Chapter-3

Proximate & Phytochemical analysis of Abutilon Indicum

3.1 Introduction:

Various medical plants contain the main sources for innovative pharmaceutical and

physical condition care constituents [83]. Also various natural herbs can attribute various

applicable medicinal properties. As history of human being are probably as old as the history of

plants as being useful for medicinal purposes. For purpose of pharmaceutical use scientists traced

to the extraction of these plants as well as development of several drugs from traditionally used

foil medicines [64]. Identification, extraction, classification and analysis of several new active

phytochemical from these plants give birth to some new activating high profile drugs [119]. In

India and other countries the use of traditional medicines is widely spread.Secondary plant

metabolite plays fundamental role for human being fitness in addition to might be nutritional

importants for growing body evidences [73]. It believes that natural compound extracts

commencing therapeutic plants are supplementary biological dynamic than separated compound

energetically for their interactive effects [96].Chemical point of view analysis of plants has

exposed the occurrence of numerous chemicals including alkaloids, tannins, steroids, saponins,

flavonoids, glycosides etc.

For different disease treatments purpose worldwide traditional medicine plant based

drugs have been used. Large amount from meditational plants are produced in India and suitably

called these as botanical garden of the world. For the past two tanners, there has been an

enhancing investigation within the significance of dissimilar extract obtain since conventional

medicil plant at the same time as possible source from some innovative disinfectant agent.

Medicinal plants constitute rich sources from which disinfectant agents may be determined.

Plants are applied tomedically with in unlike country and the sources of numerous effective and

dominant drugs [173].

The pharmacological medicine provide an substitute advance intended for the invention

of antimicrobic (activity) agentive role, specifically to consider the medical plant withalong a the

past of conventional uses as a prospective foundation of substances alongwith considerable

biological and pharmacological activities such as anticancer, antioxidant and heptagon protective

other many incurable diseases. Phytochemical studies let beconcerned the concentration of the

plant scientist develop innovative as well as advanced technique. Such type of technique brought

a considerable role in the investigation for supplementary resource from unprocessed materials

for the pharmaceutic industries (physiochemical). Although wide range from uses in traditional

medicine is often mentioned in the literature, few ethno botanical surveys and complete

physiochemical studies have been published.

In recent years, plant metabolites (phytochemical), provide with strange pharmacologic

actions, get a load widely looked into as a medicinal source agents [108]. For the bacterial

infection perquisite photochemical with adequate antibacterial efficacy will be used [25]. So

prevention of various ailment and treatment man has been using various plants part [177].

Medical plant corresponds to a prosperous foundation from which antimicrobic agentpossibly

found. As source of many powerful drugs plant is applied medicinal purposes in different

countries [173]. Nigeria is based on the claims of their effective use for the treatment of many

diseases from the interest in the scientific investigation of medicinal plants. To enhance the use

of this plant against disease caused by the test pathogen, consequently research into the effects of

these local medicinal plants is expected.

Medicinal plants having great importances to the physical condition of communities as

well as individuals, plant have been providing sources of encouragement for original drug

compound. Modern scientist have made phenomenal step in developing this heritage handed

over by our forefathers [170]. Plants usually contain physiologically active substances which are

technically referred to as drugs and greater than the year these drugs contain demoralized as

conventional medicines for the treatment of a variety of ailment afflicting man [2]. Antimicrobial

substances are those substances that are competent from inhibiting or else destroying the

development of microorganism [181]. The drugs obtained from medicinal plants are termed

crude drug of natural or biological origin as described by pharmacist and pharmacologist [170].

Contagious disease is the most important problem in while to humanity. Use of antibiotic drug

becomes a worldwide disquiet option [182]. The investigational efficiency from numerous

obtainable antibiotics be individual exposed by the emergences from multicultural challenging

pathogen [27]. Various contagious diseases contain notorious to be treated with herbal remedy

through out the history of human beings. The constant in addition to imperative require

discovering some new anti-microbial compounds with diverse probable chemical

structures.Novel mechanisms of action for new and reappearing infectious diseases. However

researcher effectively turning their attentions toward family line medicines, look for the new

ways in the direction of build up superior drug along side microbic infection [29]. To increase

the disappointment from chemotherapeutic in addition to antibiotic drug confrontation exhibit

through pathogenetic microbic communicable agent have lead in the direction of the

transmission from a number of therapeutic plant for the most part probable disinfectant activities

[36]. The therapeutic activities of most plants are allocated to the occurrence of one or more of

such mechanism such as tannins, saponins, cardiac glycosides, alkaloids etc [132].

Previous chapter deals with the Abutilon Indicum is of the main traditionally important

medicinal plant, so it is very widely used in local tribal people so this chapter deals to investigate

preliminary phytochemical screening of Abutilon indicum leaf, stem, root and mature fruit with

seed extract in different solvent like water, ethanol, chloroform, ether, acetone and methanol for

quantitative and qualitative analysis of about active secondary winding metabolite, in the

direction of determination of ethno medical claim from such extensively use medical plant.

3. 2 Required material with methods:

3. 2. 1 Plant materials:

Healthy plants materials like leaves, stem, root and matured fruits with seeds of A.

indicum were collected in month of September to November 2011 from Shirur and Khed

(Ambegaon), Baramati, Haveli and Maval Tahsil area from Pune District in State of

Maharashtra, India. Forciting the normal morphologic characteristics feature provide through

flora, used for recognition from different variety.

Chemical analysis holds an important position in our modern society. Knowledge of

analytical procedures has become essential in practically all the arts and science as well as the

technical operations of industry. The discovery of new medicinal, new fabrics, new alloys, and

other materials would not be possible without the services of the analytical chemist. Analytical

chemistry is such an important part of our industrial structure and advancing science that without

it our present day economy could not exist.

In the early days of chemistry, analysis was almost synonymous with chemistry itself.

Robert Boyle first introduced the word “analysis” to apply to the identification of individual

substances in the presence of each other. Form his interest in analysis it was recognized that

advancement in chemical knowledge could come only from the experimental method of the

laboratory. Berzelius was a master in the field of chemical analysis. He developed many new

experimental methods which include the separation of the metal constituents of a number of a

complex minerals in 1818 has published a table atomic weights based on analytical procedures

which was a model of quantitative accuracy for many years. Heinrich Rose, a pupil of Berzelius,

published one of the first texts to deal exclusively with analytical chemistry: Today there are

more than 50 elementary textbooks in the fields of qualitative and quantitative analysis that find

current use in the various colleges and universities of our country.

The last twenty years have witnessed an expanding interest in the problems and methods

of chemical analysis. This has been the result of many causes: the great increase in industrial

production, the great number and variety of industrial products, the more careful control that is

required in the manufacture of these products, and the necessity for speed in the securing of

analytical information no longer possible by the classical methods of chemical analysis. Many

companies that in the past were able to succeed without analyzing their raw materials or their

products, have recently found that it is now essential tomake analyses in order to maintain

quality and uniformity. The purchaser demands assurance that he can obtain, in the future,

materials of the quality and uniformity that he now secures. Another element in the expending

analytical picture has been the demand caused by recent wars for substances urgently needed for

military purposes as well as substitutes for scarce materials. The atomic energy project alone has

required a large amount of research in analytical methods.

There has been a vast increase in the number of analyses that are needed in the daily

problem of engineering, metallurgy, manufacturing, and many other fields. It is often necessary

to determine traces of elements so small that chemical tests fail and sensitive physical

instruments, such as the spectrograph, the mass spectrometer, and the Geiger counter, must be

used. The propertiesof our common metals, aluminum, iron, copper, zinc, lead, etc., are

tremendously influenced by the presence of other elements either added intentionally or known

as impurities. It is a vital matter, therefore, that the composition of these metals be known

accurately. Manufacturers have established large expensive laboratories, with a wealth of

equipment and with large and versatile staffs to operate them, for the sole purpose of analyzing

rapidly and accurately their raw materials and products. Between five and six million analyses

are made in a single year by one large company.

Agriculture has been aided with increased yields and enhanced soil fertility through

accurate chemical analysis, by adjusting the composition of fertilizers and by effective use of

insecticides and pesticides. In particular, the presence of proper amounts of trace elements such

as zinc, iron, manganese, molybdenum, cobalt, and boron which are essential to plant growth is

ensured by good chemical control. The cause of the disease of cattle in certain regions of western

United States was unknown until chemical analysis revealed the presence of selenium in the

vegetation. Similarly, a disease of sheep was traced to the absence of cobalt in soil and plants.

Geologists have been interested in the composition of meteorites and in the age of rocks

which they have determined by the amount of uranium, thorium, and pure isotopes of lead which

they contain. Geological prospecting has recently been aided by chemical analysis of the amount

of uranium, thorium, and pure isotopes of lead which they contain. Geological prospecting has

recently been aided by chemical analysis of the ash of vegetation.

Advances in medicine and public health would not be possible without chemical analysis.

An enormous number of chemical analyses is made daily in the laboratories of hospitals for

purposes of diagnosis and treatment. Analyses of blood, urine, etc., are absolutely essential in

order that proper treatment may be prescribed. An excess or deficiency of sugar, calcium,

potassium, iodine, or other substances may be the cause of a pathological condition. The

importance of fluoride in potable waters and its influence on dental caries was discovered only

by careful analysis of these waters; its addition to municipal water supplies is possible only with

careful analytical control.

The Federal Bureau of Investigation as well as state and municipal police departments

has found chemical analysis indispensable in identifying paint, blood, metals, and in establishing

evidence of quilt or innocence.

The historian and the archaeologist have obtain valuable information from chemical

analyses of soil, building materials, various objects of art, and coins recovered in excavations of

various sites. Certain objects of art, when associated with ancient coins, can be dated roughly

from the chemical composition of the coins which underwent progressive debasement that

paralleled increasing economic difficulties that occurred with the decline of the empire.

The progress of many a research problem has been retarded by lack of a suitable

analytical method or lack of analytical data. It has been said that a good analyst furnishes “the

eyes through which research sees its way forward.” The work of the analytical chemist, although

often unnoticed and rarely dramatized, contributes in a very important way to research, general

welfare, and progress.

Training in analytical chemistry serves as a sound basis for studies in other branches of

chemistry and other science. The laboratory work of physical chemistry required the quantitative

skills that are developed in the analytical course. Work in biology, geology, and physics may be

accomplished at a higher level of understanding and ability with a background of the principles

and techniques acquired in a course in quantitative analysis. Medical and dental schools

recognize the importance of quantitative analysis by requiring or recommending it as a

prerequisite for admission to the study of medicine.

The Scope of Quantitative Analysis: Qualitative analysis is concerned with the identification and

separation of chemical substances and the chemical principles on which such procedures are

based. Several schemes of analysis are in general use, the most common being the so-called

sulfide method of separation.

Quantitative analysis is concerned with the determination of the amount of chemical

substances. Present either alone or in a simple or complex mixture of other substance. Unlike

qualitative analysis, there is no general scheme of separation but rather a scheme of experimental

techniques that find general use after the isolation of the particular substance to be determined. In

certain physicochemical procedures the isolation may not be necessary. The literature of

quantitative analysis is extensive and there are many scientific journals devoted exclusively to

the subject. A selection of some of these sources of information is given in the appendix.

Quantitative analytical methods may be broadly divided into two general groups. Group I

consist of those methods in which the final measurement of the substance sought is made by

direct or indirect measurements of volume and weight after proper treatment of measurements of

volume and weight after proper treatment of a measured portion of the material to be analyzed.

The two most important classifications in this group are gravimetric methods and titration

methods. The latter are often referred to as volumetric or titrimetric analysis.

Gravimetric methods: In these methods the analysis is carried out by a series of weighing

operations. The most usual is the isolation of the substance sought by direct precipitation of the

substance, or a suitable compound of it, with subsequent purification before weighing. In some

methods the substance is deposited electrically on a suitable electrode and then weighed. Another

method involves the evolution of a gas. The gas or vapor may be evolved by heat or a chemical

reaction and its weight determined by the gain in weight of the vessel in which absorption takes

place. Alternatively, the loss in weight of the original substance may be used.

The most commonly used gravimetric methods for an introduction to quantitative

analysis are the determination of chloride and of sulfate in water-soluble samples in the absence

of interfering substances. The chloride is determined by precipitation as silver chloride and the

sulfate by precipitationas barium sulfate. The favored determination by electro analysis is the

deposition of copper on a platinum cathode. The usual gravimetric separations include the

analysis of brass and limestone. In the former, tin, lead, copper, and zinc are separated from each

other and determined in suitable insoluble forms. The latter involves the separation and

determination of silica, hydrous oxides, calcium, and magnesium. Loss on ignition is also

sometimes included.

Titration methods: In these methods the substance sought is determined by a carful

measurement of the volume of a solution of know concentration required to react with the

solution to be analyzed. Titration methods fall into four general group. These are: (1)

neutralization reaction in solving the determination of acids and bases. (2) Oxidation reduction

reaction involving the determination of reducing agents. (3) Precipitation reactions which are

usually limited in an introductory course. (4) complex-formation reactions which involve the

formation of a stable complex ion or a slightly dissociated molecule (other than water).

The volume of reagent required for the completion of the reaction in these methods is

usually measured by a visible color change that occurs in the reaction itself or is produced by the

presence of a chemical indicator. The completion of the reaction may also be measured by some

physicochemical change such as a change in potential, electrical conductance, absorption of

radiation, change in temperature, and many others. Most of these latter methods are used only in

advanced courses.

Group II consists of those methods in which the final measurement is made upon the

system as a whole. Almost any physical measurement may be used in this group to give the

desired analytical result. Comparison with mixtures of known composition is necessary. The

procedures are so varied that only a few illustrative examples are given here. The measurement

of specific gravity serves as the basis for the analysis of such binary mixtures as alcohol and

water. Thermal measurements such as melting points give information on the purity or

quantitative composition of substances or binary mixtures. Electrical measurements of

conductance, electromotive force, dielectric constant, etc., are used to determine the composition

of a mixture from a single physical measurement. Optical methods include: emission of spectra,

absorption of radiation, diffraction, fluorescence, refraction, and many others.

The methods outlined in Group I are those studied in the introductory course in

quantitative analysis. A suitable selection from these gives the student familiarity with

fundamental principles and laboratory techniques in the quantitative handling of materials. The

methods of Group II assume the background of those in Group I are stressed in introductory

courses because the principles and the techniques are essential in connection with other courses

because the principals and the techniques are essential in connection with other courses in

chemistry and in many field of scientific study such as biology, geology, physics and medicine,

as well as in the practical application of analytical chemistry.

3. 2. 2 Plant material processing:

The matured whole plant with root be collected, dried in shade, coarsely powdered and

pass through the mesh with proper size. It store in an airtight instrumentation for further analysis

in cold place.

3. 3 Extraction procedure:

The powder of antenna part was got rid of with the petroleum ether and successively

extracted with chloroform, alcohol, ether and water with the help of Soxhlet apparatus.

3.4Solvent extraction:

Although solvent extraction method is known for a long time to the chemists, it has

received the recognition of chemists as a method of separation recently. Such types of process

have much in common by means of partial decontamination.

Solvent extractions involve the distribution otherwise partition of a matter connecting

two non-miscible liquid in drop a line to with both others. The procedure has become

increasingly important in the analysis of metal become it furnishes clean separations in a short

span of time and has the further advantages of simplicity of technique and equipment.

The procedure from solvent removal with solvent is usually working moreover designed

for the isolations of dissolve substance commencing solution or from solidness mixture. The

latter process is known as washing.

1. Distribution law: In contact with each other if a solute is soluble in both of two immiscible

liquids, the matter will distribute itself connecting the both of two liquid therefore, the ratio of

concentrations is a constant. Mathematically, this may be expressed as follows:

Co

Kd =

Cw

Where Kd be the circulation invariable or coefficient, Co be the absorption of the extractable

(distributable, partitionable) specie in the organic phase and Cw is the concentration of the

extractable species in the water layer. The circulation fixed, Kd be moreover recognized at the

same time as the separation constant.

It is importants with the purpose of it is concentration from distributable specie that is

specified in Eq. (1). If any type of interaction takes place between the distributable species and

component in either of the phases, the distribution coefficient does not show the total amount

extracted or the total concentrations from the metal ions in both from the both phases. Due to this

analytical chemists use the distribution ratio, D, in phase of Kd the distribution, D, may be

defined.

Unsophisticated plants extract was equipped by Soxhlet descent methods. Near about

twenty gram from fine grained plants materials consistently pack within a in thimbleful in

addition to extract by means with 250ml of dissimilar solvent individually. Solvent applied be

ethanol, acetone along with methanol.Procedure for extraction continue intended for 22 hour

otherwise plow the solvents by draw off tubes of a separator get color less. Subsequent to with

the intention of the draw out be in use during a glassbeaker as well as reserved, heated on hot

plate and at 350-45ºC till all the solvents get vaporized.The dried draw out be keep in fridge at 3-

5ºC for their further use for chemical substance examination purpose.

If a solute is soluble in both of non-miscible liquid keeping in contacts with each others,

the solutemust be disseminating itself among the both liquid consequently with the purpose of

the proportion for concentrations is a constant. Mathematically, this may be expressed as

follows:

Co

Kd =Cw

Where, Kd be the probability distribution constants or coefficient, Co be concentrations of the

extractable (distributable, partitionable) genus in the organic phase and Cw is the concentration of

the extractable species in the water layer. The probability distribution constants, Kd be in

addition identified as the partitioning coefficients.

It be significant to notes with the purpose of it is concentration from distributable genus

that is specified in Equation. If any type of interaction takes place between the distributable

species and component in either of the phases, the distribution coefficient does not show the total

amount extracted or the total concentrations from the metallic ions in every from the both phase.

Due to this analytical chemists use the distribution ratio, D, in phase of Kd .the distribution, D,

may be defind as follows:

Mt0

D =

Mtw

Where Mto denotes the total molar concentration of all species having the metal ion in the

organic phase and Mtw denotes the total molar concentration of all species having the metal ion in

the water phase.

If the entire metal ion in each of the two phases is present as the partitionable species

then Kd equals D only. For systems having equal to Kd the ratio of concentration remains same as

the ratio of the solubility of the extractable species in the two phases. This has been found to be

true. For many extraction system in which little interaction takes place. However it is assumed

that concentration ratio and solubility ratio are the same especially if the system is influenced by

the pH. An example is the distribution of 8-hydroxyquinoline between water and chloroform the

value of Kd is720 but the value of D has been found to vary from 0.006 at pH zero to 60 at pH

five and 720 at pH seven. Above pH nine the values of D decrease continuously to 0.05 at pH

fourteen.

Efficiency of Extraction: The main interest in most extraction processes is the effectiveness for

the extractions underneath specified sets of condition. Thus the use of the per cent of extraction

(%E) is more meaningful then either D or Kd. the %E and D are related by the following relation.

100D

% E =

D+ Vto/V0

In the above edationV0andVto denote the volumes of organic and water phase respectively. If

these two volumes become equal the denominator in Equation, reduces to D+1. As the extraction

process tends to approach 100% the distribution ratio tends to become infinity.

Column Chromatography:

It is known that the rate of adsorption is used in column chromatography. In this method,

the mixture to be separated is dissolved in a suitable solvent and allowed to pass through a tube

containing the adsorbent. The component which has greater adsorbing power is adsorbed in the

upper part of the column. The next component is adsorbed in the lower portion of the column

which has lesser adsorbing power than the first component. This process is continued. As result

the partially separated and adsorbed in the various parts of the column. The initial separation of

the various components can be improved by passing either the original or some other suitable

solvent slowly through the column. The initial separation of the various components can be

improved by passing either the original or some other suitable solvent slowly through the

column. The various bands present in the column become more defined. The banded column of

adsorbent is termed a chromatogram, and the operation is spoken of as the development of

chromatogram, the portion of a column which is occupied by a particular substance is called its

zone. The narrower the zones, the larger the number of substances which can be separated in a

column of a definite length, and the more concentrated are the more concentrated are the eluates.

In order to separate or to estimate the various concentrated, two procedures may be

adopted:

i. After development, the column of adsorbent may be pushed out of the tube, the variouszones are cut with a knife at boundaries and the substances present in zones extractedwith a suitable solvent. This process of recovery of constituents from the chromatogramis known as elution.

ii. After development, the column may be washed with more solvent, now termed theeluent, and each component is collected separately as it reaches the end of the columnand is released. The process of separation of a mixture of two substances A and B hasbeen represented.

Apparatus: A simple straight glass tube tapered at the bottom and often fitted with a top is

still commonly used. The ratio of length to diameter should be greater than 20:1; a ratio of 40:1

can be taken as standard. This tube is about 2-3 cm in diameter with 20-30 cm long. This may

retain several grams of adsorbate. The adsorbent is supported on a plug of cotton or glass wool,

for wider tubes a perforated disc, covered by a pad of cotton or glass wool may be employed.

Long and narrow tubes are used for difficult separations. Columns fitted with cooling jackets are

used when constant temperature conditions are required. Separating columns with detachable

adapters are being used. In these, the absorbent is packed between two sintered glass discs and

small-bore Teflon tubing is used for liquid input and output.

Adsorbents: General Requirements: In order to obtain columns with good filtration properties,

the adsorbents should have following characteristics: Their particles should be spherical in shape

and uniform in size.

i. Their mechanical stability must be great enough to prevent the formation of finedust which might be deposited in the channels of the packing.

ii. They should not react chemically, either with the eluting solvents or with thesample components.

iii. They should contain as small amount of soluble components as possible.iv. They should be catalytically inactive and as a rule, have neutral surface. However,

exceptions are ion exchangers.

Aluminium oxide activated by heating to about 200-3000C in a current of air or carbon dioxide

is the most widely used adsorbent. Other adsorbents are silica gels, activated charcoal,

magnesium carbonate, calcium carbonate, calcium sulphate, barium carbonate, etc. Various

polyamides such as nylon, perlon, ultramid BM 228 and powdered polyacryl amide (particle size

ca 0.04mm) are suitable for the separation of lower fatty acids, phenols flavones, quinines and

synthetic dyes.

Powdered sugar, sieved and dried in a desiccators, has been used for the separation of the

coloring matters of leaves as well as or phenols. Starch is suitable for the separation of

racemates.

List of adsorbents in increasing orders of adsorption as bellow.

Grades Series of Adsorbents

1. Sucrose, starch2. Insulin3. Talc4. sodium carbonate5. Calcium carbonate6. Calcium phosphate7. Magnesium carbonate8. Magnesia mixtures9. Line10. Activated silicic acid11. Activated magnesium silicate12. Activated alumina13. Fuller’s earth

3.5 Phytochemical screening:

3.5.1 Extract preparation:

Fresh leaves, stem, roots, fruits with seed were wash systematically below running ware

tap two to three times, removing all soil particles and shade dried under normal temperature.

This crude material used separately for extraction process. Dried stem, leaves, roots, whole fruits

with seeds are separately homogenize to a excellent fine particles in addition to store within air

tightcotainer. 45 gm from leaf materials, roots, stem, seeds powders were extracted by means of

1000 ml of solvent (water, ethanol, chloroform, acetone, ether, methanol etc) for 42 hours by

utilizing soxhlet apparatus. These different extracts used for different phytochemical chemical

tests and other analysis purpose. The various extract after extraction were submitted to first

round phytochemicals screening purpose. A little quantity from sample is treating with means of

Dragendroff reagent and Wagner’s reagent to detect the occurrence of alkaloid. Tannins with

phenolic compound presence were tested with the help of ferric chloride solution with lead

acetate solution and aqueous bromine solution [105].

3. 6 Phyto-chemical analysis:

The commencing phyto-chemical study accepted away through typical methoddescribe

[110] with some small modification. The plant parts (root, stem, leaf and matured fruits with

seed) extract was assayed designed for the occurrence for alkaloid, phenols, terpenoids, and

saponins. The name ‘alkaloid’ is given to a group of organic bases of vegetable origins which

contain at least one nitrogen atom. All alkaloids are having marked physiological action toxic or

curative on living organisms. These compounds being similar to alkalis in their basic nature, they

were called as ‘alkaloids’. On the whole alkaloids are very poisonous but if taken in small

quantities can act as medicines, e.g nicotine, stimulates the central nervous system, morphine,

relieves pain, coniine, deadly poisonous and quinine – specific for malaria. Alkaloids’ occurs in

the leaves, roots, bark, seeds, and fruits of plants. The do not occurs in Free State but are present

as their salts with organic acids such as oxalic, tartaric, citric and maleic acids. Usually more

than one alkaloid occurs together in one plant, e.g. Opium contains about 20 different alkaloids.

The alkaloids present in one plant are generally closely related in chemical constitution and

properties.

The plant material containing alkaloids is dried, powdered and extracted with petroleum

either to remove soluble fats .The residue is then extracted with methyl alcohol so alkaloids get

dissolved in methyl alcohol and the residue containing cellulosic and insoluble material is

rejected. The filtrate is distilled off and the crude plant extracted is then treated with dil.HCl or

H2SO4 (pH=2), when alkaloids are converted into their soluble salts, which are then treated with

ether or chloroform. The ether soluble part contains non basic plant material and is rejected.

The water soluble part (containing salt of alkaloids) is treated with Na2CO3 or NaOH when

alkaloids precipitate out, which are then extracted with ether or chloroform. The either layer is

distilled off and the residue obtained contains a mixture of alkaloids. The individual alkaloids are

separated by using techniques like fractional distillation, steam distillation and chromatography.

The alkaloids are colorless, crystalline, non-volatile solids (exception coniine and nicotine).

They are soluble in organic solvents like either chloroform, alcohols but insoluble in water.

Coniine and nicotine are water soluble. Most of them are optically active (leavo rotator). They

are bitter taste. Being basic in nature, they dissolve in acid forming their salts that may be readily

crystallized.

The classification of alkaloids is not well defined due to large variety of their structures.

However on the basis of the nature nucleus present in the molecule, the alkaloids are broadly

classified as follow: A pure sample of alkaloid is subjected to qualitative analysis so that its

empirical formula is found out. Then by determining its molecular weight, its molecular formula

is determined. The presence of olefin bond (C=C) can be determined by bromination, HBr,

nitrosyl chloride consumed in addition reactions. In alkaloid the oxygen if present may be –OH,

>C=O,-COOH, ester, amide, lactone, lactum, methoxy, methylene dioxy groups, which can be

defect. The presence of hydroxyl group is indicated by the action of acetyl chloride or acetic

anhydride. It may be phenol or alcoholic –OH group. The phenol group is soluble in NaOH and

gives blue/green/violet coloration with FeCl3 solution. The alcoholic nature is determined by the

action of dehydrating agents such as H2SO4, P2O5 or oxidising agents.

Terpenoids are naturally occurring compounds obtained from various parts of plants.

Simple terpenoids occur in essential oil which are volatile and obtained from sap and tissues of

certain plants and trees. Complex terpenoids are not steam volatile and are obtained from gum

and resins of plant. Some constituents of simple terpenoids obtained from essential oils are citral,

cineol, pinene, nerol, geraniol, menthol etc. As the terpenoids occurs in the nature there is no

general method for isolation. The isolation of mono and sesqui terpenoids is effected in two steps

that is isolation of essential oil from plant and separation of individual Terpenoids from the

essential oils by using the techniques such as column chromatography, fractional distillation,

sublimation etc.

In general four method contains industrial process towards the isolation from terpenoids

from plants material i.e. expression method, steam distillation, solvent extraction, adsorption in

purified fats. The plant material is crushed and juice is screened to remove the large particles.

The juice is centrifuged when the half of the essential oil obtained in the centrifuge. The residue

is used for removing inferior oil by distillation. The crushed plant material is extracted with

solvent like petroleum ether, diethyl ether. The extract is subjected to distillation and the solvent

is get recovered. The temperature is not allowed exceed more than 50oC.The essential oil

obtained by this method has the natural odor.

3.7 Thin layer chromatography:

The technique of thinlayer chromatographic technique is primarily introduced through

Shraiber along with Izmailo in 1938. These workers used this technique for separating plant

extracts on 2mm thick and firm adhesive layer of alumina set on glass plates. Consden Grodon

and Martin started using filter papers. Their work in the field of amino acid analysis met with

considerable success Williams carried out chromatography on absorbent layer sandwiched

between two glass plate one of the latter has a small hole through which solutions and

developing solvent were applied to the layer. Attempts were made using adsorption

chromatography on impregnated filter paper and later glass fiber paper coated with silicic acid or

alumina. Kirchner in 1950 was one of the first to do this. He was able to separate and identify

terpene. TLC as a procedure for analytical adsorption chromatography was first introduced by

Stahl who was mainly responsible for bringing out standard equipment for preparing thin layers.

TLC is often named by other name such as drop, strip, spread layer, surface chromatography and

open column chromatography.

The TLC from an alcoholic extracts carried away to find the number of constituents present in

the plant [42]. The stripped eye practical spots with intense yellowish color in addition to under

uitra violet cabinet spots observed with incandescent yellowish color which having Rf values

near to flavonol and flavones.

Chromatography is relatively a new technique which was first devised a botanist in 1906

in Warsaw by M. Tswett, for the separation of individual components from colored substance.

Now a day various type of chromatography are in uses to separate almost any given mixtures

whether colorless or else colored into it’s constituent and test the purity of these

constituent.Since then the technique has undergone tremendous modification.. The name chroma

tography (Greek Chroma color and Graphy writing) means color writing. The component of a

mixtures move through a porous medium (called stationary phase) under the influence of some

solvent or gas (called moving phase), essentially the technique of chromatography is based on

the differences in the rates.

Chromatography classification: The motivating stage may be a liquid or else gas based up on

moving phase and the natures of the fixed, different type of chromatographic techniques are as

follows.

Adsorption chroma tography: The technique which is based on the differences in the adsorption

coefficient in this the fixed phase which is a solid, e.g., silica gel, magnesium oxides, alumina

etc. the solute were adsorbed in dissimilar part of the adsorptive columns.

Partition chromato graphy: it operates through mechanisms analogou in the direction of

contradict present distributions .the fixed phase may be a liquid strongly adsorbed on a solid

which acts as a support. In this case the solutes get dispersed among the moving liquid and the

fixed liquid, the method be known partition chromato graphy.

Introduction to instrumental methods of chemical analysis: In the broadest sense an

instrument for chemical analysis does not generate quantitative data but instead simply converts

compounds datas to a appearance that be more readily noticeable thus the instruments can be

visualized as a communication device the instrument does the job in various step which are as

follows.

Generation of a signal: The signal used in the instrument may be produced from the sample

itself .for example the yellow radiation emitted by heated sodium atoms acts as the source of the

single in a flame photometer. It is to be noted that with many instruments the original signal is

formed independently of the sample. However it is the modification of the signal by the species

of the interest which is related to the concentration. For examples the signal in a polarimeter is a

beam of plane polarized light: however it is the change in orientation of the plane by the sample

which provides the desired information.

Transduction: The transformation of single to one of the different nature is called

transformation. Many instrument use a transducer for converting the original analytical single to

one that is more conveniently measured. Examples of transducers are the photocells the

thermocouple and the photomultiplier tube that convert radiant energy into electrical single it is

of interest to note that a chemical coulometer is also a transducer because it transforme electrical

single into a related amount of a gas or a solid which may be subsequently measured.

Amplification of the transformed single: It is possible to increase the sensitivity of many

instruments by amplification of the original signal or its transduced form. Amplification is

generally done electronically but mechanical amplification is also a countered as in the pointer of

an analytical balance.

Presentation of the signal: the transduced and amplified signal from an instrument is generally

presented as a linear.

3.8 Qualitative andQuantitative phytochemical screening:

The qualitative in addition to quantitative phytochemicals testing for plants must be done

allowing toward typical processes. Following are some phytochemical compounds get analyzed.

3.8.1 Alkaloids:

The presence of alkaloids was shown by precipitation of salts and reaction with Mayer’s

reagent (potassium tetra-iodomercurate solution). To 10 g of powdered sample, 50 ml of 10%

sulphuric acid was added. After 24 hrs maceration at ambient temperature the mixture was

filtered on filter paper and rinsed with distilled water to obtain 50 ml of filter, one ml of which be

identified within a testtube and treated on five drop of Mayer’ reagent. A whiten yellow residue

indicates the presence of alkaloids.

Tests for alkaloid;

To the extracts mild hydrochloricacid be adds in addition to filtered out.This filtrate shall

be covered through assorted alkaloid reagent.

a) Mayer’s test:

The filter covered with Mayer’s reagent, if appearing from cream color indicates the

incidence of alkaloid.

b) Dragendroff’s test:

The filter coverby means of Dragendroff reagents appearing from reddish brownish

precipitous indicate the being there are alkaloid.

c) Hager’s test:

The strain while covered by Hager’s reagent, appearing of yellowish color precipitant

indicate the existence for alkaloid.

3.8.2 Tests for saponins:

Mixed crude extract with 5ml from distilled water in testtube in addition to it is stirred

smartly.The establishment from static foamy material gives idea about or indication for the

comportment for saponin.

Tests used for saponin:

Foam test:

In a test tube take one ml of plant extract to this add twenty ml of distilled water, shake

well the test tube material. Formation of foamy material at the top part of test tube indicates

presence saponin in given sample.

3.8.3 Terpenoids:

The presence of terpenes was demonstrated using concentrated sulphuric acid [32]. An

extract was first prepared by maceration for 24 hrs of 1 gm of powdered sample and 20 ml of

ether. The extract obtained was used to detect carotenoids and coumarins in addition to sterols.

Sterols and triterpenes were detected by adding 1 ml of chloroform to the residue left by

evaporating 10 ml of the macerate. The solution obtained was placed in two test tubes and 1 to 2

ml from conc. sulphuric acid be placed in bottom of one of them and the other acting as a

control. The formation of a brownish-red or violet ring where the two phases met indicated their

presence. Carotenoids, terpenes pigments useful for photosynthesis, were detected using

antimony trichloride [32]. Two to three drops of a concentrated solution from

antimonytrichloride (SbCl3) in the solvent chloroform were added to take up the residue left by

evaporating 5 ml of ether extract to dryness. A blue coloration indicated their presence.

The history of these compounds spans the centuries of civilization. Ancient Egyptians

knew about several essential oils particularly oil of turpentine. Terpenoids be able to obtain

commencing the flowers, leaves, fruits and roots of many plants by employing steam distillation

or solvent extraction methods. Terpenoids are most widespread, chemically interesting and

provide structures of great diversity. Although some of these compounds like camphor occur in a

sufficiently pure from to allow crystallization, majority occur as complex mixtures of isomeric

substances often difficult to separate. The dextrorotatory from of a simple terpene limonene

occurs in plants and to the extent of 95% in the citrus peel oil of sweet oranges.

Terpenoids have a simple unifying feature and can be considered to be built up or whose

structures may be divided into isoprene units. A classification of these compounds based on the

number of isoprene (2-methylbutadiene) units has been made. Significantly racemic mixture of

limonene can be synthesized by heating isoprene:

Recently biological activity of various systems has been related with terpenoids. Sesqui-

terpene and diterpenes provide a number of antibiotics. Several sesqui-terpenes are active against

experimental tumors and the plant growth hormones like gibberellins are di-terpenoids.

A large number of terpenoids are known in the literature. For example, among

sesquiterpenes over 600 members are known. The terpenoids fall into several classes possessing

different carbon framework.

Several natural products are known by their trivial names since these were isolated and

described in the literature long before their structures were known. Often these trivial names are

based on botanical origins. An example is the crystalline sesquiterpenes secondary alcohol

belonging to the codenine group which was isolated from vetiver oil (Vetiveriazizaniodes)

known as ‘khus’ oil in India. The alcohol was, therefore named, as khusinol and another

crystalline primary alcohol belonging to the same group and isolated from the same source has

been named khusol.

Istrati and Ostrogovich separated two crystalline compounds from cork and in honor of

Friedel, Istrati named the more soluble of the two compounds as friedelin. Terpenoids of

friedelin group have the following carbon framework and cerin which is a keto alcohol belongs

to this group. Even today it is easier to a compound by modification of a trivial name.

These isopropylidene double bonds play an important role in the easy cyclization reaction

for which many acyclic terpenoids are noted and result in the formation of six-member rings.

The ring closes either to give p-menthane or 1, 1, 3-trimethylcyclohexane derivatives. An

example is the acid-catalyzed cyclization of citronellal which affords isopulegol. Another related

example of great interest is the cyclization of linalool to α-terpineol acetate with acetic

anhydride:

Under acid catalysis geraniol readily undergoes a reversible allylic rearrangement to

recemic linalool which itself is known to occur naturally in both the enantiomeric forms in a

large number of essential oils. This type of a rearrangement is doubtless involved during the

biosynthesis of linalool and this isomerization had complicated the structure elucidation of

linalol.

An example where an alternative mode of cyclization occurs is that of citral where the

oxygen functions is blocked by condensation with aniline. The Schiff’ base thus obtained

undergoes cyclization and hydrolysis on treatment with acid to afford a mixture of α and β-

cyclization as shown in reaction. Another example of this type of cyclization is found during the

synthesis of vitamin A, where pseudo ionone is cyclized under acidic conditions to β-ionone in

one of the key steps.

Most of the members of this class have the p-menthane skeleton which arises from

chemical modification of NPP. The purification of the hydrocarbon members of the class had

presented a major problem to the early investigations. Use of nitrosylchoride afforded crystalline

nitrosochlorides by addition to the ethylene double bonds. This reagent, as far as its utility is

concerned, is compared with the most valuable reagent namely phenyl hydrazine used in the

study of carbohydrates.

Limonene and Carvone as an example where nitrosochlorides formation helped in

structure elucidation, mention may be made of limonene. It has one asymmetric carbon atom and

both enantiomers occur separately in Nature, as well as the race mate which is often called

dipentene. It contains two double on dehydrogenation gives p-cymene.

In confirmation with the placement of trisubstituted double bond in limonene, it afforded

a nitrosochlorides which on alkaline hydrolysis gave carvone of established structure as shown.

Like many other terpenoids Carvone affords several interesting products involving most

interesting reactions. Thus its hydro bromide on treatment with alkali undergoes a rearrangement

4 to afford a ring expanded ketone eucarvone via an intermediate care none and the latter occurs

in nature as well. Carvone gives ‘Carvone camphor’ on irradiation with u.v. light in the presence

of ethanol.

The monocyclic terpenoids are noted for their stereo chemical complexity and an

example is of menthol which has been known since the pre-Christian era, and finds use in

medicine. Its stereochemistry presented several problems and this study is of considerable

importance. 1, 8-Cineole and as caridole also find use in medicine and the latter the only known

peroxide among terpenoids.

A significant feature of the bicyclic Monoterpenoids is the facility with which they

undergo rearrangements of their carbon skeletons. These are carbonium ion rearrangements and

typical examples of the conversion of α-pinene to camphene and camphor have been presented.

Here it is sufficient to give an introduction to the various types of bicyclic Monoterpenoids and

some preliminary understanding of carbonium ion rearrangements.When a carbonium ion is

created on the bicyclic system of either a Monoterpenoids or a system related to it, a bond shift

takes place which results in the change of the carbon skeleton of the molecule.

With evidences from the occurrence from ions 1 and 2 mixture of products derived from

each ion is obtained during a reaction involving the formation of these ions. More precisely

instead of speaking about the ions separately we may have a single ion 3 called nonclassical

intermediate which is capable of reacting to afford products which are expected from either ion.

A nonclassical ion may be formed during the ionization of a molecule, if the geometry of the

molecule is such that the carbon carbon bond can act as a neighboring group. Thus such a bond

assists in the disappearance of the leaving groups.

However, in the endo isomer a similar solvolysis not be assist by the 1, 6 bond since it is

for unfavorable positions towards the back side bother attack. Thus in this case a classical ion 5

is formed first which is then converted to the more stable non-classical intermediate.

1. Solvolysis of the exo-isomer is 350 times faster as compared with the endo isomer and is in

keeping with the concept of neighboring group participation.

2. The solvently from the optically active exoisomer gaves simply recemic exoisomer, which can

be explained by considering the formation of a nonclassical ion. Thus in the nonclassical ion the

position of two with one are equivalent in addition, however only from the exo-direction in either

case therefore, it would be attacked by the nucleophile with equally.

3. In the case of an ion like Lydride shift can also take place and the most rapid of hydride shift

is the 2,6 shift which produces a mirror image ion which thus provides another route to

racemization of products:

4. When a compound of less symmetry as compared with norborneol is involved in a similar

reaction sequence the attack by the nucleophile at two positions will afford different compounds

with different carbon skeletons:

5. If a nonclassical ion is an intermediate between a secondary and a tertiary ion, the reaction

occurs more rapidly at the more substituted carbon as seen in the methanol lysis of isobornyl

chloride. Monoterpenoids with skeletons other than p-menthane group may be present.

Terpenoids in this class may be divided into two distinct groups, (a) having the 1, 1, 3-

trimethylcyclohexane skeleton and (b) a group having a five member ring to which a lactone ring

is usually attached (cyclopentanoid Monoterpenoids). The representative members are shown.

The biosynthesis of the cyclopentane ring in these compounds has been suggested to

involve internal Michael addition. Sesqui-terpenoids are a group of C15 compounds derived by

the assembly of three iso-prenoid units. Significant development in their study emerged with the

discovery that many of them could be dehydrogenated to easily recognizable aromatic system.

Thus with the nature of their carbon skeletons known, the position of double bonds and other

functional groups could be established by oxidative degradation. Use of spectroscopic methods

as an aid in their structure elucidation coupled with the Biogenetic Isoprene Rule have proved to

be the foundation-stone of sesqui-terpenoids in particular and of terpenoids chemistry in general.

The sesqui-terpenoids are classified according to their basic carbon skeletons. Sesqui-terpenoids

are a group of C15 compounds derived by the assembly of three iso-prenoid units. Significant

development in their study emerged with the discovery that many of them could be

dehydrogenated to easily recognizable aromatic system. Thus with the nature of their carbon

skeletons known, the position of double bonds and other functional groups could be established

by oxidative degradation use of spectroscopic methods as an aid in their structure elucidation

coupled with the biogenetic isoprene Rule have proved to be the foundation stone of sesqui-

terpenoids in particular and of terpenoids chemistry in general The sesqui-terpenoids are

classified according to their basic carbon skeletons. Typical sesqui-terpene skeletal is presented.

The naturally occurring primary alcohol farnesol has theoretical importance because of

its biosynthetic role. Its isomer nerolidol is related to it in the way as linalool is to geraniol. Free

lingyne a furano compound with iso-prenoid structure is the first acetylenic terpenoids isolated.

Among the monocyclic sesqui-terpenoids mention may be made of bi-sabolene and zingiberene,

the latter being the major component of oil of ginger. Interestingly, the elemane system present

in the alcohol elemol, unlike monocyclic carbon skeleton may arise by a rearrangement.

The monocyclic group is further exemplified by the important plant growth hormone

abscisic acid found in young cotton fruit. It is a growth inhibitor and one of the few terpenoids

which has been assigned a definite role in plant physiology. The absolute configuration of the

cad inane group related to codenine was determined by a study of the ORD curves of derived

ketone 1 and by the degradation to D (+) isopropyl succinic acid. This acid on the basis of

modern nomenclature belongs to (S) series. Occurrence of a distinct group of antipodal cad inane

types related to (-) cadinane has been reported in North Indian variety of vetiver oil. Structures

and absolute configuration of these terpenoids have been determined by their chemical

correlation.

A relatively small group of sesqui-terpenoids has been discovered which has the chemical

and stereo chemical characteristics of rings A and B of di- and tri terpenoids. These compounds,

therefore, represent yet another mode of biogenetic cyclization from farnesyl pyrophosphate. The

first member of this class to be discovered is frimenol. Another member of this class is the

hydroxy-Iactoneiresin which has an ‘abnormal configuration to iresin was assigned that of

steroids and tri terpenoids. This configuration to iresin was assigned on the basis of chemical

degradation followed by ORD studies.

The perhydroazulene Group: This group is represented by a variety of compounds, like

hydrocarbons, alcohols, lactones etc. which give derivatives of the bulehyddrocarbonazulene on

dehydrogenation and these compounds, therefore, have bicyclo [5.3.0] decane ring system.

Other Bicyclic Sesqui-terpenoids: The remarkable fungal toxin helminmthosporal, which

is known to destroy a substantial part of the cerel crop of North America, has been characterized

as a sesqui-terpene by de Mayo. Mention also best made here of the unusual diketoneacorone

isolated from sweet flag oil which represents the first spiran terpenoids to be discovered.

Several members of this growing class are known today and these have a ‘medium ring’,

ie, a ring having between 8 and 11 carbon atoms. As expected of medium ring compounds, all

these compounds show a strong tendency to undergo trans annular reactions, particularly in the

presence of acids and bases leading to the formation of a new ring and a typical example from

this group is caryophjuyllene with a nine member ring manifests its eagerness to cyclize in a

variety of interestionie actions. Thus on acid catalyzed cyclization two products are formed,

namely caryolannol and clovene and the formation of latter involves a 1, 2- shift with

enlargement of the four-membered ring:

Still another acid catalyzed rearrangement product of caryophyllene is neoclovene

formed by the mechanism. Among the base-catalyzed Trans annular cyclization mention may be

made of the epoxide 1, prepared by the epoxidation of caryophyllene. This on permanganate

oxidation and treatment of the resulting oxidoketone with base led to an intra molecular

cyclization. The caryophyllene epoxide 1 proved to be a useful derivative for the structure

determination of caryophyllene. The decisive step was the IR study of the derived oxido-ketone

2, (before treatment with base) which showed absorption at 1698 cm-1, a value typical of a keto

group on a medium-sized ring.

This coupled with other observations led to the presence of a nine member ring in

caryophyllene. Chemical evidence for the latterm and for the relationship of the double bonds

was obtained when the oxido-ketone afforded 3 on base treatment. Oxidation of tree gave an

enedion four which was characterized by its ultraviolet spectrum and further oxidation to the di

acid five which easily formed an anhydride.

The cedrol, a crystalline tertiary alcohol, is responsible for the odor of cedar wood

whose conformational formula shows its structure and stereochemistry. This representation also

brings out its relationship to simple sesquiterpenes like bisabolene and shows that a probable

biogenetic route to the carbon skeleton of Cedrol may involve a stereo specific cyclization of y-

bisabolene. Longifolene occurs in the essential oil from the oleoresins of different species of pine

and its structure was determined by an X-ray study of its hydrochloride and provides an

exception to the precedent that physical methods merely confirm a structure already proved by

chemical degradation, In accordance with the presence of the camphene ring system in

longifolene it undergoes facile Wagner-Meerwein rearrangement. Longiborneol, a component of

cedar wood, and longipinene which is the pinene analogue of longifolene, a constituent of

turpentine, are structurally closely related to longifolene. Lastly mention may be made of

longicyclene which is the first tetra cyclic sesquiterpenoid discovered by Sukh Dev and the well-

known patchouli alcohol.

Willstatter in 1906 found that chlorophyll is a phenyl ester since on saponification it

gives a nitrogen-free substance as oil which was identified as a monounsaturated primary alcohol

of the formula C20H39OH. Thus phytol is extremely widespread in nature. Vitamin A and the

corresponding aldehyde represent the known compounds of biological importance while

membrane is a di terpenoids. The C20 ‘resin acids’ represent a series of bicyclic and tricyclic

carboxylic acids and their main skeletal class are derived from13, 14-secopimarne, pimarane and

abitane. The currently accepted numbering system as shown in abietane is analogous with that

used for triterpenoids and steroids. The term ‘seco’ is used to designate the cleavage of a bond

between two atoms of the parent structure. Similarly the number preceding the prefix ‘nor’ is the

number of the atom eliminated.

Another class of di terpenoids of importance which have plant growth promoting

properties and thus have role in plant physiology is the gibberellins. A group of these

diterpenokds is found in the culture fluid of the fungus Gibberellafujikuroi, which is responsible

for a disease of rice common in japan and an important member is gibberellic acid. The

majority of tetra cyclic di terpenoids have the cross carbon skeleton of phyllocladene 1 and its

double bond isomer isophyllocladene 2 although considerable variation in configuration is found.

Their diastereoisomeric relative, kaurene 3 is also reported to occur in nature.

Another class of diterpenokds of importance which have plant growth promoting

properties and thus have role in plant physiology is the gibberellins. A group of these

diterpenokds is found in the culture fluid of the fungus Gibberellafujikuroi, which is responsible

for a disease of rice common in japan and an important member is gibberellic acid. The

majority of tetra cyclic diterpenokds have the cross carbon skeleton of phyllocladene and its

double bond isomer isophyllocladene although considerable variation in configuration is found.

Their diastereoisomeric relative, kaurene is also reported to occur in Nature. The phyllocladene

skeleton may in nature from the carbocationic structure formed by the cyclization of the

pimarane skeleton as shown

The compound trachylobanic acid A represents a unique group of pentacyclic di

terpenoids called as the trachylobanic and these have been isolated from the plant. Trachylobium

verrucosum, Significantly, the same plant as well contains kaurenic and isokaurenic acids B

which are formed by the action of acidic reagents on A. atisirene C also occurs in Nature and it

may be formed from the non-classical ion D which is believed to be common biogenetic

precursor of the tetracyclocabanes and probably of all the tetra cyclic di terpenoids. An

experiment in support of this is that (-)-kaurene 3 and the parent hydrocarbon of A, i.e.,

trachylobanic are all isomerizes by acid into mixtures of atisirene and kaurene and their double

bond isomers.

Triterpenoids

These are the most widely distributed terpenoids class; however, they cannot compete in

structural variety with their smaller relatives. Generally they have four or five rings fused in the

same general way. The range of their functional groups is rather restricted which presents a

barrier to their derivational studies. The applications of physical methods have almost replaced

the classical derivative approach and some of the molecular rearrangements are presented.

General Methods of Structure Elucidation of a Terpenoids: As in the case of other natural

products there are four to the determination of the structure of a terpenoids.

The Initial Characterization and Definition of the Functional Groups

This involves the isolation of a pure sample by chromatographic methods and

determination of the molecular formula by means of mass spectrometry. A few color reactions

may give valuable information, e.g., color with tetranitromethane (TNM) will show the presence

of carbon-carbon unsaturation .The identification of functional groups is done by the use of

physical methods, like IR, NMR and UV spectral methods. From this information coupled with

evidence such as micro hydrogenation and per-acid titration number of double bonds may be

found, and thus, from the analysis, the number of rings present for example as in the case of

abieticacid acid.

Determination of Underlying Carbon Skeleton:

The identification of the dehydrogenation products from a terpenoids with sulphur or

selenium coupled with biogenetic thinking allows establishing the carbon skeleton. This is seen

several cases described. Thus formation of eudalene from eudesmol accounts for fourteen out of

its fifteen carbon atoms and application of isoprene rule helped in the assignment of correct C15

carbon framework.

An identification of the products of dehydrogenation, spectral data, and biogenetic

concepts were sufficient to assign a carbon framework in the case of hardwickiic acid.

A variety of methods are employed for these purposes which are explained in the text. Here it

will be sufficient to give the outline of only a few of these methods.

Very often structure determination rests on a judicious set of interrelationships between

the known and the unknown compound in hand .one of the methods may involve selective

removed of the oxygen functions to define the parent hydrocarbon. Using this method structure

and stereochemistry was assigned to khusol and khusinol whose parent hydrocarbon was found

to be antipodal to the already known (+) cadinene Spectral data (IR & NMR) helps to define the

number of terminal pendant groups like C-CH3, primary alcohol, carboxylic acid and C=CH2

assumption that the terpenoids obeys the isoprene rule, (for example a di terpenoids) and has a

common skeleton, can help to define the series to which a compound belongs. Thus, the tricyclic

pimarane-abitane diterpenes possess five pendant groups, di terpenoids), kauranoid diterpenes

four and so on.

Dehydrogenation in a number of cases provides evidence for the location of some

functional groups on the already deduced carbon skeleton. Dehydrogenation of an alcohol may

generate a carbonium ion to involve a methyl migration useful for structure determination. An

example is found in the structure elucidation of amyrin (, dehydrogenation of triterpenoids).

Another example is found in structure elucidation of abieticacid where dehydrogenation studies

of the parent acid and the derived alcohol help in locating the position of carboxyl group. The

use of Grignard reagents to insert an extra methyl group(s) at the sites of oxygenation and their

location in the products of dehydrogenation helps in a number of cases. Structure determination

of khusilal and cassaic acid using this technique may be cited as examples.

The most important information which certainly fixed the position of the double bond in

guaiol came from dehydrogenations studies. Guaiol on ozonolisis afforded a diketone which

underwent a spontaneous intramolecularaldol condensation to give a cross conjugated dienone

(by elimination of one hydroxyl group and dehydration of other) belonging to the cadinane

group. This was proved since the dienone on dehydrogenation afforded cadalene and the related

naphthol.

Spectral methods are extremely useful in determining the position of functional groups.

Thus infrared absorption reveals the occurrence for hydroxyl groups while seen number,

positions and multiplicity from CHOH protons in the NMR gives information on whether it is a

primary, secondary or a tertiary alcohol. This is confirmed by oxidation which may give acidic

or ketonic material, whilst the carbonyl absorption in the infrared characterizes the ring size of

ketone. The number of methylene groups in the α-position(s) to the ketone is obtained by

deuteration studies, mass spectroscopy beging used to define the number of protons exchanged

with deuterium etc. Alternatively, the oxidation with selenium dioxide can give a diketone (as in

the case of camphor,

Woodward rules can predict λ max of α, β-unsaturated aldehydes and ketones thus in α-

cyperone the placement of the double bond in the α, β-position to the keto group must be as

shown in c rather than b since it exhibits λ max at 252 nm which matches with the predicted value

of 254 nm. Moreover, NMR spectrum can distinguish between b and c since the latter would

show the presence of two olefin protons as compared with four if the structure.

Oxidative Degradation:

Oxidative degradation has played a remarkable role in deducing the positions of double

bonds, functional groups and the underlying carbon skeleton. Several examples of oxidative

degradation have been presented to shown the strength of the method in structure elucidation.

These can be briefly mentioned as under.

1. The ozonolysis of the terminal methylene which is characteristic of the kaurene-

phyllocladene series gives formaldehyde and a norketone which could be identified by its

infrared spectrum as a cyclopentanone system:

2. Structure of citral is based mainly on the identification of its oxidation products.

3. Oxidative degradation of copaene involved ozonolysis, oxidative cleavage of the ozonide

with alkaline hydrogen peroxide, and Baeyer-Villiger oxidation when the terpenoids

afforded a cyclobutanone derivative. The latter was all important in the structure

elucidation of copaene.

4. Often it may be necessary to establish the structure of an intermediate by synthesis as

seen in the structure elucidation of copaene.

Α-Terpineol is an optically active Monoterpenoids with the molecular formula C10H18O.

It contains a tertiary hydroxyl group and ads on two atoms of bromine to show that it has

one double bond. Oxidation of α-terpineol with 1% KMnO4hydroxylates the double bond

to give a tri hydroxy compound. This on oxidation with chromic acid gave a keto-lactone

which is obviously formed from an intermediate ϒ-hydroxy acid B. further oxidative

degradation gave terebic acid whose structure was confirmed by synthesis.

Knowledge of a Molecular Rearrangement

The appreciation of a molecular rearrangement which may occur during degradative

work goes a long way in structure elucidation. An example of this is found during the structure

elucidation of α-pinene.

The oxidation of α-pinene with permanganate gives pinonic acid as one of the products.

Using chromic acid as an oxidizing agent pinonic acid is degraded to isoketocamphoric acid. Α-

Campholenic acid is obtained from camphor oxime 3 by the acid-catalysed dehydration and

hydrolysis. Isoketocamphoric acid is obtained by the oxidation of α-campholenic acid.

Significantly a rearrangement, subsequent to protonation of the carbonyl group (see pinacol

rearrangement, is involved in the oxidation of pinonic acid. The work thus clearly demonstrates

the relationship of camphor with α-pinene and reflects on the structure of the latter.

Steroids:

Many naturally occurring compounds containing the per hydrocyclopentenophenenthrene

nucleus are called steroids. This ring system or, in very rare cases, its modification is present in

compounds like sterols, sex hormones, bile acids, adrenal cortical hormones, cardiac glycosides,

toad poisons, some alkaloids and sapogenins. The name ‘sterol’ was given to the solid alcohol

isolated from lipid extracts of tissues. Steroids are the general name which covers all compounds

with the sterol-like skeleton. Majority of the steroids are hydroxylated at C-3 while in some the

hydroxyl group is attached in other positions of the ring system or the side chain. In the case of

steroids the principal parent hydrocarbons are named as below.

Some other parent substances include stigmastane, ergostane, cardanolide, spirostane and

bufanolide. With each name, the configuration at C-5 is indicated by a prefix 5α- or 5β. Among

the conventions used to denote configuration in the side chain (R) and (S) representation is very

reasonable. Several prefixes are employed to indicate deviations from the normal skeleton of the

parent hydrocarbon. The prefixes ‘homo’ and ‘nor’ proceeded by a capital latter indicate

enlargement or contraction respectively of a particular ring: Ring fission with addition of two

hydrogen atoms is indicated by the prefix ‘seco’. Thus the following compound is named 6α,

11€-dihydroxy-B-norpregn-4- ene -3, 20-dione.

Sterols:

Cholesterol is abundantly available and can be isolated from vertebrates and invertebrates

and has been reported to occur in plants. Several aterols have their origin in marine invertebrates

and they suggest a relationship between the stage of evolution and the nature of the nature of the

steroid formed. The sterols of marine invertebrates, as compared with the eight-carbon side chain

of cholesterol, contain a C9 or C10 unit in the form of a methyl or an ethyl group at C24 carbon.

The bile of higher animals contains bile acids in the form of their sodium salts of peptidic

conjugates with taurine and glycine. In these compounds the hydroxyl groups are α-oriented and

ring juncture is cis and the side chain bears the carboxyl group. Stereo chemically bileacid

resemble cholesterols within orientation for side chain in addition to the angular methyl groups

in the ring fusion. It has been established that bile acids are made in liver from cholesterol. The

salt of these acids are known to lower the surface tension of water and act as good emulsifying

agents.

Inspection of the conformational formula of methyl cholate shows that in it the least

hindered hydroxyl group is the α-equatorial at C-3. Of the C-7 and C-12 axial α-hydroxyl

groups, the one at C-7 is more hindered because of encumbrance by the methylene group in ring

A due to cis fusion of A, B rings. Interestingly, it was shown that upon acetylation with acetic

anhydride and pyridine, C-7 hydroxyl was acylated in preference to that at C-12. The

inaccessibility of12 α-hydroxyl to esterification is due to its intramolecular association with the

ester group in the side chain at C-17 which being sufficiently long is capable of curling around

12 α- hydroxyl closely. In confirmation with this explanation, when the C-17 side chain is

shortened the 12 α- hydroxyl group actually becomes easier to acylate as compared to the 7 α-

hydroxyl.

Steroid Hormones:

Hormones are the compounds which are secreted by specific glands, and they exert

control over various body processes. The natural estrogens are ovarian hormones which, together

with progesterone, control the sexual cycle in female mammals. In the case of animals the

estrogens produce estrus (heat) which is followed by characteristic changes of the vaginal

epithelium. Several methods of bio-assay, therefore, are based on these changes. The naturally

occurring estrogens like esteadiol, estriol differ from other steroid hormones in the presence of

an aromatic ring A and that the C3 hydroxyl group is, therefore, phenolic, the nonexistence for

the methylgroup at C10, in addition to presence for either keto or hydroxyl group at C17 or

sometimes at C16.

Estrogens can be isolated from the human pregnancy urine while the urine of pregnant

mares had been the source of all the estrogens required commercially. In urine, the compounds

are present as water-soluble glucuronides or sulphates.The naturally occuring androgens like

testosterone and androsterone are C19 compounds and cause the development of the male sex

organs or secondary sex characteristics.The adrenocortical hormones, e.g., cortisone and cortisol,

are C21 compounds produced by the cortex of the adrenal. These hormones are essential for the

maintenance of life and their deficiency produces a variety of metabolic disturbances.

The cardenolides are C23 steroids having and α, β-unsaturated-ϒ-lactone side chain and a

hydroxyl group at C14. These have the special property of exerting a particular along with

morepowerful actions to heart muscle and are often called cardiotonic principles. Many cardiac

glycosides are isolated from plants of tropical regions and were used by natives of Africa and

South America as arrow poisons. Structurally most of these compounds belong to the 5 β-series

and in all with one exception the C3 hydroxyl is β-oriented .typical examples are strophanthidin

and digitoxigenin. Ozone oxidises the, β-unsaturated-ϒ-lactone side chain to a 20:21- ketal as its

glyoxylate ester to provide a direct route to some corticoid side-chains.

The bufadienolides provide another small group of C24 steroids having as a side chain a

doubly unsaturated six membered lactone ring. They are cardio active and occur as glycosides

involving the C3 hydroxyl group, in plants of the squall family and in the venom glands of toads

occur as esters of suberylarginine involving the C14 hydroxyl group. Bufalin is a typical example.

Preparation made by the extraction and drying of the seeds and leaves of the purple

foxglove was used in the treatment of dropsy. It was in 1785 that a Scottish physician William

Withering introduced the use of digitalis in heart therapy with spectacular success. One of the

active components of digitalis is the glycoside of digitoxigenin. The modes of action of this

component of digital is the glycoside of digitoxigenin. The modes of action of these compounds

are: the glycosides exert a specific action on the heart, they increase the contractibility of the

heart muscle, diminish the heart rate and increase the cardiac glycosides have a specific affinity

for heart muscle, since when the cardiac glycoside is administered either orally or by intravenous

injection, the amount absorbed by the heart tissue is 10-40 times the quantity takes up by all

other tissues and organs. In terms of the pure glycoside, digitoxin the daily dose in heart therapy

is 0.3 mg of intravenously injected material.

The following structural features are essential for the activity.

1. Unlike the glycosides, the free aglycones or genins are only weekly active. More than

twenty sugars have been isolated as products of hydrolysis of cardiac glycosides. Of

these, only three namely D-glucose, L-rhamnose and D-fucose have been isolated

from other plant materials. Interestingly D-glucose is the only component having a 6-

hydroxyl group and several components are 2, 6-desoxy sugars while others are 3-

methyl ethers.

2. The unsaturated lactone ring and the fourteen β-hydroxyl group are both necessary for

the activity. Hydrogenation of 20-22-double bond of the glycoside results in the loss

of activity, which is again observed on isomerization with base when an isoglycoside

is formed. This reaction involves the interaction of the fourteen β-hydroxyl group

with the lactone ring, which initially involves migration of the double bond from the

20, 22-position. Subsequent attack of the fourteen β hydroxyl at C21 results in of the

formation of a new six-membered ring containing oxygen, which assumes the chair

conformation:

3. From a study of the now enormous number of these glycosides which are known, it is

established that compounds with 5α-configuration are less active as compared to 5 β-

glycosides of 3 α-ols are very weakly active. A 19-aldehyde group is favorable for

high potency. A 5 β-hydroxyl group also enhances activity, as does a 4, 5-double

bond. It is established that, unlike a 4, 5 double bonds, a 5,6-double bonds is

distinctly unfavorable to cardiac activity.

The sapogenins are steroids with twenty seven carbon atoms and are typical in having a

spiro ketal side chain and this type of spiroketal group is not found in any other natural product.

They occur naturally as glycosides in plants and are known as saponins since they have the

property of forming a soapy lather in water. Saponins are water and ethanol-soluble but are

insoluble in ether. Some of them, digitonin, in particular, form stable complexes selectively with

three β-hydroxysteroids. In addition to the saponindigitonin, an example of a sapogenin is

tokorogenin:

Alkaloids (alkali-like) are nitrogenous bases which occur in plants and several of these

produce striking physiological effects which vary greatly from alkaloid to alkaloid. These

physiological actions include paralysis, stimulation of central nervous system, blood pressure

and pain relief.

The classification of alkaloids is based on a mixture of chemical and botanical criteria

and systematic names are seldom used for alkaloids and their common names have a variety of

origins. Thus the name of the opium alkaloid, morphine, is from Morpheus, the ancient Greek

god of dreams, strychnine is from the seeds of the Strychnos plant while the name tobacco

alkaloids, nicotine comes from Nicot who was a French ambassador who sent tobacco seed to

France. However, one characteristic about alkaloids names is the common ending in, showing

that they are all amines. A reference to the structures and stereo chemical aspects of some of the

alkaloids has been made. Structures of a few other alkaloids from various groups are presented.

General Methods of Structure Determination:

The general methods of structure determination include the isolation of a pure compound

followed by the initial characterization and definition of the functional groups. The underlying

carbon skeleton is determined and the functional groups are placed on these skeletons. The final

phase involves the determination of both the relative and absolute stereochemistry.

Present day methods of structure determination are much more rigorous then those of

earlier times and involve a combination of physical and chemical methods. Thus by the

application of spectral methods, a variety of functional groups can be easily identified which is

presented here as a combination of classical and present day methods.

Molecular Formula

The molecular weight determination is done on a pure specimen in more than one

solvent, perhaps by Rast’s method (camphor, m.p. 1800; depression for 1 g. mol. Compound in

100 g camphor, 4000) and thus the molecular formula is determined. The elemental analysis and

accurate mass measurement of the molecular ion immediately points to the molecular formula in

modern times.

Use of spectral method:

In, NMR and UV spectra give extremely useful information about the functional group

and some stereo chemical features. As an example we may discuss the structure of piperine

C17H10NO3 which was established around 1882 using classical method and today an examination

of some of the spectral data immediately point to several structural features in the compound.

IR bands (cm-1) at: 1635, 1608 (C=C stretching of diene)

1608, 1580, 1495 (C=C stretching of phenyl ring)

1635 (C=O stretching of –CO-N ˂)

995 (C –H bending of trans –CH=CH-)

850,830,806 (C-H bending 1, 2, 4 - trisubstituted benzene ring)

UV: λ max 245nm (log ε 4.47) is that of a-1 phenyl butadiene in conjugation with –CO-R and

methylene dioxychromophores.The NMR spectrum along with other structural feature shows the

presence of methylene dioxy group by displaying a characteristic signal at 5.95δ.

Hydrolysis:

The nitrogen containing part is isolated sometimes by means of the gentle hydrolysis

which may leave two parts for identification thus piperine afforedpiperidine and piperic acid 1

to show that the alkaloid is the piperidine amide of pipericacid. Similarly atropine gives tropine

and tropic acid.

Unsaturation:

The presence of unsaturation is determined by the addition of bromine HBr etc and

particularly NMR spectroscopy is employed for detecting olefin protons. Thus the presence of

two double bonds in piperic acid 1 was proved by the preparation of tetrabromopiperic acid.

Oxidation:

Oxidation is one of the most valuable methods for structure determination by employing

a variety of oxidizing agents it is possible to get a variety of product whose identification gives

information about structure. Thus the structure of nitrogen free portion of piperine, i.e, piperic

acid was deduced from its oxidation with potassium permanganate which afforded oxalic acid

piperonal and piperonylic acid.

Synthesis of Intermediates:

A synthesis in logical stages of various degradation products helps considerably. Thus

the structure of piperic acid was proved by synthesis (1894) as shown in scheme 1 which

involves Rrimer-Tiemann and Claisen-Schmidt reactions.

Degradations:

Degradations general or specific are carried out in the hope of finding a recognizable

kernel. Among the vigorous disruptive reagents zinc dust or soda lime are commonly employed.

Thus the formation of phenanthrene as a product of zinc dust distillation of morphine

showed the presence of phenanthrene nucleus. Zinc dust dehydrogenation of yohimbine provides

another example to show the strength of this degradative technique in structure elucidation.

Similarly fusion of cinchonine with potassium hydroxide gives lapidine while on vigorous

oxidation Cinchoninic acid is formed. Therefore, cinchonine contains a quinoline nucleus with a

side chain atC-4.

A special method of degradation is used in the case of alkaloids having biphenyl ether

linkages e.g., thebisbenzylisoquinoline alkaloids. These are cleaved into two units by reduction

with sodium in liquid ammonia which helps in structure elucidation as in the case of o-

methyldauricine.

3.8.4 Phenols:

The total phenol contented of extract isfound out by Folinciocalteu reagent process [122]

by little change. Plants extort (1 ml) be mixed with Ciocalteu reagent (0.1 ml, 1 N) and allow to

stand for 15 min; Then 5 ml, from saturated Na2Co3 be added. The mixture allows standing for

thirty minutes at room temperature and the total phenol get determine spectrophotometrically at

760nanometers. Gallicacid applied like internal standard. Total phenols value constitutes express

in term of Gallicacid corresponding for expressed compound.

Tests to phenol compound:

a) The draw out be covered among inert ferricchloride solution; appearing from violetred

coloration indicate that presence of phenolic compound.

b) The extract is treating with 10 percent sodiumchloride solution; appearances of creamy color

indicate the existence for phenol compound.

3.9 Result and Discussion:

A) Preliminary qualitative phytochemical testing:

B) Quantitative phytochemical testing:

A) Preliminary qualitative phytochemical testing:

Country mallow is explore phytochemical through different researcher and possess to

find out number of chemical constituents various plants parts possess various active metabolites

which are used on various common and severe health symptoms. Various diseases are cure due

to its various chemicals found in that plant so necessary to carry out to find the various extraction

methods by using different solvent system. Different researcher worked on the various

parameters to find out very essential compounds.

In this present attempt, on various chemicals which are polar to non polar in nature were

used for preliminary test phytochemical testing of Abutilon indicum. Data represent in Table III-

1 extraction in water medium, only alkaloids was present in root, stem and leaf other compounds

were absent in it.

In ethanol solvent (Table III-2) alkaloids, saponins, terpenoids and phenols were present

commonly in root, stem and leaves. Basic active metabolites screening in chloroform solvent

(Table III-3) which were generally used for extraction show the in root, stem and leaves show

the positive alkaloid test. Alkaloids present in seeds, roots, bark, leaves and other parts of plants.

One species usually contain a mixture of related alkaloids. In the plant part is dried, powdered

and extracted with solution.

Table III - 1. Preliminary phytochemical screening of Abitulon indicum

Sr.

No

Plant parts Phytochemical

Aqueous.

Extract

Alkaloids Saponins Terpenoids Phenols

1 Root present - - -

2 Stem present - - -

3 Leave present - - -

4 Fruit with

seeds

- - - -

Table III - 2. Preliminary phytochemical screening of Abitulon indicum

Sr.

No

Plant parts Phytochemical

Ethanol

extract

Alkaloids Saponins Terpenoids Phenols

1 Root pose - - -

2 Stem pose pose pose pose

3 Leave pose - pose pose

4 Fruit - - - -

After descent evaporation of the solvent, the salt is decomposed by adding mineral acid to

liberate the free base. The may be directly extracted with dilute mineral acid from which the

alkaloids is liberated be neutralization of the solution. The mixture is separated, mainly by

chromatography, to isolate the pure compounds.

Table III - 3. Preliminary phytochemical screening of Abutilon indium

Sr.

No

Plant parts Phytochemical

Chloroform

extract

Alkaloids Saponins Terpenoids Phenols

1 Root pose - pose pose

2 Stem pose pose pose pose

3 Leave pose pose pose pose

4 Matured

Fruit with

seeds

- - - -

Table III -4. Preliminary phytochemical screening of Abutilon indicum

Sr.

No

Plant parts Phytochemical

Ether extract Alkaloids Saponins Terpenoids Phenols

1. Root pose - pose -

2. Stem pose pose pose pose

3. Leave pose pose pose pose

4. Matured

Fruit with

- pose - -

seeds

Terpenoids were present in all plant parts except in fruits. Table number III-4, Table

number III-5 and Table number III-6 indicate that the presence of secondary metabolites in ether,

chloroform and methanol, compare these three we observe that methanol solvent were best for

extraction of plant material.

Table III - 5. Preliminary phytochemical screening of Abitulon indicum

Sr.

No

Plant parts Phytochemical

Acetone.

extract

Alkaloids Saponins Terpenoids Phenols

1. Root Present - - -

2. Stem Present - - -

3. Leave - - - -

4. Matured

Fruit with

seeds

- - - Present

Table III - 6. Preliminary phytochemical screening of Abitulon indicum

Sr.

No

Plant parts Phytochemical

Methanol

Extract

Alkaloids Saponins Terpenoids Phenols

1 Root pose - - -

2 Stem pose - pose pose

3. Leave pose pose pose -

4. Matured - pose - -

Fruit with

seeds

By the complete analysis of the plant it contains asparagines saponins and mucilaginous

substances, hexoses, alkaloids, n-alkane mixture (C22-34

), flavonoids, alkanols ascategories of

most important compound. The important constituent report within this plants were as fumaric,

p-coumaric acid, caffeic, vanillic, β-sitosterol, (R)-N-(1’-methoxycarbonyl-2-phenylethyl)-4-

hydroxybenzamide, galacturonic, p-β-D-glycosyloxybenzoic, amino acids, p-hydroxybenzoic,

etc.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Leaves Stem Root fruits withseed

Alkaloids

Saponin

Terpenoids

Phenols

mg/gm Sample.

Graph: II-A.Quantitative phytochemical analysis.

Fumaricacid p-hydroxybenzoicacid

Caffeicacid para-coumaricacid

Vanillicacid Glutamicacid

Fumaricacid p-hydroxybenzoicacid

Caffeicacid para-coumaricacid

Vanillicacid Glutamicacid

Fumaricacid p-hydroxybenzoicacid

Caffeicacid para-coumaricacid

Vanillicacid Glutamicacid

Sitosterol Asparticacid

L-serine L-Leucine

L-Threonine Alkanols

Sitosterol Asparticacid

L-serine L-Leucine

L-Threonine Alkanols

Sitosterol Asparticacid

L-serine L-Leucine

L-Threonine Alkanols

Chemical structure of the saponins (solanine)

The plant of Abutilon Indicum contain essential oil which are mainly comprises of

geraniol, farnesol, caryophyllene, α-pinene, endesmol, caryophyllene oxide, borenol, elemene,

α-cineole and geranyl acetate along side with number of other minor constituent as shown in

Table number-2 [116], [60]. The oils from seedsof thiss plants afford 10 methylene octadeca 9-

enoicacid (sterculicacid), cis 12,13-epoxyoleic (vernolicacid), 9, as well as 8, 9 methylene-

heptadeca 8-enoicacid (Malvali acid) [89], [68]. Airborne part of the flora contains gossypetin 8-

and 7- glucosides, β-sitosterol, tocopherol oils, flavonoids as well as cyanidin-3-rutinoside. The

leave of such plants comprise sapogenins, flavonoids, steroids and carbohydrates. Flavonol,

saponins some alkaloids derivatives of kaempferol and quercetin predominate in the species from

Malvaceae family.

Gallic acid is present in the root of this plant. Essential oils of flowering parts contain α-

cineole, geraniol acetate, α-pinene, caryophyllene, geraniol, borenol, elements, caryophyllene

oxide, farnesol and eudesmol is shown in Fig. 2 [64], [121].

Terpenoid hyhrocarbons are the biggest and most various family of natural product, range

in structure starting to polycyclic to linear molecule and with diamention from the six carbon

hemi-terpenes to naturalistic rubber, with compressing thousands of isoprenoids units. By the

condensation of isoprenoids terpenoids are synthesized and classified with their present five

carbon core structure. With presence of many flavors along with aromatic molecule, such as

geraniol, caryophyllenelinol, menthol framed with two isoprenoids mono-terpenes (C10), with

Chemical structure of the saponins (solanine)

The plant of Abutilon Indicum contain essential oil which are mainly comprises of

geraniol, farnesol, caryophyllene, α-pinene, endesmol, caryophyllene oxide, borenol, elemene,

α-cineole and geranyl acetate along side with number of other minor constituent as shown in

Table number-2 [116], [60]. The oils from seedsof thiss plants afford 10 methylene octadeca 9-

enoicacid (sterculicacid), cis 12,13-epoxyoleic (vernolicacid), 9, as well as 8, 9 methylene-

heptadeca 8-enoicacid (Malvali acid) [89], [68]. Airborne part of the flora contains gossypetin 8-

and 7- glucosides, β-sitosterol, tocopherol oils, flavonoids as well as cyanidin-3-rutinoside. The

leave of such plants comprise sapogenins, flavonoids, steroids and carbohydrates. Flavonol,

saponins some alkaloids derivatives of kaempferol and quercetin predominate in the species from

Malvaceae family.

Gallic acid is present in the root of this plant. Essential oils of flowering parts contain α-

cineole, geraniol acetate, α-pinene, caryophyllene, geraniol, borenol, elements, caryophyllene

oxide, farnesol and eudesmol is shown in Fig. 2 [64], [121].

Terpenoid hyhrocarbons are the biggest and most various family of natural product, range

in structure starting to polycyclic to linear molecule and with diamention from the six carbon

hemi-terpenes to naturalistic rubber, with compressing thousands of isoprenoids units. By the

condensation of isoprenoids terpenoids are synthesized and classified with their present five

carbon core structure. With presence of many flavors along with aromatic molecule, such as

geraniol, caryophyllenelinol, menthol framed with two isoprenoids mono-terpenes (C10), with

Chemical structure of the saponins (solanine)

The plant of Abutilon Indicum contain essential oil which are mainly comprises of

geraniol, farnesol, caryophyllene, α-pinene, endesmol, caryophyllene oxide, borenol, elemene,

α-cineole and geranyl acetate along side with number of other minor constituent as shown in

Table number-2 [116], [60]. The oils from seedsof thiss plants afford 10 methylene octadeca 9-

enoicacid (sterculicacid), cis 12,13-epoxyoleic (vernolicacid), 9, as well as 8, 9 methylene-

heptadeca 8-enoicacid (Malvali acid) [89], [68]. Airborne part of the flora contains gossypetin 8-

and 7- glucosides, β-sitosterol, tocopherol oils, flavonoids as well as cyanidin-3-rutinoside. The

leave of such plants comprise sapogenins, flavonoids, steroids and carbohydrates. Flavonol,

saponins some alkaloids derivatives of kaempferol and quercetin predominate in the species from

Malvaceae family.

Gallic acid is present in the root of this plant. Essential oils of flowering parts contain α-

cineole, geraniol acetate, α-pinene, caryophyllene, geraniol, borenol, elements, caryophyllene

oxide, farnesol and eudesmol is shown in Fig. 2 [64], [121].

Terpenoid hyhrocarbons are the biggest and most various family of natural product, range

in structure starting to polycyclic to linear molecule and with diamention from the six carbon

hemi-terpenes to naturalistic rubber, with compressing thousands of isoprenoids units. By the

condensation of isoprenoids terpenoids are synthesized and classified with their present five

carbon core structure. With presence of many flavors along with aromatic molecule, such as

geraniol, caryophyllenelinol, menthol framed with two isoprenoids mono-terpenes (C10), with

three terpenoids sesqui-terpene (C15). Other bimolecular compounds, such astriterpenes (C30), di-

terpene (C20), and tetra-terpenes (C40) illustrate extremely particular properties.

Review of literature according to above results, it directly indicate that the amount of

total content depends upon on the plant parts, we used for extraction. The specific active

compounds present in that particular parts, which plants can accumulated many valuable

compounds.

Roots show the better positive results on preliminary experiments in which any solvent

show the alkaloids. Only fruits with seeds show the saponins in some solvents.

B) Quantitative Phytochemical Screening:

Generally screening is useful to perform and characterize chemical composition of

Abutilon indicum root, leaf, stem and fruits with seed samples. The screening covered mainly

nitrogenous and active compounds, Alkaloids, Saponins, Terpenoids and Phenols (Table III - 8).

All three samples leaf, stem and fruits with seed showed positive test with different

alkaloids, saponins, terpenoids and phenols on the basis of their Rf values in thin layer

chromatography. Terpenoids or terpens are a class of compounds that are found mainly in plants.

The simpler ones are liquid, generally with characteristic fragrance, present in the essential oils

of plants. Essential oils are volatile oils as distinct from the fatty oils or lipids obtained from oil

seeds which are non volatile triglycerides. The adjective, essential is related to essence as in

essence of flower, fruits and similar plant parts. They are hydrocarbon or oxygen containing

compounds having hydroxyl, carbonyl and related functional groups. The number of carbon is

terpenoids is in multiples of 5, beginning with 10. Those having 10 carbons are called mono

terpenes, those with 15 carbons are sesquiterpine, and those with 20 carbons are diterpenes.

Many terpenoids on pyrolysis give 2-methyl-1, 3-butadiene- also known as isoprene as one of

the products. The carbon skeletons of all terpenoids can be considered to be built up of isoprene

units joined head to tail. This is known as the isoprene rule.

Data depicted in the Table III - 7, Rf values of this all active compounds show

tremendous variation in it. Comparison of two parts of plants, root extract spot show four bands

on the TLC plate compare to the stem and leaf spot. Stem and leaf show only 2 bands. Total

alkaloids (Fig III - 8) present in these plants parts mainly root (0.99mg), stem (0.36mg) and

matured leaves (0.42). Among the various plant parts the root show the accumulation of

alkaloids is more compare to the other parts of plants. Saponins occurs only in fruits and leaves

extract, compare to the other plant extract it show only two spots on TLC plate.

Terpenoids and phenols show the similar results, compare these two only two spots were

more in phenols which occur in leaf and stem plant extract. The root and leaf plant extract also

show the terpenoids, but phenols present in leaf and fruits with seeds plant extracts.

Table III –7

Qualitative active metabolite screening by TLC

Contents

Name

Solvent System Plant parts Number of

Bands

Spray reagent

Alkaloids Benzene:

chloroform

Root 4 Ninhydrin

Stem 2 Dragendroff’s

Reagent

Leaves 2 Iodine vapors

Fruits - -

Saponins Toluene: acetic

acid

Root - Iodine vapors

Stem 1 Iodine vapors

Leaves - -

Fruits 2 UV light

Terpenoids Acetic acid :

acetone

Root 5 Ninhydrin

Stem 1 Ninhydrin

Leaves 1 Ninhydrin

Fruits - -

Phenols Ethanol:

Aceticacid:

water

Root 7 Folincioaiteu’s

Stem - -

Leaves - -

Fruits 2 Ninhydrin

Table III – 8

Quantitative phytochemical analyses

Name of Compound Plant parts (mg/10 g Sample)

Leaves Stem Root fruits with seed

Alkaloids (% of

alkaloids/ 10 gm of

sample)

0.42 0.36 0.99 -

Saponins 0.23 0.32 - 1.34

Terpenoids 1.45 0.12 0.76 1.32

Phenols 0.65 0.71 0.0 0.89

The prospective to favorable effect about healthiness phenolic compound have a great

attention. Numerous observational examines have been exposed pharmacological and biological

property for phenolic compound over the last few years, specially their antimicrobial activity,

cytotoxic activity, antiviral and anti inflammatory.

The majority from the medical plant is considerably predictable to enrich with phenolic

compounds as well as bio flavonoids that contain exceptional antioxidant property. Phenolic

compounds are dynamically energetic within remedial stomach in addition to kidney problem

with supportive as anti-inflammatory for actions. Total phenolic compounds in leaf, stem and

matured fruits with seed was found to be (Fig III - 8) 0.65mg, 0.71mg and 0.0 mg / 10 gm of

sample respectively (Table III - 8).

Saponins exist extensively considerably identified antitusive activity and to have

expectorant activity. Total four saponins were found to be present, two of which were observed

in leaf and other two which are common to stem and seed were found to have Rf value 0.55 and

0.65 (Table III - 8). Total Saponins content in leaf, stem and matured fruits with seed was found

to be 0.23 mg, 0.32 mg and 0.0mg per 10gm (Fig III-8)of samples severally. Saponins are

detected in lots of plants and animals. Several proletarians passed away general phytochemical

analysis of plant intended for the existence of saponin.

Alkaloids along with terpenoids were found in samples i.e. leaf, stem and roots where as

saponins some amount of phenols were found in fruits with seed samples only. In this present

work roots, stem and leaves bearing considerable amount of active metabolites and could be used

as potential source of antioxidant, antimicrobial and various other biological activity to

contribute the health benefit of the consumers.

The phytochemical analysis of the plants excerption exposed for occurrence from

constituent which be identified to the show evidence of physiological with medical activities

[168]. Occurance of phytochemicals by the analysisof the plants part revealed such as phenols,

terpenoids, flavonoids, tannins, steroids, glycosides, saponins, and alkaloid. The phenol

compound is the biggest furthermore often commonly observered group of the plants substances

[166]. Such compounds have genetic property for example antiaging, antiapoptosis, anti-

carcinogen, anti-atherosclerosis, anti-inflammation, improvement of endothelial purpose and

cardiovascular protection, in addition to cell proliferation activities and prohibition of

angiogenesis [77]. Within phenolic compounds the antioxidant properties of meditional plants

occure majorly [34], [112].

Natural antioxidants are mostly coming of the plant into the appearance for phenol like

compound for example flavonoids, tocopherols, phenolic acids, etc [4]. Tannin binds toward

prolife rich interferes and protein with proteins synthetic thinking. Flavonoid is hydroxyl group

of phenol consistence’s constitute are synthesized through plant with responses in the direction

of microbic contamination and they are establish to be antimicrobic substance beside extensive

selection of microorganisms in ex vivo. Their action is most likely because of their power to

form complexes through additional proteins and cellular which are soluble in addition to form

complexes alongwith cell wall ofbacterias [120]. For the treatment of many diseases harbal

medicines are more powerful and become more popular for accepted conviction due to this

common medicines are safe, easily available with fewer types of fallout. Certainly, public need

and the market erstwhile so immense that here is additional possibility with the intention of so

many medical plant nowadays, countenance both experimental deprivation with extinction for

genetical multiplicity [124]. The whole pharmaceutically based industry are paying attention

toward growth with design from some fresh modern native plants base drug by research with

contributes by traditional organization from medicine [137].

The plant draws out are in addition exposed to comprise saponin which is identified to

create repressive effects on tenderness [100]. Saponin has played a role of coagulating alongwith

precipitating of redblood cell. Saponins have various special properties such as hemolytic

activity bitterness, cholesterol adhering properties, formation of foam in aqueous solutions, etc

[171], [135].

Alkaloids have linked with medicinal purposes from the ancient time with individual for

totheir general genetic places for their toxic property. A number of personnel comprise report the

antispasmodic, antibacterial and analgesic [19], [71], dimensions for alkaloid [172].

Nowdays, conventional as well as ethnobotanical use for instinctive compound,

particularly to plants source experienced a large extent attentions. They tested ever since its

efficiency with in generalit are safe for human being for their use.The classic approaching within

the research for innovative molecule to the managements of several disease. By the systematic

selection for some journalism uncommitted resting on Abutilon indicum depict the information

with the purpose of a democratic medication amongst the several Ayurvedic, ethnic group,

traditional practitioner etc for the discussion of their ailments. Researchers are searching the

therapeutic possible of these plants as it has more therapeutic belongings which are unknown.

Thus the plant considered here be able to the potential origin of new utile drug. The

phytochemical delineation of the draw out, quality standards and the identification of responsible

binominal compounds are essential for further study. Abutilon indicum is consequently extract

commencing this plant might be a superior foundation intended for functional drug. The

conventional medicines exercise be suggested powerfully to such plant in addition to it's

proposed with the aim of supplementary effort must be carried away to seperate, with to

distinguish dynamic constituent which are responsible for support of the activities of such type

plant purge, furthermore supplementary function be recommended toward clarify some potential

mechanisms. The action of these extracts therefore an exciting dispute that may add to our

phototherapeutic compounds. Thus such plant can be applied like option sources for utilitarian of

drug. Additional considerations be considered necessary with this plant toward characterize,

elucidate and isolate, the structures for the binomial combines by such plants to industrial drugs

preparation.

The study has shown that the extracts of Abutilon indicum possess metabolites that

enhance itsactivity as an antimicrobial agent, which was found tobe effective against these

pathogenic microorganisms involved in causing some diseases. Therefore the results of these

studies provide to rationalise the use of the plants parts to traditional medicine practice inPune

district’s rural area (Ambegaon). The activity of this plant should further be investigated against

wide range of microorganisms. Thorough pharmacological studies should be carried out on pure

extract for the a variety of plant part from Abutilon indicum, so as to ascertain their mode of

actions and toxicological effects on various animal organs. Over dose of the plant parts extracts

should be avoided by patients. This is because high dose of the extracts may have adverse effects

on organs like liver or kidney. Also purification and toxicological studies should be carried out

with a view of sourcing antimicrobial agents for drug development.

The result obtains with the analyzed study it recommend to identify phyto chemical

compound might be the bio-active constituent along with such type of plants constitute providing

to gradually more useful source for bio-active compound to significant medical merits. So it

needs to test active metabolites activity on specific biological properties and to check the toxicity

of the active substances. Specific compounds influence to human physiology and used on various

biological activity.

3.10 Proximate Analysis for Abutilon Indicum:

3.10.1 Introduction

Most of the crude drugs (plant materials) are usually put in quarantine store and they

remain there for long time. During storage proper ventilation, humidity controls, suitable

temperature and light conditions should be ensured to maintain their original pharmacological

action. However it is observed that, crude plant materials, before being taken for processing, are

not analyzed which can lead to changes in original characteristics. To avoid this, the crude drugs

should be tested for the following tests as per the USP and Indian Herbal Pharmacopoeia (IHP).

1. Foreign organic matter.

2. Ethanol soluble extractives.

3. Water soluble extractives.

4. Total ash contents.

5. Ash which is insoluble in acid.

6. Ash which is soluble in water.

7. Loss by drying.

8. Percentage moisture content.

1) Foreign Organic Matters:

Medicinal plants part should be completely free from observable sign of contaminations,

i.e.insects’ moulds, and other animal contaminations, including animals’excretory product,

fungus and dust. To obtain marketed plant materials that is entirely free from some form of

harmless unknown subject matter. However, no toxicant, dangerous or differently dangerous

foreign material or residue should allow. Before the cutting or ground for testing plant material

sand, soil, dust, stone, and other foreign organic matter must be removed. Macroscopic

observations are conveniently being applied for determination of unknown matter in whole or

particular plants material.

I. Material consisting of foreign matter or any all of the following

II. Materials other than those named with the limits specified for the plant material

concern or parts of the medicinal plant material.

III. Any part of organism, or product of living being other than that cited in the

specification and description of the plant material concerned.

IV. Medicinal plant materials not adhering mineral admixtures such as sand, soil stones,

dust etc.

2) Sampling:

Abutilon Indicum plant material of selected plants were collected from various places

from Junnar taluka in bulk, thoroughly washsd with distilled water to eliminate the dust particle

on the surface of the plant and the soil particles adhering to the roots. Excess water was allowed

to drain off by spreading the plant material on filter papers. Then 500 gm of the washed and

drained plant material of each plant was taken and spread as a thin layer on a white, clean muslin

cloth. Using magnifying lens as wellas by visual inspection foreign matters get sorted. Portions

of the sorted foreign matter were weighed and the contents of foreign matter in grams per 100

grams of the sample were calculated. The procedure was carried out for a total of five sets.

Observations:

It was observed that the percentage foreign organic matter in Abutilon Indicum plant

material was obtained. The results are given in the following Table 1

Calculations:

(M1 - M)% foreign organic matter -------------------- x 100

M 2

Where,

M : Weight of empty dish in grams.

M1 : Weight of dish with foreign matter in grams.

M2 : weight of sample (whole plant material) in grams

3) Extractable Matters:

In this method plant material in the form of powder get extracted by using proper

solvents and analysed different phytochemical constituents. Here according to Indian Herbal

Pharmacopoeia water and ethanolbe used as common solvents for determination of extractable

matters.

Sampling:

Crude drug was prepared as described “collection and primitive drug preparation” before.

A portion of each plant powder was used for analysis.

Procedure:

Accurately five grams plant material taken in stoppered glass volumetric flask, then 100

mlof distilled water added. The flask is stirred continuosly to 6 hour, and then allow in the

direction of keep period of 18 hour. The filtered the contents quickly to avoid loss of solvent.

The filtrate was transferred to a previously weighed clean beaker and using water bath get

evaporate. After evaporation, dry the extract at 104°C for six hours and kept in a dessicator for

cooling. The beaker was weighed and percent extractable matter in water was calculated. The

above procedure was repeated thrice for determination of water soluble extractable matter.

Ethanol soluble extractable matter was determined by following the above procedure

except ethanol was used instead of water, as extracting solvent. The experiment was repeated for

three times.

Observations:

It was observed that the percentage ethanol extractable matter in (Abutilon Indicum) plant

material in powdered form was showed. Results are committed with Table No. 1

4) Ash Content

Following ignition system for ash of therapeutic plants material be found out through

three unlike method that measures the followings.

Ash amount.

Ash which is insoluble in acid.

Ash which is soluble in water.

Total amount of material remaining after the ignition is describes the totalash contents.

Which include some physiologic ash, which earned from tissues of the plant itself, and

nonphysiological ash, it represents the residues for the extraneou matters (soil and sand particals)

appearing on the surface of the plant material.

By adding dilute hydrocloric acid with the total ash is boilled, acid insoluble ash is

determined along with ignitings the residual nonsoluble thing. Such gives idea about quantity of

silica present as siliceous earth andsand.

Residue subsequent treatment for the full amount of ash alongwith distilledwater and

difference in weight gives idea about water soluble ash.

3.10.2 Total ash

Total ash is obtained with following method.

Apparatus:

Silica dish, desiccators, air oven, muffle furnace.

Procedure:

Accurately weight two grams of dried plant material in a tarred silica dish, ignite with a

glow of heating element or Bunsen burner near about 60 minutes. The detonation was completed

with holding it within mufflellar heating system at 550°C ± 20°C till grey ash was formed. It

was then cooled in desiccators and weighed. The process was repeated (ignition, weighing with

cooling) untill the differentiation within the correct weight amongst two consecutive weighing

was a lesser amount than 1 mg.

Observations:

It was observed that the percentage total ash content of each plant powder was recorded.

The results are given in following Table 1

3.10.3 Acid Insoluble Ash

Acid insoluble Ash was obtained by following method.

Chemicals:

Dilute HCl, 5 N HCl, and AgNO3 solution.

Apparatus:

Silica dish, desiccators, air oven, muffle furnace.

Procedure:

Take accurately two gm of the dried plant material in a porcelain or silica dish then ignite

it with a Bunsen burner for about one hour. The porcelain dish was kept in mufflellar furnace on

5450°C ± 20°C untill greyash obtain. The ash was moistened with concentrated HCl and

evaporated to dryness after which it was kept inside the electric aircraft maintained at 135°C ±

5°C intended for 3 hr. After cooling, do the additions of 25 cc. of dilute HCI, and then heat it on

a water bath for 10 minutes by covering with watch glass. It was then allowed to cool, filtere it

through Whatmann filter paper with number 41. Wash the residue with hot water till washings

were free from chloride (as tested with AgNO3 solution). The filter paper and the residue were

put in a dish and ignited in a mufflefurnace at 545°C ± 10°C intended for 1 hr. The process of

cooling by keeping in desiccator as well as weighing was repeated till the difference between

two successive weights was found to be less than one mg.

Observations:

It was observed that the percentage acid insoluble ash content of plant powder was

showed. The results are indicated in Table 1

Water Soluble Ash

Water soluble ash was obtained by following method.

Chemicals- Distilled water.

Apparatus -Silica dish, desiccators, air oven, muffle furnace.

Procedure

Twenty five ml of distilled water get added in a silica dish which contains the totalash

and boil intended for ten minutes. By using ashless filter paper nonsoluble matters get composed.

Then wash, it by hot water and ignite in crucible for the period of fifteen minutes at a

temperature not more than 455°C. The weight of this residues get subtracted trough the wt of

totalash, then the water soluble ash is to be calculated.

Observations:

The observation of the percentage for water soluble ash content of plant powder is

recorded. The results shown with following Table No. 1

3.10.4 Loss on Drying:

Drying percentage by ioss of water gets obtained by following method.

Apparatus:

ASTM sieve (18/BS sieve), wide mouthed stoppered weighing bottle, Dessicator, air

oven.

Procedure:

Five grams of plant powdered sample was weighed in wide mouthed stoppered weighing

bottle. The bottle was then placed with lid open within hotair automatically maintained at 95°C ±

1°C oven. The sample was kept in an oven for two hours. The bottle was then removed, covered

and placed in a dessicator. The bottle was weighed after cooling to room temperature and

weighed.

The bottle was again kept in the oven for two hrs and the above procedure was repeated

(heating, weighing andcooling) untill the differentiation in the wt. between these two successive

weighings is lower than 1 mg. Three readings for each sample were recorded.

Observations:

It was observed that the percentage of loss on drying in plant powder was recorded. The

results are shown with following table.

3.10.5 Content of moisture:

The moisture from plant powders were obtained by following method.

Karl-Fischer titrimetric method

Instrument:

Digital Automatic Karl Fischer Titrator (microprocessor based) model µAqua Cal-10

Analab Instrument Pvt. Ltd.

Reagents:

Karl Fischer (KJF) reagent, methanol K/F grade, commercial grade methanol (only for

cleaning the dispensing system), μl syringe and distilled water.

Procedure: Reaction vessel was rinsed thoroughly with methanol magnetic stirring rotor was

inserted in the vessel and placed in proper position. The large rubber cork was removed and

some K/F grade methanol was added using funnel, to the reaction vessel just enough to submerge

the metal wires of sensors in the reaction vessel. The cork was replaced immediately. The K/F

reagent and methanol bottles were placed in position. Then the instrument was turned on and the

speed of magnetic stirrer was adjusted. Methanol was neutralized and the titer factor was

determined by calibrating the K/F reagent. This was done by adding 10 µl of distilled water with

the help of a μl syringe in the reaction vessel and completing the titration. The calibration of the

reagent was done in triplicate. The readings were noted and the titer factor was calculated. The

data for determination of titer factor is given in following table number 8 for qualitative analysis

in addition to this is calculate by using the formulas.

Water added (wt. in mg)Titer factor = ---------------------------------

Reading in cm3 (vol.)

3.10.6 Sample Analysis:

Plant material in the powder form is taken with exact amount of 100 mg in weight and

added to the titration vessel and the titration was allowed to complete. The results obtained are

given in table phytochemical analysis.

Following are the parameters, which are, required for checking the quality of raw

material of plant powder selected plants before going for treatments. These character control

parameters are depending on cultivation, handling of the raw substantial and storage conditions.

3.10.7 Calculations:

(A)Proximate Analysis

1) Water soluble extractable matter:-

5g sample powder +100 ml water

(M) wt. of empty beaker = 85.13 gm

(M1) wt. of beaker +residue = 85.75 gm

(M2) wt. of powder = 5g

(M1 - M)% water soluble content = -------------------- x 100

M 2

(85.75-85.13)% water soluble content = -------------------- x 100

5

= 12%

2) Ethanol soluble extractable matter:-

5gm sample powder +100ml ethanol

(M) wt. of empty beaker = 108 gm

M1) wt. of beaker +residue = 108.52 gm

(M2) wt. of powder = 5 gm

(M1 - M)% ethanol soluble content = -------------------- x 100

M 2

(108.52-108)% ethanol soluble content = -------------------- x 100

5

= 10.4%

3) Total Ash content:-

(M) wt. of empty crucible = 17.87 gm

(M1) wt. of crucible +residue= 18.32 gm

(M2) wt. of powder = 2 gm(M1 - M)

% ethanol soluble content = -------------------- x 100M 2

(18.32-17.87)% ethanol soluble content = -------------------- x 100

2

= 22%

4) Acid Insoluble ash:-

(M) wt. of empty crucible = 19.3gm

(M1) wt. of crucible +residue = 19.6 5gm

(M2) wt. of powder = 2gm

(M1 - M)

% ethanol soluble content = -------------------- x 100

M 2

(19.6 5 - 19.3)

% ethanol soluble content = -------------------- x 100

2

= 17.5%

5) Water soluble ash content-

(M) wt. of empty crucible =20.5gm

(M1) wt. of crucible +residue= 20.95gm

(M2) wt. of powder =2gm

(M1 - M)% water soluble content = -------------------- x 100

M 2(20.95-20.50)

% water soluble content = -------------------- x 1002

= 22.5%

6) Loss of drying:-

(M) wt. of empty beaker = 55.39 gm

(M1) wt. of beaker +residue = 58.59 gm

(M2) wt. of powder = 5gm

(M1 – M)% content = -------------------- x 100

M 2

(58.59-55.39)% conte = -------------------- x 100

5= 64%

Table III-9: Proximate Analysis of Abutilon Indicum.

Sr. No ParameterPLANT

1 Foreign organic matter 0.1%

2 Ethanol soluble extractives 10.4%

3 Water soluble extractive 12%

4 Total ash content 22%

5 Acid-insoluble ash 17.5%

6 Water soluble ash 22.5%

7 Loss on drying 64%

8 Moisture content 2%

Chart, III-2: Proximate Analysis of Abutilon Indicum.

3.11 Phytochemical Analysis:

According to medicinal importance form plant contituents are extensively assorted group

of chemical compound showing superior difference in stability and solubility. These are fats,

7 Loss on drying42%

8 Moisture content1%

Proximate analysis

Chart, III-2: Proximate Analysis of Abutilon Indicum.

3.11 Phytochemical Analysis:

According to medicinal importance form plant contituents are extensively assorted group

of chemical compound showing superior difference in stability and solubility. These are fats,

1 Foreign organicmatter

0%

2 Ethanol solubleextractives

7%

3 Water solubleextractive

8%

4 Total ash content15%

5 Acid-insoluble ash12%6 Water soluble ash

15%

Proximate analysis

Chart, III-2: Proximate Analysis of Abutilon Indicum.

3.11 Phytochemical Analysis:

According to medicinal importance form plant contituents are extensively assorted group

of chemical compound showing superior difference in stability and solubility. These are fats,

3 Water solubleextractive

8%

4 Total ash content15%

5 Acid-insoluble ash12%

fixed oils, phenols, waxes, proteins, tannins, carbohydrates, alkaloids, volatile oils, glycosides,

resin combinations and resins. Inpresent study the analysis of phytochemicals from plant powder

is carried out through extraction method. The plant tissues dried obtained by conventional

chemical methodis to be continuously extracted powder material in soxhlet apparatus. The

observations of phytochemical extracts like reasonably polar extracts from phenols and

terpenoids, then basic alkaloids extract, polar extract of quaternary alkaloid with n-oxides,

neutral extract of fats and waxes and fibers is used for calculation of percent extraction. After

complete phytochemical profile of given plant material fractionation of crude extract is desirable

in order to separate the main classes of constituents from each other prior to chromatographic

analysis. The phytochemicals broadly classified as fats and waxes (lipids), fixed oils, tannins,

phenols, alkaloids, proteins, carbohydrates, glycosides, resins, volatiles oils, combinations etc.

The precise mode from extraction depends on the tenure and type of the substance isolated. The

plant material obtained by classical chemical methodis to continuously extracted powder

material by Soxhlet apparatus with assortment of solvent. The plant material were collected,

cleaned, washed and dried in shades. After complete drying they are kept in oven at 300C for one

week and then powdered (sieved by 80 mesh size). The powder is used for further analysis.

Following steps were used for phytochemical analysis:

Accurately weighed 20gm of the plant powder material packed in whatmann filter paper then

kept in Soxhlet apparatus for continuous extraction for 12 hrs. The mixture of methanol and

water 500 ml in volume ratio 4:1 was used as extract ant. Extract get cooled and filtere through

Whatmann filter paper number 41 into a dry, preweighed beaker.

1. The residue was extracted with 500 cc of (5 x 100 cc) of ethyl acetate and filtered into a

dry preweighed beaker. The residue obtained after filtration comprised plant fibers.

Weight of the residue was noted down and percent crude fiber was calculated.

2. The filterate obtained from (2) get evaporate to dryness using water bath maintained at

450 C ± 5 0C after evaporation of EtOAc the beaker get allow to cool at room temperature

in dessicator. Afterward cooling weight of beaker containing the residue was noted down.

The residue obtained was reconstituted in EtOAc to obtain a final concentration of

10mg/cc. These extracts get filtere by using Whatmann filterpaper number 41. This

filtrate is known as neutral extract. It consists of fats and waxes.

3. The filtrate obtained from (1) was evaporated to approximately 1/10th its volume by

heating in a water bath maintained at a temp less than 700C. It was acidified with 2M

H2SO4. The acidified filtrate was extracted using 150cc (3 x 50 cc) chloroform using

separating funnel. Chloroform layer get transferred to a dry preweighed beaker. The

chloroform layers evaporate to dryness using waterbath maintained at 450C ±5 0C.

4. After evaporation of chloroform the beaker get allow to cool at room temperature in

dessicator, after cooling weight of this beaker containing the residue was noted down.

The residue obtained was reconstituted in chloroform to obtain a final conc. of 10mg/cc.

These extracts getfiltere through Whatmann filter paper number 41. The filtrate obtained

was the moderately polar extract, which consists of terpenoids.

5. The aqueous layer obtained from (4) was basified to pH=10 with 2M NaOH. It was

further extracted with 120 cc (2 x 60cc) chloroform: method in volume ratio 3:1 followed

by extraction with 80 cc (2 x 40 cc) chloroform using separating funnel. Aqueous basic

layer get transferred to a dry preweighed beaker. The aqueous basic layer get evaporate to

dryness with the help of water bath maintained at 700C. After evaporation of the solvent

the beaker was allowed to cool at room temp in a dessicator. After cooling, the weight of

the beaker containing the residue was noted down. The residue obtained was

reconstituted in methanol to obtain a conc. of 10 mg/ cc. Extract is filtere through

Whatmann filter paper number 41. Methanol extract obtained the polar extract. The polar

extract consists of N. oxidesand quaternary alkaloids.

6. The organic layer (chloroform and methanol) was transferred to a dry and preweighed

beaker on a water bath maintained at 45 C ±5 0C. After evaporation of solvent the beaker is

allow to cool at room temp in adequate. After cooling, weight of beaker containing residue

was noted down. The residue obtained was reconstituted in chloroform to obtain final

concentration of 10 mg/cc. Extract then filtere through whatman filter paper number 41.

Chloroform extract constituted the basic extract, comprising of alkaloids.

7. Two grams of dried leaf powder get extracted with 100cc MeOH (4 x 25 cc) in a dry

stoppered conical flask. The extract was filtered into a dry preweighed beaker. The

filtrate obtained is evaporating to dryness with the help of water bath maintained at 700C.

After evaporation, the beaker was allowed to cool at a room temperature in a dessicator.

8. Two grams of dried leaf powder was extracted with 100cc EtOH (4 x 25 cc) in a dry

stoppered conical flask. The extract was filtered into a dry preweighed beaker. The

filtrate obtained get evaporate to dryness using water bath maintained at 700C. After

evaporation, the beaker was allowed to cool at a room temperature in a dessicator. After

cooling, the weight of the beaker containing the residue was noted down.

The schematic representation of the phytochemical analysis can be represented as

Follows:

Fig. III-3: Schematic representation of Phytochemical Analysis

Phytochemical Analysis20 gm Plant Powder + 500 cc MeOH + Water (4:1)

Soxhlet Extraction

Residue + 500cc EtOAc

Soxhlet Extraction

Residue

Evaporation 60 5ºC

Plant Fibers

Filtrate

Evaporation at45 5ºC

Fats and Waxes

FiltrateEvaporation to 1/10th Volume below70 ºC

Acidified by 2MH2SO4SimpleExtraction with 75 cc Chloroform

Chloroform Layer

Evaporation toDryness 45 5ºC

Terpenoids and Phenolics

Aqueous layer (Acid Layer)

Aqueous Layer

Evaporation at 70 ºC

Quaternary alkaloids and N oxides

Organic LayerChloroform and MeOHEvaporation at 45 5ºC

Alkaloids

Basified by 2M NaOH + 60cc(Chloroform: MeOH) 3:1+40ccChloroform

CALCULATIONS FOR PHYTOCHEMICAL ANALYSIS

(10gm powder + 200ml methanol+50ml water)

a) Fats and waxes:-

(M) wt. of empty beake =103.03 gm

(M1) wt. of beaker +residue =103.22gm

(M2) wt. of powder =20gm

(M1 - M)% Fats and waxes content = -------------------- x 100

M 2(103.22– 103.03)

% Fats and waxes content = ----------------------- x 10020

= 0.95%

b) Terpenoids and Phenols:-

(M) Wt.of empty beaker =98.34gm

(M1) wt. of beaker +residue=99.73gm

(M2) wt. of powder =20 gm

(M1 - M)% Terpenoids and Phenolic content = -------------------- x 100

M 2(99.73 – 98.34)

% Terpenoids and Phenolic content = -------------------- x 10020

= 6.95%

c) Q. Alkaloids & N-oxides:-

(M) Wt.of empty beaker = 98.2gm

(M1) wt. of beaker +residue = 98.91gm

(M2) wt. of powder = 20 gm

(M1 - M)% Q. Alkaloids & N-oxides content = -------------------- x 100

M 2(98.91-98.2)

% Q. Alkaloids & N-oxide content = -------------------- x 10020

= 3.55%

d) Alkaloids:-

(M) wt. of empty beaker = 103.20gm

(M1) wt. of beaker +residue =103.73gm

(M2) wt. of powder = 20 gm

(M1 - M)% Alkaloids content = -------------------- x 100

M 2

(103.73 – 103.20)% Alkaloids content = -------------------- x 100

20

= 2.65%

e) Fibers:-

(M1) Wt of empty beaker = 90.16 gm

(M2) Wt of empty beaker +fiber = 107.34gm

(M) Wt of powder = 20 gm

(M1 - M)% fibers content = -------------------- x 100

M 2

(107.34-90.16)% Fibers content = -------------------- x 100

20

=85.9%

Table III-10: Phytochemical analysis of Abutilon Indicum

Chart III-4: Pie graph with phytochemical analysis of Abutilon Indicum

1 Terpenoids &Phenolics

7%

5 Fibers.86%

Sr. No. PARAMETERS % COMPOSITION

1 Terpenoids &Phenolic 6.95

2 Basic extract(Most Alkaloids), 2.65

3Polar extract (Q. Alkaloids & N-

oxides)3.55

4 Neutral Extract (fats and waxes) 0.95

5 Fibers. 85.9

Table III-10: Phytochemical analysis of Abutilon Indicum

Chart III-4: Pie graph with phytochemical analysis of Abutilon Indicum

1 Terpenoids &Phenolics

7%

2 Basic extract(MostAlkaloids),

3%

3 Polar extract (Q.Alkaloids & N-

oxides)3%

4 Neutral Extract(fats and waxes)

1%

Phytochemical Analysis

Sr. No. PARAMETERS % COMPOSITION

1 Terpenoids &Phenolic 6.95

2 Basic extract(Most Alkaloids), 2.65

3Polar extract (Q. Alkaloids & N-

oxides)3.55

4 Neutral Extract (fats and waxes) 0.95

5 Fibers. 85.9

Table III-10: Phytochemical analysis of Abutilon Indicum

Chart III-4: Pie graph with phytochemical analysis of Abutilon Indicum

2 Basic extract(MostAlkaloids),

3%

3 Polar extract (Q.Alkaloids & N-

oxides)3%

4 Neutral Extract(fats and waxes)

1%

Sr. No. PARAMETERS % COMPOSITION

1 Terpenoids &Phenolic 6.95

2 Basic extract(Most Alkaloids), 2.65

3Polar extract (Q. Alkaloids & N-

oxides)3.55

4 Neutral Extract (fats and waxes) 0.95

5 Fibers. 85.9