4. standardization and evaluation of selected...

Standardization, extraction and evaluation of selected plants

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4. Standardization and evaluation of selected plants

4.1. Introduction

A system to ensure that every single medicinal plant, part of a plant or an extract, an isolate

or an enriched portion or a product thereof, being sold has the correct substances in the

correct amount and will induce its therapeutic effect, is known as standardization. Medicinal

plants, being an important aspect of various traditional systems of medicine, have been used

therapeutically around the world. From Ayurveda to Chinese traditional medicine, from

Unani to Tibetan Medicine and from Amazonian to African Medicine, all systems of

traditional medicine, although based on different theoretical and cultural models, integrate

phytotherapy into their doctrine [1]. In high-income countries, however, the widespread use

of phytotherapy declined at the end of the first part of the twentieth century, due to the

development and production of synthetic medicines. During the past few decades, however,

phytotherapy has started to be increasingly used even in industrialized countries. In low and

middle-income countries, phytotherapy never stopped being important, often representing the

only therapeutic system preferred by certain people; in the Indian sub-continent about 70%

of the population (WHO) extensively use traditional and alternative medicines for health care

[2]. The growing use of botanicals by the public has initiated evaluation of the health claims

of these agents and steps are being taken to develop standards for their safety, efficacy and

quality. In addition, the WHO has developed a series of technical guidelines and documents

relating to the safety and quality assurance of medicinal plants and herbal materials as a

minimum requirement.

The present study is hence an effort to standardize the plant materials, prepare a suitable

extract form, and to carry out its evaluation for the development of a herbal dietary

supplement. The entire plant of Cissus quadrangularis Linn., dried exudate of Commiphora

mukul, and ripe or half ripe fresh fruits of Morinda citrifolia were selected for the study. Each

one was standardized as per the WHO guidelines.


47

4.2 Experimental

4.2.1 Materials, instruments and chemicals, given separately in list of chemicals,

instruments and equipment Page no. I-III

4.2.2 Collection and authentication

The entire plant of Cissus quadrangularis Linn., was collected from areas in and around

Erode, Tamilnadu, India and from the hilly areas of Pavagada, Karnataka, India, during the

months of April and June, 2008. The fresh ripe or half ripe fruits of Morinda citrifolia Linn.,

and the exudate of the plant Commiphora mukul Engl., (Shuddha Guggul) were obtained

from Alva’s pharmacy, Mizar, near Manipal, Karnataka, during the month of September

2008.

The plant/ plant material was authenticated by Dr. Gopalakrishna Bhat, Department of

Botany, Poorna Prajna College, Udupi, Karnataka. Voucher specimens of the plant/ materials

vide Nos. 514, 563, 571 respectively; have been deposited in the Department of

Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, India.

4.2.3 Morphology and Microscopy

4.2.3.1 Morphology [3]

Macroscopic characters of the medicinal plant material were based on shape, size, colour,

surface characteristics, texture, fracture and appearance of the cut surface.

4.2.3.2 Microscopy

4.2.3.2 .1 Anatomical studies [3,4]

Free hand sections of the specified parts were boiled with chloral hydrate to remove all the

colouring matter and then carefully stained with phluoroglucinol and HCl (1:1). The sections

were mounted as per standard procedures. Photomicrographs were made at the Department

of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal, using different

magnifications.

4.2.3.2.2 Powder analysis [5]

The dried and powered plant materials which passed through sieve no. 60 were used for

powder analysis. The macroscopic characters of the powders were observed first, following


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which, the powders were examined microscopically by mounting in chloral hydrate solution,

iodine solution and by staining with phluoroglucinol: HCl.

4.2.4 Physical/physicochemical standardisation [3, 4, 5, 6,7]

4.2.4.1 Total ash

About 2 g of the powdered drug was accurately weighed in a tared silica crucible. The

powdered drug was spread as a thin layer at the bottom of the crucible. The crucible was

incinerated at a temperature not exceeding 450°C until free from carbon. The crucible was

cooled and weighed. The procedure was repeated till a constant weight was observed. The

percentage of the total ash was calculated with reference to the air-dried drug.

4.2.4.2 Acid insoluble ash

The ash obtained as described in the determination of total ash was boiled with 25 mL of

hydrochloric acid for 5 min. The insoluble ash was collected on an ash-less filter paper and

washed with hot water. The insoluble ash was transferred into a tared silica crucible, ignited,

cooled and weighed. The procedure was repeated till a constant weight was observed. The

percentage of acid insoluble ash was calculated with reference to the air-dried drug.

4.2.4.3 Water soluble ash

The ash obtained as described in the determination of total ash was boiled for 5 min with 25

mL of hot water. The insoluble matter was collected on an ash-less filter paper and washed

with hot water. The insoluble ash was transferred into a tared silica crucible and ignited at a

temperature not exceeding 450°C. The procedure was repeated until a constant weight was

observed. The weight of the insoluble matter was subtracted from the weight of the total ash.

The difference in weight was considered as water-soluble ash. The percentage of water-

soluble ash was calculated with reference to air-dried drug.

4.2.4.4 Extractive values [8]

4.2.4.4.1 Ethanol soluble extractive

5 g of previously weighed air-dried drug was taken in a stoppered flask to which 100 mL of

95% ethanol was added. It was shaken continuously for 4 h on a magnetic stirrer. Then it

was filtered rapidly taking precautions against loss of the solvent. 25 mL of this filtrate was

evaporated to dryness in a tared flat-bottomed petri dish, dried at 105°C and weighed. The

percentage of ethanol soluble extractive was calculated with reference to the air-dried drug.


49

4.2.4.4.2 Water soluble extractive

5 g of previously weighed air-dried drug was taken in a stoppered flask to which 100 mL of

chloroform water was added. It was shaken continuously for 4 h on a magnetic stirrer. Then

it was filtered rapidly taking precautions against loss of the solvent. 25 mL of this filtrate

was evaporated to dryness in a tared flat-bottomed petri dish, dried at 105°C and weighed.

The percentage of water-soluble extractive was calculated with reference to the air-dried

drug.

4.2.4.4.3 Ether soluble extractive

5 g of previously weighed air-dried drug was taken in a stoppered flask and 100 mL of ether

was added to it. It was shaken continuously for 4 h on a magnetic stirrer. Then it was filtered

rapidly taking precautions against loss of the solvent. 25 mL of filtrate was evaporated to

dryness in a tared flat-bottomed petridish, dried at 105 °C and weighed. The percentage of

ether-soluble extractive was calculated with reference to air-dried drug.

4.2.4.5Foreign organic matter [3]

The sample (100-500 g) was spread on a white tile or a glass plate uniformly to form a thin

layer without overlapping. The sample was inspected with the unaided eye or by means of a

lens (5x or above).

The foreign organic matter was separated manually. After complete separation, the matter

was weighed and percentage w/w present in the sample was determined as described in

WHO guidelines.

4.2.4.6 Moisture content by Loss on drying [3]

About 2-5g of accurately weighed drug was dried at 100-105C for 5 h, and then weighed

again. Percentage was calculated with reference to the initial weight.

4.2.4.7 Volatile content [3]

Volatile content of the plant material was determined using Clavenger’s apparatus as

described in WHO guidelines.


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4.2.5 Phytochemical evaluation

4.2.5.1Preparation of extracts

The plant/material was either extracted successively with different organic solvents from

lowest polarity to highest polarity, or exhaustively extracted with ethanol and then

fractionated successively with solvents of lowest polarity to highest polarity.

4.2.5.1.1 Procedure

A. The dried coarsely powdered plant/ material (50 g) was successively in increasing order of

polarity extracted with n-hexane, chloroform, ethyl acetate, n-butanol and methanol by the

hot extraction process using a soxhlet apparatus. After completion of each extraction process

(4 h) the solvent was removed by distillation and the extracts were concentrated in vacuo.

The marc left after each extraction process, was dried so that it was completely free from the

solvent.

B. Plant material (3 kg) was exhaustively extracted with ethanol (95%) in a soxhlet apparatus

for 6 h. After extraction the solvent was distilled from the extract under pressure, till syrupy

consistency after which it was evaporated to dryness under pressure. The solvent free extract

(50 g) was suspended in sufficient water. The suspension was fractionated with various

solvents of ascending polarity (n-hexane, chloroform, ethyl acetate, n-butanol and methanol).

Each fraction was then separated and distilled to remove solvent and concentrated in vacuo

4.2.5.2 Physico-chemical evaluation of the fractions

4.2.5.2.1 Determination of moisture content

Same method as described above in 4.2.4.6 was employed.

4.2.5.2.2 Determination of solubility [9]

100 mg each of various fractions (n-hexane, chloroform, ethyl acetate, n-butanol) obtained

from the ethanolic extract of whole plant of C. quadrangularis were accurately weighed,

dissolved in 10 mL volume of various solvents and solubility was observed.

4.2.5.2.3 Fluorescence analysis [10,11]

The hexane, chloroform, ethyl acetate, butanol fractions and total ethanol extracts were

observed under daylight and under UV[254 nm] and the color was recorded. The test material

was further treated with different reagents namely, 1N Hydrochloric acid, 1N sodium


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hydroxide (aqueous), ferric chloride, 1N nitric acid, ammonia, iodine, 1N sodium hydroxide

(alcoholic), picric acid and 1N Sulphuric acid, and then observed for any color change in

daylight as well as under UV[254 nm].The change in colour was recorded

4.2.5.3 Preliminary phytochemical screening [7]

Each of the extracts/ fractions was subjected to various phytochemical tests to detect the

presence of various phytoconstituents such as carbohydrates (Molisch’s, & Fehling’s tests),

glycosides (Borntrager’s & Modified Borntrager’s test), saponins (Foam test), flavonoids

(Shinoda’s test), alkaloids (Mayer’s, Dragendroff’s, Wagner’s & Hager’s reagent tests),

sterols (Libermann Burchard test), fixed oils (Spot & Saponification test), tannins and

phenols (Ferric chloride, Lead acetate & Aqueous bromine solution), proteins and amino

acids (Biuret test, Ninhydrin test).

4.2.5.4 Estimation of total phenolic content [12,13]

Total phenolic content was estimated by Folin–Ciocalteu colorimetric method using gallic

acid as a standard phenolic compound.

a. Reagents - 1) Folin–Ciocalteu reagent (0.2 N)

2) Saturated sodium carbonate (75 g/l)

b. Procedure - 100 µl (two replicates) of the samples (1 mg/mL) were mixed with 900 µl of

distilled water and 5mL of 0.2 N Folin–Ciocalteu reagent. After 5 min, 4 mL of saturated

sodium carbonate (75 g/l) were added. The absorbance of the resulting blue-coloured solution

was measured at 765 nm after incubation at 30°C for 1.5 h with intermittent shaking.

Quantitative measurements were performed, based on a standard calibration curve (10, 20,

40, 80, 160 and 320 µg/mL of gallic acid in 95% methanol). The total phenolic content was

calculated as gallic acid equivalents (GAE) in milligrams per gram of dry material by the

following formula.

Where T = total phenolic compounds, mg/g plant extract, in GAE;

C= concentration of gallic acid established from calibration curve, mg/mL;

V = the volume of extract, mL;

M = the weight of ethanolic plant extract, gram.

4.2.5.3 Estimation of total flavonoid content[14]

The total flavonoid content was determined using the method of Meda et al., with minor

modifications.


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a. Reagent – 1) Aluminium trichloride (2%)

b. Procedure - 5 mL of 2% aluminium trichloride was mixed with the same volume of

sample (1 mg/mL). Absorbance at 415 nm was taken after 10 min against a blank sample

consisting of 5 mL of sample solution and 5 mL of methanol without aluminium trichloride.

The total flavonoid content was determined using a standard curve of quercetin at 1–64

µg/mL. The average of three readings was expressed as quercetin equivalents (QE) on a dry

weight (DW) basis.

4.2.5.5 Estimation of tannin content [15].

Tannin content of the samples was determined using the method of Bajaj & Dev Sharma.

a. Principle [16,17]

Phosphomolybdic-phosphotungstic acid (Folin-Denis reagent) is reduced to a blue colour

complex of tungsten and molybdenum oxide in alkaline solution by phenols. The intensity of

the blue colour produced is measured using spectrophotometer at 760 nm. Tannic acid is used

as a standard to calculate the concentration of phenols in the sample and is expressed as

tannic acid equivalent.

b. Reagents – 1) Folin-Denis reagent.

2) Saturated sodium carbonate solution. (35 g in 100 mL water)

c. Extract solution 10 mg of extract was dissolved in 10 mL of methanol.

d. Procedure

1 mL (0 - 1000 µg/mL in distilled water) of the standard tannic acid solution was pipetted

into 10-mL standard volumetric flasks containing 7.5 mL of water. 0.5 mL of Folin-Denis

reagent and 1.0 mL of sodium carbonate solution was added and diluted to the mark with

water. The solution was mixed well and the absorbance was determined at 760 nm after 30

min. Absorbance was plotted against concentration of tannic acid. Determination of samples

was carried in the same manner as standard, where 1 mL of each fraction (1 mg/mL) was

taken instead of standard and total tannin content was expressed in mg tannic acid equivalent

(TAE)/g dry weight (DW) of fraction. The blank consisted of all the reagents without the

sample.


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4.2.6 Marker based standardisation of the selected plants and their extracts

by HPTLC.

4.2.6.1 Cissus quadrangularis

4.2.6.1.1 Estimation of -sitosterol and lupeol in various fractions obtained from

ethanolic extract of the whole plant of C. quadrangularis Linn.

a. Preparation of Standard solution

The stock solutions of standard β –sitosterol and lupeol were prepared by dissolving 1mg

each of accurately weighed standards in 5 mL methanol and transferring into a 10 mL

volumetric flask. The flasks were sonicated for 10 min and the final volume of the solutions

was made up to 10 mL with methanol to get stock solutions containing 100 µg/mL.

b. Sample preparation

10 mg, each of ethanolic extract, n-hexane and chloroform fractions obtained from ethanolic

extract of C. quadrangularis were accurately weighed, dissolved in 5 mL methanol and

transferred to a 10 mL volumetric flask. The flasks were sonicated for 10 min and the

contents were filtered through Whatman No. 1 paper (Merck, Mumbai, India). The final

volume was made up to the mark with methanol. This solution (1 mg/mL) was further used

for HPTLC estimation.

c. Mobile phase - Benzene: Ethyl acetate (9.5: 0.5 v/v).

d. Estimation procedure

HPTLC was performed on 10 x 10 aluminium backed plates coated with silica gel 60 F254.

Standard solutions and sample solutions were applied on the same chromatographic plate as

bands of 2 l volume using Linomat V sample applicator equipped with a 100 μL Hamilton

syringe. Ascending chromatographic development was performed at room temperature (28 ±

2°C), with the mobile phase, in Camag twin-trough glass chamber previously saturated with

mobile phase vapour for 20min.

e. Visualization and Scanning

After development, the plate was dried, sprayed with 10% methanolic sulphuric acid,

followed by heating at 105ºC for 10 min. The plate was then scanned between 254 and 554


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nm with Camag TLC Scanner- 3.The phytoconstituent(s) content was calculated by the

formula,

AUC of sample x Conc. of std x %purity

AUC of standard x Conc. of sample 4.2.6.2 Commiphora mukul

4.2.6.2.1 Estimation of -sitosterol in hexane and total ethanol extract obtained from

Commiphora mukul Engl.

Preparation of standard/ sample solutions and content estimation were similar to that

followed for the estimation of -sitosterol in C. quadrangularis (4.2.6.1.1).

4.2.6.2.2 Estimation of guggulsterone in hexane and total ethanol extract obtained from



The stock solutions containing 1mg/mL standard guggulsterone E and Z in methanol were

prepared as mentioned above (4.2.6.1.1a).


A 1mg/mL solution of each of n-hexane and ethanolic extract of Commiphora mukul Engl. in

methanol was prepared as discussed before (4.2.6.1.1b).

c. Mobile phase – Toluene:acetone (9: 1 v/v).


HPTLC was performed in accordance with the afore said procedure (4.2.6.1.1d).

e. Visualization and Scanning

After development, the plate was dried, sprayed with 10% methanolic sulphuric acid, and

then heated at 110ºC. The plate was scanned at 366 nm and the content was estimated by

using the same formula given earlier

4.2.6.3 Morinda citrifolia

4.2.6.3.1 Estimation of -sitosterol in hexane, chloroform fractions & ethanolic extract

of fruits of M. citrifoliaLinn.


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The methodology for preparation of standard/ sample solutions and content estimation was

same as followed in the estimation of -sitosterol in C. quadrangularis (4.2.6.1.1) with same

HPTLC conditions.

4.2.6.3.2 Estimation of gallic acid in various fractions of ethanolic extract of fruits of M.

citrifolia Linn.


A 100 µg/mL stock solution of standard gallic acid was prepared by dissolving 1 mg of

accurately weighed standard in methanol as per the standard procedures.


A 1 mg/mL of sample solution was prepared by dissolving 10 mg each of ethyl acetate and n-

butanol fraction obtained from the ethanol extract of the fruit and ethanol extract in methanol

as per the standard procedures. The Solutions were sonicated (for 10 min), filtered through

Whatman No. 1 paper.

c. Mobile phase – Toluene: Ethyl acetate: Formic acid: Methanol (3:3:0.8:0.2 v/v/v/v)


The estimation was carried out as per the procedure described above. The plates were

scanned as 380nm.

4.2.6.3.3 Estimation of scopoletin in hexane, chloroform fractions & ethanolic extract of

fruits of M. citrifoliaLinn.

A 100 µg/mL stock solution of standard scopoletin was prepared by dissolving 1 mg of

accurately weighed standard in methanol as per the standard procedures.


A 1 mg/mL of sample solution was prepared by dissolving 10 mg each of ethyl acetate and n-

butanol fraction obtained from the ethanol extract of the fruit and ethanol extract in methanol

as per the standard procedures. The Solutions were sonicated (for 10 min), filtered through

Whatman No. 1 paper.

c. Mobile phase – Chloroform:acetone (9.5:0.5 v/v)


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4.2.7 Heavy metal analysis

4.2.7.1 Determination of heavy metal content [3]

Heavy metal content for crude drugs was determined by using the Atomic Absorption

Spectroscopy as per the recommendations of WHO.

4.2.8 Microbiological evaluation

4.2.8.1Determination of micro-organisms [8,9]

Presence of micro-organisms were detected as per WHO guidelines.

4.2.8.1.1Total Aerobic Bacterial Count:

1 g of sample (crude drug powder) was weighed in a sterile test-tube. It was dissolved /

suspended in 9 mL of buffered sodium chloride-peptone solution pH 7.0 (PB). Then the

contents were vortexed to form a uniform suspension. Dilutions were made upto 10-3

in PB. 1

mL of each dilution was taken in a sterile petri plate and to it, 18 mL of liquefied soyabean-

casein digest medium (TSA) was added at a temperature not exceeding 45°C. The contents

were mixed and the plate was allowed to set. These plates were then incubated at 28°C for 5

days. The plates were examined daily for the bacterial count.

The numbers of colonies formed were counted and the results were expressed in terms of

cfu/g or cfu/mL.

4.2.8.1.2 Total Yeast and Mould Count

Sample (crude drug powder, 1 g) was weighed in a sterile test-tube. It was dissolved and

suspended in 9 mL of PB. The content was vortexed to form a uniform suspension. Dilutions

were made upto 10-3

in PB. To 1 mL of each dilution taken in a sterile petri plate, 18 mL of

liquefied Sabouraud’s agar (SAB) was added at a temperature not exceeding 45°C. The

contents were mixed and the plate was allowed to set.This was followed by incubation at

23°C for 5 days and daily examination for the count. Numbers of colonies formed were

counted and the results were expressed as cfu/g or cfu/mL.

NMT: Not More Than

Table 4-1: Microbial contamination limits in medicinal plant materials; WHO [9]

Plant material/

formulation

Total Aerobic

bacterial count

Total Yeast and Mould

count

Enterobacteriaceae

count

Used for internal use NMT 105 /gm NMT 103 /gm NMT 103/gm


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4.2.8.1.3 Detection of individual pathogens

4.2.8.1.3.1 Preparation of stock

1 g of each of the samples (crude drug powder) was weighed in a sterile test-tube. It was

dissolved / suspended in 9 mL of either Lactose Broth (LB) buffered sodium chloride-

peptone solution pH 7.0 or phosphate buffer (PB). The contents were then vortexed to form a

uniform suspension. The tubes were incubated at 37°C for 2-3 h.

4.2.8.1.3.2 Escherichia coli

i. Primary test

0.1 mL of the stock in LB was transferred to 10 mL of MacConkey’sbroth and incubated at

37°C for 24 h. A subculture was prepared on MacConkey’sagar plate and incubated at 37°C

for 24 h. Growth of red, generally non-mucoid colonies indicated the presence of E.coli.

Gram’s staining was carried out when any colonies were found present. Confirmation was

done by secondary test.

ii. Secondary test

A single colony was picked from the plate and a saline suspension of it was prepared.

IMViC’s test was performed as follows

a. Indole test

2-3 loops full of suspension in peptone water (3 mL) were inoculated and incubated at

37°C for 24 h. 0.5 mL Kovac’s reagent was added. A red colour ring in alcohol phase

indicated positive results.

b. Methyl red test

2-3 loops full of suspension in Glucose phosphate broth (3 mL) were inoculated and

incubated at 37°C for 24 h. Methyl red indicator was added. A red colouration after

addition of methyl red indicated positive results.

c. Voges Proskaur test

2-3 loops full of suspension in Glucose phosphate broth (3 mL) were inoculated and

incubated at 37°C for 24 h. On addition of 1 mL 40% KOH and 3 mL α-naphthol,

presence of pink colour in 2-3 minutes indicated positive results.

d. Citrate test


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Sub-culture of suspension on Citrate agar slant was prepared and incubated at 37°C

for 24 h. Growth with bluish colouration indicated positive results.

iii. Interpretation:

Positive indole and methyl red test; with negative VP and Citrate test confirmed the

presence of E.coli.

The sample being examined passed the test if colonies of the type described did not

appear in the primary test, or if the secondary tests were negative.

4.2.8.1.3.3 Salmonella spp.

i. Primary test

1 mL of the stock in LB was transferred to 10 mL of tetrathionate bile brilliant green broth

and incubated at 37°C for 24 h. A subculture was prepared on Deoxycolate Citrate Agar and

Xylose Lysine Deoxycolate agar media; and incubated at 37°C for 24 h.

Table 4-2: Salmonella colonies on different media. Medium Description of colony Deoxycolate Citrate Agar Well developed, colourless Xylose Lysine Deoxycolate Well developed, red, with or without black centres

ii. Secondary test

A single colony from the plate was taken and its saline suspension was prepared. Further sub-

culture of suspension was prepared on Triple Sugar Iron agar slant using deep inoculation

technique and incubated at 37°C for 24 h. A change of colour from red to yellow observed in

the deep culture (but not in the surface culture) with the formation of gas with or without

production of hydrogen sulphide, confirmed the presence of salmonella spp.

iii . Interpretation

The material passed the test if no such colonies were detected or if the confirmatory

biochemical tests were negative.

4.2.8.1.3.4 Staphylococcus aureus

i. Primary test

0.1 mL of the stock in PB was transferred to 10 mL of TSA broth and incubated at 37°C for

24 h. A subculture was prepared on Baird Parker’s agar incubated at 37°C for 24 h. Presence


59

of black colonies surrounded by clear zones indicated the presence of S. aureus.

Confirmation was done by secondary test.

ii. Secondary test

Suspension in TSA broth was prepared on single colony. Tubes were then incubated at 37°C

for 24 h. 1: 10 dilution of plasma in saline was prepared and from this 1 mL was taken in a

small tube and 100 μl of above grown culture was added and then incubated at 37°C and

examined for coagulation at 1h., 2 h. and 3 h. The conversion of plasma in a soft or stiff gel

(best seen on tilting the tube to horizontal position) was considered a positive result.

iii . Interpretation:


biochemical test was negative.

4.2.8.1.3.5 Pseudomonas aeruginosa

i. Primary test

0.1 mL of the stock in PB was transferred to 10 mL of TSA and incubated at 37°C for 24 h. A

sub-culture was prepared on Cetrimide agar plate and incubated at 37°C for 24 h. Growth of

colonies which are Gram negative rods on Gram’s staining colonies indicated need for

secondary test.

ii. Secondary test

2-3 drops of freshly prepared oxidase reagent was placed on filter paper strip and smear of

the colony was applied on it. The test was positive if a purple colour was produced within 5-

10 seconds.

iii. Interpretation


biochemical test was negative.

4.2.8.1.3.6 Note

For validation of these tests a control set was always run using the following standard

cultures:


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Escherichia coli ATCC 8739

Salmonella spp.

Staphylococcus aureus ATCC 6538

Pseudomonas aeruginosa ATCC 9027

4.3 Results and Discussion

4.3.1 Standardization

4.3.1.1 C. quadrangularis

4.3.1.1.1 Macroscopy

C. quadrangularis is a rambling shrub, climbing over bushes, with thick fleshy quadrangular stem, glabrous and constricted at the nodes.

Leaves, alternate coriaceous, broadly ovate to suborbicular, 6.5 x 8 cm with serrate margins.

Flowers are small umbellate cymes, calyx cup shaped obscurely lobed. Petals are greenish yellow, red tipped and berries are globose.

4.3.1.1.2Microscopy

4.3.1.1.2.1 Transverse section of fresh quadrangular stem

A transverse section of inter- nodes showed an angular outline.

Epidermis shows a single row of cells covered with thick cuticle. Numerous stomata are

found on the epidermis.

Cortex is made of several layers of thin walled parenchyma cells. Some cells contain chloroplast , starch grains or acicular raphides of calcium oxalate crystals. At the corner internal to the epidermis three to four layers of compactly arranged sclerenchymatous cells are seen.

Cork: Next to cortex are 3-4 layers of rectangular cork cells arranged compactly without intercellular spaces.

Phellogen occurs in the sub-epidermal layer. Collenchyma occurs as discrete strands in the corners. Individual collenchyma cells are isodiametric with cellulose thickening at their angles. The endodermis is not distinguishable.

Fig 4-1: Photo showing stems and leaves of Cissus quadrangularis


61

Fig 4-3: Fresh ripe or half ripe fruit of

M. citrifolia

Vascular bundles are collateral, open and arranged in a ring around the large central pith.

Pith constitutes more than half the thickness of the stem and is composed of large

parenchyma cells, compared to the cortical parenchyma.

The microscopic assessment along with the morphological characters of the plant further

confirm its identity. The characters and their description matched that of the standards given

[18].

4.3.1.2M.citrifolia

4.3.1.2.1Macroscopy

Morinda citrifolia L. (Rubiaceae), known popularly

as Noni, is a small evergreen tree or shrub, native to

South Asia that currently grows throughout the

tropics. Leaves are broadly elliptic, bright green,

glabrous; flowers are white and in dense ovoid heads. The fruits of Morinda citrifolia are

very distinct and easy to recognize. The white tubular flowers form in clusters on the young

fruit. The syncarpous fruit grows to be about 5-10 cm long and turns from a greenish to a

translucent yellowish-white colour when the fruit ripens. The surface of the fruit is covered

with polygonal segments that surround post-floral nectarines, which continue to function

during the development of the fruit.

a

f

c

ep

ck

x

cm ph

Sc

n

st

co

r

Fig 4-2:T.S of stem of C.quadrangularis and its

magnified (10X) parts and powder characteristics.a:

air cavity; c: cuticle; ck: cork; cm: cambium; co:

collenchyma; ep: epidermis; f: fibers; n: calcium oxalate

needles; r: rosettes; ph: phloem; Sc: sclerenchyma; st:

sunken stomata; x: xylem


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4.3.1.2.2 Microscopy

4.3.1.2.2.1 Transverse section of unripe fruit

Morinda citrifolia is a syncarpous many- seeded fruit. A T.S. taken through the fruits showed

the following characters.

1. Pericarp

a) Epicarp: A single layer with small

cells.

b) Mesocarp: is the bulk of the fruit and is

made up of parenchymatous cells, in

which collateral lignified vascular

bundles arranged in a radial manner

were found. Some cells of mesocarp

were filled with calcium oxalate crystals

in the form of acicular raphides.

c) Endocarp: not distinct

2. Testa

Seed of the fruit contains a hard seed coat , the testa. It is thick , lignified and made up of

sclerenchymatous bands represented by tangentially and longitudinally running fibres.

The whole layer is lignified.

3. Endosperm

Made up of thick walled polygonal colorless parenchymatous cells containing fixed oils

and aleurone grains.

4.3.1.2.2.2 Powder characteristics of unripe fruits of M. citrifolia

1. Organoleptic evaluation

Color : Brownish black

Odour: Not characteristic

Taste: Bitter

2. Microscopic characters

Ep

Mc

T

Sc En

Fig 4.4: Microscopic characters of the fresh

fruit of M citrifolia. Ep: Epicarp; Mc:Mesocarp;

Endocarp; T: Testa; En: Endosperm; Sc:

sclerenchyma [Magnification: 10 and 40 X]


63

Fig 4-5: Photograph showing

shuddhaguggul (Commiphora mukul)

Endosperm: Polygonal colorless cells filled with aleurone grains & fixed oil.

Calcium oxalate crystals: Scattered

accicularraphides were seen in the powder.

Fibres: Group of lignified sclerenchymatous fibres

of testa layer were seen.

Few layers of parenchymatous cells of the mesocarp

were seen containing calcium-oxalate crystals in the

form of acicular raphides.

The fruits were identified as Morinda citrifolia. As the

fruit of the plant is an aggregate, it was difficult to take a TS, hence the possible sections or

parts of section were taken to see and understand the tissues in the fruit. The findings were

compared with available standards for reconfirmation of the identity.

4.3.1.3C.mukul

4.3.1.3.1 Macroscopy

Commiphora mukul is an oleo-gum-resin obtained as dried exudate by making incisions on

stems of the plantain the winter seasons. It is obtained as yellowish brown to dark brown

tears which are translucent.

Color: Brown to dark brown

Odour: Agreeable, aromatic and balsamic

Taste: Characteristic bitter

Size and shape: 0.5 to 1.00 to 2.5 cm in diameter: Rounded or irregular masses or

agglomerated tears. Tears are somewhat transparent, with waxy surface and are brittle in

nature with fractured surface.. The botanical identity was confirmed by the botanist. The

morphological characters supported its identification.

4.3.2 Physical/physicochemical standardisation [3, 4,5, 6]

In the standardization of herbal material, physical and physico-chemical factors play an

important role in the establishment of purity and quality. Ash values testify for the presence

or absence of foreign matter like silica; extractive values indicate the extractable matters in a

solvent and is an indication of possible exhausted material. Moisture on the other hand


64

indicates percentage content and possible deterioration if present in larger quantity.

4.3.2.1Ash Values

The ash values (total, acid insoluble and water soluble) of all the three selected drugs were

determined in triplicate. A mean of three determinations and its standard error was

calculated. The results were expressed as Mean±SE (table 4-3).

The ash values of all the three crude drugs were within the prescribed limits. C.

quadrangularis, showed higher total ash due to the presence of calcium oxalates. C. mukul

shows higher mineral content which is acid insoluble. The total ash of M. citrifolia must be

due to physiological ash which is why the water soluble ash is greater.

4.3.2.2 Extractive values

The amount of extractable mater in each plant material, extracted by different solvents

namely, water, ethanol and ether was determined by cold maceration, the values are tabulated

below (Table 4-4)

The results suggest that all the plants /materials have a higher content of water soluble mater

as compared to the alcohol or ether soluble content. It is especially noteworthy that, the fruits

of M. citrifolia showed richer water soluble content than any other plant /material which may

be due to the high carbohydrate, amino acid and glycoside composition. The higher ether

soluble extractive of C.mukul, compared to the other extractives is probably due to the

presence of resins and volatile oils..

Table 4-3: Ash values of C.quadrangularis, C.mukul and M. citrifolia

Parameters C.quadrangularis (% w/w)

C.mukul (% w/w)

M.citrifolia (% w/w)

Total ash 10.48± 1.24 7.5 ± 0.64 5.66 ± 0.22 Acid insoluble ash 0.38± 0.02 1.7 ± 0.13 1.0 ± 0.06

Water soluble ash 2.15± 0.44 5.6 ± 0.42 4.0 ± 0.18

Table 4-4: Extractive values of C.quadrangularis, C.mukul and M. citrifolia Parameters C. quadrangularis

(%w/w) C. mukul (%w/w)

M. citrifolia (%w/w)

Water soluble extractive 10.5 ± 0.82 6.35 ± 0.45 24.9 ± 1.6 Ethanol soluble extractive 4.0 ± 0.22 8.5 ± 0.22 16.9 ± 1.4

Ether soluble extractive 8 ± 1.14 8.10 ± 0.27 8.2 ± 1.8


65

4.3.2.3 Foreign matter [3]

The foreign matter in the different plant materials was observed by the naked eye and by the

aid of a magnifying lens(Table 4.5). Guggul, being a purified oleo-gum-resin showed little

silicaceous mater. The foreign inorganic matter present in C. quadrangularis can be

attributed to the presence of few roots in addition to the whole plant material. The fruits of

noni were freshly collected hence there was no adherent inorganic mater.

Table 4-5: Weight (%) of foreign organic and inorganic mater in C. quadrangularis, C. mukul & M. citrifolia

Parameters C. quadrangularis (%w/w)

C. mukul (%w/w) M. citrifolia (%w/w)

Foreign organic matter

Nil 1.062 ± 0.53 (Adherent bark and leaves)

1.532 ± 0.42 (fruit stalk and sepals)

Foreign in-organic matter

0.55 ± 0.02 (Silicaceous material in roots)

0.62 ± 0.04 (Silicaceous material in roots)

Nil(Fruits were thoroughly washed)

4.3.2.4Moisture content [3]

The moisture content of the dried powdered material of all the crude drugs was determined

by loss on drying method and is tabulated below (Table 4-6). Strictly, the test is moisture and

other volatile mater, as it is loss on drying, the selected material showed moisture within

limits. Some of the crude drugs were observed to be highly hygroscopic, such drugs would

have to be stored in air tight containers. M. citrifolia showed slightly higher moisture than

the other two drugs.

4.3.2.5Fluorescence analysis of the powdered crude drugs [10, 11]

The powdered crude drugs were observed under day light and UV[254] light. Change in

colour was recorded. Fluorescence of the powdered crude drugs has been studied as a

pharmacognostic character to distinguish between plants and their species.The powders were

then treated with different reagents as per the descriptions of Chase et al., [10] and Kokoski

et al., [11].

Table 4-6: Percentage of moisture in dry powders of C. quadrangularis, C. mukul and M. citrifolia

Parameters C. quadrangularis (%) C. mukul (%) M. citrifolia (%)

Loss on Drying 5.2±24 6.4 ± 0.45 8.80 ± 0.32


66

4.3.3 Phytochemical evaluation

Herbal drugs are known to contain a wide variety of chemicals which include both primary as

well as secondary plant metabolites. The pharmacological activity of a plant is generally

attributed to a specific chemical entity of the plant or a group of chemicals, for instance,

isoflavonoids in soy are believed to be responsible for the estrogenic activity. It is, therefore,

important to assess the quality of a plant material by evaluating the phytochemicals. While

phenols and polyphenols are responsible for antioxidant potential of plant, certain specific

chemicals like lupeol, guggulsterone E and Z are responsible for the specific activity. The

phytochemicals of a plant tend to vary depending on the geographical source, time, method of

collection etc., hence it is essential to evaluate drugs for their constituents before use.

4.3.3.1 Extraction and fractionation

The yield of each extract/ fraction, its physical state, colour, and consistency was recorded

(Table 4.8). Of all the selected drugs, C. quadrangularis showed the highest lipid soluble

material with a yield of about 24% in n-hexane which accounts for the waxy components of

the plant. The plant was also found to contain substantial quantities of mid-polar to polar

components. Guggul being an oleo-gum-resin showed moderate proportions of non-polar,

mid-polar and polar constituents. The consistency was sticky and semi-solid.

Table 4-7 Fluorescence analysis of the crud drug powders Sr. No.

Powder + Treatment

White light UV light (254 nm) CQ CM MC CQ CM MC

1 Powder as such Green Brownish black

Black Dark green Brown Brown

2 Powder + 1N HCL Greenish black

Dark brown

Light brown

Blackish brown

Brown Green

3 Powder + 1N H2SO4

Dark brown

Brown Blackish brown

Dark brown Brown Light green

4 Powder + 1N NaOH (Aq.)

Greenish yellow

Black Light brown

Brown Black Light green

6 Powder + Ammonia

Yellowish brown

Yellow Brown Yellowish brown

Parrot green

Parrot green

7 Powder + Iodine Brown Brown Brown brown Chocolate green

Dark brown

8 Powder + 5% FeCl3

Greenish Brown Yellowish Brown

Greenish blue

Chocolate brown

Dark brown

9 Powder + Acetic acid

Green Light brown

Brown green Black Buff

10 Powder + 1N HNO3

Yellowish brown

Light brown

Light brown

Yellowish brown

Dirty green Parrot green

11 Powder + Picric acid

Greenish yellow

Brown Yellowish brown

Yellowish brown

Brown Greenish brown


67

Table 4-8: Percentage yield ,colour and consistency of extracts and fractions of C.quadrangularis, C.mukul and M. citrifolia

Sr. No. Solvent Colour and Consistency Yield (% w/w)

CQ CM MC CQ CM MC

1. Hexane Dark green ( Waxy semisolid)

Yellow (Sticky Oil)

Yellowish green (Sticky semisolid) 23.89 8.98 1.15

2. Chloroform Dark green (Sticky semisolid)

Brown (Sticky semisolid)

Dark green (Sticky semisolid) 16.37 1.97 0.62

3. Ethyl acetate

Dark brown (Sticky semisolid)

Brown (Sticky semisolid)

Dark brown (Sticky semisolid) 18.3 1.55 3.54

4. Butanol Dark brown (Sticky semisolid)

Dark brown (Sticky solid)

Dark brown (Sticky semisolid) 18.7 1.01 3.89

5. Total ethanol

Dark brown (Sticky semisolid)

Dark brown (Sticky solid)

Buff (Sticky semisolid) 7.41 11.8

5 11.2

5


68

4.3.3.2.3 Fluorescence analysis [11, 12]

Individual fractions of the selected plants, were also observed for fluorescence pre and post treatment with different reagents (Table 4-9,4-10,4-

11)

A. C. quadrangularis:

Table 4.9: Fluorescence analysis of the fractions of C. quadrangularis

S. no

Fraction + treatment

Hexane Chloroform Ethyl acetate Butanol

White light UVlight (254nm)




1 Dried fraction Dark green Dark green Yellowish

green Dark Green Dark brown Dark brown Dark brown Dark brown

2 Fraction + 1N HCl Dark green Dark green Yellowish


3 Fraction + 1N

H2SO4 Dark green Dark green

Yellowish green

Dark Green Dark brown Dark brown Dark brown Dark brown

4 Fraction + 1N

HNO3 Dark green Dark green

Yellowish green


5 Fraction + 1N

NaOH Dark green Dark green

Yellowish green


6 Fraction + 1N

Ammonia Dark green Dark green

Yellowish green


7 Fraction + 1N

Iodine Greenish brown


Brown Dark brown Dark brown Dark brown Dark brown

8 Fraction + 1N 5%

FeCl3 Dark green Dark green Dark green Dark green Dark green Dark green Dark green Dark green

9 Fraction + 1N

Picric acid Yellowish

green Yellowish green

Yellowish green

Yellowish green Yellowish


Yellowish green

Yellowish green

10 Fraction + 1N Acetic acid

Dark green Dark green Dark green Dark green Dark green Dark green Dark green Dark green


69

B. C. mukul

Table 4.10: Fluorescence analysis of the fractions of C.mukul

S. no

Fraction + Treatment

Hexane Chloroform Ethyl acetate Butanol





1 Dried fraction Yellow Green Light brown Dark green Chocolate brown

Green Chocolate brown

Buff

2 Fraction + 1N HCl Yellowish brown

Greenish Brown Yellowish


3 Fraction + 1N

H2SO4 Yellowish brown



4 Fraction + 1N

HNO3 Yellowish brown



5 Fraction + 1N

NaOH Yellowish brown



6 Fraction + 1N

Ammonia Yellowish brown



7 Fraction + 1N

Iodine Greenish brown


Brown Dark brown Dark brown Dark brown Dark brown

8 Fraction + 1N 5%

FeCl3 Dark green Dark green Dark green Dark green Dark green Dark green Dark green Dark green

9 Fraction + 1N

Picric acid Yellowish


Yellowish green

Yellowish green Yellowish


Yellowish green

Yellowish green

10 Fraction + 1N Acetic acid

Dark yellow Dark green Dark green Dark green Dark green Dark green Dark green Dark green


70

C. M. citrifolia

Table 4.11: Fluorescence analysis of the fractions of M. citrifolia

S.no Fraction+treatment Hexane Chloroform Ethyl acetate Butanol

White light UV light (254nm)




1 Dried fraction yellowish green Light green Dark green Light brown Dark brown Dark green Dark brown Dark green

2 Fraction + 1N HCl yellowish green Green Dark green Light buff Dark brown Green Brown Green

3 Fraction + 1N H2SO4 Light green Light brown Dark green Brown Light brown Light green Light brown Light green

4 Fraction + 1N HNO3 Light green Light brown Light green Light brown Brown Dark green Dark brown Green

5 Fraction + 1N NaOH yellowish green Light green Dark green Light brown Light brown Light green Brown Dark green

6 Fraction + 1N Ammonia Light green Light brown Light green Light buff Dark brown Dark green Brown Green

7 Fraction + 1N Iodine Dark green Dark brown Dark green Chocolate

brown Dark brown Earthy brown Dark brown Dark green

8 Fraction + 1N 5% FeCl3 Light orange Dark green Orange Dark green Brownish

orange Green Orange Dark green

9 Fraction + 1N Picric acid Green Yellowish green Light green Yellowish

green Green Parrot green Light green

Yellowish green

10 Fraction + 1N Acetic

acid Light green Buff Green Light brown Light brown Green Light brown Green


71

4.3.3.3 Preliminary phytochemical screening

The results of the study are tabulated below (Table 4.12),

+ sign in the table indicates presence while – sign represents absence of the phytoconstituents

Table 4-12: Preliminary phytochemical screening of various fractions and the extracts of

C. quadrangularis, C. mukul and M. citrifolia

Test

Hexane Chloroform Ethylacetate Butanol Ethanolic

Extract

C

Q

C

M

M

C

C

Q

C

M

M

C

C

Q

C

M

M

C

C

Q

C

M

M

C

C

Q

C

M

M

C

Alkaloids - - - + + + - - - - - - - - +

Carbohydrates - - - - - - - - - - + + + + +

Phytosterols + + + + + + - + - - - - + + +

Fixed oils and

fats + + + - - - - - - - - - + - +

Saponins - - - - - - - + - + + + + + +

Phenolic

compounds

and tannins

- - - - - - + - + + + + + + +

Proteins - - - - - - - - - - - + + + +

Gums,

Mucilage - - - - - - - - - - + - - + -

Flavonoids - - - - - - + + + + + + + + +


72

Fig 4-6: Standard plot of gallic acid.

Fig 4-7: Standard plot of quercetin

Preliminary phytochemical screening is an important method to determine the basic nature of

chemical constituents present in the crude drugs. It is useful in selection of a method for

extraction and carrying out any specific assay.

4.3.3.4 Estimation of total phenolic content.

[12,13,14]

Phenols and flavonoids have been known for their

antioxidant potential. Total phenolic content is an

estimate of the antioxidant capacity of a plant on

one hand and on the other is a parameter for the

quality of the plant. The total phenolic content was

estimated using gallic acid as a reference standard and the results are given below (Table 4-

13) as gallic acid equivalents.

Note: Abs = Absorbance; GAEq = mg Gallic Acid Equivalent/g dry weight of fraction

4.3.3.5 Estimation of total flavonoid content [15].

Flavonoids have especially become synonymous with

antioxidant constituents and is hence taken as an

indirect measure of the antioxidant potential of the

crude drug. The total flavonoid content was measured

using quercetin as a reference standard and results

have been expressed as quercetin equivalents (Table

4.14)

Table 4-13: Total phenolic content of the extracts and selected fractions of C. quadrangularis, C. mukul and M. citrifolia

Fraction

C. quadrangularis C. mukul M. citrifolia

Abs GAEq Abs GAEq Abs GAEq

Ethyl acetate 0.241 118.78 0.76 374.59 1.083 53.37

n-Butanol 0.24 118.29 0.93 458.4 0.573 28.24

Ethanolic extract 0.217 106.95 0.52 256.3 0.772 38.05


73

Fig 4-8: Standard plot of Tannic acid

y = 0.008x R² = 0.9942

0

0.2

0.4

0.6

0.8

1

0 50 100 150

Ab

sorb

ance

Conc. (µg/ml)

TOTAL TANNIN CONTENT

Tannic acid

Table 4-14: Total flavonoid content of the extracts and selected fractions of C. quadrangularis, C.

mukul and M. citrifolia

Fraction C. quadrangularis C. mukul M. citrifolia

Abs QEq Abs QEq Abs QEq

Chloroform 0.039 3.29 0.012 1.02 -- --

Ethyl acetate 0.066 5.57 0.052 4.39 0.032 2.704

n-butanol 0.054 4.56 0.09 7.60 0.037 3.127


Note: Abs = absorbance; QEq = mg quercetin equivalent/g dry weight of fraction

4.3.3.6 Estimation of tannin content[16].

Tannins are one of the most widely distributed

phytoconstituents which are also endowed with

antioxidant activity. Total tannin content is an

important quality control parameter for the

evaluation of botanicals and was estimated using

tannic acid as a reference standard. Results were

expressed as tannic acid equivalents (Table 4.15).

Table 4-15: Total tannin content of the extracts and selected fractions of C. quadrangularis, C. mukul

and M. citrifolia

Fraction C. quadrangularis C. mukul M. citrifolia Abs TAEq Abs TAEq Abs TAEq

Ethyl acetate 0.868 108.14 0.611 60.29 0.609 75.87

n-butanol 0.859 107.01 0.74 76.37 0.826 102.90


Note: Abs = Absorbance; TAEq = mg Tannic Acid Equivalent/g dry weight of fraction


74

4.3.4 Marker based standardisation of the selected plants and their extracts

using HPTLC.

Standardization of any plant remains incomplete until and unless the chemical composition is

assayed. Unfortunately estimation of all the chemical constituents of a plant or an extract is

next to impossible. There is hardly any method to determine the concentration of each

chemical present in an extract. As a possible solution to this problem, marker based

chromatographic assays have been developed. Biologically active phytoconstiuents have been

used as standard markers for the standardization of medicinal plants or their purified or semi

purified extracts by suitable chromatographic techniques. High performance thin layer

chromatography (HPTLC) has been successfully used for this purpose. The simplicity of

operation and reproducibility of results have made HPTLC an important tool in the quality

control of medicinal plants. In the present study all the plant extracts and their fractions were

standardized using bioactive markers.

4.3.4.1 Estimation of -sitosterol, lupeol in various fractions obtained from ethanolic

extract of the whole plant of C. quadrangularis Linn.

The amount of -sitosterol and lupeol was estimated in the hexane, chloroform fraction and

total ethanolic extract as given in Table 4.16

β-sitosterol is one of the most abundant sterols found in plants and has shown profound

biological effects in the reduction of carcinogen induced colon cancer and inflammation [19].

Lupeol, a pentacyclic lupane-type triterpene, abundant in diverse flowering plant families is

shown to have intense biological effects on inflammation, rheumatism and cancer [20].

Inflammation and its causative factors viz., certain kinases have a significant role in the

pathophysiology of osteoporosis. Of the several minor pathophysiological pathways involved

in the pathogenesis of osteoporosis, activation of pro-inflammatory kinases is one. Agents

which suppress such kinases have been shown to have antiosteoporotic activity. C.

Quadrangularis has both of these compounds which were therefore, estimated in the total

extract and in its fractions (n-hexane and chloroform), as bioactive markers.


75

Table 4-16:Estimation of -sitosterol, lupeol in various fractions and the total extract of C. quadrangularis

S.no Fraction -sitosterol Lupeol

Rf AUC %content Rf AUC %content

1 Standard 0.15 3769.3 98 0.28 8654.3 98

2 Hexane 0.15 2131.2 5.54 0.27 2504.6 2.83

3 Chloroform 0.15 667.7 1.73 0.27 527.1 0.59

4 Ethanolic Extract 0.15 2044.4 5.31 0.27 2030.1 2.29

Lupeol -sitosterol

Track 7 ID : CQ Ethanolic Extract

3D Chromatographic profile

-sitosterol

Lupeol

Lupeol -sitosterol

Track 3 ID :CQ Hexane fraction

Track 2 ID :Betasitosterol (Std)

Track 1 ID :Lupeol (Std)

Lupeol -sitosterol

Fig 4-9: Chromatograms of standard β-sitosterol lupeol, hexane fraction and ethanol extract of C.

quadrangularis


76

4.3.4.4 Estimation of -sitosterol in hexane and total ethanolic extract obtained from


The unsaponified fraction of C. mukul was found to contain -sitosterol along with myricyl

alcohol [21]. The drug has been an Ayurvedic remedy for inflammation [22] and the activity

can be partly attributed to -sitosterol among the other responsible constituents. Hence,

guggul was standardized to -sitosterol, a bioactive marker, both in the hexane and total

extract. The content of -sitosterol in the hexane and total ethanolic extracts of Commiphora

mukul was found to be 24.40 and 21.70 % w/w respectively (Table 4-18).

Table 4-18: Estimation of -sitosterol in various fractions and the total extract of C. mukul.

S.no Fraction -sitosterol

Rf AUC %content

1 Standard 0.18 1345.8 98

2 Hexane 0.18 3354.2 24.49

3 Methanol 0.19 2912 21.70

3D Chromatographic profile of TLC plate Track 1. ID Standard β-sitosterol

Track 4. ID: Hexane fraction Track 7. ID: Total Ethanol extract

Fig 4-10: Chromatograms of standard β-sitosterol, hexane fraction and ethanol extract of C. mukul


77

4.3.4.5 Estimation of guggulsterone E and Z in hexane and total ethanolic extract of


Guggul has been used since time immemorial in traditional remedies for pain and

inflammation. Extensive research on the dried exudate has revealed its potential as an

antioxidant and antihyperlipedemic agent. Two compounds namely guggulsterone E and

guggulsterone Z have been identified as bioactive constituents. Further Ichikawa and

Agarwal have demonstrated the osteoclastic suppression activity of these compounds [23].

Total extract of shuddhaguggul was therefore standardized to guggulsterone E and Z. The

results are tabulated below (Table 4.18)

Table 4-18: Estimation of guggulsterone E and Z in the total extract of C.mukul.

S.No. Sample Guggulsterone –E Guggulsterone –Z

1.

Standards

Rf AUC % Content Rf AUC % Content

0.39 10279.6 95 0.47 12675.7 95

2. Guggul extract 0.39 5284.4 2.52 0.47 1898.6 2.20

Fig 4-11: Chromatograms showing standard peaks of Guggulsterone E & Z and that of total extract of C. mukul

Track ID 2: Guggulsteron E Track ID 3: Guggulsteron Z Track ID 4:Guggl extract


78

4.3.4.6 HPTLC estimation of -sitosterol in hexane and chloroform fractions & in the

ethanolic extract of the fruits of M. citrifolia Linn.

As discussed earlier, β-sitosterol is one of the abundant sterols which is found in many plants

including M. citrifolia [24,25]. The fruits have been used as a general tonic especially in

arthritic pain. β-sitosterol can be estimated as a marker for the assessment of quality of the

drug. Thus the fruit extract was standardized to β-sitosterol by HPTLC and results are

tabulated below ( Table 4.19)

Table 4-19: Estimation of -sitosterol in various fractions and in the total extract of M. citrifolia.

Track ID Fraction Rf AUC % content

2 -sitosterol 0.23 9229.6 98 3 Hexane 0.23 5366.4 5.69 4 Chloroform 0.23 4370.3 4.64

7 Total ethanolic extract 0.23 2706.3 2.87

Chromatographic profile of standard and extract

Track 2 ID: -sitostearol Track 3 ID: MC Hexane

Track 4 ID: MC CHCl3 Track 7 ID: MC MeOH

Fig 4-12: Chromatograms showing peaks of -sitosterol in standard, hexane, chloroform fractions and ethanolic extract of fruits of M. citrifolia Linn.


79

4.3.4.7 Estimation of Scopoletin acid in the ethanol extract of fruits of M. citrifolia Linn.

Scopoletin is one of the abundant coumarin present in the fruits of M. citrifolia and it is

biologically active [26,27]. The constituent is specific to the extract and is known to inhibit

certain cytokines like TNF-α and PGE2. Tumour necrosis factors and prostaglandins are

involved in the pathogenesis of osteoporosis [28] and hence its concentration in the extract

gives vital information. The marker was therefore estimated in the total extract of Morinda

citrifolia fruit.

The total extract of the fruit was found to contain 1.56% of Scopoletin

4.3.4.8 Estimation of gallic acid in various fractions obtained from ethanolic extract of fruits of M. citrifolia Linn.

The ethanolic extract of M. citrifolia and various other fractions were standardized to their

gallic acid content by HPTLC. The ethyl acetate fraction showed the highest percentage of

gallic acid (3.23 %) as compared to the others.

Gallic acid, being an established antioxidant that scavenges free radicals,was used as a

bioactive marker for standardization. The ethyl acetate fraction being partially purified

Table 4-19A: Estimation of Scopoletin in the total extract of M. citrifolia. Track ID Fraction Rf AUC % content

2 Scopoletin 0.53 1930.4 98

4 Noni extract 0.53 614.3 1.556

The total extract of the fruit was found to contain 1.56% of Scopoletin

Fig 4-13: Chromatograms showing standard peaks of Scopoletin and that of total extract of M. citrifolia

Track ID 2: Standard Scopoletin Track ID 4: Scopoletin peak in noni extract


80

showed a better percentage of gallic acid than that of other fractions including total ethanol

extract.

4.3.5 Heavy metal analysis

Plants are known to contain many minerals along with some metals. Iron is very commonly

found in thick fleshy green leaves. Most of the traditional Ayurvedic and Siddha formulations

have been found to contain heavy metals in large quantities (much beyond permissible

limits). It is therefore imperative to find and estimate heavy metals in the herbal raw

Table 4-20: Estimation of Gallic acid in various fractions and the total extract of M. citrifolia. Track ID Fraction Rf AUC % content

1 Gallic acid 0.56 12576.4 98

7 Ethyl acetate 0.56 4150.3 3.23

8 N-butanol 0.56 734.7 0.57

6 Total ethanolic extract 0.56 1291.5 1.006

Chromatographic profile of standard and extract

Track 1 ID: Gallic acid Track 6 ID: MC ethanolic extract

Track 7 ID: Ethyl acetate Track 8 ID: MC N-butanol

Fig 4-14: Chromatograms showing peaks of Gallic acid in standard, ethanol extract and its fractions


81

materials. This was carried out by atomic absorption spectroscopy. The results are given

below.

4.3.6 Microbiological evaluation

The total aerobic bacterial yeast and mould count was found to be within the prescribed limits

(WHO guidelines), No pathogenic organisms were observed [3] and the raw material was

found to pass the tests (Table 4.22).

Herbal raw material during its processing (collection, drying, dressing and storage), tend to

get contaminated with bacteria yeast and moulds and sometimes with some potentially

pathogenic organisms. If the material is processed improperly and especially if there is

excess of moisture in the crude drugs they are prone for microbial contamination. Any crude

material, if found contaminated, must not be processed further and must be rejected. The test

is therefore very important in the evaluation and standardization of herbal raw material..

Table 4-22: Microbial load and individual pathogen count in the plant materials of C. quadrangularis, C. mukul and M. citrifolia.

Total extracts C. quadrangularis C. mukul M. citrifolia

4.3.6.1Total Aerobic Bacterial Count (NMT 1000 cfu/g):

Total bacterial count 600 cfu/g 600 cfu/g 600 cfu/g

4.3.6.2 Total Yeast and Mould Count (NMT 1000 cfu/g):

Yeast and mould 20 cfu/ g 20 cfu/ g 20 cfu/ g

4.3.6.3Detection of individual Pathogens

E.coli Absent Absent Absent

Salomonella spp Absent Absent Absent

S. aureus Absent Absent Absent

P.aeruginosa Absent Absent Absent

Table 4-21: Estimation heavy metals in individual plant material of C. quadrangularis, C. mukul and M. citrifolia.

Metals C. quadrangularis C. mukul M. citrifolia Lead (NMT 10 ppm) Absent Absent Absent Mercury (NMT 10 ppm) Absent Absent Absent Arsenic (NMT 5 ppm) Absent Absent Absent


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4.4 Conclusion

Standardization is an important aspect of any herbal formulation development. It is important

to identify and record the physical, physicochemical and chemical properties of each plant

material that is involved in product development. The entire plant of Cissus quadrangularis,

dried, purified, oleo-gum-resin of Commiphora mukul (Shuddhaguggul) and dried half ripe

fruits of Morinda citrifolia, their respective extracts and their fractions were standardized in

accordance with the WHO guidelines. All the evaluated parameters were found to be within

the prescribed limits. The extracts and various fractions of the respective extracts were

standardized using bioactive markers like, β-sitosterol, guggulsterone E, guggulsterone Z and

gallic acid.

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