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www.wjpps.com Vol 6, Issue 1, 2017.

740

Kanimozhi et al. World Journal of Pharmacy and Pharmaceutical Sciences

COMPARATIVE EVALUATION OF UN-PURIFIED AND PURIFIED

FORM OF NERVAALAM (CROTON SEEDS) BY

PHARMACOGNOSTICAL, PHYSICOCHEMICAL AND FT-IR

ANALYSIS.

J. Kanimozhi*, S. Vanathi, A.M. Dharsana and P. Shanmugapriya

Post Graduate Department of Nanju Noolum Maruthuva Neethi Noolum, National Institute

of Siddha, Tambaram Sanatoruim, Chennai 600047, Tmilnadu, India.

ABSTRACT

The World Health Organization (WHO) has now imparting an greater

effort regarding the need and importance of medicinal plants for public

health care in developing countries like India and has evolved with

specific guidelines to support the member states in their capacity to

formulate national policies on traditional medicine and to study their

potential usefulness including standardization, evaluation, safety and

efficacy. Standardization of herbs is an essential factor in order to

assess the quality, purity, safety and efficacy of drugs based on the

concentration of their active principles. It is very important to establish

a system of standardization for every plant medicine in the market,

since the scope for variation in different batches of medicine is

enormous. Still now there is no proper documentary evidence available

on purification aspect of this medicinal herb and hence this prompted us to peruse systematic

characterization of purification process of the plant Nervaalam (Croton tiglium). Croton

seeds (CS) are traditionally used in abdominal disorders constipation and dyspepsia. Croton

seeds were subjected to microscopical, physicochemical and FT-IR analysis in order to

ascertain its purity. Results of pharmacognostical evaluation of CS show the presence of well

distinguished seed coat, pericarp, cotyledon and section of seed coat reveals visible meso and

endocarp. Further presence of calcium oxalate crystals was confirmed in the endocarp. The

results obtained from physicochemical analysis of un purified CS reveals that total ash value

of CS is about 2.74% in which acid insoluble ash is 1.44%. Extraction value of drugs renders

most significant information about partition of the active component the results of extract

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 6, Issue 1, 740-754 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. J. Kanimozhi

Post Graduate Department

of Nanju Noolum

Maruthuva Neethi

Noolum, National Institute

of Siddha, Tambaram

Sanatoruim, Chennai

600047, Tmilnadu, India.

Article Received on

24 Oct. 2016,

Revised on 14 Nov. 2016,

Accepted on 04 Dec. 2016

DOI: 10.20959/wjpps20171-8308


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value study shows that alcohol soluble extract of CS is 11.50% and water soluble extract is

8.49%. The loss on drying value of CS is about 11.35%. Physicochemical analysis of Purified

CS reveals that total ash value of CS is about 2.28% in which acid insoluble ash is 1.08%.

The results of extract value study shows that alcohol soluble extract of CS is 9.87% and water

soluble extract is 2.36%. The loss on drying value of purified CS is about 10.80%. Result

obtained from the FT-IR analysis reveals that raw unpurified CS shows the presence of

functional groups like alcohol, alkane, nitro, acid and aldehyde ,whereas the purified sample

indicates the presence of alkenes and amines in addition to existing functional groups.

KEYWORDS: Nervaalam, Croton tiglium, Croton seeds, Pharmacognostical evaluation

Physicochemical, FTIR.

INTRODUCTION

In Ayurveda, siddha and unani (ASU) system of medicine plants, minerals, and animal

products are used as main drugs to cure various ailments.[1]

There is a global resurgence in

the use of these medicines along with a growing scientific interest in them as a source of new

drugs.[2]

There has been a boom in the usage of ASU drugs and export is appreciably high in

the last two decades.[3]

The usage of plants as medicines goes back to early man. Certainly the great civilization of

the ancient chinese, indians and north africans provided written evidence of man's ingenuity

in utilizing plants for the treatment of a wide variety of diseases. Herbal preparations against

human ailments are gaining importance due to the partial rejection of synthetic drugs because

of their side effects. Complementary and alternative medicines may be appealing to patients

due to their seemingly low side effects profile and optimistic evidence especially when more

treatments that are conventional have high failure rates or numerous side effects. The earliest

mention of the medicinal use of plants is to be found in the Rig-Veda which dates back as

early as 3500 BC.

Currently about 80% of the world population depends on herbs, herbo-minerals and other

plant-derived medicine for the first line of primary health care for human alleviation because

it has very minimal side effect. Herbal medicine will be broadly classified into various basic

systems: Traditional indian herbalism, which is part of traditional oriental medicine, siddha

herbalism, which is derived from siddha system of medicine.[4,5]


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Nervaalam (Croton tiglium) is an important medicinal plant of the family Euphorbiaceae

which is used for the treatment of constipation, dyspepsia, dysenteriae, gastrointestinal

disorders, intestinal inflammation, rheumatism, peptic ulcer, visceral pain and

headache.[6,7,8,9]

C. tiglium seeds oil is reported to contain phorbol esters and crotonic acid

along with the fatty acids.[10]

C. tiglium, commonly known as Kumbhinī is well known for its

severe purgative action.[11]

Croton tiglium (CT) belongs to the family Euphorbiaceae is widely used in siddha

formulation like soolai kudaram, Agasthiyar kuzhambu, Thazhamboo Mathirai, Meganatha

Kuligai, etc after purification process. Croton seeds are traditionally used in abdominal

disorders constipation and dyspepsia.[12,13,14]

Several studies are being carried towards its activities genotoxicity, M³ muscarinic receptor

and Ca²+ influx mediated muscle contraction, Anti-HIV-1 phorbol ester activity, purgative

and inflammatory activity.[15]

It consists majorly phorbol ester which has tumor enhancing property. Seeds are reported to

contains croton, a toxalbumin and crotonoside. Major known chemical constituents are

glyceryl crotonate, crotonic acid, crotonic resin. The amino acid composition of purified

lectin from seeds was reported as aspartic acid, threonine, serine, glutamic acid, proline,

glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan,

histidine, lysine, arginine and cysteine. Siddha system of medicine recommends the

administration of Nervalam only after purification.

Standardisation of drug means

confirmation of its identity, quality and purity throughout all phases of its cycle.[16]

Drug standardization and way forwards for indian system of medicine drug manufacturing

industry as the national authorities like national drug regulatory authorities should ensures

that all ASU pharmaceutical product subjected to their control are in conformity with quality,

safety, efficacy and that all premises employed the manufacturing and distribution of these

preoducts comply with god manufacturing process (GMP) standards, so as to ensure the

continued conformity of the products with these requirement until such time as they are

delivered to the end user. By understanding the need of current scenario with respect to

herbal drug purification and standardization the current work was undertake to evaluate the

purity of croton seed by systematic purification process as per siddha principles and further

study drug is subjected to microscopical, physicochemical and FT-IR analysis.


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MATERIALS AND METHODS

Purification Process

Test drug collection

Nervaalam (Croton tiglium) was obtained from reputed drug store in Chennai.

Identification and Authentication

The drug was identified and authenticated by Prof. Dr. Jayaraman, Ph.D. (voucher no

PARC/2016/3221), Plant Anatomy Research Centre, West Tambaram.

Method of Purification

3Liters of Buffalo dung juice was taken in a mud pot. 650gms of Croton seeds were knotted

in a cotton cloth and soacked in the dung juice by hanging in the above pot as

Thulayanthiram. The mud pot was heated using firewood under low flame until the dung

juice was reduced to 1 lit. The knotted seeds were taken out and cloth over the seeds were

removed. Then washed with warer, remove the outer seed coat and inner cotyledon were

removed. Remaining 350gms of seeds are again subjected to thulayanthiram by using Raw

rice with water for 3hours. After 3hrs boling the knotted seeds were taken out and cloth over

the seeds were removed, the seeds were washed with water. Again the seeds were subjected

to thulayanthiram now by using Milk for 3 hours. After 3hrs, knotted seeds were taken out

and the cloth over the seeds were removed, then washed with water and dried it. After drying,

it was fried with Castor oil. After complete of frying purified Croton seeds was collected and

stored in an air tight container (Book name- Sigicha rathna deepam).

Pharmacognostical Analysis

Macroscopical Investigation

The macroscopical evaluation of CS was performed as per the methods of Khandelwal.[17]

Various organoleptic characters such as color, shape, size, odour, taste and texture were

studied and further width of the raphe and hilum were carefully observed using simple

microscope.

Microscopical Investigation

The seeds were cut into thin transverse section using a sharp blade and the sections were

stained with saffranine. Transverse sections were photographed using LEICA trinocular

microscope attached with camera under bright field light.[18]


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For the microscopical evaluation, the fresh samples were cut free handed and immersed in

clearing reagent (chloral hydrate). The sections were dehydrated with varying strength of

absolute alcohol and then stained with the mixture of phloroglucinol and conc. HCl (1:1,

v/v).[19]

Anatomical characteristics were described from the samples which were fixed and embedded

in plastic resin.[20]

The blocks were sectioned at 10 µm on a rotary microtome with steel

knives type C. The sections were stained with Toluidine Blue 0.05% in acetate buffer with

pH 4.7.[21]

Physico-chemical analysis

Percentage Loss on Drying[22]

10gm of CS was accurately weighed in evaporating dish and was air dried at 105oC for 5

hours and then weighed.

Determination of Total Ash[23]

3 g of CS was accurately weighed in silica dish and incinerated at the furnace a temperature

400 ºC until it turns white in color which indicates absence of carbon. Percentage of total ash

will be calculated with reference to the weight of air-dried drug.

Determination of Acid Insoluble Ash

About 0.5gm of the ash obtained by total ash test will be boiled with 25 ml of dilute

hydrochloric acid for 6mins. Then the insoluble matter is collected in crucible and will be

washed with hot water and ignited to constant weight. Percentage of acid insoluble ash will

be calculated with reference to the weight of air-dried ash.

Determination of Alcohol Soluble Extractive

About 5 g of the air dried CS will be macerated with 100 ml of Alcohol in a closed flask for

twenty-four hours, shaking frequently during six hours and allowing to stand for eighteen

hours. Filter rapidly, taking precautions against loss of solvent, evaporate 25 ml of the filtrate

to dryness in a tared flat bottomed shallow dish, and dry at 105ºC, to constant weight and

weigh. Calculate the percentage of alcohol-soluble extractive with reference to the air-dried

drug.


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Determination of Water Soluble Extractive

About 5 g of the air dried CS will be macerated with 100 ml of chloroform water in a closed

flask for twenty-four hours, shaking frequently during six hours and allowing tost and for

eighteen hours. Filter rapidly, taking precautions against loss of solvent, evaporate 25 ml of

the filtrate to dryness in a tared flat bottomed shallow dish, and dry at 105ºC, to constant

weight and weigh. Calculate the percentage of water-soluble extractive with reference to the

air-dried drug.

Determination of pH

About 5 g of CS will be dissolved in 25ml of distilled water and filtered the resultant solution

is allowed to stand for 30 mins and the subjected to pH evaluation using pH meter.

Fourier Transform – Infra Red Spectroscopy[24]

Fourier Transform – Infra Red Spectroscopy Study (FTIR) IR data acquired with FT-IR

spectrometer FT/IR-4100 –Jascoasia portal. About 20 mg of the CS before and after

purification was taken on a microspatula and grounded well with required quantity of KBr

salt. Sample admixed with KBr with trituration aided by mortar and pestle until to get a

uniform fine powder of sample- KBr mixture. Further mixture was loaded in pellet die and

subjected to 5000-10,000 psi in pelletizer. Resulting pellet was placed in FTIR sample holder

and expose to IR radiation to get the spectra.

RESULTS

Macroscopic Evaluation of CS

Macroscopic examination of CS appeared as oblong, oval, approximately 12-15mm in length,

5-8 mm in width, ventrally shows longitudinally running centrally located ridge of raphe. A

small circular point located at the narrow end is the hilum. It is brown and some seeds are

black in colour. Hence the drug was confirmed as seed of Nervalam (Croton tiglium).

Microscopic Evaluation of CS

Results of pharmacognostical microscopic evaluation reveals that the seed is spherical

capsule, three valued or three deciduous, two valved coeei, seeds three, smooth, carunculate;

Testa, crustaceous; albumin copious. Cotyledon flat, ovary covered with stellate tomantum.

Further seed shows wide and thick, broadly elliptical body. It also consists of outer seed coat

and inner pericarp. These two cotyledons which are flat, long and parallel to each other


746


(Figure 1). The cotyledons have thin layers of epidermis and circular thin walled ground

parenchyma cells.

Cot - cotyledon, Pc - Pericarp, osc – Outer seed coat

Figure 1: Vertical section of SC

The outer seed coat includes thick layer palisade like vertically elongated compact

macrosclereids; this zone in the epicarp of the seed coat (Figure 2).

Ec – Endocarp, MC- Mesocarp

Figure 2: Sectional view of the seed coat of SC

Inner to the epicarp, is a thin portion of three or four layers of vertically elongated, spindle

shaped thick walled cells (Figure 3).


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EnC – Endocarp, MC – Mesocarp

Figure 3: Slide view of Endocarp and Mesocarp of SC

The endocarp portion is wide and thick. It consists of epidermal layer of squamous cells and

circular, thin walled compact parenchyma cells (Figure 4).

Cot – cotyledon, EnC – Endocarp -

Figure 4: Sectional view of cotyledon and endocarp

The cells possess dense starch grains (Figure 5). These are also sparsely distributed calcium

oxalate dense type of crystals.


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Cr - Calcium oxalate crystal

Figure 5: Crystal distribution in the endocarp

Physico -chemical Evaluation of CS

The results obtained from physicochemical analysis of un purified CS reveals that total ash

value of CS is about 2.74% in which acid insoluble ash is 1.44%. Extractive values are

representative of the presence of polar and non-polar compounds in a finished product. The

water soluble extractive value can be used to indicate poor quality, adulteration with any

unwanted material or incorrect processing of the crude drug during the process of drying;

storage etc. Extraction value of drugs renders most significant information about partition of

the active component the results of extract value study shows that alcohol soluble extract of

CM is 11.50% and water soluble extract is 8.49%. The loss on drying value of CM is about

11.35%. Physicochemical analysis of Purified CS reveals that total ash value of CS is about

2.28% in which acid insoluble ash is 1.08%. The results of extract value study shows that

alcohol soluble extract of CM is 9.87% and water soluble extract is 2.36%. The loss on

drying value of purified CM is about 10.80%. The results of physicochemical analysis were

tabulated in Table 01.

Table 1: Physico-chemical Evaluation of CS

S. No. Physico-chemical

Parameter

Before Purification

% in W/W (mg/g)

After Purification

% in W/W (mg/g)

1. Loss on drying at 105oC 11.35 % 10.80 %

2. Ash Values

Total Ash

Acid Insoluble Ash

2.74 %

1.44 %

2.28 %

1.08 %

3. Extract Values

Water soluble

Alcohol soluble

8.49 %

2.36 %

2.36 %

9.87 %

4. pH 5.2 6


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FT-IR Analysis of CS

FT-IR analysis of un-purified croton seeds shows the presence of most significant functional

groups such as alcohol, alkane, nitro, acid and aldehyde and while compared to un-purified

sample, the FT-IR spectra of purified sample shows the addition to two more functional

groups, that is alkene and amine in addition of these functional groups are might be

importance of the purification process. The FT-IR spectrum with corresponding stretching

and bending were shown in the figure 6 and 7.

Figure 6: FT- IR spectrum of CS before purification

Figure 7: FT- IR spectrum of CS after purification


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DISCUSSION

Majority of pharmaceutical companies are presently conducting extensive research on plant

materials gathered from the rain forests and other places for their potential medicinal value.

Traditional psychopharmacology has innumerable models to study the pharmacodynamic

activities.[25]

But the extrapolation of the findings of these models to clinical indication is not

easy and can be misleading too. Many medicinal plants from India have been shown to have

activity by the conventional methods of psycho neuropharmacology.[26]

According to WHO, the macroscopical and microscopical description of a medicinal plant is

the first step towards establishing the identity and the degree of purity of such plant.

Macroscopic identity of medicinal plant material is based on sensory evaluation parameters

like, shape, size, color, texture and odour. Here the macroscopic examination it appeared as

oblong, oval, approximately 12-15mm in length, 5-8 mm in width, ventrally shows

longitudinally running centrally located ridge of raphe. A small circular point located at the

narrow end is the hilum. It is brown and some seeds are black in colour. Hence the drug was

confirmed as seed of Nervalam (Croton tiglium).

Transverse section of the sample CS showed, seed is elliptical in shape, thin layer of nucleus,

centrally located cotyledons embedded in a narrow cavity and encircled by wide oily

endosperm. TS of seed coat shows on outer row of palisade like cells of testa with deposits of

carbonated and covered with thin cuticle. The cells are thick walled.

On morphological examination it was revealed that there were considerable changes in

Organoleptic character. The colour of un-purified Croton seeds was brownish black in colour,

when compared to this Purified Sample which was brown in colour. At the same time odour

of Unpurified Sample was strong irrient in nature, but in purified Sample had castor oil in

smell.

Calcium (Ca) oxalate crystals are prevalent in fungi[27]

and many higher plants[28]

, including

legumes.[29]

The shapes, locations and hydration forms of these crystals are specific for a

species and developmentally determined by the type of cell, tissue and organ in which they

occur. Presence of CaO crystals determines the nature and purity of the herbs.[30]

Microscopical evaluation of the present investigation reveals the presence of CaO crystals

which determine its purity.


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WHO and AYUSH insisted many guidelines to be followed for quality control for a better

standardization of the drugs.[31]

Siddha system of medicines comprises many numbers of safe

and valuable herbal medicines have better therapeutic efficiency either at its raw state or

purified form and are clinically used by the Siddha practitioners.

The total ash value of un-purified croton was found to be 2.74 and finally it reaches 2.28% in

purified samples which is very minimal level. It indicates the purity of drug. Acid insoluble is

designed to measure the amount of ash insoluble to diluted hydrochloric acid. Acid insoluble

ash of un-purified CS was found to be 1.44% and it reduced to 1.08% after purification.

Strongly acidic nature of the drug may cause the harmful effects to the body and poorly

absorbed from gut, so the screening for pH is important for drug. It represents the chemical

nature of the drug and the site of absorption.[32]

The pH of un-purified CS was 5.2,but after

purifying with buffalo dung, raw rice, milk and castor oil it was observed that the pH of 6. It

is weekly acidic and it may aids in quicker absorption.

Loss on drying test is designed to measure the amount of water and volatile matter in a

sample when sample is dried under specified conditions. Moisture is one of the major factor

that responsible for the deterioration of the drugs. Low moisture content is always desirable

for higher stability of drug. The percentage of loss on drying of CS before and after

purification (11.35% to 10.80%) was found within acceptable limit (1- 20%).

Water soluble extractive value of un-purified CS is 8.49% and in purified sample it was

2.36% it shows the possibility of water soluble constituents such as tannins, sugar and alcohol

soluble substance such as alkaloids may be present in the drug. Alcohol soluble extractive

value of un-purified CS is 11.50% and in purified sample it was 9.87%.

FTI-R analysis of un-purified croton seeds shows the presence of bio-active functional groups

such as alcohol, alkane, nitro, acid and aldehydes. Whereas the report of purified CS shows

the presence of two functional groups, that is alkene and amine along with the existing ones.

Addition of these functional groups justifies the importance of the purification process.

The quality and therapeutic efficacy of herbal drugs is dependent on the active constituents

which are present in the plant cell. Survey denotes that newly approved drugs reported

between 1983 and 1994, drugs of natural origin predominated (78%) in the antibacterial area,

while 61% of the 31 anticancer drugs approved in the same period were either natural


752


products, nature derived products or compounds modeled on natural product parents or

“leads”. In addition, 50% of the bestselling pharmaceuticals in 1991 were either natural

products or their derivatives.[33,34]

CONCLUSION

Quality control for efficacy and safety of herbal products is of paramount importance. Quality

can be defined as the status of a drug that is determined by identity, purity, content and other

chemical, physical, or biological properties, or by the manufacturing processes. From the

data’s of the present investigation it was concluded that the siddha drug Nervaalam (Croton

seeds) was purified and analyzed according to the standard procedures. Results of the study

generated an evidence based data with respect to macroscopical, microscopical,

physicochemical and functional group parameters of the study drug CS. There were notable

changes was found between un-purified and purified form of CS. Further studies have to be

carried with special emphasis on phytochemical and characterization aspect of the drug and

its future.

ACKNOWLEDGEMENTS

I wish to acknowledge my thanks to Dr. Annop Austin and IIT Madras for their analytical

support in this research work and The Noble research solutions, Chennai for their technical

support for preparing this manuscript.

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