Need Of Standardization For Herbal Formulation Biology Essay

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Herbal medicines has grow to be a popular form of health care. Ayurveda is the oldest healing system of medicine. Main formulations used in ayurveda are based on herbs. According to WHO herbal drugs are defined as herbal medicine that are administered to patients and are mixture of herbal substance and other constituents.

The importance of herbal medicine practices is indicated by fact that about 80% of the world population depends on traditional medicine for their active constituents. Currently the popularity of herbal medicine is in demand, so the usage of them is increasingly day by day. WHO supports the usage of herbal medicine because of availability of more raw materials.

From the past to present psychologically villagers having more trust on herbal medicine due to their lesser side effects. The formulation in ayurveda is based on two principles used as a single drug and more than two drugs. Two or more herbs are used in formulation is called as Polyherbal formulation. From many centuries Polyherbal formulation are used for curing several diseases. No official standards are available for the herbal preparation. It is Very difficult to identify the presence of all the ingredients in such a formulation .

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.ADVANTAGES OF HERBAL MEDICINES:

Herbal medicine have long history of use and better patient tolerance as well as acceptance.

Medicinal plants have a renewable source, which is our only hope for sustainable supplies of cheaper medicines for the world growing population.

Availability of medicinal plants is not a problem especially in developing countries like India having rich agro-climatic, cultural and ethnic biodiversity.

The cultivation and Harvesting of these herbal medicines is ecofriendly.

Prolong and apparently uneventful use of herbal medicines may offer indication of their safety and efficacy.

STANDARD IZATION OF HERBAL DRUGS:

In developing countries there has been great demand for herbal products. Standardization is an essential measurement to ensuring quality control of herbal product. To obtain quality oriented herbal products, care should be taken from the extraction and purification process in case of poly herbal drugs.

Standardization of herbal drug is not easy job as several factors influence the bio efficacy and reproducible therapeutic effect. The herbal formulation is systamatically formulate the medicament using raw materials collected from different batches of formulation are to be observed. Several studies have shown that quantitative variations in marker constituents in herbal preparations.

Standardized herbal products of consistent quality and containing well defined constituents are required for realiable clinical trials and to provide consistent beneficial therapeutic effects. To scientists, it is a major challenge to development of authentic analytical methods which can be realibly profile the phytochemical composition, including quantitative analysis of marker/ bioactive compounds and other constituents.

NEED OF STANDARDIZATION FOR HERBAL FORMULATION:

It is the fundamental responsibility of the regulatory authorities to make sure that the consumers get the medication, which assurance. Purity, safety, potency and efficacy. Herbal product has been enjoying regeneration among the customers throughout the world. However, one of the impediments in the approval of the Ayurvedic formulation is the lack of standard quality control profile. The quality of herbal medicine i.e. the profile of the constituents in the final product has inference in efficacy and safety. Due to complex nature and natural variability of the constituents of plant based drugs, it is difficult to launch quality control parameter and new analytical technique are expected to help in circumventing this problem. The quality control of crude drugs and herbal formulations is of supreme importance in justifying their satisfactoriness in modern system of medicine. But one of the major problems faced by the herbal drug industry is non availability of rigid quality control profile for herbal formulations.

In polyherbal formulation it is very difficult to estimate each and every ingredient of their chemical constituents, compared to when single based formulation. Various chromatographic and spectophotometric methods are available like, HPLC, HPTLC, UV, FLOURIMETRY, to identifying active constituents of medicinal herbs.

ANALYTICAL TECHNIQUES:

Separation of individual components from the herbal mixture is the key step to enable identification and bioactivity evaluation.

Chromatography is the powerful and suitable analytical method for the separation and quantitative determination of a considerable number of compounds. these includes paper chromatography(PC), thin layer chromatography (TLC), HPLC, HPTLC,GC methods mostly used for detection of separated compounds. However UV absorption has been the most commonly used for the preliminary identification of the separated components various detectors like flame ionization(FID), fluorescence (FD),are also available for specific cases. these detection methods allows the quantification of chemical compounds that are present in plant material or herbal product.

HPTLC Analysis on Herbs

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HPTLC is the most simple separation technique today available to the analyst. HPTLC is a qualitative tool for separation of simple mixtures where speed, low cost and simplicity are required and it is also a tool for quantitative analysis .

High-Performance Thin-Layer Chromatography for the analysis of medicinal plants presents the hypothetical and technical information needed to perform reliable and reproducible results in order to establish the identity, purity, quality, and stability of raw materials, extracts, and finished botanical products.

Major features

High performance thin layer chromatography (HPTLC) is valuable quality assessment tool for the evaluation of botanical materials.

It allows for the analysis of a broad number of compounds both efficiently and cost effectively.

Additionally, various samples can be run in a single analysis thereby radically reducing analytical time.

With HPTLC, the same analysis can be viewed using different wavelengths of light thereby providing a more complete profile of the plant than is typically observed with more specific types of analyses.

HPLC ANALYSIS ON HERBS:

High performance liquid chromatography is a accepted method for the study of herbal medicines because it is easy to use and easy to learn and is not limited by the stability or volatility of the sample compound. Generally, HPLC can be used to analyze all the compounds in the herbal medicines. The most popular reverse-phase columns may be used in the analytical separation of herbal medicines. It is necessary to detect that the optimal separation condition for the HPLC involves many factors, such as the different mobile phase compositions, pH adjustment, pump pressures etc.

ADVANTAGES:

HPLC is most poular technique because its versataility, precision and its reproducibility, relatively low cost

2) Mainly it is helpful in comparision of one or two markers by quanitative approach.

Analysis of herbal formulation done by recently detectors such as coloumetric array detection and charged aerosol detection .eg chiconic acid in posodonia ocenamica.

USE OF MARKER COMPOUNDS FOR IDENTIFICATION AND STANDARDIZATION OF HERBAL DRUGS:

Marker compounds plays a major role in fingerprinting analysis of herbs. It may be used to identify herbal materials, specification for raw materials chemical and chromatographic methods to be used to aid in identification of herbal material or extract.

Markers are chemically defined constituents or groups of constituents of herbal substance, a herbal preparation or a herbal medicinal product which are of interest for control purposes independent of whether they have any therapeutic or pharmacological activity. Markers serve to calculate the quantity of herbal substances or herbal preparations in the Herbal Medicinal Product if the marker has been quantitatively determined in the herbal substance or herbal preparation. There are two categories of markers Active markers are constituents or groups of constituents which are generally accepted to contribute to the therapeutic activity. Analytical markers are constituents or groups of constituents that serve for analytical purpose.

BIOANALYTICAL METHOD DEVELOPMENT FOR PHYTOCHEMICAL CONSTITUENTS:

Large number of phytochemical constituents were isolated & identified from herbal drugs. Hence they are available as markers, for the analysis of formulation containing them and as well as to establish pharmacokinetic and pharmacodynamic data. Which is most necessity of the current pharma scenario.

Analysis of drug in biological fluids plays an important role in the pharma field. Generally after the administration of drug it will reaches the systemic circulation and undergoes metabolism, finally it will excreted through urine in the form of drug metabolite or in the form of drug as such. So the quantitative determination of drug and their metabolite in the biological fluids used to find out the therapeutic efficacy of the drug. Plasma or serum and urine samples are usually used in clinical analysis because these samples contain fewer interfering substances than whole blood. Some of the pharmokinetic parameters mainly involved in this studies are Cmax, tmax, AUC.

SIGNIFICANCE OF DRUG ANALYSIS IN BODY FLUIDS:

Bio equivalence studies

Therapeutic drug monitoring

Bioavailability

Drug-drug interaction

Adverse effect

Overdose

EXTRACTION:

For quantitative determination, the direct analysis of biological sample is impossible. Because it may contains interferences. So for the quantitative determination, the active analyte should be extract from the biological sample. This is achieved by various type of extraction process. It mainly involves,

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Protein precipitation

Liquid-liquid extraction

Solid-phase extraction

Membrane filteration

Quantification procedure

Peak-height or peak-area measurements only provide a response in terms of detector signal. This response must be related to the concentration or mass of the compound of interest. To accomplish this, some type of calibration must be performed.

They are

Normalised peak area method

External standard calibration method

Internal standard calibration method

Method of standard addition

IMPORTANCE OF INVITRO METHODS:

The importance of bioanalysis is most valued in different fields of pharmaceutical sciences. In pre-clinical and clinical stages extensive studies are performed in new drug development. Animal fluids were analysed in pre clinical study. Whereas, human samples were examined in clinical studies. Parent drug and its metabolite, toxicological and therapeutic concentrations of it were determined, in grouping with the pharmacodynamic properties of the potential drug. At last, the formulation of the drug must be optimized. Normally, biological samples can't be injected directly into the analyzing system without sample preparation. Pretreatment of sample was having maximum importance for the adequate analysis of drugs. However, it was a time consuming process, this can limit the sample throughput. The proper selectivity can be attained during the sample preparation for the separation & detection. In the first step major separation between the analyte(s) of interest and the other compounds was frequently done. Large extent of sensitivity was obtained by the detector. However, pre-concentration of the analytes at some point in sample preparation will also add to sensitivity. Thus, sample pretreatment is necessary for achieving sufficient sensitivity. several sample preparation techniques have been developed for bioanalytical purposes. Most classical sample preparation techniques are liquid-liquid extraction (LLE), solid phase extraction, precipitation method. Wide range of solvent polarity and its general applicability made this technique well-liked. However, from an environmental point of view, the use of large amounts of organic and frequently chlorinated solvents was not favourable. Furthermore, LLE has often limited separation efficiency. Protein precipitation was the simple method of extraction when compared with that of LLE and SPE. This can be done by using the appropriate organic solvents which has good analyte solubility and protein precipitating properties. First choice of solvent was acetonitrile in protein precipitation method due to its absolute precipitation of proteins. Methanol was the second choice of organic precipitant which offers more solubility of the analyte in these solvents. After protein precipitation the supernatant obtained can be used for analysis.

LITERATURE SURVEY OF MARKERS

EMBELIN

1) R. paul chowdhury, et al., reported on quantitative analysis of embelin in myrisine Africana L.(myrisinaceae) using HPLC and HPTLC. In HPLC mobile phase sytem consist of methanol:water: acetic acid: tetra hydro furan (85:15:3:0.1v/v/v/v) detection was carried out at 288nm. In HPTLC solvent system consist of n-propanol:n-butanol: ammonia(7:1:2v/v/v) detection was carried out at 333nm.

2) S. Gopinath, et al., reported on simultaneous estimation of embelin and plumbagin by reverse-phase high performance liquid chromatographic method. Resolution achieved by using mobile pjhase Acetonitrile:50mM potassium dihydrogen ortho phosphate (45;55)detection wavelength 290nm.

3) Shelar R, Maurya C, et al., reported on Embelin -An HPLC Method for Quantitative Estimation in Embelia ribes Burm. F. Embelin was separated by using isocratic mode consisting of 0.1 % trifluoroacetic acid in water and methanol (in proportion of 88:12). Detection carried out at 288nm.

4) Babu Ganesan , reported on optimization of extraction conditions for embelin in embelia ribes by UV spectrophotometry. Extraction conditions optimized for best possible extraction of embelin from the fruits of Embelia ribes in different solvents. Detection wavelength is 291nm.

5) Indian Herbal pharmacopeia, vol-1 part II, For Embelia ribes identification test was done by shaking the seeds powder with solvent ether and filtered to a filtrate portion 5%v/v sodium hydroxide, deep violet colour is developed in aqueous layer.

BERBERINE:

Shiv Shankar Shukla, et al., reported on TLC densitometric development and validation of Berberine as markers in poly-herbal Unani formulations. solvent system consist of methanol:acetic acid:water(8:1:1v/v/v).detection wavelength used 350nm.

Pi-Lo Tsai, et al., reported on HPLC Determination of berberine in medicinal herbs and a related traditional Chinese medicine. Mobilephase of acetonitrile:methanol:20mMPhosphate(35:20:45,v/v/v) and wavelength used as 346nm.

Chi-Ming Chen, et al., reported on determination of berberine in plasma, urine and bile by high- performance liquid chromatography. Mobile phase consist of 60-63%acetonitrile in 0.1%phosphoric acid with UVdetection at 267nm.

Li Yi , et al., reported on simultaneous determination of baicalin , rhein and berberine in rat plasma by coloumn- switching high- performance liquid chromatography.

Indian herbal pharmacopeia, part I, vol II. Mentioned the plant profile of Berberis aristata.

PROFILE OF MARKERS

Berberine hydrochloride

Synonym : Berberine chloride form, natural yellow 18;

Structure:

IUPAC name: 6-dihydro-9, 10-dimethoxy-, chloride;

Molecular formula : C20H18NO4Cl

Molecular weight : 371.82

PROPERTIES:

Melting point : 200oc

Solubility : soluble in cold water. In soluble in benzene, ether, chloroform

Characterstic: Isoquinoline alkaloid. It is present in roots ,rhizomes, stems of various Eg: Berberis aristata, Berberis aquifolium

Category: used in treatment of trachomas (eye infection), bacterial diarrhoea.

Embelin

Structure :

IUPAC name : 2,5 -di hydroxy 3- undecycyl -2,5- cyclo hexadiene- 1,4-benzoquinone

Molecular formula : C17H2604

Molecular weigh : 294.391 g/mol

Melting point : 142-143oc

Solubility : soluble in 10MM ethanol, alcohol, chloroform, benzene.

Category : It is effective in treatment of ascariasis. And shows antimicrobial activity against bacteria.

AIM AND PLAN OF WORK:

Berberine and embelin are potent plant constituents of medicinal herbals and they are well established for their structure and medicinal uses. Polyherbal formulation containing berberine and embelin are available in the market and are used as antibacterial agents in the treatment of ascariasis, trachomas (eye infection) and bacterial diarrhoea.

As per herbal pharmacopeia Embelin assay was done by taking the powder & transferring to glass stoppered flask. This was shaken occasionally for 30 minutes with solvent ether. Maceration was done in percolator for 30 minutes and solvent ether was used for extraction, till the ethereal solution gives pink colour with a drop of dilute ammonia solution. Whereas, for Berberine no assay method was mentioned in herbal pharmacopeia.

Literature survey reveals various analytical methods for their individual estimation from formulation and plant extracts. Where as no analytical method has been reported for the combined estimation of the Berberine and embelin from polyherbal formulation and human plasma.

chromatography is the powerful analytical method for separation and quantitative determination of considerable compounds.

Hence the aim of work is,

Quantitative estimation of BERBERINE and EMBELIN from polyherbal formulation by using HPLC and HPTLC.

Bioanalytical estimation of embelin and berberine in human plasma by high performance thin liquid chromatography.

DEVELOPMENT OF HPTLC METHOD FOR MARKERS

selection of initial separation conditions:

1) Precoated silica gel 60F254 on aluminium sheets was used for the separation of selected biomarkers.

2) The solvent for markers preparation was selected to be methanol. They were easily soluble in methanol.

Selection of wavelength

An ideal wavelength is one that gives maximum absorbance and good response for the markes to be detected .The sensitivity of the HPTLC method that uses UV detection depends upon the proper selection of wavelength. Both these markers were scanned individually at 200-400nm from which a wavelength 347nmwas selected for the studies(.Fig) Absorption spectrum of berberine and embelin (347nm)

Development of optimum Mobile Phase

A solvent system that would give dense compact spots and good separation from solvent front and application position was to be selected. Initially, different solvent systems were tried and observations were as given below.

Solvent system tried

Observation

Methanol:acetic acid:water

Only Berberine is identified as yellow spot

Methanol:propanol:butanol:ammonia

Methanol:npropanol:butanol:ammonia

Propanol:nbutanol:ammonia

Both Berberine and Embelin gave good separation with symmetric peak

In above mobile phase system Propanol: n-butanol: ammonia was selected because the spots was compact and well retained and hence it was chosen for further optimization.

EFFECT OF RATIO:

Different ratios of Propanol: n-butanol: ammonia like 6:2:2, 7:1:2 were tried in order to achieve an optimum separation with good peak shape. A ratio of (6:2:2%v/v) was selected because it gave compact spots that are well resoluted from solvent front and sample application positions

Optimization of chamber saturation

The above fixed mobile phase was added to one side of a twin trough chamber and different saturation times from 5 to 30 minutes were tried. It was found that a saturation time of less than 10 min caused fluctuations in Rf value and edge effects. Saturation times of 15 min and above were devoid of fluctuations and edge effects and hence a saturation time of 15 min was fixed.

Optimization of plate saturation

The above fixed mobile phase was added to one side of a twin trough chamber and the spotted TLC plate was kept in the empty side. Different plate saturation times from 5 to 30 minutes

were tried. It was found that a saturation time of 15 min gave compact spot and hence fixed.

Fixed Experimental Conditions

Stationary Phase : Pre-coated silica gel 60F254 on aluminium sheets.

Mobile phase : Propanol: n-butanol: ammonia(6:2:2%v/v)

Chamber saturation time : 15 minutes

Migration distance : 80 mm

Band width : 6 mm

Slit dimension : 5 Ã- 0.45 mm

Source of radiation : Deuterium lamp

Detection wavelength : 347 nm

Rf value : 0.42&0.54

VALIDATION OF THE METHOD

The validation of the developed method was carried out in terms of linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), inter and intraday precision, repeatability of sample application and measurement, stability studies and selectivity.

Preparation of standard stock solution of Berberine and embelin :

Standard stock solution of both berberine and embelin (50g/ml) were prepared in methanol.

Linearity and Range

From the above prepared stock solution 1-3.5l was applied on a 10Ã-10 pre coated TLC plate with Linomat 5 applicator. With the fixed chromatographic conditions the plate was developed and analysed photometrically.

Linear regression data showed a good linear relationship over a concentration range of 50-175ng/spot for berberine and embelin. The slope, intercept and correlation co-efficient values were found to be -630.858,20.99, 0.9997 for embelin, 870.076 and 23.983 0.999 for berberine respectively. (Table1)

TABLE 1 : CALIBRATION GRAPH

Concentration(ng/spot)

PEAK AREA

BERBERINE EMBELIN

50

2689.4

757.6

75

3874.1

1379.4

100

4770.2

2177.1

125

5560.3

2690.9

150

6123.1

3348.5

175

6747.6

4002.5

Lineargraph of (Berberine 50 to 175ng/spot) Linear graph of Embelin

G:\emb and ber\EMBLIN\BER.bmp G:\emb and ber\EMBLIN\EMB.bmp

CHROMATOGRAM OF STANDARD- 1

G:\emb and ber\50 ng.bmp

CHROMATOGRAM OF STANDARD- 11

G:\emb and ber\75ng.bmp

CHROMATOGRAM OF STANDARD- 111

G:\emb and ber\100ng.bmp

CHROMATOGRAM IV :

G:\emb and ber\125ng.bmp

CHROMATOGRAM V :

G:\emb and ber\150ng.bmp

CHROMATOGRAM VI

C:\Users\kishore\Desktop\emb and ber\175ng.bmp

Limit of Detection (LOD) and Limit of Quantification (LOQ)

The LOD and LOQ of the drug were determined by applying decreasing amounts of the drugs on the plate. The lowest concentration at which the peak is detected is called the 'limit of detection (S/N=3) which was found to be 30 ng/spot for embelin and 40 ng/spot for berberine. (Fig2) The lowest concentration at which the peak is quantified is called 'Limit of Quantification (S/N=10) which was found to be 50 ng/spot.

LOD OF EMBELIN

G:\emb and ber\EMBLIN\LOD OF EMB.bmp

LOD OF BERBERINE

G:\emb and ber\LOD OF BER 0.4.bmp

Recovery

Recovery studies of the drugs were carried out for determining accuracy parameter. It was done by mixing known quantity of standard drug at 100% level and the contents were re analyzed by the proposed method The percentage recovery and its % RSD were calculated. (table 2)

Formulation

%Recovery

% RSD

Berberine

97%

Embelin

90%

Precision

Precision of the method was demonstrated by

Intra day precision

Inter day precision

Repeatability

Repeatability of sample application

Repeatability of measurement

Intra day precision

Intra day precision was found out by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drug for three times on the same day. Each concentration was applied in duplicate and % RSD was calculated (table 3 )

Table 3: Intra day precision

Concentration (ng/µl)

Peak area

% RSD

BERBERINE

EMBELIN

B

E

2

4770.2

2177.7

1.36

1.09

4654.9

2168.2

4664.3

2132.9

3.5

6747.6

4002.5

0.43

0.44

6723.1

3987

6694

3967

Inter day precision

Inter day precision was found out by carrying out the analysis of the standard drug at three different concentrations in the linearity range of drugs for three days and % RSD was calculated (table3)

Table 4: Inter day precision

Concentration

(ng/µl)

Day

Peak area

%RSD*

BERBERINE

EMBELIN

2

1

4770

2177

1.89

1.82

2

4689

2133

3

4593

2055

3.5

1

6747

4002.5

1.5

1.7

2

6697

3987

3

6897

3876

*RSD of six observationsRepeatability

Repeatability of sample application

Repeatability of sample application was assessed by spotting 6l of drug solution six times on pre coated TLC plate followed by development of plate and %RSD was calculated (table 5 )

Table5: Repeatability of Sample application

Concentration(ng/ul)

Peak area

%RSD

Berberine

Embelin

Berberine

Embelin

2

4770

4754

4733

4743

4723

4738

2177

2167

2172

2156

2168

2247

0.34

1.5

Repeatability of measurement

Repeatability of measurement of peak area was determined by spotting 5 l of standard drug solution on pre coated TLC plate. After development of the plate the spot was scanned six times without changing position of the plate and %RSD was calculated (table 6)

Table 6 : Repeatability of Measurement

Concentration(ng/spot)

Peak area

%RSD*

BERBERINE

EMBELIN

Berbrine

Embelin

3

6123

6122

6111

6126

6115

6120

3348

3347

3323

3343

3321

3318

0.09

0.42

Stability Studies

The drug may undergo degradation in solution or on the plate when exposed to atmosphere. Hence it is necessary to conduct stability studies for berberine and embelin solution and drug on the plate.

The stability of both marker solution was studied by chromatogram the standard solution at different time intervals and comparing the peak area with that of the freshly prepared solution. A 10 % reduction in peak area was considered as loss of stability. The solution was found to be stable up to 5 hours. The results are shown in (table 7)

Concentration(ng/µl)

Time (hrs)

Peak area

Berberine

Embelin

3

0

1

2

3

4

5

6123

6011

5875

5782

5689

5500

3348

3296

3223

3156

3123

3014

Robustness

In order to demonstrate the robustness of the method, the following optimized conditions were slightly varied.

± 3 min in chamber saturation time

± 0.1% change in mobile phase ratio

± 2 min in plate saturation time

± 2 mm in solvent front

The response factors for these changed chromatographic parameters were almost same as that of fixed chromatographic parameters and hence the developed method is said to be robust.

FORMULATION ANALYSIS :

Poly herbal formulation containing berberine and embelin named as LUMITAL TABLETS and ENTOSTALTABLETS collected from the market used for dysentry and bacterial diarrhoea.

Composition

Label claim(mg)

Oroxylum indica

100

Embelia ribes

50

Acacia catechu

25

Berberis aristata

300

Aloe barbadens

100

Santalum album

25

Vanga bhasma

10

Composition

Label claim(mg)

Holarrhena antidysenterica

600

Oroxylum indica

500

Berberis aristata

300

Cassia aungstifolia

150

Embelia ribes

50

Bombax malabricum

25

Eclipta alba

50

Zingiber officinale

25

Formulation I Formulation II

The above two poly herbal formulations containing berberine in Berberis arista and embelin in Embelia ribes.

EXTRACTION OF BERBERINE AND EMBELIN FROM POLYHERBAL FORMULATIONS:

To estimate the content of berberine and embelin from above two formulations, with three different batches were tried. 10 tablets were weighed and powdered. Powder equivalent to one tablet was taken and transferred into a 25 ml volumetric flask containing 10 ml methanol and sonicated for 10 min, and finally made up to 25 ml with same solvent and This solution was filtered.

RECORDING OF CHROMATOGRAM:

With the fixed chromatographic conditions, various volumes of extracted solution were applied on precoated TLC plate. The plate was analyzed photometrically and chromatograms were recorded, fig (2). This was followed by application of extracted solution obtained from the polyherbal formulation.

Peak areas of formulation chromatograms were compared with that of standard chromatograms and amount of Berberine and Embelin in formulation was calculated from the calibration graph. The results are shown in ( table 8).

RESULTS OF FORMULATION ANALYSIS

Formulation

Batch

no's

Label claim(mg)

Amount present(µg)

Berberine

Embelin

Berberine

Embelin

I

Sf0309004

Sfo301004

Sf0302009

50

50

50

50

50

50

14.75

21

18.3

31

25

21

II

Sf049001

Sf048005

Sf049801

300

300

300

50

50

50

26

29

27

54

48

37

Formulation chromatogram

H:\emb and ber\FORMULATION PEAKS.bmp

Quantification OF BERBERINE AND EMBELIN FROM POLYHERBAL FORMULATION BY HPLC METHOD

Selection of Chromatographic Method for Separation

The nature of the sample (Polar or Nonpolar), its molecular weight and solubility are the main factors to be considered in the selection of the method. Berberine and embelin were polar in nature; hence reverse phase chromatography was selected.

Selection of Solvent

Both the markers were freely soluble in methanol. The drugs were solubilised in the mobile phase itself to avoid any possible disturbances in the chromatogram due to solvent incompatibilities.

Selection of Wave length

An ideal wave length at which both the markers (berberine and embelin) gives good response is to be selected. Absorption spectra of berberine and embelin were recorded and shown in fig.(1) As both the drugs have optimum absorbance at 286 nm it was chosen for the detection of compounds.

Selection of Initial Chromatographic Conditions

Sl. No.

Mobile phase

Observation

Retention time (min)

Fig

1.

Methanol: ortho phosphoric acid(70:30% v/v)

No peak for embelin

Berberine

Embelin

1

4.5

-

2.

Acetonitrile: KH2PO4 (50:50% v/v)

Tailing is observed for two peaks

4.8

11.2

2

3.

Acetonitrile: KH2PO4 (40:60% v/v)

Tailed peak

4.6

11.3

3

4.

Acetonitrile: KH2PO4 (70:30% v/v)

Tailed peak

3.3

4.5

4

5.

Acetonitrile: NaH2PO4 (40:60% v/v)

Good symmetrical peak

0.53

7.63

5

Separation using-KH2PO4: ACN

Initially separation was tried to achieve with combinations of KH2PO4 Buffer and Acetonitrile (ACN), with different ratios were tried. In this condition tailing is more for two peaks. To reduce this different buffers were tried.

Separation using NaH2PO4: ACN

As The KH2PO4 used for the berberine and embelin combination didn't show the satisfiable separation, the NaH2PO4 with acetonitrile was tried as next choice. The mobile phase composition such as 60:40,this buffer shows peaks of berberine and embelin good. The chromatograms are shown below.

Fig 1( separation with methanol:orthophosphoric acid(70:30)

Fig 2( Acetonitrile:potassium dihydrogen ortho phosphate(50:50)

Fig 3(Acetonitrile:potassium dihydrogen ortho phosphate(60:40)

Fig 4(Acetonitrile:potassium dihydrogen ortho phosphate(60:40)

Fig 5(Acetonitrile:sodium di hydrogen orthophosphate(40:60)

Selection of Mobile Phase Ratio

The different concentration of NaH2PO4 tried with different ratios such as 40:60, and 70:30 %v/v with ACN were tried. It was found that the retention of Berberine is merged with solvent peak with increase in percentage of organic phase. Hence 40:60v/v was selected.

Selection of pH of the Mobile Phase

Different pHs for mobile phases were tried such as 3, 3.2, 3.5 and There was no considerable difference in retention. At pH 3.2 peak shapes of drugs were found better and hence selected.

Fixed Chromatographic Conditions.

Column : RP-C18 (MERCK 5 cm) column

Mobile Phase : ACN+20M NaH2PO4(60:40)

pH of aqueous phase : 3.2

Detection Wavelength : 286 nm ( PDA Detector)

Flow Rate : 1ml/min

Temperature : Ambient Temperature

VALIDATION OF RP-HPLC METHOD

The developed HPLC method was validated in terms of parameters like linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), precision, accuracy, robustness, specificity and stability

Linearity and range:

Preparation of standard solution of Berberine and Embelin:

Stock solution containing concentrations of 50 µg/ml of Berberine and Embelin were prepared using methanol. This solution was suitably diluted and mixed to get aliquots of standard solutions.

Berberine and Embelin were found to be linear in the range of 1-5 µg/ml respectively. The standard chromatograms are shown below (fig1-5). Calibration graphs were plotted using peak area of standard drug Vs concentration of drugs in standard solutions, (fig 20-21). The Slope, intercept and correlation coefficient values were found to be, and -6647.800,67878.200 respectively for berberine,5667.700,64352.900 and respectively for embelin .

Calibration graph of berberine and embelin

CONCENTRATION (µg/ml)

PEAK AREA

Berberine

Embelin

1

62679

56352

2

136411

12135

3

209298

199408

4

265190

282457

5

320054

318582

Standard chromatogram(1µg/ml)

Standard chromatogram (2µg/ml)

Standard chromatogram (3µg/ml )

Standard chromatogram (4µg/ml)

Standard chromatogram(5µg/ml)

Calibration graph of berberine

Calibration graph of embelin

Precision

Precision of method was demonstrated by

Intra day precision

Inter day precision

Repeatability of injection

1) Intra day precision

Intra day precision was found out by carrying out analysis of standard drug solutions at three different concentrations in the linearity range of the drugs for three times on the same day. Each concentration was applied in duplicate and % RSD was calculated table(1)

CONCENTRATION

(µg/mL)

PEAK AREA

%RSD

BERBERIN

EMBELIN

BERBERINE

EMBELIN

2

2

62679

61823

60832

56352

55882

54978

1.4

1.25

5

5

320054

328702

317893

318582

318492

309462

1.7

1.6

2) Inter day precision

Inter day precision was found out by carrying out the analysis of standard drug solutions at three different concentrations in the linearity range for three days and % RSD was calculated (table 2)

CONCENTRATION

(µg/ml)

DAY

PEAK AREA

%RSD

BERBERINE

EMBELIN

BERBERIN

EMBELINE

2

2

1

62679

56352

1.5

1.6

2

61097

55482

3

60891

54379

5

5

1

320054

318582

1.7

1.5

2

318854

317821

3

309615

309862

3)Repeatability of injection

A standard solution of mixture of drugs was injected 6 times and its % RSD was calculated (table3 )

Concentration(µg/ml)

Peak area

%RSD

Berberine

Embelin

Berberine

Embelin

3

209298

208768

208329

208903

207859

207323

199408

198938

198501

198203

197881

197420

0.34

0.36

4)Recovery

To study the reliability, suitability and accuracy of the method, recovery studies were carried out. To the formulation, 100% of standard berberine and embelin were added. The solution was made up to mark with solvent itself. The chromatograms were recorded. The concentration of drugs present in resulting solution was determined using assay method; percentage recovery and percentage RSD was calculate (table4)

Drug

% Recovery (100 % level)

% RSD

BERBERINE

95%

0.87

EMBELIN

98.7%

0.78

Robustness

In order to demonstrate the robustness of the method, the following optimized conditions were slightly varied.

 0.5 % in ratio of Acetonitrile in mobile phase

 0.1 units in pH of buffer

 0.1 ml of flow rate

The response factors for these changed chromatographic parameters were almost same as that of the fixed chromatographic parameters and hence developed method is said to be robust.

6. Specificity

Specificity was studied by injecting the mobile phase and monitored for any additional peaks. No additional peaks were found at the retention time of two drugs .So the method was said to be specific.

7. Stability

Sample solutions were subjected to stability studies under room conditions. Stabilities were studied by looking for any change in retention time, resolution, peak shape etc, when compared with chromatogram of freshly prepared solution. A 10 % reduction in peak area was considered as loss of stability. The solution of Berberine and embelin was stored under room temperature was found to be stable up to 5 hrs( Table 6)

CONCENTRATION

(µg/mL)

TIME

PEAK AREA

BERBERIN

EMBELIN

BERBERINE

EMBELIN

4

4

0

201026

199480

1

197289

197225

3

187654

180257

5

172356

172268

System suitability studies

System suitability parameters like number of theoretical plates (N), peak asymmetry factor (As), capacity factor (K1), resolution (Rs) were studied( table 8)

Table 8: System suitability studies

Drug

RT

N

RS

As

Berberine

0.53

-

-

Embelin

7.3

3588

17

1.187

ANALYSIS OF FORMULATION

Fixed chromatographic conditions were used for the analysis of formulation.

Poly herbal formulation containing berberine and embelin named as LUMITAL TABLETS and ENTOSTALTABLETS collected from the market used for dysentry and bacterial diarrhoea.

Composition

Label claim(mg)

Oroxylum indica

100

Embelia ribes

50

Acacia catechu

25

Berberis aristata

300

Aloe barbadens

100

Santalum album

25

Vanga bhasma

10

Composition

Label claim(mg)

Holarrhena antidysenterica

600

Oroxylum indica

500

Berberis aristata

300

Cassia aungstifolia

150

Embelia ribes

50

Bombax malabricum

25

Eclipta alba

50

Zingiber officinale

25

Formulation I Formulation II

The above two poly herbal formulations containing berberine in Berberis arista and embelin in Embelia ribes.

EXTRACTION OF BERBERINE AND EMBELIN FROM POLYHERBAL FORMULATIONS:

To estimate the content of berberine and embelin from above two formulations,10 tablets were weighed and powdered. Powder equivalent to one tablet was taken and transferred into a 25 ml volumetric flask containing 10 ml methanol & DMSO(50:50) and sonicated for 10 min, and finally made up to 25 ml with methanol and This solution was filtered.

Recording of chromatogram

A steady baseline was recorded with the fixed chromatographic conditions. This was followed by injection of extracted solution prepared from the formulation. The chromatogram is shown in (fig). The amount of berberine and embelin in the formulation was calculated from the standard graphs were plotted using peak area of standard drugs to the concentration of corresponding standard( table 9)

Table 9 ( HPLC FORMULATION)

FORMULATION

LABEL CLAIM(mg)

AMOUNT PRESENT(µg)

BERBERINE

50

1.51

EMBELIN

50

4.68

Chromatogram of formulation

RESULT AND DISCUSSION:

RP-HPLC method is described for the quantitative estimation of berberine and embelin from poly herbal formulation.

Several mobile phase systems were tried with buffers and various organic solvents to achieve good retention time and resolution. and the wave length selected was 286nm. In sodium di hydrogen ortho phosphate and Acetonitrile(40:60) at pH 3.2 gave good separation from each other, and the retention time is 0.53&7.3 mins respectively for berberine and embelin.

Linear response was obtained in the range of 1-5µg/ml. for both markers of berberine and embelin. Quantification was achieved by plotting calibration graph using peak area of standard against concentrations. the coefficient values was found to be (r=0.997,0.998) for Berberine and Embelin respectively.

The slope and intercept values was found to be 5667.700, 64352.900 for Berberine -6647.800,67878.200 for Embelin respectively. precision is demonstrated by intra day and inter day is measured at three concentrations and three determinations were performed at each concentration instrument precision was studied by replicate injections of the sample and %RSD was calculated for each concentration. The RSD was found to be 0.34% for Berberine and 0.36 for Embelin respectively.

Recovery studies were performed by known amount of standard is added to the formulation containing Berberine and Embelin. Recovery is determined at 100% level and repeated three times. The average accuracy was found to be 95% for Berberine 98.7% for Embeline.

statistical analyses proves the method is accurate , precise and reproducible and selectivity.

CONCLUSION:

The proposed HPLC method is useful for quantification of markers in poly herbal formulation. In a reported HPLC method Embelin content was quantified from Embelia ribes. and hence quantification of Embelin from poly herbal formulation were not established. All the standards and samples can be analyzed simultaneously by the current HPLC method. However it makes advantageous compared with existing UV spectroscopic method. This method is used for routine quality- control testing.

Results of analysis of formulation, recovery and precision studies

Drug

% Recovery

Precision (% RSD*)

Intra

day

Inter

day

Repeatability

Of injection

Berberine

95

0.3

0.2

0.36

Embelin

98.7

0.53

0.4

0.4

ESTIMATION OF BERBERINE AND EMBELIN FROM POLY HERBAL FORMULATION BY USING HPTLC METHOD

A HPTLC method was developed for the combined estimation of berberine and embelin from poly herbal formulation.

For the estimation of both berberine and embelin by HPTLC method, methanol was employed as the solvent based on solubility of the drug and volatility of the solvent. The wavelength of 347 nm was selected for the estimation of markers shown maximum absorbance at this wavelength. Mobile phase system consisting of n-propanol:n-butanol :ammonia(6:2:2v/v) was selected based on optimum resolution and peak shape. Chamber saturation time and plate saturation time was fixed as 15 min. Rf value of both berberine and embelin was found to be 0.42 ,0.50 respectively.

The HPTLC method was validated in terms of parameters like linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), accuracy, precision, robustness, specificity and stability. The linearity was found to be in the range of 50-175ng/spot (of berberine and embelin .The correlation co-efficient value (0.999,0.997) proves a good linearity. The LOD and LOQ were found to be 20ng/µl and 40 ng/spot respectively Recovery was found to be 95% and 101.2% with a %RSD of 1.89, 1.92 Low %RSD values for precision indicate that the method is good and reproducible. Results are shown in table. The stability studies were carried out. The developed plate was found to be stable for about five hours and the solution stability was for about five hours under room temperature. To study robustness, the conditions were slightly changed and the method was found to be robust.

From the marketed formulation, sample solution was made and the peak response was used to calculate the concentration of the drug. The amount of drug present was calculated and the percentage recovery was found to be 95.2, 90% respectively.

Results of precision studies (%RSD*)

Drug

Precision

Repeatability

Berberine

Intra day

Inter day

Sample application

Measurement

1.89

1.92

1.23

0.71

Embelin

0.40

0.86

1.06

0.8

*RSD of six observations