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CHAPTER 5

PHYTOCHEMISTRY

5.1 INTRODUCTION

The whole plant or organism serves as an active laboratory for the production of

natural products from primary metabolites such as proteins, amino acids, carbohydrates, fats

and oils, which are mostly obtained from food items. The primary metabolites are basic

biological molecules also called biochemicals, which are functional compounds found

virtually in all plants and organisms. Secondary metabolites are varieties of simple to

sophisticated bizarre molecules, also called natural products. They are fascinating chemical

molecules, very useful and of great importance in nature, as well as highly diversified in

structures, properties, uses, chemistry etc. These varied properties and characters emerge

from their biological generation, production and formation from basic primary metabolite

sources and origin. Natural products are in restricted taxonomic groups and species of

organisms. They are from secondary metabolic processes and express individualities of

organisms [123].

5.1.1 Phytoconstituents

Phytochemicals are chemicals derived from plants and the term is often used to

describe the large number of secondary metabolites found in plants. Phytochemical

compounds usually exert peculiar, unique and specific active physiological effects

responsible for their therapeutic and pharmacological functions. Activities of such

naturally occurring compounds are generally responsible for changes, which are utilized to

satisfy human being‘s desires. These complex substances of diverse nature occur mostly in

plant based foods; they are in very small amounts in gms or mg or µg/Kg of samples. They

do not add to body calorie and are numerous in types. These phytochemicals are applied

mostly for preventive and healing purposes.

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5.1.2 Functions of phytoconstituents

Bioactive compounds in plants can be defined as secondary plant metabolites

eliciting pharmacological or toxicological effects in man and animals. Secondary

metabolites are produced within the plants besides the primary biosynthetic and metabolic

routes for compounds associated with plant growth and development and are regarded as

products of biochemical ―side tracks‖ in the plant cells and not needed for the daily

functioning of the plant. Several of them are found to hold various types of important

functions in the living plants such as protection, attraction or signaling. Most species of

plants seem to be capable of producing such compounds [124].

Phylogenetically, the secondary bioactive compounds in plants appear to be

randomly synthesized. Several of them are found to hold important functions in the living

plants. For example, flavonoids protect against free radicals generated during

photosynthesis. Terpenoids attract pollinators or seed dispersers or inhibit competing plants.

Alkaloids usually ward off herbivore animals or insect attacks (phytoalexins). Other

secondary metabolites function as cellular signaling molecules or have other functions in the

plants. Those plants producing bioactive compounds seem to be the rule rather than the

exception. Thus, most plants even common food and feed plants are capable of producing

such compounds. However, the typical poisonous or medicinal plants contain higher

concentrations of more potent bioactive compounds than food and feed plants [125].

5.1.3 Extraction and isolation of phytoconstituents

Phytochemical methods mainly involve extractions, purifications and isolations of

active compounds from the plants. Preliminary tests and screenings on plant extracts are

faster and easily done following standard procedures and methods in manuals and literature.

They detect the presence and amount of basic phytoconstituents like terpenoids, alkaloids,

flavonoids, saponin, glycosides, steroids, tannins, phlobatannin and anthraquinones to

mention few. Phytochemical screening assay is a simple, quick, and inexpensive procedure

that gives the researcher a quick answer to the various types of phytochemicals in a mixture

and an important tool in bioactive compound analyses [126].

Phytochemicals are active metabolites that necessarily require extraction and

isolation from their natural sources with many unwanted materials. The phytochemical can

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come singly or as a mixture of important substances to form active principle

responsible for its activity (synergetic activity).When singly active, the processes of their se

parationis of great practical advantages, which in many cases the isolated phytochemical

have better and higher activity. Once preliminary separations and detections have confirmed

the presence of active secondary metabolites, their characterizations as they are separated

follows, chromatographic techniques are utilized in separations and purifications to isolate

bioactive constituents based on polarity or other gradient factors. The first step in the

process of obtaining secondary metabolites from biogenic material is to release them

from the matrix by means of extraction [127].

Due to the complex composition of the material and the minute amounts of some of

the constituents present, the choice of extraction method is of great importance. Obviously,

an incorrect choice will cause the entire isolation process to fail if some or all of the desired

components of the material cannot be released satisfactorily from the matrix. The initial

crude extract is usually a more or less complex mixture. Quite often there are certain target

compounds or compound groups of interest. A logical next step in the isolation process is to

separate the target compounds from the crude extract. This can be achieved e.g. by liquid-

liquid partition or by some low-resolution chromatographic isolation. The aim of these step

is to concentrate the desired components and make the sample amenable to the final

purification steps. The third step in the isolation process usually involves some type of high-

resolution method to separate the compounds of interest from the other compounds still

remaining in the extract. As the undesired components of the mixture are likely to bear some

resemblance to the target compounds, this stage usually involves optimization of the

separation method to achieve sufficient resolution in the final preparative isolation. Often

the final isolation step involves liquid chromatography, especially HPLC or TLC, although

other separation methods have been successfully applied [128].

5.1.4 Characterization of phytoconstituents

The isolated compound is characterized by spectroscopic methods. The four basic

types of spectroscopy utilized in the characterizations of purified natural product

compounds. They are ultraviolet (UV), infrared (IR), mass-spectroscopy (MS) and nuclear

magnetic resonance (NMR) techniques. MS is an instrumental technique, while the other

three utilizes different parts of the broad electromagnetic radiation spectrum. UV

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spectroscopy discovered and utilized in 1930s gives detailed information on detecting the

presence of conjugation in molecules and the extents of conjugation. By 1940s the infrared

(IR) region of electromagnetic radiation was utilized to detect different vibration frequencies

of different chemical bonds present in the molecule. Combination of these two types of

spectroscopy [UV & IR] gave information about the functional groups present in the

molecule. MS was introduced a decade after by 1950s, involving three important steps:

Ionization and vaporization; Separation of ions by m/z; and detections. The analytical

technique provides information which determines the molecular ion. Compounds are ionized

for analysis and also fragments are produced useful for structural characterizations. Almost

all compounds can be analyzed by MS, but modes of ionization and type of instruments

determine the results.

NMR is a type of absorption chromatography which reveals connectivity of

nuclei in the metabolite. Superficially and most common, 1H and

13C NMR [1D] techniques

[earlier used] are unambiguously and widely utilized in elucidation of structures of naturally

occurring metabolites usually isolated and purified from their natural sources. Recently the

2D and 3D-NMR are utilized [as in use of HSCQ, TOCSY, COSY, HMBC and NOESY

etc.,]. Fundamentally NMR reveal information on types of chemical environments in the

metabolite from the frequency absorption chemical shift values; (b)number of protons in

each type of environment from integral values; (c) details on type of nuclei/protons on

adjacent and neighboring positions in the metabolite, giving details on the stereochemistry

and 3-dimentional structure of metabolites.

The theory of NMR is based on magnetic atomic nuclei with net nuclear spin ‗I‘,

capable of having (2I+1) patterns of orientations. Such NMR-active atomic nuclei have odd

atomic number and/ or odd mass number. An internal standard, usually TMS [Si (CH)3] with

equivalent twelve protons and arbitrarily have absorption at d0, is used in calibrating NMR

spectrum for easy interpretations and evaluation of resonances and absorptions. Most used

unit is δ (delta), the other unit is T (tau). Relationship between both is expressed thus: δ =10-

T or T=10-δ.

.

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5.1.5 Finger print analysis

Fingerprint in essence is chemoprofiling, which means establishing a characteristic

chemical pattern for the material or its cut or fraction or extract, which help in its

identification. A chromatographic fingerprint is commonly applied method for qualitative

and quantitative analysis of low-molecular mass compounds from complex biological,

pharmaceutical and environmental samples. Instrumental chromatographic methods like GC

and HPLC are extensively replaced by TLC [129]. ‗‗The colorful picture-like TLC image

manifested vividly the specific pattern of the given species that cannot be described properly

by words‘‘ [130] the classical fingerprint by TLC is done by visual inspection of the

chromatogram and comparison to a reference standard. The analyte and reference standard

are chromatographed together on the same plate under the optimized chromatographic

conditions. Comparison can also be made to the results obtained from other plates or their

images (book, electronic library, etc.) or to a verbal description of the expected results or

both. The advantage of TLC technique is reflected, when the identity of the analyte is not

known or uncertain and in cases when reference standards are not available. An important

characteristic of HPTLC fingerprint analysis is the large number of samples that can be

analyzed in parallel. Also, it could be used to establish proper extraction parameters, to

standardize and normalize extracts and to detect any changes or degradation in the material

during formulation, i.e. to monitor the production of extracts and finished products. It is

important to preserve the composition of the raw material during process development

[131].

High performance thin layer chromatography, as a method of chemical fingerprinting, is

a suitable method for rapid assessment of the authenticity of the food products as a chemical

composite. As such, the analysis will enable to distinguish the presence of aberrant chemical

components from adulterants, as well as favorable or unfavorable chemical changes arising

from varied treatments or storage of the product [132]. HPTLC is; also, useful in

determination of constituent of different pharmaceutical dosage forms in the presence of

their degradation products and additives and it is sometimes the only technique of choice for

the determination of drugs in mixtures due to its high resolution power [130, 133]. For

conventional identification of pharmaceuticals, HPTLC has been used in almost all

Pharmacopoeias worldwide.

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Gas chromatography (GC) and GC-MS with high specificity, high sensitivity,

stability and small amount of sample characteristics, are unanimously accepted as the

method for the analysis of volatile constituents of herbal medicine [134]. GC can detect

almost all the volatile chemical compounds with high sensitivity, which is especially true for

the usual FID detection and GC–MS. Moreover, the high selectivity of capillary columns

enables separation of many volatile compounds simultaneously within very short time.

However, it is not convenient for the analysis of samples of polar, non-volatile and heat-

labile ingredients [135]. The samples must be gasified by the tedious sample pretreatment

such as derivatization, but the ingredients in most herbal instances are high polar

compounds, which limits Gas chromatography‘s application in the chemical identification

and authentication of herbal medicines. To solve this problem and to expand the gas

chromatography in the identification of herbal medicine, Chinese University of Hong Kong

and Shanghai Innovative Research Center of Traditional Chinese Medicine combined to

firstly set up off-line pyrolysis- gas chromatography - mass spectrometry fingerprint method

to obtain the fingerprints of herbal medicines [136].

5.1.6 Biological fingerprint

Recently, biological fingerprinting analysis, as a method of screening the natural

samples for the presence of most active compounds, has been introduced. It was originally

developed with the use of HPLC, but Cies‘la et al. [137] applied this concept

in TLC. They constructed a ‗‗binary chromatographic fingerprint‘‘ combining chemical

and biological detection systems. In the former case, the plates were sprayed with

vanillin reagent; while in the case of biological fingerprint methanolic solution of a stable

free DPPH radical was applied [138]. The biological detection in liquid chromatography

gives an opportunity for comprehensive herbal sample analysis that is being able to

distinguish the bioactive compounds from among the set of chromatographic and

spectroscopic signals.

Due to the complexity of the composition of natural extracts, separating each

antioxidant compound and studying it individually is costly and inefficient, notwithstanding

the possible synergistic interactions among the antioxidant compounds in a mixture.

Therefore, it is advantageous for researchers to have a convenient method for the rapid

quantification of antioxidant effectiveness. The concept of coupling chromatographic

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fingerprints with biological finger printing analysis has gained much attention for the quality

control of plant extracts. Thin layer chromatography with post chromatographic

derivatization using a methanol solution of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) can be a

valuable tool in such analyses [139,140]. However, the identification of the free radical-

scavenging activity of each compound in a complex mixture is a difficult task. Consolidating

chromatographic separation and the determination of antiradical activity allow analysts to

evaluate and to quantify the effect of the free radical scavenging activity of the herbal

extracts using HPTLC test with post chromatographic derivatization.

5.1.7 Automated Multiple Development (AMD)

Automated multiple development (AMD) is performed using a specially designed

apparatus that permits stepwise gradient elution on a TLC plate (Figure 5.1a). The method

was developed in the mid-eighties and has some significant advantages over traditional

capillary TLC [141]. The development is carried out in a controlled atmosphere thus

enabling the achievement of more reproducible results. The plate is dried in a vacuum

between successive runs and the developments are carried out under a nitrogen atmosphere,

oxidation of the analytes can be avoided during the chromatographic separation. Moreover,

as stated earlier, AMD permits gradient elution [142]. Multiple developments with an

incremental increase in the development length and a decreasing solvent strength gradient is

the basis of separation by automated multiple developments (AMDs).

AMD is a technique that uses repeated development of HPTLC plates with

decreasing solvent strength on the increasing distance. After each development, the plate is

carefully dried by vacuum. The development starts with the most polar solvent (for the

shortest development distance) and concludes with the least polar solvent (for the longest

migration distance) [142]. Gradient development with linear eluotropic profile leads to a

band reconcentration improving the separation. A successful separation depends mainly on

the choice of the solvent components, optimization of the shape of the gradient, the stepwise

movement of the elution front, and the repeated developments [143]. AMD is highly

recommended in case of samples containing substances of wide polarity or those being

structural analogs. For the best resolution of constituents spanning wide polarity range, steep

gradient is especially beneficial, while shallow gradient with small increases of developing

distance provides good results in case of the analogs [144].

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AMD provides a more certain approach to optimize a gradient separation, when

compared to other non-automated TLC gradient methods [145]. In case of non-automated

gradient elution, the formation of multiple zones of different solvent strength in the direction

of chromatography can be observed as a result of solvent demixing [145]. When compared

to manual methods, AMD provides a high degree of gradient reproducibility. One of the

disadvantages of AMD is the possibility of losing volatile as well as less volatile

constituents present in the analyzed samples, during repetitive drying under vacuum.

5.1.8 Validation

Specificity is the ability to assess unequivocally the analyte in the presence of

component which may be expected to be present. Typically these might include impurity,

degradants, matrix, etc. Lack of specificity of an individual analytical procedure may be

compensated by other supporting analytical procedure(s).

This definition has the following implications: Identification ensures the identity of

an analyte. Purity Test ensures that, all the analytical procedures performed allow an

accurate statement of the content of impurities of an analyte, i.e. related substances test,

heavy metals, residual solvents content, etc. Assay (content or potency) provides an exact

result, which allows an accurate statement on the content or potency of the analyte in a

sample.

(a) Accuracy

The accuracy of an analytical procedure expresses the closeness of agreement

between the value which is accepted either as a conventional true value or an accepted

reference value and the value found. This is sometimes termed trueness.

(b) Precision

The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple sampling of the

same homogeneous sample under the prescribed conditions. Precision may be considered at

three levels: repeatability, intermediate precision and reproducibility. Precision should be

investigated using homogeneous, authentic samples. However, if it is not possible to obtain

a homogeneous sample it may be investigated using artificially prepared samples or a

sample solution. The precision of an analytical procedure is usually expressed as the

variance, standard deviation or coefficient of variation of a series of measurements.

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(c) Repeatability

Repeatability expresses the precision, under the same operating conditions over a

short interval of time. Repeatability is also termed intra-assay precision.

(d) Intermediate precision

Intermediate precision expresses within-laboratory variations: different days,

different analysts, different equipments, etc.

(e) Reproducibility

Reproducibility expresses the precision between laboratories (collaborative studies,

usually applied for standardization of methodology).

(f) Detection Limit

The detection limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be detected but not necessarily quantitated as an exact value.

(g) Quantitation Limit

The quantitation limit of an individual analytical procedure is the lowest amount of

analyte in a sample which can be quantitatively determined with suitable precision and

accuracy. The quantitation limit is a parameter of quantitative assays for low levels of

compounds in sample matrices, and is used particularly for the determination of impurities

and/or degradation products.

(h) Linearity

The linearity of an analytical procedure is its ability (within a given range) to obtain

test results, which are directly proportional to the concentration (amount) of analyte in the

sample.

(i) Range

The range of an analytical procedure is the interval between the upper and lower

concentration (amounts) of analyte in the sample (including these concentrations) for which

it has been demonstrated that the analytical procedure has a suitable level of precision,

accuracy and linearity.

(j) Robustness

The robustness of an analytical procedure is a measure of its capacity to remain

unaffected by small, but deliberate variations in method parameters and provides an

indication of its reliability during normal usage.

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5.2 MATERIALS AND METHODS

5.2.1 Preliminary phytochemical analysis

Phytochemical examinations were carried out for all the extracts as per the standard methods

[145].

(a) Detection of alkaloids

Each 0.5gm of extract was dissolved in 5ml of 1N HCl and filtered.

(i) Mayer’s Test: Filtrates were treated with Mayer‘s reagent (Potassium Mercuric

Iodide). Formation of a yellow coloured precipitate indicates the presence of

alkaloids.

(ii) Wagner’s Test: Filtrates were treated with Wagner‘s reagent (Iodine in

Potassium Iodide). Formation of brown/reddish precipitate indicates the presence

of alkaloids.

(iii) Dragendroff’s Test: Filtrates were treated with Dragendroff‘s reagent (solution

of Potassium Bismuth Iodide). Formation of red precipitate indicates the

presence of alkaloids.

(iv) Hager’s Test: Filtrates were treated with Hager‘s reagent (saturated picric acid

solution). Presence of alkaloids was confirmed by the formation of yellow

coloured precipitate.

(b) Detection of sugars

0.5gm of extracts were dissolved individually in 10 ml distilled water and filtered.

The filtrates were used to test for the presence of carbohydrates.

(i) Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol

solution in a test tube. Formation of the violet ring at the junction indicates the

presence of monosacharides.

(ii) Benedict’s test: Filtrates were treated with Benedict‘s reagent and heated gently.

Orange red precipitate indicates the presence of reducing sugars.

(iii) Fehling’s Test: Filtrates were hydrolyzed with dil. HCl, neutralized with alkali

and heated with Fehling‘s A & B solutions. Formation of red precipitate indicates

the presence of reducing sugars.

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(c) Detection of glycosides

0.5gm of the extract was Hydrolyzed with ml of 2N HCl on a water bath, filtered

and the filtrates were subjected to test for glycosides.

(i) Modified Borntrager’s Test: Filtrates were treated with Ferric Chloride solution

and immersed in boiling water for about 5 minutes. The mixture was cooled and

extracted with equal volumes of benzene. The benzene layer was separated and

treated with ammonia solution. Formation of rose-pink colour in the ammoniacal

layer indicates the presence of anthranol glycosides.

(ii) Legal’s Test: Filtrates were treated with sodium nitroprusside in pyridine and

sodium hydroxide. Formation of pink to blood red colour indicates the presence

of cardiac glycosides.

(d) Detection of saponins

(i) Froth Test: 0.5gm of extracts was diluted with distilled water to 20ml and this

was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam

indicates the presence of saponins.

(ii) Foam Test: 0.5 gm of extract was shaken with 2 ml of water. If foam produced

persists for ten minutes it indicates the presence of saponins.

(e) Detection of phytosterols

(i) Salkowski’s Test: 0.5gm of extracts were treated with chloroform and filtered.

The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to

stand. Appearance of golden yellow colour indicates the presence of triterpenes.

(ii) Liebermann Burchard test: 0.5gm of extracts were treated with chloroform and

filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled.

Concentrated Sulphuric acid was added. Formation of brown ring at the junction

indicates the presence of phytosterols.

(f) Detection of phenols

(i) Ferric Chloride Test: 0.1gm of extracts were treated with 3-4 drops of ferric

chloride solution. Formation of bluish black colour indicates the presence of phenols.

(g) Detection of tannins

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(i) Gelatin Test: To 0.1gm extract, 1% gelatin solution containing sodium

chloride was added. Formation of white precipitate indicates the presence of

tannins.

(h) Detection of flavonoids

(i) Alkaline Reagent Test: 0.1gm extract were treated with few drops of sodium

hydroxide solution. Formation of intense yellow colour, which becomes colourless

on addition of dilute acid, indicates the presence of flavonoids.

(ii) Lead acetate Test: 0.1gm extract were treated with few drops of lead acetate

solution. Formation of yellow colour precipitate indicates the presence of

Flavonoids.

(i) Detection of proteins and amino acids

(i) Xanthoproteic Test: 0.1gm of extracts were treated with few drops of conc.

Nitric acid. Formation of yellow colour indicates the presence of proteins.

(ii) Ninhydrin Test: To 0.1gm of extracts, 0.25% w/v Ninhydrin reagent was added

and boiled for few minutes. Formation of blue colour indicates the presence of amino

acid.

(j) Detection of diterpenes

(i) Copper acetate Test: 0.1gm of extracts were dissolved in water and treated with

3-4 drops of copper acetate solution. Formation of emerald green colour indicates the

presence of diterpenes.

5.2.2 Determination of Bioactive Contents

(a) Determination of total phenolic content (96-Well plate method)

Total phenolic content (TPC) was determined according to the method developed by

Zhang et al., 2006 [146]. Briefly 100µL of Folin-Ciocalteau reagent (1N) was added to the

20 µL standard Gallic acid (100, 50, 25, 12.5, and 6.25µg/ ml)/ samples (1mg/ml) in the 96

well plate and kept for 6 min, followed by the addition of 80 µL of sodium carbonate. The

solutions were mixed and left in dark for 90 min. The absorbance was measured at 765 nm

with spectrophotometric micro plate reader (set to shake for 60s before reading). Each stand

ard and sample solution was analyzed in triplicate and the later was assayed against sample

control. Total phenolic content was expressed as mg gallic acid/100g of the dry weight of

the extract.

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(b) Determination of total flavonoid content (96-Well plate method)

Total flavonoid content (TFC) was determined according to the method described by

Herald et al., 2012 [147]. In 96 well plate the required concentration of the standard/

samples were mixed with sodium nitrite and aluminium chloride and plates were kept aside

for 30 min and the absorbance was measured at 510 nm. All samples and standards were

measured against a reagent blank. Total flavonoid content was expressed as mg quercetin

equivalent/100g of dry weight of the extract.

(c) Determination of total sterol content (UV method)

Total sterol content (TSC) was determined according to Liebermann-Burchard (LB)

colorimetric method [148] with minor modifications, using cholesterol as standard. The LB

colour reagent was freshly prepared and added to the extracts and kept at room temperature

for 13 min, by adding 50 µL concentrated sulphuric acid to 2 mL acetic anhydride. Then,

1ml extract in chloroform was added to the LB colour reagent, stirred for 1min and kept at

room temperature for 13 min. The absorbance of the mixture was measured using UV

Spectrophotometer at 650 nm. Results were expressed as mg cholesterol equivalent/100g of

dry weight of the extract.

(d) Determination of total saponin content (UV method)

Total saponin content (TSAC) was determined based on the method described by Xu

and Chang, 2009 [149].To 250 µL of the standard solution or sample solution, 250 µL of

80% methanol was added, followed by 250 µL of vanillin reagent and 2.5mL of 72% (v/v)

sulphuric acid was slowly added along the sides of the test tube. The solution was mixed

well and the tubes were placed in a water bath at 60°C for 10 min, the tubes were cooled in

ice-cold water for 3 to 4 min and then the absorbance was measured at 544 nm against the

reagent blank. The results are expressed as mg diosgenin equivalent /100g of dry weight of

the extract.

(e) Determination of total triterpenoid content (UV method)

The total triterpenoid content (TTC) of the sample was determined according to the

method of Ni et al [150], with some modifications, using urosolic acid as standard. Briefly,

0.3mL of each extracts were transferred to a tube and heated to dryness in a water bath at

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100°C, then 0.50 mL vanillin-acetic acid solution (5:95, w/v) and 0.8 mL perchloric

acid were added and then incubated in a water bath at 60°C for 15 min. Cooled in an ice

water bath and 5mL of acetic acid was added slowly and placed in a room temperature for

15 min. With a blank solution as reference, the absorbance was measured at 548 nm. Results

were expressed as mg urosolic acid equivalent/100g of dry weight of the extract.

5.2.3 Fingerprint analysis by HPTLC-AMD

HPTLC was performed on aluminium sheets coated with Silica gel 60 F, 20 cm

×10cm (Merck,254 Darmstadt, Germany). Plates were activated before use by heating in an

oven for 30 min at 110°C. 2, 4 and 8µL of the hydro-alcoholic extracts of MVL, MVS,

MVR, MHL, MHS and MHR were sprayed with compressed air, as 8 mm narrow bands

using a 100µL syringe with a Linomat 5 semi-automatic sample applicator (Camag,

Muttenz, Switzerland), 8 mm from the lower edge, with the 10 mm distance from each side

and track distance of 7 mm, i.e. 18 applications per plate. HPTLC plates were developed in

Automated Multiple Development Chamber (AMD2, Camag) with six steps gradient elution

method. Images of plates were captured using a TLC-Visualizer (Camag, Muttenz,

Switzerland) with a 12 bit camera (Camag) under UV light 254 nm, 366nm before

derivatization and at 540 nm, after derivatization with anisaldehyde sulphuric acid.

winCATS planar chromatography manager software was used for quantitative evaluation

of plates and to transform images into chromatogram.

5.2.4 Fingerprint analysis by GC-MS

GC-MS analysis for phytoconstituents of hydro-alcoholic (70%) extracts was

performed with Bruker GC-MS SCION TQ equipped with a Finnigan Trace DSQ and an

electron impact (EI) ion source. The analytes were separated on a BR-5MS capillary column

(30 m×250 μm×0.25 μm film thickness; Agilent, USA) coated with phenyl arylene polymer.

Column temperature program: from 50°C (3min isothermal) increased to 110°C at 7°C (5

min isothermal) and 200°C at 8°C ( 2min isothermal), increased to 280°C (10 min

isothermal). Carrier gas was high purity helium (flow rate 1mL min-1

) the instrument was

operated in electron impact (EI) mode at 70 eV. Analysis was performed in full scan mode

scanning in the mass range of 40- 700m/z at 1 scan S-1

.

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The injection was performed by split mode with a split ratio of 10: 1. Solvent delay

time was set for 3 min for all samples generated by different methods. MSWS V 8.0

workstation was used to process data. Interpretation on mass spectrum of GC-MS was done

using the database of in-built libraries like NIST 8 (National Institute of Standards and

Technology) and WILEY 9 having more than 62,000 patterns.

5.2.5 Biological fingerprinting by DPPH

The plates were developed by the optimized AMD method. After development, the

plates were air-dried for 15 min and immersed in the DPPH reagent (0.05 % [2, 2-diphenyl-

1-(2, 4, 6-trinitrophenyl) hydrazyl radical] DPPH in methanol) for 1 sec and then dried for 1

min at room temperature in the fume hood. The dried plates were wrapped in an aluminium

foil and kept in dark for 30 min. The antiradical activity of each component was estimated

from the intensity of disappearance of the violet/purple background of the plate and was

quantified by densitometric scanning at 517 nm. Free radical scavenging zones were readily

identified as yellow areas against a light violet/purple background.

5.2.6 Isolation and Characterization of Phytoconstituents

Hydro-alcoholic extracts of all the parts were dissolved in methanol and analyzed

chemically, to determine the presence of different chemical constituents, MHL, MHS, and

MHR extracts were selected for column chromatography. Standard procedure of column

chromatography was followed for all the selected extracts. Briefly, viscous dark brown

extract was adsorbed on silica gel (60-120 mesh) for column, after being dissolved in little

quantity of methanol for preparation of slurry.

The slurry was air-dried and chromatographed over silica gel column packed in

hexane, the column was eluted successively with hexane, ethylacetate and methanol and

three fractions were obtained. The ethylacetate fraction was further subjected to column

chromatography and successively eluted with increasing polarity with various ratios of

hexane and ethylacetate followed by different ratios of ethylacetate and methanol;

various fractions were collected separately and matched by TLC to check homogeneity. Frac

tions (having same Rf values) were combined and crystalized. The isolated compounds were

recrystallized to get the pure compound (s). Spectral data (from IR, MS, NMR) were

obtained to characterize and to elucidate the structure of isolated compounds.

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5.2.7 Quantification of Stigmasterol and Quercetin by HPTLC

5.2.7.1 Sample preparation

All the chemicals, including solvents, were of analytical grade from E.

merck, India.The HPTLC plates Si 60F254 (20cmX10cm) were purchased from Merck

(India). Standards Quercetin (99% purity), Stigmasterol (99% purity) were purchased from

Sigma (New Delhi, India). 100 mg/ml of hydro-alcoholic extracts of selected parts of

M.vaginalis and M.hastata were taken for analysis.

The extracts were filtered and vacuum dried at 45ºC.The dried extracts were

separately redissolved in 1ml of methanol and sample of varying concentration (2-6µl) for

Quercetin and (5-30µl) for Stigmasterol were spotted for quantification. 1 mg of standards

(Quercetin, Stigmasterol) were prepared separately in 1ml of methanol and different

amounts of (5000-10000ng) Quercetin and (1000-6000ng) Stigmasterol were loaded on

HPTLC plate to get the calibration curve.

HPTLC was performed to quantify the presence of Stigmasterol and Quercetin in the

hydro-alcoholic extracts. The method was validated according to the current International

Conference on Harmonization (ICH) guidelines. The method was assessed based on

linearity, specificity, precision, limit of detection (LOD) and limit of quantification (LOQ).

The crude extracts were re-dissolved in methanol, filtered and transferred quantitatively to a

10 ml volumetric flask, adjusted the volume with methanol and shaken to mix thoroughly.

Calibration curve was established using 5 analyte concentrations of the TLC standard

representing µg of Stigmasterol and Quercetin respectively. Standard and sample solutions

were applied in the form of bands on pre-coated HPTLC silica gel plates 60 F-254

(10×10cm with 250µm thickness) by means of Linomat V automated spray-on band

applicator. The mobile phase consisted of Chloroform: Methanol (10ml) (8:2v/v) and

Chloroform: Methanol: Formic acid (8.5ml), (7:1:0.5 v/v) for Stigmasterol and Quercetin

respectively.

Ascending development of the plates was carried out in 10×10 cm Camag

HPTLC twin trough chamber saturated with mobile phase for 15min at room temperature

Plates were developed to a distance of 7cm beyond the origin. Development time was 10

min. After development, the plates were air dried for 5min and derivatized with

84

anisaldehyde-sulphuric acid reagent for Stigmasterol, heated at 105°C for 5 min and without

derivatization for Quercetin. Densitometric scanning was performed on Camag TLC scanner

III in the reflectance mode at 540nm for Stigmasterol and 366nm for Quercetin. Slit

dimension was kept 6×0.1mm in absorbance mode using tungsten lamp. The entire

programme was operated using winCATS planar chromatography manager.

5.2.8 Quantification of Stigmasterol by RP- HPLC

Quantification of stigmasterol in the hydro-alcoholic extracts of MVL, MVS, MVR,

MHL, MHS and MHR were performed through reverse-phase high performance liquid

chromatography system (RP-HPLC). The HPLC system consist of Shimadzu (Shimadzu

corporation, Kyoto, Japan) binary HPLC pump, a prominence-7725i injection valve

(USA) with a sample loop of 20 μL, a UV- Visible dual wavelength detector and

the max-plot containing the peaks were obtained using Lab solutions software. A Phenomen

ex -Luna (Torrance, CA, USA) C18 column (250mm×4.6mm, 5µm particle size) was used as

the stationary phase. Isocratic mobile phase consisting of methanol: acetonitrile (30:70) at

1ml/min. The column was optimized and maintained at ambient temperature throughout

analysis and detection wavelength was set at 208 nm for Stigmasterol. HPLC grade

methanol and acetonitrile were procured from Merck (Mumbai, India) were used.

Stigmasterol was purchased from Sigma Chemical Co, St Louis, Mo, USA. Hydro-

alcoholic extracts were dissolved in methanol in the concentration of 1mg/ml and were

filtered through MILLEX FG (Millipore), 13mm, 0.2μM, non-sterile membrane sample

filter paper before injecting into system. Validation of the developed method was carried out

according to ICH guidelines.

5.2.9 Quantification of Quercetin by LC-MS

A Shimadzu LC-MS 2020 (Shimadzu, Japan) equipped with a binary solvent

delivery system, column compartment and photo diode array detector (PDA) was used for

the quantification and validation of Quercetin in the hydro-alcoholic extracts of Monochoria

vaginalis leaf and Monochoria hastata leaf. The chromatographic separation was performed

on Phenomenex C18 column (i.d. 250mm × 4.6 mm, 5µm) and the column oven temperature

was maintained at ambient temperature. Isocratic mobile phase consisting of Methanol:

Acetonitrile: Water (0.01% formic acid) (40:15:45).

85

The instrument was operated by switching electrospray ionization (ESI) source in

positive ion mode. High purity nitrogen was used as collision gas (flow rate 1.5L/min),

capillary voltage at 1.6kV and temperature of curved desolvation line (CDL) and heat block

at 250 and 300°C were used. All instrumentation data were collected and synchronized by

Lab solutions software (version 7.1) from Shimadzu.

5.2.10 Method Validation

This method was validated as per the ICH guidelines (1994, 1996 and 2005). The method

validation parameters checked were linearity, precision, accuracy and recovery, limit of

detection, limit of quantification, specificity, robustness and ruggedness. All measurements

were performed in triplicates.

(a) Calibration Curve and Linearity

The calibration were performed by analysis of working standard solutions of Quercetin

(5000 to 10000 ng), Stigmasterol (1000 to 6000ng) were spotted on precoated TLC plate,

using semiautomatic spotter under nitrogen stream. The TLC plates were developed, dried

by hot air and photometrically analyzed as described earlier. The calibration curves were

prepared by plotting peak area versus concentration (ng/spot) corresponding to each spot.

(b) Recovery

To determine the recovery, known concentrations of standards were added to a preanalyzed

sample of hydro alcoholic extracts. The spiked samples were then analyzed by the proposed

HPTLC method and the analysis was carried out in triplicate.

(c) Precision

A stock solution containing Quercetin and Stigmasterol compounds were prepared in

methanol and six 10µl (1000ng /spot) bands were applied and analyzed by the developed

method to determine instrument precision. Six different volumes of same concentration were

spotted on a plate and analyzed by the developed method to determine variation arising from

method itself. To evaluate intra-day precision, six samples at three different concentrations

(1000, 2000 and 3000 ng/ spot) for Quercetin and Stigmasterol were analyzed on the same

day. The interday precision was studied by comparing assays performed on three different

days.

86

(d) Limit of Detection and Limit of Quantification

Limit of detection (LOD) of an individual analytical procedure is the lowest amount

of analyte in a sample, which can be detected but not necessarily quantitated as an exact

value. LOD was calculated using the following formula,

LOD = 3.3 x Standard Deviation of the y-intercept/ Slope of calibration curve

Limit of quantification (LOQ) of an individual analytical procedure is the lowest

amount of analyte in a sample, which can be quantitatively determined with suitable

precision and accuracy. LOQ was calculated using the following formula,

LOQ = 10 x Standard Deviation of the y-intercept/Slope of calibration curve

(e) Specificity

The specificity of the method was ascertained by analyzing standard compound

Quercetin and Stigmasterol present in the hydro-alcoholic extracts.

(f) Method Specifications

Silica gel 60 F254 precoated plates (20x 10 cm) were used with Chloroform:

Methanol (10ml) (8:2v/v) and Chloroform: Methanol: Formic acid (8.5ml), (7:1:0.5 v/v) for

stigmasterol and quercetin respectively. Sample was spotted on precoated TLC plates by

using Linomat 5 applicator. Ascending mode was used for development of thin layer

chromatography. TLC plates were developing up to 70 mm and scanned in reflectance mode

at 540 nm for stigmasterol and fluorescence mode for quercetin at 366 nm. The contents of

Quercetin and Stigmasterol in the extracts were determined by comparing area of the

chromatogram of standard Quercetin and Stigmasterol.

5.3 RESULTS AND DISCUSSION

5.3.1 Phytochemical Evaluation

Preliminary phytochemical evaluation showed the presence of various

phytochemicals like flavonoids, polyphenols, saponins and glycosides in the hydro-alcoholic

extracts of all the parts (Table 5.1). The results of total phenolic content in the selected

extracts were given in Table 5.2. The content of total phenols in the extract expressed as

gallic acid equivalents (GAE) varied between 134.8 and 64.75mg/100g of dry extract. As

shown in the table, the pattern of variation in TFC was similar with TPC, with the highest

content of TFC in rootstock of both the plants and lowest in leaf extract. The content of total

87

flavonoids in the extract was expressed as quercetin equivalents (QE) varied between 98.5

and 27.4mg/100g of dry weight of the extract. The triterpenoid content of the extracts were

determined were given in the Table 5.2 equivalent to ursolic acid. The content of total

triterpenoids varied between 2137.2 and 1637.4mg/100g of dry weight. The total sterol

content of the extract was expressed as cholesterol equivalents. The total sterol content

varied between 429.5 and 214mg/g of dry weight of the extract. The total sterol content of

the extracts varied significantly. The highest content was found in rootstock followed by

stem and leaf of both the species. M.vaginalis contains higher sterol content than M.hastata.

The result of the total saponin content determination was given in Table 5.2. The contents of

total saponins in the extract were expressed as diosgenin equivalent (Ds). The saponin

content varied between 110.3 and 53.4 mg/100g of dried weight. Highest saponin content

was found in MHS (110.3) and the lowest in MVL (53.4).

Table 5.1 Preliminary phytochemical report of hydro-alcoholic extracts of M.hastata and

M.vaginalis

Phytochemical Tests Monochoria hastata Monochoria vaginalis

Leaf Stem Root Leaf Stem Root

Carbohydrates + + + + + +

Proteins and amino acids + + + + + +

Alkaloids - - + - - +

Glycosides + + + + + +

Flavonoids + + + + + +

Steroids + + + + + +

Phenols + + + + + +

Tannins + + + + + +

Saponins + + + + + +

88

5.3.2 Finger print analysis by HPTLC-AMD

Since the extracts showed a wide range of polarity, automated multiple develop-

ment was preferred. A simple, sensitive and reproducible HPTLC-AMD method for the

simultaneous fingerprint analysis of the hydro-alcoholic extract was developed. A six step

development programme with different solvent composition was optimized to separate the

maximum number of phyto-constituents on a single plate (Table 5.3, Figure 5.1 a, b).

Table 5.2 Bioactive Contents of the hydro-alcoholic extracts of M.hastata and M.vaginalis

Samples TPCa TFC

b TSC

c TSAC

d TTC

e

MHL 117.33±1.97 76.85±2.72 310.02±3.67 57.32±1.22 1732.50±13.02

MHS 64.75±1.47 27.42±3.26 382.12±2.12 110.35±2.10 2137.21±9.88

MHR 134.8±2.56 98.58±3.15 419.72±2.06 94.12±1.88 1966.08±16.32

MVL 104.50±2.01 68.74±2.88 214.05±2.44 53.47±1.36 1637.44±11.76

MVS 68.83±3.13 33.87±3.92 360.55±3.18 86.66±2.72 1918.02±21.51

MVR 106.44±2.35 57.56±2.44 429.52±1.68 107.89±3.17 1873.28±16.33

MHL-M.hastata leaf, MHS- M.hastata stem, MHR- M.hastata rootstock, MVL-

M.vaginalis leaf, MVS- M.vaginalis stem, MVR- M.vaginalis rootstock. TPCa-

expressed as mg gallic acid/100g of dry extract, TFCb- expressed as mg quercetin/100g of

dry extract, TSCc- expressed as mg cholesterol/100g of dry extract, TSAC

d-expressed as

mg Diosgenin/100g of dry extract, TTCe- expressed as mg urosolic acid/100g of dry extract.

Values are mean of three replicate determinations (n = 3) ± standard deviation

89

The fingerprint chromatographic technology was introduced and accepted by WHO

as a strategy for identification and quality evaluation of herbal medicine [31]. The

chromatograms indicated clear separation of all the constituents without tailing and

diffuseness. Automated multiple development is an instrumental technique which can be

used to perform normal-phase chromatography with solvent gradients on normal-phase

chromatography with solvent gradients on HPTLC plates. Most of the AMD applications

reported have used ―Universal‖ gradients; starting with a very polar solvent, the polarity is

varied by means of ―base‖ solvent of medium polarity to a non-polar solvent. A maximum

of 25 steps were used in ―Universal‖ gradient system. The developing system increases

while the solvent polarity is decreasing. The repeated development compresses bands on the

plate, resulting in increased sensitivity and resolution.

Table 5.3 Gradient table for Automated Multiple Development (AMD)

Gradient

steps

Solvent concentration (Vol %) Migration

distance

Drying

time

Preconditioning

with ammonia

Methanol

+Formic

acid

Ethyl

acetate

Toluene Hexane

1 50.0 20.0 30.0 0.0 20 3 Yes

2 40.0 35.0 25.0 0.0 30 3 Yes

3 30.0 40.0 30.0 0.0 40 2 Yes

4 20.0 40.0 30.0 5.0 50 2 No

5 10.0 40.0 40.0 10.0 60 2 No

6 5.0 35.0 50.0 15.0 70 2 No

Simultaneous AMD separation and comparison of six extracts from different parts

of Monochoria species, containing different classes of compounds; fatty acids, saponins,

flavonoids, phenols, alkaloids etc., were carried out. Owing to the large number of

phytoconstituents, which coexist in a plant extract, the separation of an unknown number of

90

unidentified compounds being sensitive to small structural changes and the wide differences

between the polarities of the unknown compounds, normal phase HPTLC with suitable

gradient was required.

Universal gradient system with various mobile phase compositions was checked for

separation of phytoconstituents of crude extracts, but universal gradient system did not give

an optimum separation for the selected extracts, due to high polarity fractions. In order to

improve the separation ethylacetate was included in the eluent composition of all the steps.

The gradient best separation was obtained in six step gradient composition. Preconditioning

of the plate with modifier (25% ammonia solution) was carried out before each step to

prevent peak tailing, which frequently occur in plant samples with wide polarity

phytoconstituents. AMD gradients with eluent composition and the time sequence with

migration distance is shown in Table 5.3

The corresponding densitograms obtained from the plates were shown in Figure

5.2a, b, c and d. The developed plates with various derivatization reagent and under UV are

shown in Figure 5.1c, d, e and f. AMD –HPTLC provided a good separation for polar

substances in the lower part of the plate and for the less polar compounds in the upper part,

hence an appropriate gradient system for the fingerprint analysis of the crude extracts of

Monochoria species with better simultaneous separations.

5.3.3 Finger print analysis - GC-MS

Qualitative analyses of various hydro-alcoholic extracts using GC-MS showed that,

there were different types of high and low molecular weight compounds. Most of the

identified compounds by GC-MS in the crude extracts were biologically important. The

name, RT value, percentage peak area and structure of the components were ascertained.

Identification was based on the molecular structure, molecular mass and calculated

fragments. Interpretation on mass spectrum GC-MS was conducted using the database of

National Institute Standard and Technology (NIST) having more than 62,000 patterns. The

name, molecular weight and structure of the components of the test materials were

ascertained. The relative percentage amount of each component was calculated by

comparing its average peak area to the total area.

91

The spectrum of the unknown component was compared with the spectrum of the

component stored in the NIST library version (2005), software, Turbomas 5.2. This was

done in order to determine whether this plant species contains any individual compound or

group of compounds, which may substantiate its current commercial and traditional use as

an herbal medicine. Further it helps to determine the most appropriate methods of extracting

these compounds. These results will consequently be discussed in the light of their putative

biological or therapeutic relevance. GC-MS is one of the best techniques to identify the

constituents of volatile matter, long chain and branched chain hydrocarbons, alcohols, acids,

esters etc. The GC-MS analysis of the hydro-alcoholic extracts of the selected parts of

Monochoria species revealed the presence of several compounds (phytochemical

constituents) that could contribute the medicinal quality of the plant (Figure 5.3-5.8). The

identification of the phytochemical compounds was confirmed based on the peak area,

retention time and molecular formula. The active principles with their Retention time (RT),

Molecular formula, Molecular weight (MW) and peak area in percentage are presented in

Table. 5.4 -5.9

5.3.4 Biological fingerprint-HPTLC-DPPH

The radical scavenging activity of DPPH is suitable for detecting antioxidant

properties of crude extracts substances from medicinal plants or pure compounds. DPPH

radical scavenging compounds appeared as yellow bands against a purple background on the

plate (Figure 5.2 e). Derivatization of the plate with DPPH revealed the presence of various

phytoconstituents with radical scavenging property, presence of phenolic compounds,

flavonoids in all the extracts may contribute to the radical scavenging property. Among the

crude extracts of different parts of Monochoria species, MHL and MVL showed many

phytoconstituents with radical scavenging property.

92

Figure 5.2 HPTLC plate at 254nm (c), 366nm (d), HPTLC plate derivatized in anisaldehyde

–sulphuric acid (e), HPTLC plate derivatized in DPPH (f).

93

94

95

96

97

98

99

100

101

102

103

5.3.5 Isolation of Phytoconstituents

Isolation of phytoconstituents from the hydro-alcoholic extracts were carried out

by standard column chromatography method, five different compounds, Compound I

( MVR-ET11 -14), compound II (MHR-ET-14-18), compound III (MHL 7), compound IV

(MHR-ET- 27) and compound V (MHS-50) were isolated and characterized by spectral

analysis like, IR, Mass and NMR spectroscopy. These compounds were first time reported

in Monochoria genus.

5.3.5.1 Spectral Data of Isolated Compounds

(i) Compound- I (MVR-ET-11-14)

Compound–I was isolated from the ethylacetate fraction of rootstock of M.vaginalis.

Fractions11-14 (single spot by TLC) was collected and pooled together. Recrystallized

using methanol. Characterization of the compound by IR, NMR and MS revealed the

structural identity and molecular formula of the compound.

IR spectrum of Compound-I

3428 cm -1 (OH stretching); 2937cm

-1and 2860cm

-1 (aliphatic C -H stretching);1618

cm-1 (C=C absorption peak); other absorption peaks 1458cm

-1 (CH2); 1376cm

-1(OH),

1048 cm-1(cycloalkane) (Figure 5.9).NMR Spectrum of the compound-I

The white solid compound was dissolved in CDCl3 and the chemical shifts of proton and

carbon were observed by analyzing the sample in Bruker 500MHZ NMR spectrometer.

Assignments of the 1H and

13C NMR spectrum is given in Table 5.10 Figure 5. 10, 5.11

represents the 1H and

13C NMR spectrum of the compound – I.

Structure of compound I

Figure 5.13 Structure of Stigmasterol

104

Physicochemical properties of Compound I

Physical state- white solid

Chemical test- Purple with Anisaldehyde sulphuric acid reagent

Solubility- Chloroform

Molecular formula- C29 H48O

Molecular weight- 412

Melting point- 154-156˚c

Rf - 0.5 (Chloroform: Methanol- 8: 2)

The compound was identified as Stigmasterol (Figure 5.13) with a molecular

formula of C29 H48O, based on the mass spectrum exhibiting a molecular ion peak at m/z

413.25 (M+1)+ (Figure 5.12).

(ii) Spectral Data of isolated compound- II ( MHR-ET-14-18)

Compound–II was isolated from the ethylacetate fraction of rootstock of M.hastata.

Fractions14-18 (single spot by TLC) was collected and pooled together. Recrystallized

using methanol. Characterization of the compound by IR, NMR and MS revealed the

structural identity and molecular formula of the compound.

The spectral data of the compound-II (Figure 5.14- 5.17) was matching with stigmasterol;

hence, the compound was identified as Stigmasterol (Figure 5.13), with a molecular

formula of C29 H48O, based on the mass spectrum exhibiting a molecular ion peak at m/z

413.25 (M+1)+ (Figure 5.17).

105

Table 5.10 1H and

13C NMR assignments of compound – I.

106

107

108

109

110

111

112

113

114

(iii) Spectral data of Compound-III (MHL-7)

IR spectrum of Compound-III (MHL-7)

3312 (OH stretching), 1667 (C=O), 1609, 1518, 1454 (aromatic-C=C-), 1374 (aromatic -

CH), 1258 (Aromatic-C-O-), 1201 (C-O), 1154(-C-CO-C-), 934, 815,649, 602 (aromatic-H).

(Figure 5.18).

NMR spectrum

The yellow semi solid mass was isolated from ethyl acetate fraction number 7-10,which

was successively washed excess amount of pet.ether (at 60-80°C) to remove the sticky

mass and recrystallized with methanol to get yellow solid, dissolved in CDCl3 and the

chemical shifts of proton and carbon were observed by analyzing the sample in Bruker 500

MHz NMR spectrometer. Assignments of the 1H and

13C NMR spectrum is given in Table

5.11, Figure 5.19, 5.20 represents the 1H and

13C NMR spectrum of the compound – III.

Table 5.11 1H and

13C NMR assignments of compound – III.

Atom 1H (δ, ppm)

13C (δ, ppm)

2 - 147.67

3 - 135.68

4 - 175.79

5 6.19 (1H, s) 160.66

6 - 98.16

7 - 163.91

8 6.42 (1H, s) 93.32

9 - 156.09

10 - 102.94

1‘ - 121.91

2‘ 6.89 (1H, s) 115.01

3‘ - 145.02

4‘ - 146.75

5‘ 7.55 (1H, d) 115.57

6‘ 7.67 (1H, d) 119.93

115

Structure of Compound-III (MHL-7)

Figure 5.22 Quercetin

Physicochemical properties

Physical state- Yellow semisolid

Melting point-315˚C

Chemical test- UV active compound, Yellow fluorescence with Aluminium nitrate

Molecular formula- C15H10O7

Molecular weight- 302

Rf- 0.56 (chloroform: Methanol-3:2)

The compound was identified as quercetin (Figure 5.22) with a molecular formula of

C15H10O7, based on the mass spectrum exhibiting a molecular ion peak at m/z 300.75

(M-1)+ (Figure 5.21)

116

117

118

119

120

(iv) Spectral Data of Compound-IV (MHR-ET-27)

The off white flaky compound was isolated from ethyl acetate fraction number 25- 27.

IR Spectrum

The IR spectrum showed characteristic absorptions at 3528 (-O-H stretching), 1714 (-CO-

O) and 1258cm-1 (C=C) (Figure 5.23).

NMR Spectrum

The off white flaky compound was dissolved in equal ratio of CDCl3 and CD3OH.

Chemical shifts of proton and carbon were observed by analyzing the sample in Bruker

500MHZ NMR spectrometer. Assignments of the 1H and

13C NMR spectrum is given in

Table 5.12 Figure 5.24, 5.25 represents the 1H and

13C NMR spectrum of the compound –

IV.

Structure of Compound-IV (MHR-27)

Figure 5.27 32-hydroxy dotriacontanyl ferulate

Physiochemical properties

Physical state- off white flakes

Solubility- Choloroform: methanol (1:1)

Chemical test- Purple with Vanillin sulphuric acid reagent

Molecular formula- C42 H74 O5

Molecular weight- 658

Melting point- 87˚C

Rf - 0.41 (Chloroform: Methanol: formic acid- 3:2: 0.5)

121

The compound was identified as 32-hydroxy dotriacontanyl ferulate (Figure 5.27), with a

molecular formula of C42 H74 O5, based on the mass spectrum exhibiting a molecular ion

peak at m/z 573.10 (M-OH)+

(Figure 5.26).

Table 5.12 1H and

13C NMR assignments of compound – IV.

Atom 1H (δ, ppm)

13C (δ, ppm)

1‘ - 127.15

2‘ 7.26 (1H, s) 109.41

3‘ - 147.96

4‘ - 146.81

5‘ 7.03(1H, d ) 115.80

6‘ 6.92 (1H, d) 123.05

7‘ 7.58 (1H, d) 144.62

8‘ 6.30 (1H, d) 114.75

-C=O - 167.38

-OCH3 3.93 (3H, s ) 55.99

1 4.18 (2H, t) 64.63

2 1.69 (2H, m ) 28.82

3 1.53- 1.58 (2H, m ) 26.03

4 - 29 1.25 – 1.37 (52H, m) 29.33 – 29.71

30 1.53- 1.58 (2H, m ) 25.77

31 1.53- 1.58 (2H, m ) 32.86

32 3.64 (2H, t) 63.14

122

123

124

125

126

(v) Spectral Data of isolated compound-V (MHS-50)

IR spectrum

Infrared (IR) spectroscopic analysis, absorptions bands at 3570.36 – 3186.51 cm-1

(OH stretching), 2864.39 cm 1

(CH stretching), 1833.22 cm-1

(C=O stretching) and

2353.23cm-1

[(CH)n bending] (Figure 5.28).

NMR spectrum

The white solid compound was isolated from ethanol fraction number 46-50 and

dissolved in CDl3 and the chemical shifts of proton and carbon were observed by analyzing

the sample in Bruker 500MHZ NMR spectrometer. Assignments of 1H and

13C NMR

spectrum is given in Table 5.13. Figure 5.29, 5.30 represents the 1H and

13C NMR spectrum

of the compound – V.

Table 5.13 1H and

13C NMR assignments of compound – V.

Atom 1H (δ, ppm)

13C (δ, ppm)

1 - 179.14

2 2.34 (2H, t) 33.89

3 1.63(2H, m ) 24.70

4-13 1.28 – 1.29 (20H, m) 29.07 – 29.70

14 1.31(2H, m ) 31.93

15 1.33((2H, m ) 22.70

16 0.88 (3H, t) 14.12

-COOH 10.83 (1H, br s) -

127

Structure of Compound-V (MHS-50)

Figure 5.32 Hexa decanoic acid

Physicochemical properties

Physical state- White solid

Chemical test- purple with anisaldehyde sulphuric acid reagent

Molecular formula-C16H32O2

Molecular weight-256.42

Melting point- 63˚C

Rf- 0.6 (Hexane: Ethyl acetate- 4:1)

The compound was identified as Hexadecanoic acid (Figure 5.32), with a molecular

formula of C16H32O2, based on the mass spectrum exhibiting a molecular ion peak at m/z

266.10 (M-1)+

(Figure 5.31).

128

129

130

131

132

5.3.6 Quantification of Stigmasterol and Quercetin

HPTLC could provide adequate information and parameters for comprehensive

identification and differentiation of the two closely related herbal medicines. Experimental

conditions, such as mobile phase composition, scan mode, scan speed and wavelength

detection were optimized to provide accurate and precise results for the quantification of

Stigmasterol and Quercetin individually. Development with the mobile phase, Chloroform:

Methanol (10 ml) (8:2 v/v) on the pre coated HPTLC plates produced compact, flat, bands

of stigmasterol (Rf 0.3), when derivatized with anisaldehyde – sulphuric acid reagent (Figure

5.33.). The content of stigmasterol varied in all the extracts (Figure 5.33a) and the results

were summarized in Table 5.14. Preliminary TLC experiments showed the presence of

Quercetin in the hydro-alcoholic extracts of MVL and MHL (Figure 5.34), hence Quercetin

content in these extracts were quantified and validated by HPTLC, the optimized mobile

phase was found to be Chloroform: Methanol: Formic acid (7:0.5:0.5). Quercetin content of

MVL and MHL was found to be 0.1616 % w/w and 0.0597% w/w respectively (Table 5.15).

Validation data for the developed quantitative HPTLC method meet the acceptance criteria

for accuracy, precision, linearity, detection and quantification limits set by ICH (Table 5.16).

Table 5.14 Quanification report of Stigmasterol by HPTLC and HPLC

S.No Samples Concentration (%w/w)

HPTLC HPLC

1 MHL 0.214 0.256

2 MHS 2.017 2.044

3 MHR 2.592 2.616

4 MVL 0.176 0.192

5 MVS 2.131 2.179

6 MVR 1.927 2.24

The described method is suitable for routine use by manufacturers for product

quality control. It is simpler than HPLC and faster, because up to six samples (applied in

133

duplicate singly with a minimum of three standard concentrations) can be analyzed on each

plate, rather than performing sequential injection of the samples and standards in HPLC. Cost

of solvent purchase and disposal is very low because not more than 15 mL of mobile phase for

development is required in the chamber trough containing the plate and an additional 10 mL

for vapor saturation in the other trough. The processing of samples and standards together at

the same time (in-system calibration) leads to improved reproducibility and accuracy.

The amount of quercetin present in the hydro-alcoholic extract of MVL and MHL

were quantified and validated by LC-MS method (Figure 5.36). Quercetin content in the

hydro alcoholic extracts of MHL and MVL were found to be 0.0903% w/w and 0.749 %

respectively (Table 5.15). Slight variation in the content of Quercetin was observed in the

HPTLC and LC-MS method. The summary of validation by LC MS was given in Table

5.17.

Table 5.15 Quanification of Quercetin by HPTLC and LC MS

S.No Samples Concentration (%w/w)

HPTLC LC MS

1 MHL 0.1614 0.0903

2 MVL 0.0597 0.0749

HPLC is the preferred analytical tool for quantification of marker compounds in

herbal drugs, because of its simplicity, sensitivity, accuracy; suitability for thorough

screening etc., RP-HPLC-PDA analysis was conducted to quantify the content of

Stigmasterol in the hydro-alcoholic extracts of different parts of Monochoria species (Table

5.14). At detection wave length of 205nm. The quantity of stigmasterol was calculated from

the respective peak areas according to individual standard curves. Figure 5.35, 5.35a, 5.35b,

shows the retention time and percentage of Stigmasterol present in different extracts

respectively. The validation summary of stigmasterol by HPLC was given in Table 5.17.

134

Table 5.16 Validation summary of Stigmasterol and Quercetin by HPTLC

Parameters Values

Stigmasterol Quercetin

Linearity range 1000-5000ng 1000-5000ng

Correlation Coefficient (R) 0.9950 0.9970

LOD (ng/Spot) 80 80

LOQ (ng/spot) 200 500

RSD (%) of Intraday precision (n=3) 2.43 2.43

RSD (%) of Interday precision (n=3) 2.94 2.94

Recovery (%) 99.77±0.92 99.48±0.58

Table 5.17 Validation summary of Quercetin by LC MS and Stigmasterol by HPLC

Parameters Values

Quercetin Stigmasterol

Linearity range 100-500 ng ml-1

1.6-25 µg ml-1

Correlation coefficient (R) 0.9931 0.9892

LOD 57.5 ng ml-1

3.66 µg ml-1

LOQ 174.2ng ml-1

11.09 µg ml-1

RSD (%) of Intraday Precision (n=3) 6.7 3.12

RSD (%) of Interday Precision (n=3) 10.4 4.43

Recovery 96.74 97.43

135

Figure 5.33 Quantification of Stigmasterol by HPTLC- HPTLC chromatogram of plate

derivatized in anisaldehyde- sulphuric acid (a), Spectra of standard and sample

(b),Chromatogram of MHL (c),Chromatogram of MVL(d).

136

Figure 5.33a HPTLC Chromatogram of MHS (a), Chromatogram of MVS (b),

Chromatogram of MHR (c), Chromatogram of MVR (d).

137

Figure 5.34 HPTLC Chromatogram of plate at 366nm (a), Spectra of standard and samples

(b), Linearity graph of quercetin (c), Chromatogram of standard quercetin (d),

Chromatogram of MHL (e), Chromatogram of MVL (f)

138

Figure 5.35 HPLC Chromatogram of MHL and MVL- Stigmasterol structure (a),

HPLC chromatogram of standard (b) HPLC Chromatogram of MHL (c), HPLC

Chromatogram of MVL (d).

139

Figure 5.35a HPLC Chromatogram of MHS (a), HPLC Chromatogram of MVS (b).

140

Figure 5.35b HPLC Chromatogram of MHR (a), HPLC Chromatogram of MVR (b).

141

Figure 5.36 LCMS report of quercetin- Quercetin structure (a), Chromatogram of

standard (b), Chromatogram of MHL (c), Chromatogram of MVL (d).