7. Formulation development and...

Formulation development and evaluation

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7. Formulation development and evaluation

7.1. Introduction

Hippocrates, the father of medicine insisted, "Let food be thy medicine and medicine be thy

food," nearly 2,500 years ago, which has found a renewed interest in the present health

conscious world. Prevention and management of chronic diseases has been prime focus today

and hence an explosion of consumer interest in the health enhancing role of specific foods or

physiologically-active food components, so-called functional foods [1]. Today herbs and

animal based products are therefore in a new shoe of segment called functional food and

dietary supplements. A dietary supplement is defined as “a product taken by mouth that

contains a „dietary ingredient‟ intended to supplement the diet that may include: vitamins,

minerals, herbs or other botanicals [2]. On the other hand Functional foods are defined by

health Canada as “ordinary food that has components or ingredients added to give it a

specific medical or physiological benefit, other than purely nutritional effect”.

Dietary supplements and functional foods make a huge market across the globe about $ 120

billion in 2007 while in India it was Rs. 18.75 billion market which was expected to grow at

33% and estimated to reach Rs.55,000 crore by 2015. While India and China still recognizing

the potential of nutraceuticals, the US has a well-established market for the botanical dietary

supplements (Cygnus Business Consulting & Research 2008) especially after DSHEA

(1994), making it a $17.1 billion market (estimated in the year 2000) [3].

A dietary supplement can be a tablet or a capsule or can simply be a powder [2].

Development of any of these formulations involve various stages first being, selection of herb

or a part of herb, its collection, authentication followed by drying and storage. The next stage

is to select a suitable dosage form which is followed by processing of the stored plant

material which includes, grinding of the plant material, extraction of the material by suitable

method and drying of the extract. The raw material and the extracts are subjected to stringent

quality control tests and evaluated for safety and efficacy of the selected raw material by in

vitro and in vivo methods and LD50 and ED50 are established. The third step is to incorporate

the standardized extract in the selected dosage form and the last stage is to evaluate the

developed formulation.

The preliminary steps for the present study, namely selection, collections, authentication and

standardization have already been carried out (chapter 4). The standardized plant material


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was extracted and the extracts were characterized and standardized for specific markers. The

extracts were subjected to intense in vitro and in vivo biological studies for antioxidant and

antiosteoporotic potentials and effective dose of each extract was established along with their

safety profile (chapter 5). Based on the efficacy of individual extracts a blend was formulated

and an elaborate study has been carried out which is described in chapter 6. The present

work, therefore, aims at development and evaluation of a tablet dosage forms as herbal

dietary supplement. In the foregoing chapter the development of tablet dosage form on the

test blend will be explained.

7.2 Experimental

7.2.1 Analytical methods

7.2.1.1 Preparation of hydrochloric acid buffer (pH- 1.2)

To the 50 ml of 0.2 M KCl in a 200 ml volumetric flask, 85 mL of 0.2M HCl was added and

then water was added to volume.

7.2.1.2 Preparation of phosphate buffer saline (pH- 7.4)

To the 50 ml of 0.2 M potassium dihydrogen phosphate in a 200 mL volumetric flask, 39.1

mL of 0.2 M sodium hydroxide was added and water was added to volume

7.2.1.3 Standard plots of lupeol, scopoletin and guggulsterone

1 mg/mL solution of each of the standards, lupeol, scopoletin and guggulsterone E was

prepared in methanol. Different concentrations 10 to 100 µg/mL solutions of each of the

standards were prepared and the absorption was measure at 365 nm (for lupeol), 228 nm (for

scopoletin) and 281 nm (for guggulsterone E). A linearity curve was then plotted

concentration vs. absorbance for each of the standard.


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7.2.2 Dose Selection

A dose of 500 mg/kg b.w. of the test combination was found effective in our previous study.

A human equivalent dose (HED) was calculated according to FDA requirement [4] using

following formula.

HED = Animal dose (mg/kg) x [Animal Km/Human Km]

Table 7-1Concentratins and absorbance of standards.

Concentration

(µg/ml) Absorbance of

lupeol at 365 nm. Absorbance of scopoletin

at 228 nm.

Absorbance of guggulsterone-E

at 281 nm.

10 0.071 0.124 0.133

20 0.173 0.207 0.227

30 0.273 0.297 0.327

40 0.381 0.389 0.423

50 0.497 0.471 0.541

60 0.613 0.567 0.648

70 0.738 0.658 0.761

80 0.853 0.769 0.893

90 0.976 0.879 1.012

100 1.106 0.997 1.139


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Table 7-2: conversion of Animal dose to HED and BSA.

The above values are based on data from FDA Draft Guidelines [4]. To convert dose in

mg/kg to dose in mg/m2, multiply by Km value.

7.2.3 Pre-formulation studies

7.2.3.1 Pre-formulation studies include studies of-

Assessment of physicochemical properties of herbal extracts and their relevance to the

final formulation.

The physical and chemical stability of the herbal extracts.

Compatibility studies of the herbal extracts with potential excipients.

7.2.3.2 Compatibility studies

7.2.3.2.1 Sample preparation for physical compatibility studies

Binary/ tertiary blends of extracts and excipients were stored at 40C (refrigerator) as control,

and at 250C at room temperature for 4 weeks. The observations (colour, flow, and sticking)

were recorded every week.

7.2.3.2.2 Sample preparation for chemical compatibility studies by HPTLC

Compatibility between herbal extracts and between extracts and excipients was determined

by HPTLC using specific markers and the finger print. β-Sitosterol, lupeol, guggulsterones E

& Z and scopoletin were used as markers for Cissus quadrangularis, Commiphora mukul and

Morinda citrifolia respectively. Individual extracts, their binary mixture in all combinations

and tertiary mixtures were mixed together at least 1 hour before analysis. The mixtures were

then ground and known quantities of the mixtures were sonicated in quantity sufficient


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methanol to dissolve the ingredients thoroughly. A known strength of the mixture and

individual extracts was then prepared. Known concentrations of the standards were also

prepared and HPTLC was carried out as described in chapter 4 for individual markers.

7.2.3.2.3 Sample preparation for solid state compatibility studies by DSC-

Drug-drug and drug- excipients mixtures were prepared in 1:1 ratio and pure drug samples

were weighed and all the samples were subjected for the DSC. The thermograms obtained

were compared for any differences.

7.2.4 Tablet formulation

7.2.4.1 Preparation of blend and characterization

Various batches of the blends namely F1 to F9 were prepared keeping active composition

constant and varying the excipients as per the formula.

Active Extracts: Ethanol extract of C. quadrangularis and ethanol extract of M. citrifolia

were converted to dry powder by Lyophilisation. The ethanol extract of C. mukul was oily

lyophilisation was not possible and hence adsorbed on known quantity of diluent or filler to

form a free flowing powder.

The Cissus and Morinda dry extracts absorbs moisture (highly hygroscopic) hence the

powder was pulverized with known weight of diluent/ filler to minimize moisture absorption.

The extract diluent mixture was passed through sieve 60 on a tray and rest of the ingredients

were added by passing through sieve 60 as well. The mixture was then mixed thoroughly or

pulverized. .Different blends were packed in airtight containers separately and labelled until

further use.

The blends were then characterized for various parameters like flow property, moisture

content and the batch that meet the parameters was selected for final formulation.

7.2.4.2 Characterization of the powdered blend

Bulk density [5]

Tapped density [5]

Angle of repose [6]

Carr‟s index IC% = Density Tapped Density Bulk/Density Tapped

Hausner‟s ratio = Density Tapped / Density Bulk


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Table 7-3: Relation of flow property with powder parameters.

Flow property Angle of repose

(degrees)

Carr’s index Hausner Ratio

Excellent 25-30 10 1.00-1.11

Good 31-35 11-15 1.12-1.18

Fair 36-40 16-20 1.19-1.25

Passable 41-45 21-25 1.26-1.34

Poor 46-55 26-31 1.35-1.45

Very poor 56-65 32-37 1.46-1.59

Very very poor >66 >38 >1,60

7.2.4.3 Evaluation of Dosage Form

7.2.4.3.1 Weight variation [7]: For carrying out this test, 20 tablets were taken randomly and

weighed. The average weight was calculated, and then each tablet was weighed individually

and their weights were compared with the average weight.

7.2.4.3.2 Hardness [6]: The hardness of tablets is defined as the force required to break the

tablet in diametric compression test. The hardness of three tablets from each formulation was

determined by Monsanto hardness tester. Hardness was expressed in kg.

7.2.4.3.3 Friability [6] Tablet hardness is not absolute indicator of strength since some

formulations, when compressed into hard tablet tend to cap on attrition, losing their crown

positions. Six weighed tablets were place in friabilator and operated for 4 min at 25 rpm. The

tablets were made free from dust and reweighed. The % friability is determined as difference

in weight *100. The acceptable limit of friability is less than 1%.

7.2.4.3.4 Disintegration [8] Six tablets were placed in the tubes along with a plastic disk

over the tablets. The disk imparts pressure on the tablets. The tubes were allowed to move up

and down in the media as 29- 32 cycles per minute in water media maintained at 37°C. Time

required to pass all tablets through the mesh is determined as its disintegration time.

7.2.4.3.5 Drug content (Estimation of Lupoel, Scopoletin and Guggulsterone E.)- Three

tablets from different batches were randomly selected and crushed separately in a mortar. The


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contents were sonicated with suitable quantity of methanol and filtered. The mark was

washed carefully and the volume of the extract was made up to produce a 1 mg/ml solution.

Assay for the reference standards was performed by the method described in chapter 4 by

HPTLC as per the procedure given earlier.

7.2.4.3.6 Dissolution [8]: The drug release is studied by using six panel USP XXIII

dissolution apparatus 2 at 50 rpm. The dissolution study was studied in 900 ml of acidic

buffer (pH 1.2) and phosphate saline buffer (pH 7.4) maintained at 37 ± 20C. Samples were

taken at appropriate time intervals for analysis. After every withdrawal an equal amount of

fresh dissolution medium was added to the flask. The study was carried out over a period of

24 hrs. All the tests were performed in duplicate. The release of markers were determined by

UV/Vis spectrophotometer

7.2.4.3.7 Real time stability studies

The formulation being herbal tablet containing no thermo labile substances the prescribed

storage conditions for the formulation are, room temperature (25 °C ± 2 °C) with relative

humidity of about 60 ± 5 %. The tablet being herbal generally no degradation is expected and

hence, a real time or long term stability testing was carried out at end one year as per the ICH

guideline [9] by HPTLC estimation of drug (Lupeol, scopoletin and guggulsterone E)

content. The content of reference standard was measured and compared with that of original

values.

7.3 Results and discussion

7.3.1 Dose selection

As described in the previous chapter, a 500 mg/kg b.w of the combined mixture of the three

ethanol extracts of C. quadrangularis, M. citrifolia and C. mukul was found safe and effective

in the reversal of osteoporotic conditions. To develop a formulation the animal dose was

converted to human equivalent does (HED) as per US FDA recommendations [10]. The

calculations for determining starting dose in humans as extrapolated from animals should use

the more appropriate normalization of body surface area (BSA) [10, 11] which can be made

by multiplying the animal dose in mg/kg with Km factor of the animal [12]. In our study the

effective dose being 500 mg/kg an equivalent HED would be 81.08 mg/kg which by

calculation a 600 mg was decided as unit dosage. Calculations are given below


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500 (animal dose in mg/kg) x 6 (Km factor for 200 g rat)/ 37 (Km Factor for 60 kg human) =

81.08 mg/kg = 4864.86 mg/day for a 60 kg individual.

It was accordingly decided to make it in 4 divided doses 4864.86/4 = 1216.21.

1216.21 mg of the blend must therefore be carried by 2 tablets. 1216.21/2 = 608.108 mg

Hence the dose of blend for the tablet was decided as 600 mg.

7.3.2 Pre-formulation studies

7.3.2.1 Compatibility studies

Incompatibility is generally chemical interactions or reactions, between the active ingredients

or some times between the active ingredients and excipients [13], if the content (actives/

excipients or both) are reactive or made reactive in presence of each other or in the conditions

provided. These reactions may manifest into change in colour, degradation of the formulation

or development of foul smell or sometimes formation of newer substances which may be

harmful [14] and most importantly loss of activity. The physical interactions can be observed

by change in colour, consistency, odour and taste of the mixture while the chemical changes

can be assessed through FTIR or HPLC or HPTLC. HPTLC is a sensitive instrument that can

be used as tool for determination of chemical interactions. Howida et al. [15] used TLC for

the determination of interactions, HPTLC is more sophisticated instrument hence it was used

for the purpose in our study.

In the herbal preparations, a number of active ingredient are involved, understanding the

reactivity of theses extracts in solid state when mixed together and with excipient is critical to

commercial formulation development [16]. In determining the drug-drug and drug excipient

interaction, thermoanalysis offers significant advantages in saving both time and substance

[17]. Thus differential scanning colorimeter (DSC) can provide valuable information as

thermal transitions associated with the active ingredients can be observed [18] and hence it

has been used most frequently for detecting such incompatibilities. In the present study the

drug or actives being the plant extracts which are multi component semisolid mixtures, the

thermal data must be supported with other methods like FTIR [19]. In our study HPTLC has

been used as a supportive method for identifying possible incompatibilities.

7.3.2.2 Chemical incompatibility studies by HPTLC

HPTLC is a reliable tool for the detection of chemical incompatibility especially when herbal

extracts are involved. The individual extracts used in the formulation have been standardized


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to specific marker compound(s). The markers and the chromatogram of a specific extract

were taken as a standard. The presence of both marker as well as the undesignated peaks in

the chromatogram at least, the major peaks when two or more extracts or excipients were

combined and eluted in homogeneous chromatographic conditions signifies the compatibility.

Our HPTLC results showed no interactions between any of the combinations after mixing in

1:1 (binary mixture) or 1:1:1 (tertiary mixture) ratios of the active extracts and with the

excipients.

7.3.2.2.1 Compatibility studies using β-sitosterol and lupeol as reference standards

Solvent System: Benzene: Ethyl acetate (9.5: 0.5 v/v).

Visualization: 10% methanolic sulphuric acid followed by heating at 110 °C for 5 min

Scanning wave length: 366 nm

Fig 7.4A Standard chromatograms.

Fig 7-4A a: Chromatogram of standard β-sitosterol Fig 7-4A b: Chromatogram of standard Lupeol

Fig 7.4B Chromatograms of individual extracts and mixtures in the solvent system.

Fig 7.4B a: Chromatogram of C. quadrangularis

Fig 7.4B b: Chromatogram of C. mukul

Fig 7. 4B c: Chromatogram of M. citrifolia


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Mixture of the extracts 1:1

Fig 7. 4B d: Chromatogram of mix of C. quadrangularis and C. mukul

Fig 7. 4B e: Chromatogram of mix of C. quadrangularis and M. citrifolia

Fig 7. 4B f: Chromatogram of mix of M. citrifolia and C. mukul

Tertiary mixture and blend with excipients

Fig 7. 4B g: Chromatogram of mixture of all three extracts C. quadrangularis, M. citrifolia and C. mukul

Fig 7. 4B h: Chromatogram of mixture of 3 extracts C. quad, M. citrifolia and C. mukul and excipents (Blend)

Fig 7. 4B i: UV Spectral matching of the standards

Table 7-4 Compatibility chart with reference to different peaks.

Peaks Cq Cm Mc Cq+Cm Cq+Mc Cm+Mc Cq+Cm+Mc Blend

Unknown -0.01 0.02 0.01 0 0 0.01 -0.02 -0.02

Unknown 0.01 ** 0.08 ** ** ** 0.02 0.02

Unknown 0.17 0.17 ** 0.17 0.17 0.17 0.18 0.19

β-sitosterol 0.25 0.26 0.25 0.25 0.25 0.26 0.26 0.26

Unknown ** 0.31 ** 0.31 ** 0.32 0.32 0.32

Lupeol 0.4 0.4 0.4 0.41 0.41 0.41 0.42

Unknown ** ** ** ** ** ** ** 0.83

Unknown ** ** 0.86 ** 0.88 0.88 0.89 0.89

β-sitosterol and lupeol are the reference standards used for the compatibility study for Cissus

quadrangularis. β-sitosterol was present in all the extracts and their combinations in all ratios

as well as the blend showed the peak. Lupeol being specific to the C. quadrangularis at

appeared only in those combinations in which C. quadrangularis was present. The


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chromatograms (major peaks) of each extract could be located in the combination (Table 7-

4). Thus no sign of incompatibility was found in the combinations.

7.3.2.2.2 Compatibility studies using Scopoletin

as reference standards

Solvent system: Chloroform: acetone (9.5:0.5)

Visualization: UV 366 nm

Fig 7-5B: Chromatograms of individual extracts.

Fig 7-5B a: Chromatogram of C. quadrangularis

Fig 7-.5B b: Chromatogram of C. mukul

Fig 7-5B c: Chromatogram of M. citrifolia

Fig 7-5C: Chromatograms of binary mixtures (1:1) of the extracts.

Fig 7-5C a: Chromatogram of C. quad + C. mukul

Fig 7-5C b: Chromatogram of C. quad + M.citrifolia

Fig 7-5C c: Chromatogram of M.citrifolia + C. mukul

Fig 7-5D: Chromatograms of Tertiary mixtures (1:1:1) of the extracts and the blend with excipients.

Fig 7-5D a: Chromatogram of C. quad+C.mukul+M.citrf

Fig 7-5D b: Chromatogram of blend along with Xcpients

Fig 7-5D c: UV absorption spectra of Scopoletin

Fig 7-5A Standard chromatogram.


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Table 7-5: Compatibility chart with reference to different peaks.

Peaks C.quad C. mukul M. citri C.q+Cm C.q + Mc Cm+Mc Cq+Cm+Mc Blend

Unknown ** 0.06 ** ** 0.1 0.1 0.03 **

Unknown ** ** ** ** ** ** 0.06 0.06

Unknown ** 0.11 0.11 0.11 ** ** 0.11 0.11

Unknown ** 0.28 ** 0.28 ** 0.28 0.28 0.28

Scopoletin ** ** 0.36 0.36 0.36 0.37 0.37

Unknown ** 0.67 ** 0.67 ** 0.68 0.68 0.68

Unknown ** 0.73 ** 0.72 ** ** 0.73 0.73

Scopoletin is a specific marker for M. citrifolia. The marker and it associated chromatogram

in the M. citrifolia extract was taken as a standard for the compatibility study. All the peaks

of the standard chromatogram could be observed in all combinations involving M. citrifolia

extract (Table 7-5) convincing that there is no sign of any incompatibility between any

combination(s).

7.3.2.2.3 Compatibility studies using Guggulsterone E and Z as reference standards

Solvent system: Toluene:acetone (9: 1 v/v). Visualization: UV 366 nm

Fig 7-6A Standard chromatograms.

Fig 7-6A a: Chromatogram of Gst E

Fig 7-6A b: Chromatogram of Gst Z

Fig 7.6B: Chromatograms of individual extracts.

Fig 7-6B a: Chromatogram of C. quadrangularis

Fig 7-6B b: Chromatogram of C. mukul

Fig 7-6B c: Chromatogram of M. citrifolia


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Fig 7-6C: Chromatograms of binary mixtures (1:1) of the extracts.

Fig 7-6C a: Chromatogram of C. quad + C. mukul

Fig 7-6C b: Chromatogram of C. quad + M. citrifolia

Fig 7-6C c: Chromatogram of M. citrifolia + C. mukul

Fig 7-6D: Chromatograms of Tertiary mixtures (1:1:1) of the extracts and the blend with excipients.

Fig 7-6D a: Chromatogram of C. quad+C. mukul+M. citrf

Fig 7-6D b: Chromatogram of blend along with Xcpients

Fig 7-6D c: UV absorption spectra of Scopoletin

Table 7-6: Compatibility chart with reference to different peaks.

Peaks C.quad C. mukul M. citri C.q+Cm C.q + Mc Cm+Mc Cq+Cm+Mc Blend

Unknown ** 0.1 ** ** 0.09 0.11 0.1 0.11

Unknown ** ** 0.16 0.17 ** 0.17 0.16 0.18

Unknown ** 0.27 0.25 0.26 0.25 0.29 0.28 0.28

Unknown ** 0.3 0.28 0.29 0.28 0.32 0.31 0.31

Gug. E ** 0.4 ** 0.41 ** 0.43 0.43 0.42

Gug. Z ** 0.48 ** 0.48 ** 0.52 0.53 0.5

Unknown ** 0.59 ** 0.59 ** 0.63 0.66 0.61

Unknown 0.77 0.78 ** 0.78 0.77 0.74 86 0.78

Guggulsterone E and Z are specific markers for Commiphora mukul. The markers and

associated chromatogram in the C. mukul extract was taken as a standard for the

compatibility study. The chromatogram (major peaks) of the C. mukul extract and that of

other extracts located in all combinations (Table 7-6) evidencing that there is no sign of any

incompatibility between any combination(s).


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The HPTLC analysis of the extracts and their combinations along with excipients using

standard markers convincingly suggested that there is no chemical incompatibility between

the extracts; extracts and excipients.

7.3.2.3 Solid state incompatibility studies by DSC

The incompatibility studies between the multi component mixtures (extracts) using DSC

showed no sign of incompatibility. In our study all three pure extracts showed a common

exothermic peak between 132°C and 147°C apart from other peaks suggesting a common

phytoconstiuent in all three extracts. Literatures as well as our HPTLC analysis showed that

β-sitosterol is present in all three extracts. The melting point of β-sitosterol was found to be

136 °C, while the common peak fluctuate between 132°C and 147°C . The peak can therefore

be assigned as β-sitosterol and fluctuations can be attributed to the presence of other

constituents which alter the melting point. The peak and the thermographic pattern could be

observed in all the combinations indicating no physical incompatibility between the extracts.

Fig 7-7: Thermograms of individual extracts C. quadrangularis, C. mukul and M. citrifolia.

Fig 7-8: Thermograms of mixture of extracts C. quadrangularis, C. mukul and M. citrifolia.

Table no. 5.19:- Peak

100.00 200.00 300.00

Temp [C]

0.00

5.00

10.00

mW

DSC

148.09x100COnset

152.74x100CEndset

149.49x100CPeak

-1.29x100mcalH eat

124.47x100COnset

135.49x100CEndset

130.11x100CPeak

-6.93x100mcalH eat

44.39x100COnset

66.87x100CEndset

54.55x100CPeak

-16.30x100mcalH eat

Cissus+Noni

100.00 200.00 300.00

Temp [C]

-10.00

0.00

10.00

mW

DSC

122.58x100COnset

135.08x100CEndset

125.95x100CPeak

-26.77x100mcalH eat

Guggal+Noni


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Table: 7-7: Thermal events of individual extracts and their mixtures.

S. No. SAMPLE PEAK VALUE(0c) ONSET

VALUE(0c) ENDSET

VALUE(0c) HEAT

(m cal.)

1 Cissus Peak 1 55.46 46.78 73.15 -9.77

Peak 2 147.94 142.66 148.89 -42.17

2 Guggul Peak 1 33.08 28.94 42.66 -6.12

Peak 2 132.40 120.64 133.76 -16.88

3 Noni

Peak 1 126.92 123.31 133.40 -99.28

Peak 2 141.85 140.13 146.94 -9.13

Peak 3 170.67 169.45 174.15 -2.53

4 Cissus+Noni

Peak 1 54.55 44.39 66.87 -16.3

Peak 2 130.11 124.47 135.49 -6.93

Peak 3 149.49 148.09 152.74 -1.29

5 Cissus+Guggul Peak 1 85.48 81.06 93.04 -4.68

Peak 2 134.04 124.71 142.9 -94.55

6 Guggul+Noni Peak 1 125.95 122.58 135.08 -26.77

7.3.3 Tablet formulation

7.3.3.1 Preparation of blend and its characterization

Until late 1950s the vast majority of tablets produced in the world were manufactured by a

process requiring granulation of the powdered constituent prior to tableting. The advent of an

alternative technology called “Direct compression” with many advantages made tableting

economical and hazel free [20]. In the present study, direct compression was therefore

adopted for punching of the tablets.

The technology though looks simpler it requires critical approach to the sections of raw

material, their physical and physicochemical properties. It requires optimum flow properties

of the blend and knowledge of the effects of formulation variable on compressibility index of

the powdered blend [20]. In the current study the active raw material is a combination of

three biologically active ethanol extracts. Two of them (ethanol extract of Cissus

quadrangularis and that of Morinda citrifolia) were converted to dry powder by

lyophilisation while the third extract (ethanol extract of Commiphora mukul) was oily and


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remained semisolid. The dried powdered extracts were highly hygroscopic and absorbing

moisture rapidly.

Mannitol DC is specialized filler for direct compressible tablet which is insensitive to

moisture and retains its flow properties [20]. Anhydrous lactose is important direct

compressible filler most commonly used in tableting. It has excellent fluidity and is relatively

non hygroscopic [21] and hence was used. Both excipients were tried in separate batches.

Known weights of the dried lyophilised extracts were homogenized in a mortar together with

known weight of a diluent (mannitol or lactose) and the mixture was passed through mesh 60

to minimize moisture absorption. Guggul extract being semisolid, a known weight of the

extract was adsorbed on a known quantity of diluent (mannitol or lactose) by trituration in

mortar till a free flowing powder was obtained.

Microcrystalline cellulose (Avicel) and starch are specialized dry filler binders frequently

used in direct compression. Avicel is the most compressible binder and has highest dilution

potential, however it is a hygroscopic powder that absorbs moisture. Starch on the other hand,

has been a subject of argument with respect to its fluidity. Various modifications have been

done to improve its fluidity. Starch 1500 has better fluidity and compressibility. PVP (Poly

vinyl pyrolidone) is also used in the direct compression as a filler binder. It is a very strong

binder which forms stable complex with active ingredients [22]. Starch has been known to

disintegrate tablets by swelling and so is MCC which dissolves easily in water [20]. Fluidity

is an important criteria for the direct compression, for which talc is best used in the oral

dosage forms, however, it is a known retarder of dissolution rate [23,24,25] and hence its use

must as minimal as possible.

Table 7-8: Composition of the blend for a 600 mg tablet (in weight).

Ingredient

Different trial batches of 50 tablets each

Quantity per tablet in mg

F1 F2 F3 F4 F5 F6 F7 F8 F9

Actives

Cissus 225 225 225 225 225 225 225 225 225

Noni 225 225 225 225 225 225 225 225 225

Guggul 150 150 150 150 150 150 150 150 150

Excipients

MCC 24 39 42 50 30 - - - -

Mannitol 60 72 60 55 60 - 75 62 50

Talc 36 9 18 15 30 32 22 22 18

Starch - - - - - 30 20 20 30

PVP - - - - - 22 5 5 10

Lactose - - - - - 36 - 10 20

Weight/tab 720 720 720 720 720 720 722 719 728


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Different batches (9 batches) with varying ratios of these excipients were formulated and a

blend was prepared. As the direct compressibility depends on various factors, the blend was

characterized for flow properties by measuring, angle of repose, compressibility by Cars‟

index and moisture content. Of 9 batches F7 and F9 showed good flow properties and better

compressibility, F7 was found best in its flow properties.

The flow properties and compressibility of each batch of the blends was tested as per the

standard procedures. The bulk and tap densities of a solid represent inter-particulate

interaction and friction in the powder that determine the flow and compressibility [26]. Lesser

the friction/ interaction better is the flow and compressibility. Compressibility index and

Husners‟s ratio are the measures of the propensity of powder to be compressed. Both are the

relative to inter-particulate interactions given by density measurement. Angle of repose or

critical angle of repose [27] is the coefficient of static friction [28] which is a function of

fluidity of the powder and the moisture plays a significant role in the flow of powder. Hence

the measurement becomes crucial for the success of a formulation.

Nine different batched were made by varying the excipients. Mostly the excipients play a

vital role in the flow and compressible properties of the powder. In our study Batch F7

showed excellent characteristics. While F9 showed good fluid and compression

characteristics, F2 and F8 showed acceptable range. Rest of them were found to be poor or un

acceptable. All batches irrespective of their properties were punched into a tablet using 12

mm dye in a manually operated single punch machine to get a round convex tablet of

approximately 0.75 cm2

thickness.

Table 7-9: Composition of the blend for a 600 mg (mg%).

Ingredients Different trial batches of 50 tablets each

Quantity per tablet in mg%

F1 F2 F3 F4 F5 F6 F7 F8 F9

Cissus 31 31 31 31 31 31 31 31 31

Noni 31 31 31 31 31 31 31 31 31

Guggul 21 21 21 21 21 21 21 21 21

MCC 3 5 6 7 4 -- -- -- --

Mannitol 8 10 8 8 8 -- 10 7 7

Talc 5 1 2 2 4 4 3 3 3

Starch -- -- -- -- -- 8 3 3 4

PVP -- -- -- -- -- 3 1 1 1

Lactose -- -- -- -- -- -- -- 1 3


157

Table 7-10: Characterization of the various blends.

Tablet

batch

Bulk

density

(g/ml)

Tapped

density

(g/ml)

LO

D

%

Hausne

r

Ratio

Carr’s

Index

%

Angle

of

repose

Flow pr—

operty

F1 0.33 0.44 2 1.3 25 26 Poor

F2 0.29 0.46 2.2 1.6 37 32 Passable

F3 0.28 0.43 1.89 1.5 35 28 Poor

F4 0.34 0.47 1.8 1.4 28 42 Poor

F5 0.31 0.58 1.3 1.9 47 23 Poor

F6 0.22 0.52 1.36 2.4 58 47 V. poor

F7 0.38 0.48 1.2 1.3 21 21 V. Good

F8 0.34 0.44 1.8 1.3 23 24 Passable

F9 0.37 0.49 2 1.3 24 28 Good

7.3.3.2 Tablet evaluation

The obtained tablets were evaluated for pharmacopoeial physicochemical tests like; tablet

hardness, friability, weight variation and dissolution studies. Disintegration was omitted

because the tablets were showing sustained release properties in the dissolution test.

Table 7-11: Tablet evaluation.

Tablet

batch

Color

&

Shape

Thickness (cm2)

Hardness

(kg/cm2)

Friability (%)

Weight

variation

(mg%)

F1

Dar

k b

row

n;

rou

nd

con

vex

0.75±0.031 2.5 ± 0.22 0.92±0.046 4.6 ± 0.002

F2 0.73±0.053 4.3 ± 0.14 0.44±0.052 3.9 ± 0.14

F3 0.73±0.031


158

variation must be least. In the current study all batches showed minimum variation in the

weight mostly because the punching was done in a manually operated single punch machine.

Of all the batches batch number F2 and F7 showed best results in evaluation. In addition, the

blend of the F7 batch showed excellent flow properties and good compressibility prompting

us to select this batch for further studies.

7.3.3.2.1 Estimation of drug content by HPLTC

The drug content in our study was carried out by estimation of the specific markers in the

tablet formulation by HPTLC. Each extract has already been standardized to specific marker

by its estimation using HPTLC.

7.3.3.2.1.1 Estimation of lupeol

Each tablet contained 225 mg of ethanol extract of Cissus quadrangularis standardised to

2.29% of lupeol. Thus each tablet is expected to contain 5.15 mg of lupeol

Fig 7-9 Chromatograms of Standard Lupeol and the formulation.

Fig 7-9a Chromatograms of Standard Lupeol Fig 7-9b Chromatogram of the formulation

Table 7-12: Estimation of Lupeol in the formulation.

Rf Strength Inj. vol Conc. Content per tablet

Standard 0.4 100 µg/ml 2 µl 98%

Formulation 0.42 1 mg/ml 2 µl 2.29% 5.15 mg

Assay result reveals 5.15 mg lupeol in the formulation which is same as expected 5.15 mg.

7.3.3.2.1.2 Estimation of Scopoletin

Each tablet contained 225 mg of ethanol extract of Morinda citrifolia standardized to 1.66%

of Scopoletin which means each tablet contained 3.41mg of Scopoletin


159

Fig 7-10 Chromatograms of Standard scopoletin and the formulation.

Fig 7-10a Chromatograms of Standard scopoletin Fig 7-10b Chromatograms of the formulation

Our assay results showed 3.54 mg of Scopoletin in the formulation as against 3.41 mg

expected which is about +3% variation and it is with in accepted limit of variation.

7.3.3.2.1.3 Estimation of Guggulsterone E

Each tablet contained 150 mg of ethanol extract of Commiphora mukul standardized to

2.52% of Guggulsterone E which is equivalent to 3.78 mg.

Table 7-13: Estimation of Scopoletin in the formulation. Rf Strength Inj. vol Conc. Content per tablet

Standard 0.45 100 µg/ml 6 µl 98 3.92 mg

Formulation 0.43 1 mg/ml 2 µl 2.61

Fig 7-11 Chromatograms of Standard Guggulsterone E and the formulation.

Fig 7-11a Chromatograms of Standard Guggulsterone E

Fig 7-11b Chromatograms of the formulation


160

The assay revealed 3.92 mg of guggulsterone E as against incorporated quantity of 3.78 mg

which is about +4% variation which is well within the accepted limit of variation.

HPTLC has been used as tool in herbal drug standardization for estimation of specific

phytoconstituents. Sullivan and Sharma developed method for assay of glucosamine in herbal

dietary supplement tablets and capsules [30]. Curcumin has been determined as an stability

indicator in bulk drugs and formulations [31] and a number of such studies have been

published. Hence the method was adopted in in our study. Our results showed a minimum

variation in the content which was well within the permissible limits.

7.3.3.2.2 Dissolution studies

The formulation under investigation is a poly herbal formulation for which there is no set

dissolution procedure. In contrast to the synthetic medicinal products poly herbal

formulations are complex mixtures of active and inactive principles which makes its

dissolution all the more complex and difficult [32]. However the marker based assays have

been used in studying dissolution patterns of herbal drugs. Bhope et al. [32] used

androgrpholide as a marker for dissolution of a poly herbal formulation containing

Andrographis paniculata. Compendial standards for the dissolution of herbal formulation are

not available, currently only 4 monographs on herbal drugs published in USP have

dissolution specification [33]. In the current study, three specific markers namely, lupeol,

Scopoletin and Guggulsterone E, were used for estimation of drug release in vitro. Each

marker has its biological relevance in the management of osteoporosis and therefore their

selection is justified. Among the markers, lupeol and guggulsterone are steroidal tripterpenes

which are highly lipophilic in nature and very poorly soluble in water. As there no standard

procedure both hydrochloric acid buffer (pH- 1.2) and phosphate saline buffer (pH7.4) media

were selected for the study.

Scopoletin a coumarin from Morinda citrifolia is partially soluble in water and in our study a

complete release of scopoletin was witnessed within 4 h in both media suggesting least role

of pH in its dissolution. USP [34] requires a minimum of 75% release for botanical dietary

Table 7-14: Estimation of guggulsterone E in the formulation

TABLE

Rf Strength Inj. Vol Conc. Content per tablet

Standard 0.53 100 µg/ml 4 µl 98% 3.51 mg

Formulation 0.53 1 mg/ml 2 µl 1.56%


161

supplement in vitro. Our formulation complies with the requirement as far as scopoletin

concerned, however, lupeol and guggulsterone E being water insoluble constituent showed

slow release. \About 44% of guggulsterone E was released in the acid pH while lupeol was

the least which showed only 11% release in 24 h.

The release pattern was appeared to change in the saline buffer (pH 7.4) which showed a

definite increase in the release percentage of both guggulsterone E (60%) as well as lupeol

(18%). The plant extract contains number phenols; total phenol content of C. quadrangularis,

M. citrifolia and C. mukul was determined to be 107, 38.05 and 256 mgGAEq respectively.

The phenols being mild acids, gets destabilize in basic pH and forms an anion which

actsufactants. An ionic and non-ionic surfactants have commonly been used to improve the

dissolution of poorly water soluble drugs [35]. Therefore, it can be hypothesized that the

formation anionic surfactant in the basic pH has facilitated the dissolution of both lipophilic

constituents. The dis advantage of this study is, it was carried out on only one batch and no

effort was made to improve dissolution of lupeol and guggulsterone E due to time constraint.

Table 7-15: Cumulative percentage drug release in Hydrochloric acid buffer (pH 1.2).

Time (h) Guggulsterone E Scopoletin Lupeol

5 min 0 38.4±4.2 0

15 min 3.2 ± 0.025 47.8 ±1.168 3.1±0.02

30 3.92±0.079 66.81±2.34 4.47±0.06

1 11.18±0.68 83.0±3.25 5.9±0.026

2 22.2±0.65 96.31±2.87 7.1±0.24

4 26.71±1.17 104.5±3.28 8.14±0.16

6 29.6±0.93 -- 8.39±0.28

8 31.89±1.3 -- 9.89±0.48

10 33.66±1.22 -- 10.48±0.36

12 33.35±1.93 -- 10.6±0.27

16 37.6±1.53 -- 10.4±0.49

20 41.57±3.66 -- 10.8±0.67

24 44.29±4.55 -- 11.10±0.78


162

7.3.3.2.3 Real time stability studies

Formulations are pharmaceutical dosage forms that are intended to be used orally or

parentally over a period of time. The formulations should therefore have certain shelf life to

be used for long period of time. Solid dosage forms usually have longer shelf life than liquid

dosage forms as solid dosage forms offer little scope for microbial contamination or

Table 7-16: Cumulative percentage drug release in phosphate buffer saline (pH 7.4).

Time (h) Guggulsteron E Scopoletin Lupeol

5 min 8.3±0.69 40.4±2.8 0

15 min 17.95±1.24 52.6±4.25 6.5±0.82

30 min 22.94±1.65 66.4±4.68 7.02±0.46

1 26.56±2.84 76.21±3.67 8.26±1.04

2 28.89±4.24 97.05±6.84 9.48±1.46

4 32.72±2.26 99.45±4.63 11.62±0.98

6 33.95±3.54 -- 11.6±0.85

8 36.0±2.94 -- 12.45±1.28

10 36.87±3.48 -- 12.59±1.86

12 46.72±4.36 -- 13.73±1.74

16 51.42±4.98 -- 13.92±2.24

20 55.01±4.22 -- 15.95±2.68

24 60.0±4.69 -- 18.38±2.72


163

oxidation unless the drugs are highly reactive. Stability study can be carried out either as a

long term or real time stability study or under accelerated conditions as accelerated stability

study. Our formulation is a herbal formulation that are known to have longer shelf life and

hence we carried out a real time stability study of the product at room temperature (25 °C ± 2

°C) with relative humidity of 60±5 for a period of 1 yr. The tablet at end of one year were

examined physically for any sign of degradation or damage and evaluated for colour, shape,

thickness, friability, weight and hardness.

In our study no striking change could be observed in the physical characteristics. Colour,

shape, thickness and weight of the tablet remained same while a marginal increase in

hardness was observed which is negligible. The estimation of marker constituents also

revealed no significant changes.

Thus it is concluded that the tablet formulation is stable up to 1 year or more.

Table 7-17 Physical characteristics of tablets after one year.

Color and shape Thickness Friability Weight Hardness(kg/cm2)

Dark brown 0.74±0.011 0.47±0.086 721 mg 5.2

7.3.3.2.3.1 Estimation of lupeol after 1 year

Fig 7-14 Chromatograms of Standard Lupeol and the formulations.

Fig 7-14a Chromatogram of lupeol Fig 7-14b Chromatogram of the formulation before 1

yr.

Fig 7-14c Chromatogram of the formulation after 1 yr.

Table 7-18 Peak area and drug content of Lupeol.

Rf Strength Inj. vol Peak area Conc.

Content per tablet

Standard 0.4 100 µg/ml 2 µl 2674.9 98% 5.15 mg

Formulation 0.44 1 mg/ml 2 µl 685.9 2.29%

Formulation after 1 year 0.42

1 mg/ml

2 µl 618.1 2.26% 5.1 mg


164

7.3.3.2.3.2 Estimation of Scopoletin

Fig 7-15 Chromatograms of standard scopoletin and the formulation after 1 yr.

7.3.3.2.3.3 Estimation of Guggulsterone E

Fig 7-16 Chromatograms of standard Guggulsterone E and the formulation after 1 yr.

Fig 7-15a Chromatogram of Standard scopoletin

Fig 7-15b Chromatogram of the formulation before 1 yr.

Fig 7-15c Chromatogram of the formulation after 1 yr.

Table 7-19 Peak area and drug content of Scopoletin.

Rf Strength Inj. vol Peak area Conc. (%) Content per tablet

Standard 0.45 100 µg/ml 6 µl 10279.6 98 3.92 mg

Formulation before 1 yr.

0.43 1 mg/ml 2 µl 915.8 2.61

Formulation after 1 yr.

0.43 1 mg/ml 4 µl 1665.3 2.38 3.57 mg

Fig 7-16a Chromatogram of Standard Guggulsterone E

Fig 7-16b Chromatogram of the formulation before 1 yr. Fig 7-16c Chromatogram of the formulation after 1 yr.


165

7.4 Conclusion The present study was undertaken to develop a herbal formulation for the pharmacologically

developed dietary supplement combination. Development of a formulation from herbal

extracts is a challenge, and in our study three ethanol extracts were involved. The physical

and physicochemical properties of the extracts cause hurdles in the development of a

formulation. Herbal extracts contain a number of phytoconstituents besides the active

principles which makes dissolution a difficult task. Biologically active constituents have been

estimated in the in vitro dissolution studies. In our study we estimated three biologically

active constituents as reference standards for dissolution studies.

Development of a formulation from an extract or a combination of extracts deserves an entire

length of the study. Our study is a small part of a full length dietary supplement development

program and hence only preliminary work is done on the formulation. A detailed study on the

physical characteristics of extracts, their possible modification different formulations and a

detailed in vitro release study is required for the formulation.

Nevertheless, tablets of the combination of extracts were prepared as per human dose (600

mg) and their physical and chemical characteristics were evaluated. Round convex, dark

brown tablets containing 225 mg of ethanol extract of Cissus quadrangularis equivalent to

5.15 mg of lupeol; 225 mg Morinda citrifolia extract equivalent to 3.92 mg of scopoletin and

150 mg of ethanol extract of Commiphora mukul equivalent to 3.21 mg of guggulsterone E.

The formulation releases scopoletin within 4 hours while the lupeol and guggulsterone

release is slow and sustained which extends over a period of 24 h or more.

Table 7-20 Peak area and drug content of Guggulsterone E.

Rf Strength Inj. vol Peak area Conc.

Content per tablet

Standard 0.53 100 µg/ml 4 µl 1930.4 98% 3.54 mg

Formulation 0.53 1 mg/ml 2 µl 654.3 1.56%

Formulation after 1 yr.

0.53 1mg/ml 4 µl 298.5

1.52% 3.41 mg


166

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