lymphatic or skin cancer and are better for Pitta and Kaphavarieties. The second

categories of herbs are circulatory stimulants that promote blood circulation and aid in the

healing of tissues. Breast and uterine cancer are treated with these herbs which will affect all

the three elements. The third categories of herbs are immune strengthening tonics which are

better in debility conditions and usually work on Vata. The fourth category of herbs includes

special expectorants. They are used for thyroid, neck or lymphatic cancer. Roylea cinerea (D.

Don) Baillon Aerial parts – precalyon shows antitumor

Activity.

Gaur R. D., (2011), Indigenous knowledge on the utilization of medicinal plants diversity in

Siwalik region of Garhwal Himalaya, Uttarakhand.Ever since the dawn of civilization, the

ambient vegetation and the resources constituted major source of humanexistence for various

substantial requirements. Our present knowledge on plant resources emerged from the

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traditional heritable knowledge descended from generation to generation. However, traditional

knowledge pertaining to several aspects remained untapped from various remote localities or

populations. Furthermore, with the present trends of excessive exploitation of natural resources

and degradation of habitats, conservation and ecological management require coherence of

traditional skills and modern approaches. Therefore, the present study is to record traditional

plant based knowledge among the inhabitants of Siwalik region of Uttarakhand Himalaya.

Extensive field survey was made for the collection of data on the medicinal aspects of plant

species in the study area covering the parts of districts Pauri, Dehradun and Haridwar. During

the course of study 130 plant species belonging to 65 families are reported, used as traditional

medicine by the local inhabitants of this region. Roylea cinerea (D.Don) Baill.Kadwi, TiunaS C

400-1500 Lv YT Fever, Malaria Diabetes.

Sharma J. et al; (2012), The treatment of jaundice with medicinal plants in indigenous

communities of the Sub-Himalayan region of Uttarakhand, India. A total of 40 medicinal plants

belonging to 31 families and 38 genera were recorded to be used by the studied communities in

45 formulations as a remedy of jaundice. Bhoxa, nomadic Gujjars and Tharu communities used

15, 23 and 9 plants, respectively. To our knowledge eight plants reported in the present survey

viz., Amaranthusspinosus L., Cissampelospareira L., EhretialaevisRoxb., Holarrhenapubescens

Wall., Ocimum americanum L., Physalisdivaricata D. Don, Solanum incanum L. and

Trichosanthescucumerina L. have not been reported earlier as remedy of jaundice in India.

Literature review revealed that a total of 214 (belonging to 181 genus and 78 families), 19

(belonging to 18 genus and 12 families)and 14 (belonging to 14 genus and 11 families) plant

species are used as internal, external and magicoreligious remedies for jaundice, respectively

by various communities in different parts of India. Most widely used hepatoprotective plant

species for treatment of jaundice in India is Boerhaviadiffusa L. followed by

Tinosporacordifolia (Willd.) Miers, Saccharumofficinarum L.,

PhyllanthusamarusSchumach.&Thonn.,Ricinuscommunis L., Andrographispaniculata (Burm.

f.) Nees.,Oroxylumindicum (L.) Kurz, Lawsoniainermis L. and Ecliptaprostrata (L.) L. Roylea

cinerea (D. Don) Baill. Local community Garhwal district (Uttarakhand) Branch Pieces of

branches made into beads and garlanded by infants to avoid jaundice.

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RESEARCH METHODOLOGY

1. EXTRACTION

2. STANDARDIZATION PARAMETERS FOR TOTAL EXTRACT

3. ISOLATION OF FLAVONOIDS

3.1 TLC

3.2 HPTLC

4. PHYTOCHEMICAL SCREENING OF THE ISOLATED COMPOUND

5. COMPOUND CHARACTERIZATION

5.1 IR

5.2 NMR

5.3 MASS

6. METHODS FOR ANTIMICROBIAL ACTIVITY

1. DIFFUSION METHOD

1.1 Agar Disk Diffusion Method

1.2 Antimicrobial Gradient Method (E Test)

1.3 OTHER DIFFUSION METHOD

1.3.1 Agar Well Diffusion Method

1.3.2 Agar Plug Diffusion Method

1.3.3 Cross Streak Method

2. THIN LAYER CHROMATOGRAPHY- BIOAUTOGRAPHY

2.1 Agar Diffusion

2.2 Direct Bioautography

2.3 Agar Overlay Bioassy

3. DILUTION METHODS

3.1 Broth Dilution Method

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3.2 Agar Dilution Method

7. ANTIBACTERIAL ACTIVITY

7.1 Agar diffusion well-variant

7.2 Agar diffusion disc-variant

7.3 Bioautographic method direct-variant (chromatogram layer)

8. ANTIFUNGAL ACTIVITY

19

ACTION PLAN

1 st semester Finalize topic & searching literature - 4 months

Synopsis Preparation - 2 months

2 nd and 3 rd semester

Lab work&extensive research work submission of at least research paper in journal of repute -

1 year

4 th semester Compilation of data - 3 months

Writing of thesis work - 2 months

Submission of thesis - 1 month

20

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