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Herbal Medicine and its Importants

By: Mitul Shah | Views: 16161 | Date: 14-Jun-2010

Herbal Medicine is defined as branch of science in which plant based formulations are used to alleviate the diseases. It is also known as botanical medicine or phytomedicine. In the early twentieth century herbal medicine was prime healthcare system as antibiotics or analgesics were not available.

Herbal Medicine and its Importants

Herbal Medicine and its Importants


1. INTRODUCTION:

1.1 HERBAL MEDICINE AND ITS IMPORTANT
Herbal Medicine is defined as branch of science in which plant based formulations are used to alleviate the diseases. It is also known as botanical medicine or phytomedicine. In the early twentieth century herbal medicine was prime healthcare system as antibiotics or analgesics were not available. With inceasing use of allopathic system of medicine, herbal medicine gradually lost its popularity among people and it was based on the fast therapeutic actions of synthetic drugs. Almost a century has passed and it has witnessed limitations of allopathic system of medicine. Lately herbal medicine has gained momentum and it is evident from the fact that certain herbal remedies are more effective as compare to synthetic drugs.
Substances derived from the plants remain the basis for a large proportion of the commercial medications used today for the treatment of heart disease, high blood pressure, pain, asthma, and other problems. For example, ephedra is a herb used in Traditional Chinese Medicine for more than two thousand years to treat asthma and other respiratory problems. Ephedrine, the active ingredient in ephedra, is used in the commercial pharmaceutical preparations for the relief of asthma symptoms and other respiratory problems. It helps the patient to breathe more easily.
Another example of the use of a herbal preparation in modern medicine is the foxglove plant. This herb had been in use since 1775. At present, the powdered leaf of this plant is known as the cardiac stimulant digitalis to the millions of heart patients it keeps alive worldwide.

1.2 SCOPE OF THE HERBAL THERAPY AND INDIAN HERBAL MARKET
Traditional Indian practice held that certain drugs should be formulated through the addition of chosen substances that enhances bioavailability of anti-TB drug RIFAMPICIN. Formulation of piperine with rifampicin will save the drug counter effects.
Herbal oriented pharmaceutical companies are investing crores of rupees on researching, developing and popularizing OTC remedies. India can be a major player in the global market for herbal product based medicines. Exports of herbal materials and medicines can jump from just Rs. 456 crore in 2000 to Rs.3000 crore in 2005 and with a “grand strategic plan” exports can shoot to Rs.10,000 crore by 2010. 

1.3 THE SIGNIFICANCE OF QUALITY FOR EFFICACY AND SAFETY OF HERBALDRUGS                                                                                                                                            The efficacy and safety of any pharmaceutical product is determined by the compounds (desired and undesired) which it contains. The purpose of quality control is to ensure that each dosage unit of the drug product delivers the same amount of active ingredients and is, as far as possible, free of impurities. As herbal medicinal products are complex mixtures which originate from biological sources, great efforts are necessary to guarantee a constant and adequate quality. By carefully selecting the plant material and a standardized manufacturing process the pattern and concentration of constituents of herbal medicinal products should be kept as constant as possible as this is a prerequisite for reproducible therapeutic results.
The major problem in quality control is the batch to batch variations in the quality of medicinal plants. This is mainly due to the existence of ecotype pharmacological variations in the case of many medicinal plants. Therapeutic value of medicinal plants could differ depending on soil conditions, nutritional status, climatic conditions, seasonal variations, diurnal variations and their association with other organisms.


2. STANDARDISATION OF HERBS
The World Health Assembly emphasized the need to ensure quality control of medicinal plants with appropriate moral techniques and suitable standards, as it is estimated that about 80% of the people living in developing countries mainly depend upon herbal drugs for their preliminary health care needs. So, to deliver a good quality and safe medication standardization of hers and formulations is become very important. WHO gives different quality control parameters to standardize the raw material as well as finished products. 
For the standardization and quality assurance purposes three attributes viz. authenticity, purity and assay are desirable. Authenticity corresponds to the right identity which involves many parameters like morphology, microscopy, and chemical analysis. Purity pertains to evaluating that there are no adulterants present in the plant material. It can be evaluated by pharmacognostic evaluations like qualitative and quantitative microscopy, physical constants like, ash values, extractive values etc. Assay part of standardization is chemical and biological profiling by which the chemical and biological effects could be assessed and curative values get established. Chemical assay gives the quantitative evaluation of active constituents by using different techniques like HPTLC, HPLC, and Spectroscopy etc. In biological assays the drug activity is evaluated through a pharmacological model. For example the effectiveness of hepatoprotective drugs can be evaluated by its action on liver.  

2.1 Problems in standardization:
Problems in standardization arises form the complex chemical composition of herbal drug. Standardization of certain marker compound of herbal drug in general does not serve the purpose of standardization since activity of drug does not depend upon one or few components. In most of the cases, it is the result of concerted activity of several active compounds as well as of inert accompanying substances. Though these inert components do not directly affect the activity of drug, they might influence bioavailability and excretion of active component/s. Further, these inert components may also play a role in the stability of the active component and minimize the rate of side effects. If there are several active components present in a herbal drug, they may have additive or potentiating effect. The quantity of the active constituents may influence by several factors such as age and origin, harvesting period, and so on. To eliminate at least some of the causes of inconsistency, in terms of active ingredients, it was suggested that one should use cultivated plants rather than wild plants which are often heterogeneous with respect to the above factors and consistently in their content of active principles.

2.2 Standardization of finished products:
The process of standardization of finished product starts with the standardization of the raw materials. Certain parameters have been listed in different pharmacopoeias, Including Ayurvedic pharmacopoeia of India, WHO standards are Organoleptic, Microscopic examination, Morphology, Moisture Content, Foreign Matter, Ash Values, Bitterness Values, Hemolytic Index, Foaming Index, Volatile Matter, Pesticide Residue, Microbial Contamination, Metal Analysis, Radioactive Contamination and Chromatographic Evaluation are required for maintaining the quality of herbal raw material.

After the standardization of the raw material, next step is to monitor the process of preparation of the formulation and set parameters for in house quality control testing and finally the standardization of the finished product. Quality assurance of the herbal product relies upon good manufacturing facilities with adequate batch analysis and standard methods for preparation. Various processes use in the manufacture of the herbal drugs lacks standardized methods. Large scale commercialization of herbal drugs necessitates scientifically evolved standardized methods of plant drug production.

2.3 Standardization of herbs using marker compound analysis:
Every herb has a range of chemical constituents, which are produced as a result of metabolic activity in the plant. These compounds either alone or their combination are mainly responsible for the pharmacological activities or therapeutic action in the human body. Hence it would be more practical to test for the presence of these compounds. For example, Ashwagandha (whithania somnifera) can be assayed for whethanolides, Guggul (Commifora mukul) for guggulusterones, Neem (Azadirachta indica) for azadirachtine or nimbidine, Harida (Curcuma longa) for curcuminoids. For testing purpose these compounds are refered to as bio marker compounds. On the other hand, where the chemical composition of the herbs has been worked out but it is not clearly established whether these chemical entities are responsible for some particular action, any compound which is predominantly present in the herbs can be utilized as marker compound for the purpose of standardization. This group represents compounds like aegelin in Bilva (Aegle marmelose), Shatavarine in Shatavri (Asparagus racemosus), Fistuline in Aragvadha (cassia fistul ) 

One of the best methods of the standardizing herbs and herbal formulation based on the modern scientific tool is using chromatography. It helps not only in the establishing the current identity but also in regulating chemical sanctity of the herbs. For quantitative work HPLC is preferred generally. This is used mainly for volatile compound like essential oils and perfumes. In the past few years, HPTLC has emerge as a potential tool for rapid and efficient analysis of extract of herbal drugs and formulations.


3. INTRODUCTION OF HPTLC ANALYSIS
High performance thin layer chromatography (HPTLC) is an invaluable 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, numerous samples can be run in a single analysis thereby dramatically reducing analytical time. With HPTLC, the same analysis can be viewed sing different wavelengths of light thereby providing a more complete profile of the plant than is typically observed with more specific types of analyses.
The AHP has developed a network of analytical labs who will perform HPTLC analyses according to AHP protocols. Additionally, AHP reviews all of the data to assure the SOPs have been met and that the findings of the analysis are accurate. Materials that pass will be provided a AHP Certificate of Authenticity. For those that fail, an explanation of why the product failed will be provided.
Cost may fluctuate depending on the needs, costs, and availability of reference standards. PDFs of the chromatograms with a documentation report shall be provided for each analysis.
Main Difference of HPTLC and TLC - Particle and Pore size of Sorbents. 

The other differences are 
HPTLC  TLC 
Layer of Sorbent  • 100µm  • 250µm 
Efficiency • High due to smaller particle size generated  • Less 
Separations • 3 - 5 cm  • 10 - 15 cm 
Analysis Time  • Shorter migration distance and the analysis time is greatly reduced  • Slower 
Solid support • Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes  • Silica gel , Alumina & Kiesulguhr 
Development chamber  • New type that require less amount of mobile phase  • More amount 
Sample spotting  • Auto sampler  • Manual spotting 
Scanning  • Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer  • Not possible 

3.1 Advantage of HPTLC :
The Analysis of herbals and herbal preparations is challenging for several reasons :
 As Analytes, herbs are extremely complex. Even herbal preparations such as extracts contain numerous compounds in concentration that can cover several orders of magnitude.
 In many instances, chemical composition of the herb is not completely known. For several of the Ayurvedic and Chinese herbs, there are no established methods of analysis available.
 The requirements ofr a fingerprint analysis can be completely different from those for a quantitative determination of marker or key compounds, although the herbal preparation separated for fingerprints, but for quantitative determination of maker compounds. It is necessary to fully separate those compounds from all others.
 Constituents of herbals that belong to very different classes of chemical compounds can often create difficulties in detection. With this in mind, TLC and especially HPTLC can offer many advantages.

3.2 Features of HPTLC
1. Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard 
2. Several analysts work simultaneously 
3. Lower analysis time and less cost per analysis 
4. Low maintenance cost 
5. Simple sample preparation - handle samples of divergent nature 
6. No prior treatment for solvents like filtration and degassing 
7. Low mobile phase consumption per sample 
8. No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination 
9. Visual detection possible - open system 
10. Non UV absorbing compounds detected by post-chromatographic derivatization 
3.3 Steps involved in HPTLC 
1. Selection of chromatographic layer 
2. Sample and standard preparation 
3. Layer pre-washing 
4. Layer pre-conditioning 
5. Application of sample and standard 
6. Chromatographic development 
7. Detection of spots 
8. Scanning 
9. Documentation of chromatic plate 

3.4 Selection of chromatographic layer 
• 80% of analysis - silica gel60 F254 GF are used.
•For Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18 are used.

3.5 Activation of pre-coated plates 
Freshly open box of plates do not require activation 
Plates exposed to high humidity or kept o¬n hand for long time to be activated 
By placing in an oven at 110-120ºc for 30 mins prior to spotting 
Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes. 

3.6 Application of sample and standard 
• Usual concentration range is 0.1-1µg / µl 
• Above this causes poor separation
• Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe o¬n TLC plates as bands 
• Band wise application - better separation - high response to densitometer 

3.7 Selection of mobile phase 
- Trial and error 
- one’s own experience and Literature 
- Normal phase 
- Stationary phase is polar 
- Mobile phase is non polar
- Non-polar compounds eluted first because of lower affinity with stationary phase
- Polar compounds retained because of higher affinity with the stationary phase
• Reversed phase 
- Stationary phase is non polar 
- Mobile phase is polar 
- Polar compounds eluted first because of lower affinity with stationary phase 
- Non-Polar compounds retained because of higher affinity with the stationary phase
- 3 - 4 component mobile phase should be avoided 
- Multi component mobile phase o¬nce used not recommended for further use and solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100
- Twin trough chambers are used o¬nly 10 -15 ml of mobile phase is required 
-• Components of mobile phase should be mixed introduced into the twin - trough chamber 

3.8 Pre- conditioning (Chamber saturation) 
• Un- saturated chamber causes high Rf values 
• Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of solvent vapours - less solvent for the sample to travel - lower Rf values. 

3.9 Chromatographic development and drying 
• After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere
• Dry in vacuum desiccator - avoid hair drier - essential oil components may evaporate 

3.10 Detection and visualization 
• Detection under UV light is first choice - non destructive 
• Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length) 
• Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF
• Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution
• When individual component does not respond to UV - derivatisation required for detection 

4. CAMAG-HPTLC INSTRUMENTATION
Stationary Phase 
Precoated layers  HPTLC plates-
silica gel 60 F254 (mostly used)
Sample Application
Instrument for semi-automatic band wise spray-on sample application: 
Linomat 5 operated with Software "winCATS - Planar Chromatography Manager”.Chromatogram Development
Automatic Development Chamber ADC2 
For reproducible conditions in the development of the HPTLC plates, complete incl. option »Humidity Control« that allows reproducible chromatography at defined activity of the layer, incl. EquiLink for winCATS basic software.
Derivatization
CAMAG Chromatogram Immersion Device III  with
TLC Plate Heater III for heating plates to a given temperature, ensuring homogenous heating across the plate.
Chromatogram Evaluation 
Detection with Scanner 3
The most advanced workstation for densitometric evaluation of thin-layer chromatograms.
Documentation
CAMAG documentation system DigiStore2 
With high-resolution 12bit CCD camera, consisting of: Reprostar 3 lighting unit, 12bit CCD camera with built-in UV & IR filter, all connecting cables and 16 mm lens for optimal imaging of object  EquiLink for winCATS basic software.

5. QUANTIFICATION:
5.1 Estimation of Glycyrrhizic Acid by HPTLC.
a) Sample preparation :
- 0.25 g dry extract is taken in 50 ml 1N HCL and refluxed for four hours. After cooling to room temperature, it is extracted with 20 ml chloroform 5 times. The combined chloroform extract, after washing with water and filtration is evaporated at maximum temperature of 300 C and residues is dissolved in chloroform-methanol (1:1) and make up to 25 ml.
b) Sample Application : 
- Bandwise with Linomat 2 , 3 and 4 ul of standard and sample solution, band length 6 mm, distance between bands 4mm. 
C) Chromatography :
- Two step chromatogram development : 
1st Run  :-   Chloroform-Acetone 9:1
2nd Run  :- Chloroform-Diethyl Ether-Formic Acid 80:15:1
d) Quantitative evaluation :
- Scanning by absorbance at 254 nm (Hg lamp) monochromator band width 10 nm. Slit dimension 4 x 0.3, evaluation via peak height or peak area. 1ul of the calibration standard solution contains 400 ng glycyrrhetinic acid corresponding to 700 ng glycyrrhizic acid in the extract. 

5.2 HPTLC Method For Estimation Of Curcuminoids From Curcuma Longa.
Curcuma longa (Zingiberaceae), commonly called Haldi, is a well-known plant drug in Ayurvedic and Unani It has been used for the treatment of various diseases and disorders particularly for urticaria, skin allergy, viral hepatitis, inflammatory condi-tions of joints, sore throat, and for wound.Curcumin, demethoxycurcumin and bis-demetho-xycurcumin, three major pharmacologically important curcuminoids, have been isolated from C. longa : and has been shown to possess anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-mutagenic, anti-fungal, anti-viral and anti-cancer ..Methods, so far available for the determination of these alkaloids, are very cumbersome and time-consuming and also not economically viable. Therefore it was thought worthwhile to develop a simple and high-precision HPTLC method for simultaneous analysis of curcumin, demethoxy curcumin and bis-demethoxycurcumin occurring in roots of C. longa. 

Rf values by HPTLC and linear regression equa-tions for the determination of curcumin, demethoxycurcumin and bis-demethoxy-curcumin.
Compound     Rf             value   
Curcumin  0.67   
Demethoxycurcumin  0.47   
Bis-demethoxy-curcumin  0.29   

Chromatographic conditions:
Chromatography was performed on glass-backed silica gel 60 GF254 HPTLC layers (20 x 20 cm, 
300 μm layer thickness).

Methanolic solutions of samples and standard compounds curcumin, demethoxycurcumin and bis-demethoxycurcumin of known concentrations were applied to the layers as 7 mm-wide bands positioned 15 mm from the bottom and 20 mm from the side of the plate, 

Mobile Phase:  chloroform: methanol (48:2, v/v) for one hour. 
25 ± 50C and 50 % relative humidity. Quantitative analysis of the compounds was done by scanning the plates using,
Slit width:   6 x 0.45 mm, 
Wavelength (λmax):  425 nm.
In order to prepare calibration curves, stock solutions of curcumin, demethoxycurcumin and bis-demetho-xycurcumin (1 mg/5 mL each) were prepared and various volumes of these solutions were analyzed by HPTLC exactly as described above. Then calibration curves of peak area vs. concentration were prepared.
Results and Discussion: 
The wave length of 425 nm was found to be optimal for the highest sensitivity. The calibration curves for the alkaloids curcumin, demethoxy-curcumin and bis-demethoxycurcumin were linear in the range 100-1,000 ng 

5.3 HPTLC method for estimation of charantin:
Diabetes mellitus has recently been identified by Indian Council of Medical Research (ICMR) as one of the refractory diseases for which satisfactory treatment is not available in modern allopathic system of medicine and suitable herbal preparations are to be investigated

Momordica charantia Linn. Cucurbitaceae is a well known to possess antihyperglycemia, anticholesterol, immunosuppressive, antiulcerogenic, anti seperma -togenic and androgenic All the three selected polyherbal formulations contain karela as one of the plants. The reference standard charantin had to be isolated, purified and the structure authenticated by various spectral analysis. There are no titrimetric, colorimetric, spectrophotometric or chromatographic methods available for quantitative estimation of charantin in different marketed antidiabetic PHFs. Therefore an attempt has been made to develop a HPTLC method because this method is fast, precise, sensitive and reproducible with good recoveries for standardization of polyherbal formulations. 

Chromatographic conditions:
Chemicals: Analytical grade chloroform, benzene, methanol, formic acid ethyl acetate aluminium. 
Stationary Phase: Plates pre-coated with silica gel 60 F 254 (10x 10 cms, 0.2 mm thick).
Rf Value: 0.32 was visible and scanned under 536 nm.
Wavelength (λmax): 536 nm.
Procedure:
One milligram of working standard charantin was dissolved in 100 ml of chloroform to yield stock solution of 100μg/ml concentration. Calibration curve from 20-600 ng /spot was prepared and checked for reproducibility, linearity and validating the proposed method. The correlation coefficient, coefficient of variance and the linearity of results were calculated.
Sample preparation:
200 mg of PHFs were taken and extracted in 10 ml of chloroform then the chloroform extract was filtered through Whatmann no. 42 filter paper. The final volume of the extract was made to 10ml with chloroform in volumetric flask. The small and big karela were dried under shade and finely powdered. From that 50 mg of fine powder was taken and extracted by chloroform and filtered dried extract the volume make up to 2 ml with chloroform.
Method Specifications:
Silica gel 60 F254 precoated plates (10 x 10 cm) were used with benzene: methanol (80:20) as solvent system. 
plates were developing up to 8 cms. The plates were sprayed with 10% sulphuric acid in alcohol and the reagent was prepared freshly, heated at 1300 C for 2-3 min and brought to room temperature. 


HPTLC chromatogram of standard Charantin:

Results and Discussion:
Standard charantin showed single peak in HPTLC chromatogram. The calibration curve of charantin was obtained by spotting  standard charantin on HPTLC plate. After development the plate was scanned at 536 nm the calibration curve was prepared by plotting the concentration of charantin versus average area of the peak. The amount of charantin was computed from calibration curve and calibration curve was shown in Fig. 98.89.
The lowest detectable limit of charantin in different formulations was found upto 20 ng/spot.

5.4 HPTLC method for estimation Sennosides:
Cassia angustifolia (Family: Caesalpiniaceae), popularly known as senna, is a valuable plant drug in ayurvedic and modern system of medicine for the treatment of constipation  Sennoside A and B are the two anthraquinone glycosides that are responsible for purgative action of senna. A variety of poly-herbal formulations containing senna leaves are available in India to relief constipation and allied troubles. Senna is a strong purgative that should be taken in proper dosage otherwise it may lead to gripping and colon problem  Different analytical techniques, viz, thin layer chromatography, spectrophotometry, column chromatography have been reported in the literature for estimation of sennosides . 
Chromatographic conditions:
Stationary Phase:
Chromatography was performed on glass-backed silica gel 60 GF254 (20 cm x 20 cm; 0.30 mm layer thickness) 
Samples and standard compounds 1 and 2 of known concentrations were applied as 8 mm wide bands with nitrogen flow providing delivery speed 150 ηl/s from the syringe. These parameters were kept constant through out the analysis. 
25 ± 2oC and 60% relative humidity
Slit width: 6 x 0.45 mm.
Wave length (λmax): 350 nm.

Analytical standards of sennoside A and B were obtained from  Solvents (methanol, 2-propanol, ethyl acetate, formic acid) used in entire study.
Preparation of standard solutions:
Standard solutions of sennoside A and sennoside B (1 mg\5 ml) were prepared in methanol. 
Extraction of samples:
Each 4 g of different branded formulation was sonnicated with 70% methanol (3 x 20 ml) for about 45 min. Then the extract was filtered in a Buchner funnel using Whatman No. 1 filter paper and was concentrated under vacuum in a rotary evaporator at 50ºC, redissolved in methanol and finally reconstituted in 20 ml methanol prior to HPTLC analysis. 
Samples and standard compounds 1 and 2 of known concentrations were applied as 8 mm wide bands with nitrogen flow providing delivery speed 150 ηl/s from the syringe. These parameters were kept constant through out the analysis. 

Rf values by HPTLC for the determination of sennoside A and B 
Compound  Rf value   
Sennoside A  0.84   
Sennoside B  0.63   
Sennoside A and B in selected herbal formulations 
Herbal formulations  Sennoside A 
(in mg/g of formulation)  Sennoside B 
(in mg/g of formulation) 
Formulation-1  2.50  25.90 
Formulation-2  2.30  12.70 
Formulation-3  2.10  2.60 
Formulation-4  1.80  1.87 
Formulation-5  1.62  1.85 
Formulation-6  1.58  1.72 
Formulation-7  1.40  4.53 
Formulation-8  1.20  1.50 
Formulation-9  0.92  1.50 
Formulation-10  0.91  1.40 

Results and Discussions:
Scan (at 350 nm) showing the separation of sennoside A and B in the extract of a laxative formulation and limit of quantification of sennoside A and B were determined to be 0.05 and 0.25 μg/g. 
The result  showed that the relative amount of sennoside A and B in formulation-1 was highest and in formulation-10 it was lowest. Theresult reflected a 2.75 times higher concentration in formulation-1 compared to that of formulation-10. The minimum content of sennoside B (considering the highest content as 100%) was shown only by 5.41% in case of formulation-10 and less than 17.5% in case of formulation number ‘3’ to ‘9’.
Sennoside A and B was found in the concentration range 200-1000 ng. Regression analysis of the experimental data points showed a linear relationship with excellent correlation coefficient(r) of sennoside A and sennoside B of 0.991 and 0.997, respectively The average recovery rate was 95% for sennoside A and 97% for sennoside B.

5.5 HPTLC method for estimation of glucosamine :
Chromatographic conditions:
Stationary Phase: Glucosamine was separated from the plant extracts on a silica gel 60 F254
Chemicals: HPTLC plate using a saturated mixture of 2-propanol–ethyl acetate–ammonia solution (8%) (10:10:10, v/v).
wave length (λmax) : 415nm. 
The plates were developed vertically up to a distance of 80 mm. For visualization, the plate was dipped into a modified anisaldehyde reagent and heated at 120 °C for 30 min in a drying oven. Glucosamine appeared as brownish-red chromatographic zones on a colourless background. 
Result:
The relative standard deviations for repeatability and intermediate precision were between 4.9 and 8.6%. Moreover, the method was found to be accurate, as the two-sided 95% beta-expectation tolerance interval did not exceed the acceptance limits of 85 and 115% on the whole analytical range (800–1200 ng of glucosamine).

5.6 HPTLC method for estimation of trans-Resveratrol :
Ttrans-resveratrol is obtain from Polygonum cuspidatum root extracts . [(E)-5-[2-4(hydroxyphenyl)ethenyl]-1,3- benzenediol] is a naturally occurring polyphenolic compound belonging to a group called stilbenes, found in grapes, peanuts and other plants.2) trans-Resveratrol is a strong antioxidant and reported to have a protective effects against atherosclerosis, coronary heart disease, postmenopausal problems, inhibits platelet aggregation and a broad spectrum of degenerative diseases and also possess cancer chemopreventive properties. The roots of Polygonum cuspidatum, (Polygonaceae). 
Chromatographic conditions:
Stationary Phase : Silica gel 60F-254.
Mobile phase: Eluted with chloroform–ethylacetate–formic acid (2.5 : 1 : 0.1).The plates were prewashed by methanol and activated at 60 °C for 5 min prior to chromatography. The length of chromatogram run was 8 cm.
Wave length (λmax): 313nm.  
Result:
A good linear regression relationship between peak areas and the concentrations was obtained over the range of 0.5—3.0 mg/spot with correlation coefficient 0.9989. 
The limit of detection and quantification was found to be 9 and 27 ng/spot  at Rf  value 
of 0.40.
The spike recoveries were within 99.85 to 100.70%. 
The RSD values of the precision in the range 0.37— 1.84%.


5.7 HPTLC method for estimation of Phyllanthin and Hypophyllanthin in Phyllanthus Species.
The genus Phyllanthus (Euphorbiaceae) contains 550–750 species in 10–11 subgenera that are distributed in all tropical regions of the world from Africa to Asia, South America and the West Indies. Phyllanthus amarus is the most widespread species and is typically to be found along roads and valleys, and on riverbanks and near lakes in tropical areas. Other species found in India are P. fraternus, P. urinaria, P. virgatus, P.maderaspatensis and 
P. debilis. The genus Phyllanthu has a long history of use in the treatment of diabetes, intestinal parasites and liver, kidney and bladder problems. P. amarus is highly valued in the treatment of liver ailments and kidney stones and has been shown to posses anti-hepatitis B virus surface antigen activity in both in vivo and in vitro studies.
The major lignans of the genus, namely, phyllanthin (1) and hypophyllanthin (2), have been shown to be anti-hepatotoxic against carbon tetrachloride- and galactosamine-induced hepatotoxicity in primary cultured rat hepatocytes . Thus, an appropriate analytical procedure for the quantitative determination of these lignans in different Phyllanthus species is of considerable importance.  
Mobile Phase:
The phase finally chosen, namely, hexane:acetone:ethyl acetate (74:12:8, v/v/v) gave good resolution of phyllanthin (1) and hypophyllanthin (2) from other closely related lignans.
Rf values: 0.24 and 0.29, respectively.
Sample Collection
All the Hypericum species were collected from various locations in the region of Pinar del Río, Cuba. Specimen samples were all collected at blossoming time with most of the flowers opened from April to November in 2000 and 2001. The samples were dried at room temperature for seven days and powdered. Voucher specimens of H. tetrapetalum, 
H. nitidum and H. styphelioides.
Sample preparation
Powered samples (0.5 g) were extracted with 10 ml of methanol using an ultrasonic bath for 10 min. For  and HPTLC analysis the extracts were first filtered.
Standard preparation
Commercial hypericin formulation tablets  were used. Three tablets (0.9 g) equivalent to 2.7 mg of hypericine were powdered and extracted with 10 ml of methanol using an ultrasonic bath for 10 min. 
Extraction procedure. 
Air-dried leaves (1 g) of P. amarus were separately extracted with either hexane, chloroform, ethyl acetate or methanol. In each case, the extraction was carried out three times with 10 mL of solvent for 10 h at room temperature (25 ± 5°C), and the solvent was removed from the combined extract under reduced pressure to yield the respective crude residue. In order to determine the appropriate extraction solvent, each of the crude residues was dissolved separately in 1 mL of methanol and the contents of the two lignans in each sample was determined by HPTLC. The maximum content of lignans 1 and 2 was obtained by extracting with methanol    .


Analytical procedure:
Air-dried leaves (1 g) of different Phyllanthus species were extracted separately at room temperature (25 ± 5°C) with methanol (3 × 10 mL; 10 h for each extraction), and the combined extracts were filtered, dried under vacuum and made up to 1 mL with methanol prior to HPTLC analysis. 
Result:
The content of the latter (0.858%) was reportedly higher than that of the former (0.709%). 
It was observed that the reported higher concentration of 2 was due to other lignans present at the  same Rf as that of hypophyllanthin. The phase finally chosen, namely, hexane:acetone:ethyl acetate (74:12:8, v/v/v) gave good resolution of phyllanthin (1) and hypophyllanthin (2) from other closely related lignans (Fig. 1).

5.8 Estimation Of β-Asarone In Acorus Calamus Dry Extract By HPTLC.
1) Chromatographic condition:
Mobile phase : Toluene-Ethyl Acetate (93:7).
Wave length : 290nm.
2) Standard and Sample Preparation :
a) Standard Preparation :
A 60 μg/ml solution of β-asarone reference standard is prepared in toluene. 
b) Sample Preparation:
About 1 g of Acorus calamus dry extract is accurately weighed and dissolved in 5-7 ml of toluene, sonicated for about 2 minutes and volume made upto 10 ml. The solution is filtered.1 ml of this solution is further diluted to 50 ml and used for further analysis. 
3) Procedure :
- Four spots of the standard preparation and four spots of the sample preparation are applied about 1 cm form the edge of the TLC plates.
- The plate is developed upto 9 cm in the mobile phase, dried at room temperature and scanned. 
4)  Results and discussions :
- Under the chromatographic conditions described above the Rf & β- asarone is calculated. The chromatogram of standard and sample are studied on following basis.
ii. Calibration curves
iii. Inter-day coefficient of variation for analysis.
iv. Average recovery.
The method gives the best resolution of β-asarone from other constituents of Acorus calamus. Thus this newly developed HPTLC method is quick and reliable for quantitative monitoring of β-asarone in Acorus calamus and in herbal preparations containing Acorus calamus. 


6. Chromatographic Fingerprint Analysis for Herbal Medicines: A Quality Control Tool
Introduction
The construction of chromatographic fingerprints plays an important role in the quality control of complex herbal medicines. Chemical fingerprints obtained by chromatographic techniques are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the “chemical integrities” of the herbal medicines and therefore be used for authentication and identification of the herbal products. Based on the concept of phytoequivalence, the chromatographic fingerprints of herbal medicines could be utilized for addressing the problem of quality control of herbal medicines. By definition, a chromatographic fingerprint of a herbal medicine is, in practice, a chromatographic pattern of pharmacologically active and or chemically characteristic constituents present in the extract. This chromatographic profile should be featured by the fundamental attributions of “integrity” and “fuzziness” or “sameness” and “differences” so as to chemically represent the herbal medicines investigated. This suggest that chromatographic fingerprint can successfully demonstrate both “sameness” and “differences” between various samples and the authentication and identification of herbal medicines can be accurately conducted even if the number and/or concentration of chemically characteristic constituents are not very similar in different samples of herbal medicine. Thus chromatographic fingerprint should be considered to evaluate the quality of herbal medicines globally considering multiple constituents present in the herbal medicines. 
Need for development of chromatographic fingerprints:
Herbal medicines have a long therapeutic history and are still serving many of the health needs of a large population of the world. But the quality control and quality assurance still remains a challenge because of the high variability of chemical components involved. Herbal drugs, singularly and in combinations, contain a myriad of compounds in complex matrices in which no single active constituent is responsible for the overall efficacy. This creates a challenge in establishing quality control standards for raw materials and standardization of finished herbal drugs. Traditionally only a few markers of pharmacologically active constituents were employed to assess the quality and authenticity of complex herbal medicines. However, the therapeutic effects of herbal medicines are based on the complex interaction of numerous ingredients in combination, which are totally different from those of chemical drugs. Thus many kinds of chemical fingerprint analysis methods to control the quality of herbal drugs have gradually come into being, such as thin layer chromatography, gas chromatography, high performance liquid chromatography etc. chromatographic fingerprint analysis of herbal drugs represents a comprehensive qualitative approach for the purpose of species authentication, evaluation of quality and ensuring the consistency and stability of herbal drugs and their related products. The entire pattern of compounds can then be evaluated to determine not only the presence or absence of desired markers or active constituents but the complete set of ratios of all detectable analytes. The chemical fingerprints obtained by chromatographic and electrophoretic techniques, especially by hyphenated chromatographies, are strongly recommended for the purpose of quality control of herbal medicines, since they might represent appropriately the “chemical integrities” of herbal medicines and therefore be used for authentication and identification of the herbal products. 
Difficulties in development of chromatographic fingerprints for herbal medicines
When herbal drugs are considered for analysis, a large number of chemical components are involved and many of them are in low concentration. Chromatographic instruments and experimental conditions are difficult to reproduce during real analysis. Thus, the baseline and retention time shifts surely will be in existence from one chromatogram to another.  Many other problems associated with chromatographic fingerprints such as the occurrence of abnormal chromatograms from outlying herbal samples or experiments inevitably will be encountered. As a result, in order to obtain reliable chromatographic fingerprints, several data treatments would be needed during fingerprint analysis.  
Chemometric approaches and data processing for chromatographic fingerprint of herbal medicines:
Due to complexity of the chromatographic fingerprint and the irreproducibility of chromatographic instruments and experimental conditions, several chemometric approaches such as variance analysis, peak alignment, correlation analysis, and pattern recognition were employed to deal with the chromatographic fingerprint. Many mathematical algorithms are used for data processing in chemometric approaches. The basic principles for this approach are variation determination of common peaks/ regions and similarity comparison with similarity index and linear correlation coefficient. Similarity index and linear correlation coefficient can be used to compare common pattern of the chromatographic fingerprints obtained. In general, the mean or median of the chromatographic fingerprints under study is taken as the target and both are considered to be reliable. To facilitate the data processing, a software named Computer Aided Similarity Evaluation (CASE) has been developed. All programs of chemometric algorithms for CASE are coded in METLAB5.3 based on windows. Data loading, removing, cutting, smoothing, compressing, background and retention time shift correction, normalization, peak identification and matching, variation determination of common peaks/regions, similarity comparison, sample classification, and other data processes associated with the chromatographic fingerprint can be investigated with this software. 
EXAMPLES:
6.1 HPTLC fingerprinting analysis of Adhatoda vasica Nees. :  
Vasicine and vasicinone type alkaloids are separate from the Adhatoda vasica Nees.
HPTLC fingerprinting shows the presence of five peaks. Two of them, which are major, correspond to vasicine and vasicinone  with superimposable UV spectrum. 
a) System :
CAMAG Instrument
Automatic Plate Quoter
Linomat-5
Chromatogram Scanner with Wincats 
Software 
b) Operating Conditions: 
- Mobile Phase :          Methanol-Toluene-Dioxane-Ammonia (2:2:5:1)
- Wavelength  : 270 nm
- Slit Width : 5 x 0.45 nm
- Lamp Used    : D2 and W
- Scan Mode : Absorption-Reflection 
HPTLC fingerprint shows the presence of five peaks. Two of them, which are major, correspond to vasicine and vasicinone ( Rf 0.99 and 1.17) with superimposable UV-Spectrum. 


6.2. Finger printing profile on Laboratory and market formulation of Rajanyadi churna was estimated by using HPTLC. 
Both Photographs showing the comparative account of presence of various phytoconstituents in direct Methanolic extract of  different plants incorporated to that of the lab and marketed formulations.  Rf values of different constituents of individual plant Methanolic extracts are matching with the Rf values of formulation Methanolic extracts. 

6.3. HPTLC Fingerprint Identification of Commercial Ginseng Medicine : 
- The roots of Ginseng have held the esteem of the Chinese as a “ cure-all medicinal herb for thousands of years. It turns out. Nowadays, in single or multi component pill, tablet, capsule, oral liquid and even cosmetics besides the crude drug itself. Commercial Ginseng is Classified in to white ginseng (dried naturally), red ginseng (steam-processed) (Panax ginseng, family; Araliaceae) produced mainly in china and Korea ( it can therefore be called ‘ Asian ginseng’); American Ginseng (P. quinquifolium) exported from eastern U.S.A and Canada via Hongkong as well as Notoginseng (Sachi) (P.notoginseng) native of South west China. A booming Market in Asian Ginseng, American Ginseng and various kinds of their preparations recent years put forward before the analysis a task for quality control with an effective, rapid and economic analysis method. As routine drug control, TLC/ HPTLC does undoubtedly meet the requirements and the fingerprint differentation taps further the potential of TLC from the view- Point of methodology.
- As routine quality control of commercial Ginseng medicine, TLC/HPTLC is no doubt a rapid, effective and low-cost analytical method. HPTLC of Asian ginseng, American Ginseng, Notogineseng (Sanchi) and some of their preparations have been reported by Xie and Yan reliable experimental data and the reproducible chromatograms.
- It has been reported that upon comprehension of more than hundreds of specimens of commercial radix Asian Ginseng [ Panax ginseng] and American Ginseng [ P. quinquifolium], as a whole, the HPTLC pattern of Ginseng are always simpler than that of Asian Ginseng. The fluorescence intensity of mani ginsenosides sports is much stronger than the miner saponin spots. In comtrast with American Ginseng, the minor ginsenosides in Asian Ginseng (red ginseng in particular) are easier to observe and the patterns are therefore mor complicated.
- To optimize the condition of HPTLC and ginsenosides it has been reported that the solvent system, chloroform-Ethyl acetate-Methanol-Water (15/40/22/10, stand over night at 8-100C(lower phase) has a highr resolution, better reproducibility of Rf values and more impact spots by comparison with the solvent systems established by the previous investigators and used in common. Detection and scanning in fluorescence mode after visualization with 5 % sulphuric acid/EtOH reagent by dipping technique improved and enhanced the sensitivity than that in absorbance mode most commonly used. 
- Sample pretreatment through adsorption clean-up step via a small basic alumina column followed by 1-butanol extraction instead of only butanol-extraction step made the chromatogram more clear, less background contamination and reduced the trailing of some ginsenosides spots. The experimental data demonstrated that the relative humidity(RH) has significant influence on the chromatographic behavior of ginseosides. The optimum RH for pre-equilibration of the precoated HPTLC plate is 42-47 % and the optimum temperature of development is at 25-280 C.
- HPTLC fingerprints of ginseng preparations revealed the instability of ginsenosides in liquid dosage forms such as “shuan Bao Su” oral liquid (SBS-Liq) containing royal jelly and honey in admixture with ginseng extract. These findings raise questions about assessment of the stability of ginseng preparations from the standpoint of drug quality control. The results of an accelerated stability study reported by Xie and Yan elucidate the varius degradation processes of ginsenosides in the presence of royal jelly and/or honey. It also demonstrates that the HPTLC ginsenosides fingerprint can serve as stability indicating method, even when the chromatogram is not otherwise differentiated. 

7. CONCLUSION:
The problem of quality assurance of herbal medicines has been solved to a great extent with the help of chromatographic fingerprint analysis. The variation determination of common peaks/regions in a set of chromatographic fingerprints could provide useful qualitative and quantitative information on the characteristic components of herbal medicines investigated. On the other hand, whether the real samples were identified as the herbs with the same quality grade could be determined successfully by way of comparing the chromatographic fingerprints with the similarity index and linear correlation analysis. Furthermore, pattern recognition can be used to discriminate different kinds of samples of herbal medicines investigated. Thus chromatographic fingerprint analysis serves as a promising quality control tool for herbal medicines.

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