ENCLOSURE-I
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 GENERAL DISCUSSION :
Chebulic myrobalan[8] (Terminalia chebula Retz, Combretaceae) is called the
“King of Medicines” in the Tibet and is always listed first in the Ayurvedic materia medica
because of its extraordinary powers of healing with a wide spectrum of biological
activity[12]. During the last five decades, considerable progress has been achieved
regarding biological activity and medicinal applications of Terminalia chebula. It is now
considered as a valuable source of unique natural products for development of medicines
against various diseases[8]. Fruits of Terminalia chebula are credited with laxative,
stomachic, astringent and tonic properties[14]. It has been extensively used in Ayurveda,
Unani and Homeopathic medicine and has become a cynosure of modern medicine[8].
Terminalia chebula is rich in tannin (30-32%)[13,14]. The chief phytoconstituents of tannin
are chebulic acid, chebulagic acid, chebulinic acid, corilagin, gallic acid and ellagic
acid[13]. Therefore, the estimation of chebulagic acid and chebulinc acid would be an
important parameter for the quality control of polyherbal formulations.
Chebulagic acid[1,25] is a benzopyran derivative. It has been found to be
immunosuppressive, hepatoprotective and a potent alpha-glucosidase inhibitor, a human
gut enzyme useful in diabetic studies. It has been shown to be active against
Staphylococcus aureus and Candida albicans.
Molecular structure :
Chebulagic acid
Nomenclature
: Diethyl biphenyl[2,21]– Dicarboxylate 2-isoboxethylbenzoate(s)-2Isobutoxethylbenzoate(s)-2-isoprpylsuccinic acid-1-Methyl ester.
Molecular formula : C41H30O27.
Molecular weight
: 954.661 g / mol.
Characteristics
: White to light yellow crystalline powder.
Solubility
: Sparingly soluble in water, freely soluble in methanol, ethanol,
ethyl acetate and acetone.
Chebulinic acid[1,24] is one of the ellagitannins in the fruits of Terminalia
chebula, which has many potential uses in medicine.
Molecular structure :
Chebulinic acid
Nomenclature
: 1, 3, 6-Tri-o-galloyl-2,4-chebuloyl-β-D-glucopyranoside.
Molecular formula
: C41H32O27.
Molecular weight
: 956.67 g/mol.
Characteristics
: Faint yellowish crystalline powder.
Solubility
: Sparingly soluble in water, soluble in ethanol, methanol and
ethyl acetate.
6.2 NEED FOR THE STUDY :
Quantification of bioactive principles through modern analytical tools is essential
for establishing the authenticity and creditability of prescription and usage of herbal
drugs[16,17]. The use of herbal drugs and products made from them are increasing every
passing day. So, there is a demand for standardardized extracts. This necessitates the
development of suitable methods for the analysis of phytoconstituents of therapuetic
importance in formulations.
In the present scenario, HPTLC[19] is very important, essential and valuable tool for
qualitative and quantitative analysis of herbal products. HPTLC is a widely accepted
technique for its high accuracy, precision and reproducibility of results[18]. Therefore, an
attempt has been made to quantify chebulagic acid and chebulinic acid in polyherbal
formulations.
Extensive literature survey reveals that, a very few analytical methods have been
reported for the quantification of chebulagic acid and chebulinic acid which includes High
performance capillary electrophoresis (HPCE)[2,3], RP- HPLC[4], TLC-UV Densitometry[5]
and High speed counter current chromatography[7]. Apart from above methods, no HPTLC
method has been developed or found from literature for the quantification of chebulagic
acid and chebulinic acid.
Hence, there is a need for the development of suitable HPTLC method for routine
analysis of chebulagic acid and chebulinic acid in polyherbal formulations. So, an attempt
is being made to develop a simple, precise, rapid and cost-effective HPTLC method for the
quantification of chebulagic acid and chebulinic acid in polyherbal formulations.
6.3 REVIEW OF LITERATURE :
1. Ding et al[3]., have studied on Analysis of tannins in Fructus chebulae and its
confusion varieties by HPCE.
2. Pawar et al[4]., have studied on Development of a RP-HPLC Method for Analysis of
Triphala curna and its applicability to Test Variations in Triphala curna preparations.
3. Juang et al[2]., have studied on Determination of hydrolysable tannins in the fruit of
Terminalia chebula Retz. by HPLC and capillary electrophoresis.
4. Ratnayake et al[5]., have studied on Uptake of Terminalia chebula in Tannin
components by Hide:Analysis using TLC-UV Densitometry.
5. Takashi Yoshida et al[6]., have studied on New methods of analyzing tannins.
6. HanQ Sonq et al[7]., have studied on Preprative isolation of hydrolysable tannins
(chebulagic acid and chebulinic acid) from Terminalia chebula by High-speed counter
current chromatography.
7. Hartzfeld et al[9]., have studied
on Determination of hydrolysable tannins after
reaction with potassium iodate.
8. Jadhav et al[10]., have studied on Estimation of gallic acid and ellagic acid from
Terminalia chebula Retz.
9.
Jayaram et al[23] ., have studied on Effect of Geographical variation on contents of
Tannic Acid, Gallic Acid, Chebulinic acid and Ethyl Gallate in Terminalia chebula
Fruits.
6.4 OBJECTIVES OF THE STUDY:
In view of the need for a suitable HPTLC method for routine analysis of
chebulagic acid and chebulinic acid in polyherbal formulations, attempts are being made to
develop a simple, precise, rapid, accurate, sensitive and cost-effective HPTLC method for
the estimation of chebulagic acid and chebulinic acid in polyherbal formulations.
The plan of work for the proposed study is designed as follows:
 Method development and optimization of chromatographic conditions/parameters.
 Validation of developed HPTLC method in accordance with the analytical parameters
mentioned in the ICH guidelines[21,22] .
 Application of newly developed, validated HPTLC method for the quantification of
chebulagic acid and chebulinic acid in polyherbal formulations.
ENCLOSURE-II
7.
MATERIALS AND METHODS:

All the chemicals and reagents used, will be of analytical grade.

Pure sample of chebulagic acid and chebulinic acid will be procured from M/s Natural
Remedies Pvt.Ltd., Bangalore.

The polyherbal formulations will be purchased from the local market.

CAMAG Linomat 5 sample applicator and CAMAG TLC Scanner 3 equiped with win
CATS Software will be employed for the development and validation of new HPTLC
method for the estimation of chebulagic acid and chebulinic acid in polyherbal
formulations.

HPTLC method will be developed by using different solvent system and ratio
depending upon the chemical nature of chebulagic acid and chebulinic acid.

All the method development and validation will be performed on HPTLC(CAMAG)
which is available in our college laboratory.
7.1. Source of Data:
1. Library, Bharathi College of Pharmacy.
2. E-library, Bharathi College of Pharmacy.
7.2. Method of Collection of Data:
All the information noted here is compiled from the following:
JOURNALS:
1. Journal of Natural Remedies.
2. Indian Journal of Traditional Knowledge .
3. Phytopharma.
4. Indian Drugs.
5. Indian Journal of Pharmaceutical Sciences.
6. Pharmacognosy Magazine.
7. Research Journal of Phytochemistry.
8. Current science.
RELATED LINKS:
http://www.sciencedirect.com
http://www.pubmed.com
http://www.google.com
http://www.wikipedia.com
http://www.chemicalbook.com
http://www.nextbio.com
http://www.scialert.net
http://www.chemworld.com
http://www.onlinelibrary.wiley.com
7.3. DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO
BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS?
NOT APPLICABLE.
7.4. HAS ETHICAL CLEARENCE BEEN OBTAINED FROM YOUR INSTITUTION
IN CASE OF 7.3?
NOT APPLICABLE.
ENCLOSURE-III
8.
REFERENCES:
1. www.wikipedia.com.
2. Juang LJ, Sheu SJ and Lin TC. Determination of hydrolysable tannins in the fruit of
Terminalia chebula Retz. by HPLC and Capillary Electrophoresis. J Sep Sci,
2004;27:718-24.
3. Ding G, Lu YR, Ji CR and Liu YZ. Analysis of tannins in Fructus chebulae and its
confusion varieties by HPCE. J Sep Sci, 2008;26:612-25.
4. Pawar V, Lahorkar P and Ananth Narayana DB. Development of a RP-HPLC Method
for analysis of Triphala curna and its applicability to test variations in Triphala curna
preparations. Indian J Pharm Sci, Jul-Aug 2009;71(4):382-86.
5. Ratnayake S and Jan SZ. Uptake of Terminalia chebula tannin components by
Hide:Analysis using TLC-UV Densitometry. J Nath Sci, Coun Srilanka,
1984;12(1):92-103.
6. Takuo Okuda, Takashi Yoshida and Tsutomu Hatamo. New methods of analyzing
tannins. J nat prod, Jan-Feb 1989;52(1):1-31.
7. Han Q, Song J, Qiao C, Wong L, Wong L and Xu H. Preparative isolation of
hydrolysable tannins (chebulagic acid and chebulinc acid) from Terminalia chebula
by High-Speed Counter-Current Chromatography. J Sep Sci, July 2006;29(11):165357.
8. Chattopadhyay RR and Bhattacharya SK. PHCOG REV:Plant Review Terminalia
chebula:An update. Pharmacognosy Reviews, Jan-May 2007;1(1):151-55.
9. Hartzfeld PW, Forkner R, Hunter MD and Hagerman AE. Determination of
hydrolysable Tannins (Gallotannins and Ellagotannins) after reaction with Potassium
Iodate. J Agri Food Chem, 2002;50:1785-90.
10. Jadhav PD and Laddha KS. Estimation of gallic acid and ellagic acid from Terminalia
chebula Retz. Ind Drugs, April 2004;41(4):200-20.
11. Manimaran S, Subburaju T, Raja SS, Alok S, Vaibhav G, Chinnasamy K, Nanjan MJ
and Suresh B. Comparative study of gallic acid content in marketed Siddha
Formulations of Triphala Chooranam and tablets by HPTLC. Ind Drugs, Sep 2003;
40(9):532-34.
12. The Wealth of India - Raw materials, Publication and Information Directorate, CSIR,
New Delhi, 1950;10:171-77.
13. Ansari SH. Text Book of Essentials of Pharmacognosy, Birla Publications, 20052006;1:351-53.
14. Kokate CK, Purohit AP and Gokhale SB. Text Book of Pharmacognosy, Jan
2007;27:258-59.
15. Anonymous,
Indian
Herbal
Pharmacopoeia,
Regional
Research
Laboratory,
Jammutwai and Indian Drugs Manufacturers Associaton, Mumbai, 1999;p.51.
16. Markers Compounds of Ayurvedic Drugs, WHO Country Office for India, New
Delhi, July 2009.
17. HPTLC-Finger print atlas of Ayurvedic Single Plant Drugs mentioned in Ayurvedic
Pharmacopoeia, Central Council for Research in Ayurveda and Siddha, Dept. of
AYUSH, New Delhi.3&4.
18. Sethi PD. Text Book of HPTLC, CBS Publishers and Distributers, New Delhi, 1996;
1:p.3-71.
19. CAMAG BIBLOGRAPHY SERVICE, Planar Chromatography, Analytical Tasksefficiently solved by HPTLC. CBS 104, March 2010.
20. Harbone JB. Phytochemical methods, Chapman and Hall, New York, 1984;2:1-31.
21. ICH, Q2A Text on Validation of Analytical Procedures, Oct 1994.
22. ICH, Q3B Validation of Analytical Procedures: Text and Methodology, Nov 1996.
23. Jayaram Kumar K. Effect of geographical variation on contents of Tannic Acid,
Gallic acid, Chebulinic Acid and Ethyl Gallate in Terminalia chebula Fruits. Nat Prod
– An Ind J, 2006;2(4).
24. URL: http://en.wikipedia.org/wiki/File:Chebulinic_acid.PNG
25. URL: http://en.wikipedia.org/wiki/File:Chebulagic_acid.PNG