18_chapter 10.pdf - shodhganga

Simultaneous estimation of kaempferol, rutin and quercetin in herbal products 10

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques 240

10.1. INTRODUCTION

10.1.1 Plant profile of Phyllanthus niruri

Phyllanthus is a large genus of shrubs, trees and rare herbs of the family

Euphorbiaceae, comprising more than 600 species, of which P. accuminatus,

P. amarus, P. pulcher, P. niruroides, P. anisolobus, P. orbiculatus, P.

emblica, P. oxyphyllus, P. flexuosus, P. raticulatus, P. fraternes, P. simplex, P.

mullernus, P. urinaria, P. mytrifolis, P. virgatus, P. niruri and P. watsonii

were investigated for their phytochemical and pharmacological properties.

The genus is found in almost over all warmer parts of the world.

Phyllanthus niruri 331

(a) Classification:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliophyta

Order: Malpiales

Family: Phyllanthaceae

Genus: Phyllanthus

Species: Niruri

(b) Vernacular names:

Sanskrit: Bhumyamlaki

Hindi : Jar amla or Jangli amla

Kannada : Nela nelli,

Malayalam : Keelar nelli

Tamil : Kizhanelli

Marathi: Bhuiamla



Other common names: Stonebreaker and Seed-Under-Leaf (English), Chanca

Piedra (Spanish) and Quebra Pedra (Portuguese) 332

.

(c) Part used: Whole plant

(d) Botanical description: Phyllanthus niruri is an erect annual herb, 10 to

50 cm high and is indigenous to the Amazon rainforest and other tropical

areas, including South East Asia, Southern India and China. It has smooth

cylindrical stem 1.5 to 2 mm thick and deciduous horizontal branchlets 4 to

12 cm long and about 0.5 cm thick, with 15 to 30 leaves. The leaves are

alternate, elliptic, oblong or obovate, 5 to 11 mm long and 3 to 6 mm wide,

rounded to slightly pointed at the tip, scarcely oblique on one side at the base,

petioles 0.3 to 0.5 mm long,. It has small off-white-greenish flowers, which

are solitary, auxiliary, pedicellate, apetalous and monoecious.The flowers are

alone or usually one male and one (larger) female are in each leaf axil

together.

The seed capsules on stalks are 1 to 2 mm long, round, smooth, 2 mm wide,

with 6 seeds. When the fruits burst open the seeds are hurled away. Seeds are

triangular (like an orange segment), light brown, 1 mm long, with 5 to 6 ribs

on the back 2.

P. amarus and P. sellowianus are closely related to P. niruri in appearance,

phytochemical contents and history, but they are found in drier regions of

India and Brazil, and even in Florida and Texas. In a recent report, cladistic

analysis indicated that the Phyllanthus genus is paraphyletic and therefore the

two problematic and confusing species, P. niruri and P. amarus, are two

individual species.



(e) Origin and distribution: Phyllanthus niruri is widely distributed in all

tropical regions of the planet. Paleobotanical studies have not found the exact

geographic origin of this plant. This plant may be indigenous to the tropical

Americas, the Philippines or India.

Plants in the genus Phyllanthus can be found around all tropical regions of the

world: from Africa to Asia, South America and the West Indies. P. niruri can

be found in all the tropical regions of the world: through the roads, valleys, on

the riverbanks and near lakes. This plant is a common arable weed of

disturbed ground in Southern Florida, the Bahamas, the West Indies and

tropical America and is naturalized in the Old World tropics.

(f) Anatomy of the Plant333

:

Leaf : Epidermal walls wavy, stomata anisocytic, which is distributed mainly

on the lower epidermis. Upper epidermis has a thin cuticle. Stomata are

followed by respiratory cavities beneath.There is a single layer of palisade

cells, which occupy nearly half of the space between the two epidermis.

Below the palisade there is a row of broad collecting cells, each of which

occur in relation to 3 or 4 palisade cells. Reduced vascular elements are

clearly seen running on long stretch beneath the collecting cells. The palisade

ratio has been determined to vary between 13 and 17.

Branchlet :- Rounded in transverse section, cortex 6-8 cells thick most of the

cells contain chloroplast and few druses crystals. After 3 -4 rows, there is a

row of cells containing starch grains followed by 2-3 layers of fiber cells

which are interrupted by cortex parenchyma. Phloem 5 - 7 cell thick, xylem

vessels 8 - 33 mm in diameter, pith cells contain chloroplasts.

Stem :- Epidermal cells and some of the cortical cells contain tannin, cortex



about 15 cells thick, some contain calcium oxalate druse crystals, inner cortex

contains groups of 7 -10 thick walled cells interrupted at regular intervals by

parenchyma cells on the outer side of the group of thick walled cells there is a

row of parenchymatous cells containing starch grains. Phloem 7 - 10 cells

thick, thin walled, without any contents. Xylem vessels 16 -54 mm in

diameter, pith cells thin walled may contain a few druse like crystals.

Root :- Cork cells 6 - 8 cells thick, contain dark brown tannin, cortex 10 - 15

cells thick, some filled with tannin and some with starch, phloem 4 - 6 cells

thick, xylem vessels 12 - 53 mm in diameter.

(g) Traditional uses333

: In many countries around the world, plants in the

genus Phyllanthus are used in folk remedies; therefore this genus is of great

importance in traditional medicine. P. niruri has a long history in herbal

medicine systems such as Indian Ayurveda, Traditional Chinese Medicine and

Indonesian Jamu.

The genus Phyllanthus has a long history of use in the treatment of liver,

kidney and bladder problems, diabetes and intestinal parasites. Some related

species in this region with medicinal significance are P. epiphyllanthus, P.

amarus, P. urinaria, P. acuminatus, P. emblica. P. niruri, P. amarus and P.

urinaria are used in the treatment for kidney/gallstones, other kidney related

problems, appendix inflammation, and prostate problems. The whole plant is

used as remedies for many conditions such as dysentery, influenza, vaginitis,

tumours, diabetes, diuretics, jaundice, kidney stones and dyspepsia. The plant

is also useful for treating hepatotoxicity, hepatitis B, hyperglycaemia and viral

and bacterial diseases.333



In India, where it is called Pitirishi or Budhatri, it is a common household

remedy for asthma, bronchitis, cough, extreme thirst, anaemia, liver diseases,

jaundice, tuberculosis and cardiovascular problems. P. niruri has been used in

Ayurvedic medicine for over 2000 years and has a wide number of traditional

uses for jaundice, gonorrhoea, frequent menstruation and diabetes.

It is an important medicinal plant in Jamu (traditional medicine in Indonesia),

a well-known Indonesian traditional herbal medicine to treat various diseases.

In Jamu preparations, the plant is used as antiviral and hepatoprotective agent.

In Malaysia, P. niruri is known as Dukong anak. It is used internally for

diarrhoea, kidney disorders, gonorrhoea and cough. 331,333

This plant is traditionally used around the world in the treatment of liver

ailments and kidney stones. The Spanish name ‘chanca piedra’ means “stone

breaker or shatter stone.” In South America, ‘chanca piedra’ has been used to

eliminate gall bladder and kidney stones, and to treat gall bladder

infections334

.

In Brazilian herbal medicine, it is called “Quebra Pedra” and is considered an

excellent remedy for hydropsy, urinary and bladder infections. It is also used

to cure kidney disorders, hepatitis and diabetes. 335-337



(h) Pharmacology and Clinical Studies334-336

:

I. Hepatoprotective Effect –

Hepatitis B is one of the major diseases inflicting human population.

Alternative herbal medicine using extracts of Phyllanthus niruri has been

reported to be effective against hepatitis B and other viral infections. A study

reports quantitative determination of the anti viral effect of these herbs in

well-defined in vitro systems338

.

In one study, 37 patients with chronic viral hepatitis B were treated with a

daily dose of 600 mg of Phyllanthus niruri for 30 days. HBsAg had lost in 59 %

of the patients with in the two weeks after the end of the treatment.

Furthermore, none of the cases when followed for up to 9 months had any

symptoms of HBsAg339-342

.

II. HIV Replication Inhibition–

Aqueous extract of Phyllanthus niruri is reported to have inhibitory effect on

human immunodeficiency virus. The investigation examines the anti-HIV

Fig 10.1: Young plant of Phyllanthus niruri



effects of the alkaloidal extract of Phyllanthus niruri in human cell lines. The

inhibitory effect on HIV replication was monitored in terms of inhibition of

virus induced cytopathogenecity in MT-4 cells. The alkaloidal extract of

Phyllanthus niruri showed suppressing activity on strains of HIV-1 cells

cultured on MT-4 cell lines. The alkaloidal extract of Phyllanthus niruri was

thus found to exhibit sensitive inhibitory response on cytopathic effects

induced by both the strains of human immunodeficiency virus on human MT-

4 cells in the tested concentrations. 343, 344

III. Lipid Lowering Activity -

Lipid lowering activity of Phyllanthus niruri alcoholic extracts in triton

induced hyperlipidaemia was examined in rats. In an experiment with

cholesterol fed rats, Phyllanthus niruri at a dose of 100 mg/kg lowered the

elevated level of low-density lipoprotein lipids in hyperlipidemic and drug fed

animals.345

IV. Antidiabetic Activity –

An alcoholic extract of Phyllanthus niruri was found to significantly reduce

the blood sugar in normal rats and in alloxan diabetes rats. In normal rats,

administration of Phyllanthus niruri 200 mg/kg body weight reduced the

blood sugar by 34.5 percent and to 47.4 percent at the concentration of 1000

mg/kg by weight within 1 hour. The results indicate potential antidiabetic

action of Phyllanthus niruri.346

V. Antimalarial Activity –

The ethanolic, dichloromethane and lyophilized aqueous extracts of whole

plants of Phyllanthus niruri were evaluated for their antimalarial activity in



vivo, in 4-day, suppressive assays against Plasmodium berghei ANKA in

mice. No toxic effect or mortality was observed in treated mice, orally, with

any of the extracts as a single dose, of 500 mg/kg body weight, or as the same

dose given twice weekly for 4 weeks (to give a total dose of 4 g/kg). The most

active ethanolic extract, that of Phyllanthus niruri, reduced parasitaemia by

73%347, 348

.

VI. Antispasmodic activity –

Research done in Brazil at the Federal University of Santa Catarina in 1984

on Phyllanthus niruri revealed an alkaloid (phyllanthoside) in the leaves and

stem with strong antispasmodic activity. It served as a relaxing agent for

smooth muscles and they concluded that its spasmolytic action probably

accounted for the efficacy of Phyllanthus niruri in expelling stones.349

VII. Analgesic activity –

Methanol extract of dried callus tissue of P. niruri at a concentration of 10

mg/kg, administered intraperitonially to mice was active vs. acetic acid

induced writhing and vs. formalin – induced pedal edema showed a good

analgesic activity. 350, 351



(i) Phytochemistry:

Table 10.1: Phytoconstutients of Phyllanthus niruri with their

pharmacological effects352-424

Sr.

No

Chemical

constituents

Parts of plant Pharmacological effects

1.

Flavonoids:

a. Rutin Whole plant Antioxidant

b. Quercetin Leaf, Whole

plant

Anti-aggregant, Anticancer,

Antifungal, Anti-

inflammatory, Antispectic

Antioxidant

c. Quercitrin Leaf Antidiarrhoel activity, Anti

leishmanial,

antinociceptive, Anti

inflammatory

d. Astragalin Leaf Diuretic, Anti inflammatory

e. Catechin Root culture Anti tumor

f.Prenylated

flavanone glycoside

Whole plant Antioxidant

g. Nirurin Whole plant Antioxidant

h. Niruriflavone Antioxidant

2.

Terpenes

a. Limonene Anticarcinogenic

b. p- Cymene Leaf Antioxidant, antimicrobial

c.Lupeol, Lupeol

acetate

Root culture Anti inflammatory, Anti

tumor

3.

Coumarins

a.Ellagic acid Whole plant Anti carcinogenic and Anti

viral

b.Methyl

brevifolincarboxylate

Vaso relaxant effect



4. Lignans

a.Phyllanthin

Leaf and aerial

parts

Hepatoprotective and Anti

genotoxic , Anti viral

activity

b. Hypophyllanthin Whole plant Hepatoprotective, Anti

genotoxic

c. Niranthin , Hydroxy

niranthin ,

Demethylenedioxy

niranthin

Leaf

Anti-inflammatory,

Hepatoprotective

d. Phylltetralin Leaf Anti inflammatory

e. Nirtetralin Leaf

Anti inflammatory,

Hepatoprotective

f. Isolintetralin and

Lintetralin

Leaf Anti tumor activity

5.

Tannins

a. Repandusinic acid Whole plant Anti- HIV activity

b. Geranin Whole plant Anti-nociceptive activity

c. Corilagin Whole plant Inhibits Plasminogen –

activator- inhibitor-I,

antifungal

6.

Saponins

a. Diosgenin Whole plant Anti fungal and

cardiovascular activity

7.

Alkaloids

a. Norsecurinine Whole plant Strong anti spasmodic

activity

b. Nirurine Aerial

c. Phyllanthine Leaf , root

culture, stem

d. Phyllochrysine Leaf, stem



8.

Other compounds

a. β – glucogallin Whole plant Effective in haemolytic

disease

b.1-O-galloyl-6-O-

luteoyl-α- D-glucose

Whole plant Effective in haemolytic

disease

Linear and complex

hetero xylans

Whole plant Immunomodulators and

Anti- tissive

a. Niruriside Whole plant Anti-HIV activity

9.

Hydrocarbons

a. Triacontanal Aerial Hepatoprotective

b. Tricontanol Aerial



10.1.2 Kaempferol, Rutin and Quercetin as marker compound for

Standardization

Flavonoids are polyphenolic compounds that occur ubiquitously in foods of

plant origin. They are extremely important because of their health effects. It

has been predicted that average intake of all flavonoids is several grams per

day. They occur in foods as O-glycosides with sugars bound at the C3

position.

Flavonoids with a diphenylpropane skeleton (C6–C

3–C

6) are known to be

antimutagenic and anticarcinogenic. They also have antioxidant properties

and inhibit the oxidation of LDL. They also have anti-inflammatory and anti-

allergic effects. Flavonoids consist mainly of flavonols, flavones, catechins

and flavanones.

Kaempferol is an example flavonol. It is a strong antioxidant and helps to

prevent oxidative damage of our cells, lipids and DNA. Kaempferol seems to

prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein

and the formation of platelets in the blood. Studies have also confirmed that

kaempferol acts as a chemopreventive agent, which means that it inhibits the

formation of cancer cells.

Rutin is a member of bioflavonoids, a large group of phenolic secondary

metabolites of plants that include more than 2,000 different known chemicals.

Rutin has ability to strengthen and modulate the permeability of the walls of

the blood vessels including capillaries. Rutin is known to offer nutritional

support to the circulatory systems including the capillaries in eyes. It has

proved to be especially helpful in preventing recurrent bleeding caused by

weakened blood vessels, and has been used in treatment of hemorrhoids and

Simultaneous estimation of kaempferol, rutin and quercetin in

Standardization of Some Plant

varicose veins, helping to prevent blood vessel walls to become fragile. Rutin

is safe and effective for poor circulation, high blood pressure

Quercetin has a common flavone nucleus composed of two benzene rings

linked through a heterocyclic pyrone ring.

scavenge damaging particles in the body known as free radicals, which

damage cell membranes,

can neutralize free radicals and may reduce or even help to prevent some of

the damage they cause. Quercetin also acts like an antihistamine and an anti

inflammatory.

Simultaneous estimation of kaempferol, rutin and quercetin in herbal

Standardization of Some Plant-Based Formulations By Modern Analytical Techniques


is safe and effective for poor circulation, high blood pressure, chilblains, etc.


linked through a heterocyclic pyrone ring. Quercetin is antioxidants. They


damage cell membranes, tamper with DNA, and even cause cell death. Thus,


the damage they cause. Quercetin also acts like an antihistamine and an anti

inflammatory.

Fig.10. 2:Structure of Kaempferol

herbal products 10

Based Formulations By Modern Analytical Techniques 252


, chilblains, etc.


Quercetin is antioxidants. They


tamper with DNA, and even cause cell death. Thus,


the damage they cause. Quercetin also acts like an antihistamine and an anti-

Kaempferol



These three compounds are pharmacologically and biologically very active.

Thus, they can be considered as biomarkers. These compounds are also found

in many plants together. Hence, a single method which can determine the

content of these markers simultaneo

manufacturer and analysts of plant based formulations.



Fig.10. 3: Structure of Rutin

Fig.10. 4: Structure of Quercetin




content of these markers simultaneously will be a very beneficial tool for the

manufacturer and analysts of plant based formulations.

herbal products 10





usly will be a very beneficial tool for the



Thus, the objective of the present work was to develop and validate a

simultaneous HPTLC method for estimation of Kaempferol, Rutin and

Quercetin in various herbal formulations. We even aim at to use this

method for estimation of any of the three markers individually. Thus,

this method will show wider applicability in analysis of many different

plant-based formulations with three different biomarkers.



10.2 ISOLATION OF MARKER COMPOUNDS

10.2.1 Procurement of dried whole plant of Phyllanthus niruri

The dried whole plant of Phyllanthus niruri was supplied by Amsar Pvt. Ltd.

10.2.2 Preparation of extracts

Approximately 100 g of air-dried and powered whole plant of P. niruri was

successively extracted in a soxhlet extraction apparatus with 1000 ml of each

of hexane, chloroform, butanol and water for 18 hours. The respective

extracts were dried and the nature of the extracts and percentage yield was

recorded (Table 10.2).

Table10. 2: Different extracts of P. niruri by successive extraction

Extracts Nature Yield (g/100 g of

crude drug)

% Yield

Hexane Dark greenish sticky

solid

1.68 1.68

Chloroform Greenish solid 1.59 1.59

Butanol Brownish solid 7.83 7.83

Aqueous Brownish solid 12.07 12.07

10.2.3 Isolation of Kaempferol, Rutin and Quercetin

Kaempferol, rutin and quercetin are some of the major flavonoids present in

P.niruri and these can be used as marker compounds for standardization of

formulations containing P.niruri or any other plant based formulations

containing these compounds.



10.2.3.1 Isolation of Kaempferol and Quercetin

a) Preparation of column:

500 gm of silica gel (60-120 mesh) was activated for 30 min at 105 ºC. Slurry

of silica gel was prepared in 1000 ml petroleum ether (60-80 °C) and was

transferred to glass column; precaution was taken to avoid air entrapment.

The length of the column was approximately kept to 110 cm.

b) Preparation of sample:

The chloroform extract was adsorbed on silica and loaded on the column in

form of thin band. Petroleum ether was added slowly to the column,

precaution was taken to avoid air entrapment.

c) Elution

Following combinations of solvents were used to elute the column. 100 ml of

each mobile phase was used at a time to run the column.

Table 10.3: Pattern of column chromatographic elution for Kaempferol

and Quercetin

Sr.

No

Solvents composition

(%)

TLC studies of eluents

with mobile phase CHCl3 :

MeOH: Formic acid

(9:1:0.5)

Inference

Pet.

Ether

Chloroform

1. 100 0 No spot --

2. 90 10 No spot --

3. 80 20 No spot ---

4. 70 30 There were three spots very

close to each other with Rf

range from (0.95 to 0.8.1)

with a blackish blue color

after spraying with 10 %

alcoholic sulphuric acid.

The spots

where difficult

to separate.



5. 60 40 No spot ---

6. 50 50 One single spot was

observed with Rf value 0.81

with a blackish blue color

after spraying with 10 %

alcoholic sulphuric acid.

The amount of

obtained

compound was

too less to be

considered.

7. 40 60 No spot ---

8. 30 70 No spot ---

9. 20 80 One single spot detected at

254 nm with Rf 0.77

Sticky dark

yellow

compound

was obtained

(Component

V)

10. 10 90

11. 0 100 No spot ---

Chlor

oform

Methanol

12. 90 10 No spot ---

13. 80 20 No spot ---

14. 70. 30 One Spot detected at 254

nm with Rf 0.70

The compound

was oily in

nature.

15. 60 40

16. 50 50 One single spot detected at

254 nm with Rf 0.64

Sticky yellow

compound

was obtained

(Component

VI)

17. 40 60 The same compound was

detected

The compound

was very less

quantity.

50 ml aliquots were collected, concentrated and subjected to TLC studies. 9th



and 10th

fractions [eluted with Petroleum ether: Chloroform (60:40), (50:50)]

gave an intense spot at a dark brown sticky mass Rf 0.81 under UV lamp

under 254 nm (Fig.10.5) with mobile phase chloroform: methanol: formic

acid (9:1:0.5) (v/v). 15th

and 16th

fractions [eluted with chloroform: methanol

(60:40), (50:50)] gave an intense spot at a dark brown sticky mass Rf 0.64

under UV lamp under 254 nm (Fig.10.5) with same mobile phase. Both the

fractions were washed successively with excess amount of petroleum ether

(60 – 80°C) to remove the sticky matter and recrystallized from diluted

alcohol to obtain a dark yellow crystalline powder (component V) and light

yellow crystalline powder (component VI). The yield of component V and VI

obtained was 0.170% w/w and 0.151%w/w.

10.2.3.2 Isolation of Rutin

Butanol extract of P.niruri was used for the isolation of further marker

compounds. Column chromatographic technique was used. The above

mentioned procedure was followed. The butanol extract (700 mg) was

dissolved in methanol and adsorbed on silica gel for loading on column. The

column and extract slurry was prepared in chloroform. The column was eluted

successively with chloroform and methanol in order of increment of polarity.



Table 10.4: Pattern of column chromatographic elution for Rutin

Sr.

No

Solvents composition

(%)

TLC studies of

eluents

with mobile phase

CHCl3 : MeOH:

Formic acid (8:2:0.5)

Inference

Chloroform Methanol

1. 100 0 No spot --

2. 90 10 There were two spots

with Rf values 0.83 and

0.78 with blue color

after spraying with 10

% alcoholic sulphuric

acid.

One more spot at Rf

0.75 gave a blackish

blue color with 10 %

alcoholic sulphuric

acid.

The spots

were very

light in color

and total

amount was

too less to

be

considered.

3. 80 20 One Spot detected

under UV lamp at 254

nm with Rf 0.71 and

another been was

observed at Rf 0.68 at

366 nm.

This fraction

was very

sticky and in

less amount.

4. 70 30 There was a violet

colored spot at Rf 0.62

detected under UV

lamp at 254 nm. Other

spot with fluorescent

white color was

detected under UV

lamp at 366 nm 0.59

The fraction

had many

spots with

closely

related Rf.



and two more bands of

Rf 0.54 and 0.50 with a

blackish blue color

after spraying with 10

% alcoholic sulphuric

acid.

5. 60 40 No spot ---

6. 50 50 No spot ---

7. 40 60 One single spot was

observed with Rf

value 0.34 with a

blackish blue color

after spraying with

10 % alcoholic

sulphuric acid.

Yellowish

compound

was

obtained.

(Componen

t VII)

8. 30 70

9. 20 80 No spot ---

10. 10 90 No spot ---



7th

and 8th

fractions [eluted with chloroform: methanol (30:70)] gave an

intense spot under UV at R

(Fig.10.5) with mobile phase chloroform: methanol: formic acid (8:2:0.5) and

recrystallized from diluted alcohol to obtain a yellow crystalline powder

(component VII)

yield of component VII obtained was 0.214%

Fig.10.5 Video

Track 1: Component V

Track 2: Component VI

Track 3: Component VII

Component V, VI and VII showed single violet colored spots on TLC plate

under UV lamp.




intense spot under UV at Rf 0.34 (yellow colored compound

with mobile phase chloroform: methanol: formic acid (8:2:0.5) and


component VII) and light yellow crystalline powder (component VII)

yield of component VII obtained was 0.214% w/w.

5 Video-images of TLC plates showing Component V, VI and VII

Track 1: Component V

Track 2: Component VI

Track 3: Component VII


under UV lamp.

1 2 3

herbal products 10



0.34 (yellow colored compound) on TLC plate

with mobile phase chloroform: methanol: formic acid (8:2:0.5) and


component VII). The

images of TLC plates showing Component V, VI and VII




All three components were evaluated for physicochemical parameters such as

melting point, solubility, elemental analysis. The parameters were compared

with those of the reference standard.

10.2.4 Physicochemical of component V

The melting point of component V was found to be 277 °C which was

matching with the standard (277 °C). Solubility was tested in different

solvents such as chloroform, methanol and water. It was observed that

component V was readily soluble in methanol and chloroform. The

Lassaigne’s sodium fusion test was carried out for detection of elements.

Carbon, hydrogen is present and nitrogen, halogen and sulphur were found to

be absent. The yield obtained was 0.170% w/w from chloroform extract by

column chromatography. The summarized data is mentioned in table 10.5.

Table 10.5: Physicochemical analysis of component V

Sr. No Parameters Component V Standard

1. Color Dark yellow Dark yellow

2. Melting point 277°C 277 °C

3. Solubility

Methanol,

Chloroform

Methanol,

Chloroform

4. Element present C, H,(O) C,H, (O)

5. Yield (%w/w) 0.170% -

.



10.2.5 Chromatographic and Spectral Studies of Component V:

� HPTLC Studies: Standard Kaempferol and the isolated component V

were dissolved in chloroform and the HPTLC analysis was carried out using

the following densitometric conditions:

� HPLC Studies: Standard kaempferol and component V were analyzed

by HPLC technique using the following conditions:

• Column: C18 Phenomenex (250 x 4.60mm) - 5µ

• Mobile phase : Acetonitrile (70) : Water (30)

• Flow rate: 1.0 ml/min

• Wavelength : 254nm

• Injection loop capacity: 20µl

• Concentration of Samples: 1mg/ml of standard and component

V

� Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)

� Mobile phase : Chloroform : methanol: formic acid (9:1:0.5)

� Saturation time :15 min

� Development time :15 min

� Band width :7 mm

� Solvent front : 8 mm

� Wavelength :254 nm

� Lamp : Deuterium



Fig. 10.6: HPTLC chromatogram of standard

Kaempferol

Fig. 10.7: HPTLC chromatogram of Component V



Fig. 10.8: HPLC profile of reference standard Kaempferol:

RT=12.099 min

Fig. 10.9: HPLC profile of Component V: RT=12. 107 min



The HPTLC fingerprints (Fig.10.6 and Fig.10.7) and HPLC profile (Fig. 10.8,

Fig. 10.9) of component V matched with reference standard of kaempferol (Rf

=0.77 and RT=12.099). Thus component V was confirmed to be kaempferol.

� UV Spectroscopy: The UV spectrum was recorded in methanol for the

standard kaempferol and isolated component V.

The UV λ max of standard kaempferol was recorded to be 254 nm and 368 nm.

The component V also gave 254 nm and 368 nm of λ max.

� NMR Spectroscopy: The NMR (proton and 13

C) spectra were recorded

by dissolving the component V in deuterated methanol.



Fig. 10.10: Proton NMR spectrum of Component V

Fig. 10.11: 13

C NMR spectrum of Component V



NMR INTERPRETATION

Compound V was analyzed by 1H and

13C NMR and the data was compared

with the reported literature.

PMR spectrum (400 MHz, MeOD): 8.08 (d, J=7.8Hz, 2H, H3’/5’), 8.10 (d,

J=7.8Hz, 2H,H2’/6’), 6.41 (s,1H,H6), 6.19 (s,1H,H8)

13C spectrum (100 MHz, MeOD): 176.4 (C=O, C-4), 161.0 (C-5), 159.1(C-

9), 156.8 (C-7), 147.0 (C-4’), 146.6 (C-2), 135.0 (C-3), 129.2 (C-2’/6’), 114.9

(C-3’/5’), 122.3 (C-1’), 97.8 (C-6), 93.0 (C-8).The 1H- NMR (400 MHz,

MeOD) spectrum of V shows five proton signals, shifted at 6.00 – 8.50 ppm

which represents a polyphenol skeleton. The doublet signals, 8.08 (d, 2H,

J=7.8Hz, H3’/5’), 8.10 (d,2H,J=7.8Hz, H2’/6’), were integrated of two

protons for each, so it proves the symmetry positions in compound structure.

13C- NMR exhibit fifteen carbon signals, including two signal of symmetry

carbons 129.2 (C-2’/6’), 114.9 (C-3’/5’), six oxygenated carbons 161.0 (C-5),

159.1(C-9), 156.8 (C-7), 161.0 (C-4’), 146.6 (C-2), 135.0 (C-3) and a

carbonyl 176.4(C=O, C-4). Thus, compound V was elucidated as

Kaempferol.

Thus from the comparison with reference standard of kaempferol and from

chemical and spectral studies, component V was confirmed to be kaempferol.



10.2.6 Physicochemical analysis of component VI

The melting point of component VI was found to be 314 °C which was

matching with the standard (315 °C). Solubility was tested in different


component VI was readily soluble in methanol and chloroform. The

Lassaigne’s sodium fusion test was carried out for detection of elements.

Carbon, hydrogen is present and nitrogen, halogen and sulphur were found to

be absent. The yield obtained was 0.151%w/w from chloroform extract by

column chromatography. The summarized data is mentioned in table 10.8.

Table 10.8: Physicochemical analysis of component VI

Sr. Parameters Component VI Standard

1. Color Yellow Yellow

2. Melting point 314 °C 315 °C

3. Solubility

Methanol,

Chloroform

Methanol,

Chloroform

4. Elements present C, H,(O) C,H,(O)

5. Yield (%w/w) 0.151% w/w -

.



10.2.7 Chromatographic and Spectral Studies of Component VI:

� HPTLC Studies: Standard Quercetin and the isolated component VI

was dissolved in chloroform and the HPTLC analysis was carried out using

the following densitometric conditions:

� HPLC Studies: Standard quercetin and component VI were analyzed

by HPLC technique using the following conditions:







VI

• Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)

• Mobile phase : Chloroform : methanol: formic acid (9:1:0.5)

• Saturation time :15 min

• Development time :15 min

• Band width :7 mm

• Solvent front : 8 mm

• Wavelength :254 nm

• Lamp :Deuterium



Fig.10.13: HPTLC chromatogram of Component VI

Fig. 10.12: HPTLC chromatogram of standard Quercetin



Fig. 10.14: HPLC profile of reference standard

min

Fig. 10.15



: HPLC profile of reference standard Quercetin

15: HPLC profile of Component VI : RT=8.134

herbal products 10


Quercetin : RT=8.107

8.134 min



The HPTLC fingerprints (Fig.10.12 and Fig.10.13) and HPLC profile

(Fig. 10.14, Fig. 10.15) of component VI matched with reference standard of

quercetin (Rf =0.64 and RT=8.107). Thus component VI was confirmed to be

quercetin

� UV Spectroscopy: The UV spectrum was recorded in methanol for the

standard quercetin and isolated component VI.

The UV λ max of standard quercetin was recorded to be 257 nm and 369 nm.

The component VI also gave 257 nm and 369 nm of λ max.



by dissolving the component VI in deuterated chloroform.



Fig.



Fig. 10.16: Proton NMR spectrum (2) of Component VI

Fig. 10.17: 13

C NMR spectrum of Component VI

herbal products 10


Component VI

Component VI



NMR INTERPRETATION

PMR spectrum (400 MHz, MeOD): 7.81 (s, 1H, H- 2’), 7.70 (d, J= 8.0, 1H,

H-6’), 6.99 (d, J=8.0, 1H, H-5’), 6.53 (s, 1H, H-8), 6.27 (s, 1H, H-6).

13C spectrum (100 MHz, MeOD): 177.3 (C=O, C-4), 163.8 (C-7), 160.8 (C-

5), 156.1 (C-9), 155.3 (C-2), 148.2 (C-4’), 144.6 (C-3’), 133.0 (C-3), 122.1

(C-6’), 121.0 (C-1’), 115.7 (C-5’), 115.2 (C-2’), 103.7 (C-10), 98.4 (C-6),

93.4 (C-8).

The 1H- NMR spectrum of VI showed five proton signals, shifted at 6.00 –

8.0 ppm which represents a polyphenol skeleton. Two doublet signals 7.70 (d,

J= 8.0, 1H, H-6’), 6.99 (d, J=8.0, 1H, H-5’), and three singlet 7.81 (s, 1H, H-

2’), 6.53 (s, 1H, H-8), 6.27 (s, 1H, H-6), correspond to flavonol skeleton. The

fifteen signals in 13

C- NMR spectra including seven oxygenated carbons

{163.8 (C-7), 160.8 (C-5), 156.1 (C-9), 155.3 (C-2), 148.2 (C-4’), 144.6 (C-

3’), 133.0 (C-3)} and carbonyl 177.3 (C=O, C-4) signify the quercetin. Thus

from the analysis of NMR data verify that compound VI is Quercetin.



The NMR Spectra revealed presence of aromatic, hetrocyclic and protons of

phenolic group it was found to be almost identical to the NMR spectra of

Standard Quercetin (Fig. 10.16.1, Fig. 10.16.2, Fig. 10.17, Table 10.9, Table

10.10). Thus from the comparison with reference standard quercetin and from

chemical and spectral studies component VI was confirmed to be quercetin.

10.2.8 Physicochemical analysis of component VII

The melting point of component VII was found to be 212 °C which was

matching with the standard (211°C). Solubility was tested in different


component VII was readily soluble in methanol. The Lassaigne’s sodium

fusion test was carried out for detection of elements. Carbon, hydrogen is

present and nitrogen, halogen and sulphur were found to be absent. The yield

obtained was 0.214%w/w from chloroform extract by column

chromatography. The summarized data is mentioned in table 10.11.

Table 10.11: Physicochemical analysis of component VII

Sr. Parameters Component VII Standard

1. Color Yellow Yellow

2. Melting point 211°C 315 °C

3. Solubility

Methanol,

Chloroform

Methanol,

Chloroform

4. Element present C, H,(O) C,H,(O)

5. Yield (%w/w) 0.214% w/w -

.



10.2.9 Chromatographic and Spectral Studies of Component VII:

� HPTLC Studies: Standard Rutin and the isolated component VII was

dissolved in methanol and the HPTLC analysis was carried out using the

following densitometric conditions:

� HPLC Studies: Standard rutin and component VII were analyzed by

HPLC technique using the following conditions:







VII

• Stationary phase : Precoated plates of Silica Gel 60 GF254 (Merck)

• Mobile phase : Chloroform : methanol: formic acid (8:2:0.5)

• Saturation time :15 min

• Development time :15 min

• Band width :7 mm

• Solvent front : 8 mm

• Wavelength :254 nm

• Lamp : Deuterium



Fig.10.18: HPTLC chromatogram of standard Rutin

Fig. 10.19: HPTLC chromatogram of component VII



Fig. 10.20: HPLC profile of reference standard

Fig. 10.



: HPLC profile of reference standard rutin : RT=

Fig. 10.21: HPLC profile of Component VII: RT=4.419

herbal products 10


: RT=4.422 min

4.419 min



The HPTLC fingerprints (Fig.10.18 and Fig.10.19 ) and HPLC profile

(Fig. 10.20 and Fig. 10.21) of component VII matched with reference

standard of rutin (Rf =0.34 and RT=4.422). Thus component VII was

confirmed to be rutin.

• UV Spectroscopy: The UV spectrum was recorded in methanol for the

standard rutin and isolated component VII.

The UV λ max of standard quercetin was recorded to be 257 nm and 369 nm.

The component VII also gave 257 nm and 369 nm of λ max.



by dissolving the component VII in deuterated methanol.



Fig. 10.22: Proton NMR spectrum of Component VII

Fig. 10.23: 13

C NMR spectrum of Component VII



NMR INTERPRETATION

PMR spectrum (300 MHz, MeOD): 7.57 (s, 1H, H- 2’), 7.554 (d, J= 6.2, 1H,

H-6’), 6.80 (d, J=6.1, 1H, H-5’), 6.31 (s, 1H, H-8), 6.21 (s, 1H, H-6), 4.78

(overlap H-1”), 4.43 (H-1”’), 3.54 (m, H-3”), 3.70 (d, J=8.1, H-6”a), 3.45 (d,

J=7.08, H-6”b), 3.38 (m, H-2”/4”/ 5”), 3.32 (m, overlap, H-2”’/3”’/4”’), 1.03

(d, J=4.3, 3H, H-6”’).

13C spectrum (100 MHz, MeOD): 177.9 (C=O, C-4), 164.6 (C-7), 161.5 (C-

5), 157.9 (C-2), 157.0 (C-9), 148.4 (C-4’), 144.4 (C-3’ ), 134.1 (C-3), 122.1

(C-1’), 121.6 (C-6’), 116.2 (C-5’), 114.6 (C-2’), 104.1 (C-1”’), 103.2 (C-10),

100.9 (C-1”), 98.57 (C-6), 93.5 (C-8), 76.7 (C-3”), 75.7 (C-5”), 74.3 (C-2”),

74.2 (C-4”’), 72.4 (C-3’’’), 70.8 (C-4”), 70.6 (C-2”’), 69.9 (C-4”’) 68.3 (C-

5’’’),67.1 (C-6”), 16.4 (C-6”’).

The 1H- NMR spectrum of VII demonstrates five proton signals, shifted at

6.00 – 8.0 ppm which represents a polyphenol skeleton. Two doublet signals

7.554 (d, J= 6.2 , 1H, H-6’), 6.80 (d, J=6.1, 1H, H-5’), and three singlet 7.57

(s, 1H, H- 2’), 6.31 (s, 1H, H-8), 6.21 (s, 1H, H-6) correspond to flavonol

skeleton. The fifteen signals in 13

C- NMR spectra including seven oxygenated

carbons {164.6 (C-7), 161.5 (C-5), 157.9 (C-2), 157.0 (C-9), 148.4 (C-4’),

144.4 (C-3’), 134.1 (C-3)} and carbonyl 177.9 (C=O, C-4) signify the

quercetin aglycone.



The 1H- NMR and

13C- NMR spectra illustrate that compound VII posses two

sugar moieties with enomeric positions shift at {4.78 (overlap H-1”), 100.9

(C-1”)}, { 4.43 (H-1”’), 104.1 (C-1”’)}. Among two sugars, one is glucose

{3.70 (d, J=8.1, H-6”a), 3.45 (d, J=7.08, H-6”b) 67.1 (C-6”)}, and other is

rhamnose {1.03 (d, J=4.3, 3H, H-6”’), 16.4 (C-6”’)}. Based on PMR and 13

C-

NMR data compound VII was confirmed as Rutin.

The NMR spectra revealed presence of aromatic, hetrocyclic and protons of

phenolic group it was found to be almost identical to the NMR spectra of

standard rutin. (Fig. 10.22 and Fig. 10.23, Table 10.12 and Table 10.13).

Thus from the comparison with reference standard of rutin and from chemical

and spectral studies, component VII was confirmed to be rutin.



10.3 METHOD DEVELOPMENT

10.3.1 Preparation of standard solutions

Standard stock solutions of Kaempferol, standard were prepared by dissolving

2.1 mg of Kaempferol in methanol, yielding 10 ml of a concentration stock =

2.1 mg ml-1

(210.0 µgml-1

). From this 0.1, 0.3, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8,

3.2, 3.6, 4.0 µL of this solution were applied using LINOMAT 5 applicator

with the band width of 8 mm, which gave different concentration ranging 21-

840 ng / spot.

Similarly, working standard concentration (280 µgml-1

) of rutin and (200

µgml-1

) of quercetin was prepared by dissolving 2.8mg of rutin in and 2.0 mg

of quercetin in 10 ml of methanol. From this different volumes were applied

using LINOMAT 5 applicator in the form of band of width of 8 mm, in such a

manner to get different concentrations ranging from 56 to 840 ng/spot and 60-

1800 ng/ spot of rutin and quercetin respectively.

10.3.2 The different parameters of HPTLC such as mobile phase, band width

and detection wavelength were tried and then were optimized.

Stationary phase: The commonly used stationary phase i.e, Silica Gel 60

GF254 was used. The plates were pre-washed with methanol and activated at

60°C for 5 min prior to chromatography. As all three marker compounds were

well separated this stationary phase was optimized.

Mobile phase: Number of mobile phases combinations were tried. Such as

chloroform: ethyl acetate: formic acid, chloroform: methanol: formic acid

with different combinations. Mobile phase comprising of chloroform:

methanol : formic acid (8.2:1.5:1) gave better resolution as compared to other

mobile phases. All three compounds gave well separated and resolved peaks



with Rf 0.12, Rf 0.53 and Rf 0.69 of rutin, quercetin and kaempferol

respectively.

Band width: Band width of three different sizes 6 mm, 7 mm and 8mm were

tried. Band width of size 7 mm gave good separation without the over

saturation of the spot with sample and with maximum number of tracks were

spotted in one 20 x 10 cm TLC plate.

Detection: The TLC plate was scanned at 254nm using deuterium lamp.

The sample solutions were applied with a Camag microlitre syringe using

Camag Linomat V (Switzerland) applicator. The plates were pre-washed with

methanol and activated at 60°C for 5 min prior to chromatography. The slit

dimension was kept at 5 mm × 0.45 mm and scanning speed was 10 mm/s.

The slit bandwidth was set at 20 nm, each track was scanned thrice and

baseline correction was done.

The following densitometric conditions were used for HPTLC studies:

Stationary phase : : Precoated plates of Silica Gel 60 GF254 (Merck)

Mobile phase : Chloroform: methanol: formic acid (8.2:1.5:1)

Saturation time : 15 min

Development time :15 min

Wavelength : 254 nm

Lamp : Deuterium

Band width : 7 mm

Length of

chromatogram

: 8 cm



Linear ascending development was carried out in 20 cm x 10 cm twin trough

glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile

phase. Densitometric scanning was performed with Camag TLC scanner III in

the reflectance-absorbance mode and operated by Win CATS software (1.3.0

Camag). Concentrations of the compound chromatographed were determined

from the intensity of diffusely reflected light. Evaluation was carried out by

comparing peak areas with linear regression.

The proposed method gave very good separation and resolution of all the

three markers as indicated in Table 10.14 and Fig. 10.24.

Table10.14. Marker compounds and respective Rf Values.

Sr. No Marker compounds Rf value

1. Kaempferol 0.69

2. Quercetin 0.53

3. Rutin 0.12



Thus, the optimized HPTLC method gives a very well resolved and separated

the three marker compound’s chromatogram.

Fig 10.24: HPTLC chromatogram of Kaempferol, quercetin and

rutin using optimised parameters



10.4 METHOD VALIDATION

Validation of HPTLC method as per ICH guidelines

Analytical method validation is a process of performing several tests designed

to verify that an analytical test system is suitable for its intended purpose and

is capable of providing useful and valid analytical data. The developed

method was validated for various parameters like linearity, limit of detection

(LOD), limit of quantitation (LOQ), accuracy, precision, robustness and

system suitability as per ICH guidelines.

10.4.1 Linearity studies

Different concentrations of markers were spotted and analyzed. The analysis

was done in triplicate and the concentration range showing regression

coefficient (r2) near to one with précised value of r

2 in all triplicate analysis

was chosen.

Linearity was evaluated in the range of 21-840 ng / spot, 56-1008 ng / spot

and 60-1800 ng / spot for kaempferol, rutin and quercetin respectively. Peak

area versus concentration was subjected to least square linear regression

analysis and the slope, intercept and correlation coefficient for the calibration

curve were determined.

The concentration was found to occur in the concentration range of 84-504 ng

ml-1

for Kaempferol, 168-1008 ng ml-1

for Rutin, and 800 -1800 ng ml-1

for

Quercetin. The correlation coefficient of Kaempferol, Rutin and Quercetin

was found to be 0.9997, 0.9998 and 0.9998 respectively. The peak area (y) is

proportional to the concentration of respective marker and the regression

equations as following:



y = 12.047x + 1489.3

R² = 0.9997

0

1000

2000

3000

4000

5000

6000

7000

8000

0 100 200 300 400 500 600

y = 3.59x + 170.22

R² = 0.9998

0

500

1000

1500

2000

2500

3000

3500

4000

0 200 400 600 800 1000 1200

For Kaempferol, (x1) y = 12.047x1 + 1489.3 (Fig.10.25)

For Rutin(x2) y = 3.59 x2 + 170.22 (Fig.10.26)

For Quercetin (x3) y = 14.255x3 - 4219.3 (Fig.10.27).

Fig 10.25: Calibration curve of Kaempferol

Fig 10.26: Calibration curve of Rutin



y = 14.255x - 4219.3

R² = 0.9998

0

5000

10000

15000

20000

25000

0 500 1000 1500 2000

Fig 10.27: Calibration curve of Quercetin

10.4.2 Limit of detection (LOD) and limit of quantitation (LOQ):

LOD and LOQ were determined by using standard deviation method. A

calibration curve was prepared using concentrations in the range of 3.45-

10.35 µg/spot which is below the linearity range. Standard deviation of

residuals was measured and kept in the following equation for determination

of detection limit and quantitation limit. Detection limit =3.3σ /S and

quantitation limit=10 σ /S where σ is the residual standard deviation of a

regression line and S is the slope of the calibration curve.

The experimentally derived LOD and LOQ for all three markers were

determined as mentioned in Table 10.15.

Table 10.15: LOD and LOQ of all the three markers.

Markers LOD (ng) LOQ (ng)

Kaempferol 21 63

Rutin 56 168

Quercetin 300 600



10.4.3 Precision

Precision of the method was evaluated by repeatability (intra-day) and

instrumental precision. Each level of precision was investigated by three

sequential replicates of injections of kaempferol, rutin and quercetin at

concentrations of 168, 252 and 336 ng/spot, 336, 504 and 672 ng/spot and

1200, 1600 and 1800 ng/spot respectively.

Precision data on repeatability (intra-day) and instrumental variation for three

different concentration levels are summarized in Table 10.16.1, Table

10.16.2 and Table 10.16.3. Precision studies showed R.S.D. less than 1%,

indicating a sufficient precision

Table 10.16.1: Results of Precision Studies of Kaempferol

Type of

Precision

Intra-day Inter-day

AUC for concentration of

Kaempferol (ng/µl)


Kaempferol (ng/µl)

Sr. No 168 252 504 168 252 504

1 3515.41 4548.99 5584.16 3518.9 4556.7 5562.69

2 3527.69 4584.17 5545.18 3499.15 4547.89 5579.09

3 3536.12 4571.02 5512.22 3521.45 4569.12 5548.17

Mean 3526.40 4568.06 5547.18 3513.16 4557.90 5563.31

% RSD 0.29 0.38 0.64 0.34 0.23 0.27



Table 10.16.2: Results of Precision Studies of Rutin

Type of

Precision

Intra-day Inter-day


Rutin (ng/µl)


Rutin (ng/µl)

Sr. No 336 504 672 336 504 672

1 1409.78 1997.14 2598.16 1391.48 1987.15 2579.18

2 1387.46 1975.13 2587.78 1378.09 1974.15 2544.15

3 1389.02 1968.18 2611.1 1370.11 1990.12 2569.18

Mean 1395.42 1980.15 2599.01 1379.89 1983.80 2564.17

% RSD 0.89 0.76 0.44 0.78 0.42 0.70

Table 10.16.3: Results of Precision Studies of Quercetin

Type of

Precision

Intra-day Inter-day


Quercetin (ng/µl)


Quercetin (ng/µl)

Sr. No 1200 1600 1800 1200 1600 1800

1 15812.15 18601.2 21666.45 15845.12 18599.15 21645.16

2 15789.02 18701.11 21412.99 15877.12 18545.16 21745.18

3 15834.24 18540.33 21515.24 15909.54 18478.16 21500.69

Mean 15811.8 18614.21 21531.56 15877.26 18540.82 21630.34

% RSD 0.14 0.43 0.59 0.20 0.32 0.56



10.4.4 Accuracy

In order to evaluate the validity of the proposed method, accuracy of the

method was determined the percentage recoveries of known amounts of

mixture of kaempferol, rutin and quercetin added to sample containing a

mixture of all three standards. The analyzed samples were spiked with 80, 100

and 120 % of median concentrations (336 ng kaempferol, 672 ng rutin and

1400 ng quercetin) of standard solution in a solution containing the 100 % of

respective concentration of three standards. Accuracy was calculated from the

following equation:

[(spiked concentration − mean concentration)/spiked concentration] × 100.

The sample containing 336 ng of kaempferol, 672 ng of rutin and 1400 ng of

quercetin were spiked with the known amount of standard, and the percent

ratios between the recovered and expected concentrations were calculated.

Recoveries were obtained in the range of 81.80-118.97 %, depicting the

HPTLC proposed method for simultaneous estimation is accurate for the

quantification of all three marker compounds. (Table 10.17.1, Table 10.17.2,

Table 10.17.3)

Table 10.17.1: Recovery studies of Kaempferol

In 336 ng

Kaempferol

AUC of kaempferol

Recovery ±

S.D. (%) In sample

In

standard

In spiked

samples

268.8 (80%) 5537.09 4727.53 10729.60 104.53

336 (100%) 5537.09 5562.6 13001.06 117.13

403.2 (120%) 5537.09 7556.25 14980.09 114.41



Table 10.17.2: Recovery studies of Rutin

In 672 ng

Rutin

AUC of rutin

Recovery ±

S.D. (%) In sample

In

standard

In spiked

samples

537.6 (80%) 2579.01 2104.41 3841.80 82.03

672 (100%) 2579.01 2590.80 4195.81 81.16

806.4 (120%) 2579.01 3077.20 4626.77 81.80

Table 10.17.3: Recovery studies of Quercetin

In 2000 ng

Quercetin

AUC of Quercetin

Recovery ±

S.D. (%) In sample

In

standard

In spiked

samples

1120 (80%) 15789.01 11746.3 30299.85 110.04

1400 (100%) 15789.0 15737.7 30930.84 98.11

1680 (120%) 15789.0 19729.1 42255.88 118.97

10.4.5 Robustness

For the determination of the robustness of method, chromatographic

parameters, such as mobile phase composition and detection wavelength,

were intentionally varied to determine their influence on the retention factor

and quantitative analysis.

The mobile phase composition was altered by ± 5 % changes in the

composition of methanol. The two composition of methanol were tried 1.625

(+5% of 1.5) and 1.375 (-5% of 1.5) were tried. The chamber saturation time

was altered from 15 min to 30 min.



No changes were observed in retention time and peak shape of all three

markers with the changes made with mobile phase and chamber saturation

time (Table 10.18.1, Table 10.18.2, Table 10.18.3, Table 10.18.4, Table

10.18.5 and Table 10.18.6). The resolution and the separation of markers

were also unaltered.

10.4.6 Stability studies

Stability of the sample solutions was tested after 24, 48 and 72 hours after

preparation and storage at 4.0 °C and 25.0 °C separately. Stability was

assessed by comparing the chromatographic parameters of the solutions after

storage with the same characteristics of freshly prepared solutions of all the

three markers.

Methanol solution containing the mixture of all three markers showed 1.81 %

of degradation after 72 hrs at 4 °C and 2.59 % of degradation after 72 hrs at

25 °C of kaempferol. The rutin marker showed maximum degradation after 72

hrs of 1.41% at 4 °C and of 1.17% at 25°C. The percent of degradation of

quercetin after 72 hrs was 2.19 % and 2.91% at 4 °C and 25 °C respectively

(Table 10.19).

Table 10.18.1: Robustness (Mobile phase variation) studies of

Kaempferol

Sr.

No

Mobile phase composition (v/v)

Rf AUC

Chloroform Methanol Formic acid

1. 8.2 1.5 1 0.69 5531.03±0.45

2. 8.2 1.625 1 0.69 5531.51±0.23

3. 8.2 1.375 1 0.69 5531.86±0.42

S.D - - - 0.0 0.42



Table 10.18.2: Robustness (Chamber saturation time) studies of

Kaempferol

Sr. No Chamber saturation time (min) Rf AUC

1. 15 0.69 5531.03±0.45

2. 20 0.69 5530.99±0.31

3. 25 0.69 5531.54±0.28

4. 30 0.69 5531.05±0.14

S.D. - 0.0 0.26

Table 10.18.3: Robustness (Mobile phase variation) studies of Rutin

Sr.

No.

Mobile phase composition (v/v) Rf AUC


1. 8.2 1.5 1 0.12 2580.41±0.15

2. 8.2 1.625 1 0.12 2580.79±0.28

3. 8.2 1.375 1 0.12 2581.07±0.51

S.D - - - 0.0 0.33

Table 10.18.4: Robustness (Chamber saturation time) studies of Rutin

Sr. No. Chamber saturation time (min) Rf AUC

1. 15 0.12 2580.41±0.15

2. 20 0.12 2580.97±0.80

3. 25 0.12 2581.06±0.24

4. 30 0.12 2581.25±0.57

S.D. - 0.0 0.36



Table 10.18.5: Robustness (Mobile phase variation) studies of Quercetin

Sr.

No.

Mobile phase composition (v/v)

Rf AUC


1. 8.2 1.5 1 0.53 15787.08±0.31

2. 8.2 1.625 1 0.53 15787.48±0.24

3. 8.2 1.375 1 0.53 15788.05±0.44

S.D - - - 0.0 0.49

Table 10.18.6: Robustness (Chamber saturation time) studies of

Quercetin

Sr. No. Chamber saturation time (min) Rf AUC

1. 15 0.53 15787.08±0.31

2. 20 0.53 15787.15±0.24

3. 25 0.53 15788.06±0.14

4. 30 0.53 15787.54±0.11

S.D. - 0.0 0.45

Table 10.19: Stability Studies of solution containing three markers

Marker

compounds

Temperature

4 °C 25 °C

24 h 48 h 72 h 24 h 48 h 72 h

Kaempferol 99.87 98.10 98.19 98.93 97.39 97.41

Rutin 99.76 98.91 98.59 99.63 98.56 98.23

Quercetin 99.83 98.87 97.81 99.60 98.25 97.09



10.5 Quantitative analysis for simultaneous estimation of Kaempferol,

Rutin and Quercetin content by HPTLC

10.5.1. Procurement of plant based formulations

The following formulations were procured from the local market.

Sr. No Formulations containing

Bhuiamla Amla

1. Bhuiamla Vati Amla Vati

2. Bhuiamla Capsules Amla Tincture

10.5.2. Sample solutions

10.5.2.1 For Phyllanthus niruri Linn. extract and Emblica officinalis Gaertn

(Amla) extract

2 g of commercial methanol extracts of Phyllanthus niruri Linn. and Emblica

officinalis Gaertn each was transferred to 100 ml volumetric flask containing

50 ml of methanol and was macerated on a shaker for 24 hrs at room

temperature and volume was made upto 100ml. Then 1.0 ml of each extract

was diluted to 10 ml with methanol.

10.5.2.2 For Bhuiamla, Amla vati

2 g of powdered vati of Bhuiamla and Amla was transferred to 100 ml

volumetric flask containing 50 ml of methanol separately was macerated on a

shaker for 24 hrs at room temperature and volume was made upto 100 ml.

Then 1.0 ml of each extract was diluted to 10 ml with methanol separately.

10.5.2.3 For Bhuiamla Capsule

Sample solutions of capsule formulation were prepared same as that of vati by

transferring 2 g of capsule contents of both the plants.



10.5.2.4 For Amla tincture

10 ml of of Amla tincture (homeopathic preparation) was evaporated to

dryness. 10 mg of residue was dissolved in 10 ml methanol in a volumetric

flask.

A constant application volume of 10.0 µl/spot was employed for all the

sample solutions.

Validity of the proposed method was applied to standardization for both

traditional and modern dosage forms of three plants viz. Bhuiamla (extract,

vati and capsule), Amla (vati and tincture). The shape of the peaks was not

altered by other substances present in the matrix. The developed and validated

HPTLC method gave well resolved peaks of three respective markers in all

the formulations (Fig.10.28 and Fig.10.29). The percent content of

kaempferol, rutin and quercetin in all three products each of two plants are

indicated in Table 10.20.

The rapid, simple, precise, accurate and reproducible HPTLC method was

successfully developed and validated for simultaneous analysis of kaempferol,

rutin and quercetin in formulations containing Bhuiamla and Amla. The

various plant based products analysed were vati (an ayurvedic preparation),

capsule (a modern based formulation) and Amla tincture (homeopathic

medicine). Thus, this method can be applied for a wide range for plant based

medicines ranging from traditional to modern dosage form.

This method enabled to detect and quantified the amount of all three markers

as least as 0.199% of kaempferol to as high as 0.501 % of rutin in analyzed

plant-based products.



Table 10.20: Percent Content of Kaempferol, Rutin and Quercetin in

Bhuiamla and Amla products

Extract &

Formulati

on

Percent Content

± S.D. (%)

Weight of

formulation

per unit

(mg)

Content per unit of

dosage form (mg)

K R Q K R Q

Methanol

extract of

Bhuiamla

0.298

±0.81

0.441

±0.46

0.407

±0.30

- - - -

Bhuiamla

Vati

0.301

±0.24

0.420

±0.12

0.367

±0.88

250 0.602 1.05 0.9175

Bhuiamla

Capsule

0.278

±0.11

0.470

±0.15

0.389

±0.75

200 0.556 0.94 0.778

Methanol

extract of

Amla

0.219

±0.17

0.439

±0.060

0.322

±0.15

Amla Vati 0.212

±0.55

0.472

±0.31

0.314

±0.10

250 0.530 1.18 0.785

Amla

Tincture

0.199

±0.42

0.501

±0.15

0.306

±0.22

1000

(10 ml)

2 5 3.1

* K=Kaempferol, R=Rutin, Q=Quercetin



Fig 10.28: HPTLC chromatogram of Bhuiamla extract showing

all three markers using optimized parameters

Fig 10.29: HPTLC chromatogram of Amla extract showing all

three markers using optimized parameters



10.6 Quantitative analysis of formulations containing Clitoria ternatea

Linn (Aparajita) by HPTLC for Kaempferol content

The Clitoria ternatea Linn known as the Aparajita is an Indian Miracle Plant

which facilitates Normal Chilbirth even when a Cesarean is predicted.

Aparajita means "The Undefeated". This plant is a trailing creeper with the

usual Indigo Blue colour flowers and the rare White ones which is more of a

pale cream with a hint of green at the edges.

Leaves contain glycosides of kaempferol. The leaves are useful in otalgia,

hepatopathy and eruptions.

10.6.1. Procurement of plant based formulations

Two formulations of Clitoria ternatea Linn (Aparajita) namely Vati and

Capsules were procured from the local market.

10.6.2 Sample solutions

10.6.2.1 For Aparajita vati

2 g of powdered vati of Aparajita was transferred to 100 ml volumetric flask

containing 50 ml of methanol separately was macerated on a shaker for 24 hrs

at room temperature. Then 1.0 ml of each extracts were diluted to a 10 ml

with methanol separately.

10.6.2.2 For Aparajita Capsule

Sample solution of capsule formulation were prepared same as that of vati by

transferring 2 g of capsule contents of both the plants.

A constant application volume of 10.0 µl/spot was employed for all the

sample solutions.

The developed and validated HPTLC method for simultaneous estimation of

kaempferol, rutin and quercetin was employed to determine the content of



kaempferol alone in Aparajita containing traditional (vati) as well as modern

(capsule) dosage form. The method was well adapted and gave well resolution

of kaempferol peak in the extracts of formulations containing Aparajita

(Fig.10.30).

The percent content of Kaempferol in formulations (vati and capsule)

containing Aparajita are calculated and shown in Table 10.21.

Table 10.21: Percent Content of Kaempferol in Aparajita formulations

Aparajita

Formulations

Percent Content ±

S.D. (%) of

Kaempferol

Weight of

formulations per

unit

Content per

unit of

dosage form

Aparajita Vati 1.032±0.34 250 mg 2.58 mg

Aparajita Capsule 2.101±0.52 200 mg 4.202 mg

Fig 10.30: HPTLC chromatogram of Aprajita extract showing

Kaempferol marker using optimized parameters



The same method was successfully employed for estimation of single marker

i.e, Kaempferol in two formulations containing Aparajita (vati and capsule)

(Fig.10.24). The vati contained 1.032% of Kaempferol whereas 2.101% of

kaempferol was found to be present in capsule.

Thus, the method has a broad range of applicability to various plant products

and even various preparations of different system of medicines.

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