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HPTLC ANALYSIS OF COPTIS TEETA

K
KipaPape

This document summarizes a research study that developed and validated a high-performance thin-layer chromatography (HPTLC) method for quantifying berberine in the rhizomes of Coptis teeta, an endangered plant species found in Arunachal Pradesh, India. The method involved collecting and preparing extracts of C. teeta rhizomes, developing the extracts on a TLC plate using a mobile phase, and detecting berberine under UV light. The method was validated based on parameters like linearity, accuracy, precision, limit of detection, limit of quantification, and repeatability. The validated method was used to quantify berberine in C. teeta rhizome extracts, with results showing the

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Presented by : Kipa Pape M.Pharma Pharmacognosy & Phytochemistry 82/MPG/SPS/21 Under the Supervision of: Prof.(Dr.) Ajay Sharma HOD (Pharmacognosy & Phytochemistry) Co-guide: Dr. Foziyah Zakir Assistant Prof. Development and Validation of High-Performance Thin- layer Chromatographic Method for quantification of Berberine in Rhizomes of ,an Endangered Species Collected from Arunachal Pradesh, India Coptis teeta Wall. 1
LOCATION: Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004, Assam, India PUBLISHER: Oxford Academic AUTHORS: Ashis Kumar Goswami, Neelutpal Gogoi, Anshul Shakya, and Hemanta Kumar Sharma. Journal of Chromatographic Science, 2019, 17 doi: 10.1093/chromsci/bmz009 IMPACT FACTOR: 1.645 2
Introduction Methods and Materials used Results 1 2 3 4 CONTENTS : 5 Discussion and Conclusion Validation of HPTLC method 3
INTRODUCTION Arunachalee has its traditional methods of treatment, which have been reported earlier but their scientific study has not been carried out in most of the plants to validate the traditional claims. One such plant is Coptis teeta Wall. Synonym: Mamira, Golden thread root Biological Source: Obtained from the dried rhizomes of Common Name: Tiktmoola, Rangkeh ( in Arunachalee) Family: Ranunculaceae Chemical Constituents: Berberine, palmitine, epiberberine, jatrorhizine uses SCABIES ANTI- INFLAMMATOY DYSENTERY ANTIBACTERIAL SEVERE HEADACHE Coptis teeta Wall. Coptis teeta Wall. 4
This study was designed to develop and validate a thin layer chromatography (TLC) densitometric-based method using high - performance thin layer chromatography for quantification of berberine. Lv. and his co-workers reported that high-performance liquid chromatography (HPLC) is a versatile technique in determination of protoberberine alkaoids But Ding and his co-workers reported that HPLC analysis for these alkaloids gives poor peak and resolution and it requires long time, which leads to column contamination and damage. History Aim and Objectives 5
METHODOLOGY AND MATERIALS USED The rhizomes of C.teeta were collected from Roying, Lower Dibang Valley dist. A.P and identified and authenticated by Dr. A.A Mao ( BSI, ERC, Shillong) Collection of plant material Chemicals and standards Purchased from Merck ( Mumbai, India) Berberine procured from Sigma-Aldrich TLC fingerprint and chromatography TLC plate: Pre-coated with silica gel 60F254 Mobile phase : Butanol: ethyl acetate: formic acid: water 3.0:5.0:1.0:1.0 Spotter: CAMAG Linomat Vsemi-automatic sample spotter. Chamber: CAMAG twin through chamber Preparation of plant extract 100 mg of powdered rhizomes of C.teeta was sonicated for 15 min with 1mL of methanol. After centrifugation, the supernatant was used as the sample solution. TLC densitometric quantification of berberine using HPTLC C.teeta Quantittative analysis of the compound was done in CAMAG Scanner IV by scanning at 351 nm. The Rf value of methanol extract of rhizomes of (0.70) is same as that of standard berberine The band at 0.70 nm was identified as berberine. 6
Validation Parameters Linear range (ng/band) 90-210 ng/band Intraday (n=6) : 0.77-1.63 Percentage recovery: 93.32 (Within 90-110%) Correlation coefficient : 0.997386 (0.9-1.1) LOD : 30ng/band LOQ: 70ng/band Interday (n=6) : 1.19-1.88 Repeatability(%CV, n=10) : 1.0582 Method Validation Parameters for the Quantification of Berberine in Methanol extract of by TLC Densitometric Method C.teeta 7
The validated method gave compact bands for berberine at an Rf of 0.70. RESULTS The Rf value of berberine matched with the Rf value of methanol extract of rhizomes of which was found to be 0.70. C.teeta 1) 2) 3) The berberine concentration in the methanol extract of coptis teeta was found to be 30.97賊0.55 mg in 100 mg of the crude drug. 8
The spectra generated for berberine and methanol extract of rhizomes of at wavelength range 200-400 nm also matched. C.teeta 9
CONCLUSION DISCUSSION The amount of berberine found in (30.97%) in this method was highest in comparison to earlier studies. Clear separation was obtained through this validated HPTLC method. Requires urgent attention for its preservation and protection. The method developed was reproducible and selective for quantifying berberine in herbal drug formulation. The abundance of berberine in rhizomes of can be used for further modification to produce lead molecules with low toxicity and high potency. C.teeta C.teeta 10
References : Goswami AK, Gogoi N, Shakya A, Sharma HK. Development and Validation of High-Performance Thin-layer Chromatographic Method for Quantification of Berberine in Rhizomes of Coptis teeta Wall, an Endangered Species Collected from Arunachal Pradesh, India. J Chromatogr Sci. 2019 May 1;57(5):411-417. doi: 10.1093/chromsci/bmz009. PMID: 30753457. Bajpay, Anurag & Nainwal, Rakesh & Singh, Devendra. (2019). Coptis teeta: A potential endemic and endangered medicinal plant of Eastern Himalayas. 245- 248. Rathee, D., Rathee, S., Rathee, P., Deep, A., Anandjiwala, S., Rathee, D.; HPTLC densitometric quantification of stigmasterol and lupeol from Ficus religiosa; Arabian Journal of Chemistry, (2015); 8: 366371 1) 2) 3) 11
THIS IS THE END OF MY PRESENTATION. WISHING YOU ALL A VERY HAPPY NEW YEAR IN ADVANCE ! THANK YOU! 12

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HPTLC ANALYSIS OF COPTIS TEETA

  • 1. Presented by : Kipa Pape M.Pharma Pharmacognosy & Phytochemistry 82/MPG/SPS/21 Under the Supervision of: Prof.(Dr.) Ajay Sharma HOD (Pharmacognosy & Phytochemistry) Co-guide: Dr. Foziyah Zakir Assistant Prof. Development and Validation of High-Performance Thin- layer Chromatographic Method for quantification of Berberine in Rhizomes of ,an Endangered Species Collected from Arunachal Pradesh, India Coptis teeta Wall. 1
  • 2. LOCATION: Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh 786004, Assam, India PUBLISHER: Oxford Academic AUTHORS: Ashis Kumar Goswami, Neelutpal Gogoi, Anshul Shakya, and Hemanta Kumar Sharma. Journal of Chromatographic Science, 2019, 17 doi: 10.1093/chromsci/bmz009 IMPACT FACTOR: 1.645 2
  • 3. Introduction Methods and Materials used Results 1 2 3 4 CONTENTS : 5 Discussion and Conclusion Validation of HPTLC method 3
  • 4. INTRODUCTION Arunachalee has its traditional methods of treatment, which have been reported earlier but their scientific study has not been carried out in most of the plants to validate the traditional claims. One such plant is Coptis teeta Wall. Synonym: Mamira, Golden thread root Biological Source: Obtained from the dried rhizomes of Common Name: Tiktmoola, Rangkeh ( in Arunachalee) Family: Ranunculaceae Chemical Constituents: Berberine, palmitine, epiberberine, jatrorhizine uses SCABIES ANTI- INFLAMMATOY DYSENTERY ANTIBACTERIAL SEVERE HEADACHE Coptis teeta Wall. Coptis teeta Wall. 4
  • 5. This study was designed to develop and validate a thin layer chromatography (TLC) densitometric-based method using high - performance thin layer chromatography for quantification of berberine. Lv. and his co-workers reported that high-performance liquid chromatography (HPLC) is a versatile technique in determination of protoberberine alkaoids But Ding and his co-workers reported that HPLC analysis for these alkaloids gives poor peak and resolution and it requires long time, which leads to column contamination and damage. History Aim and Objectives 5
  • 6. METHODOLOGY AND MATERIALS USED The rhizomes of C.teeta were collected from Roying, Lower Dibang Valley dist. A.P and identified and authenticated by Dr. A.A Mao ( BSI, ERC, Shillong) Collection of plant material Chemicals and standards Purchased from Merck ( Mumbai, India) Berberine procured from Sigma-Aldrich TLC fingerprint and chromatography TLC plate: Pre-coated with silica gel 60F254 Mobile phase : Butanol: ethyl acetate: formic acid: water 3.0:5.0:1.0:1.0 Spotter: CAMAG Linomat Vsemi-automatic sample spotter. Chamber: CAMAG twin through chamber Preparation of plant extract 100 mg of powdered rhizomes of C.teeta was sonicated for 15 min with 1mL of methanol. After centrifugation, the supernatant was used as the sample solution. TLC densitometric quantification of berberine using HPTLC C.teeta Quantittative analysis of the compound was done in CAMAG Scanner IV by scanning at 351 nm. The Rf value of methanol extract of rhizomes of (0.70) is same as that of standard berberine The band at 0.70 nm was identified as berberine. 6
  • 7. Validation Parameters Linear range (ng/band) 90-210 ng/band Intraday (n=6) : 0.77-1.63 Percentage recovery: 93.32 (Within 90-110%) Correlation coefficient : 0.997386 (0.9-1.1) LOD : 30ng/band LOQ: 70ng/band Interday (n=6) : 1.19-1.88 Repeatability(%CV, n=10) : 1.0582 Method Validation Parameters for the Quantification of Berberine in Methanol extract of by TLC Densitometric Method C.teeta 7
  • 8. The validated method gave compact bands for berberine at an Rf of 0.70. RESULTS The Rf value of berberine matched with the Rf value of methanol extract of rhizomes of which was found to be 0.70. C.teeta 1) 2) 3) The berberine concentration in the methanol extract of coptis teeta was found to be 30.97賊0.55 mg in 100 mg of the crude drug. 8
  • 9. The spectra generated for berberine and methanol extract of rhizomes of at wavelength range 200-400 nm also matched. C.teeta 9
  • 10. CONCLUSION DISCUSSION The amount of berberine found in (30.97%) in this method was highest in comparison to earlier studies. Clear separation was obtained through this validated HPTLC method. Requires urgent attention for its preservation and protection. The method developed was reproducible and selective for quantifying berberine in herbal drug formulation. The abundance of berberine in rhizomes of can be used for further modification to produce lead molecules with low toxicity and high potency. C.teeta C.teeta 10
  • 11. References : Goswami AK, Gogoi N, Shakya A, Sharma HK. Development and Validation of High-Performance Thin-layer Chromatographic Method for Quantification of Berberine in Rhizomes of Coptis teeta Wall, an Endangered Species Collected from Arunachal Pradesh, India. J Chromatogr Sci. 2019 May 1;57(5):411-417. doi: 10.1093/chromsci/bmz009. PMID: 30753457. Bajpay, Anurag & Nainwal, Rakesh & Singh, Devendra. (2019). Coptis teeta: A potential endemic and endangered medicinal plant of Eastern Himalayas. 245- 248. Rathee, D., Rathee, S., Rathee, P., Deep, A., Anandjiwala, S., Rathee, D.; HPTLC densitometric quantification of stigmasterol and lupeol from Ficus religiosa; Arabian Journal of Chemistry, (2015); 8: 366371 1) 2) 3) 11
  • 12. THIS IS THE END OF MY PRESENTATION. WISHING YOU ALL A VERY HAPPY NEW YEAR IN ADVANCE ! THANK YOU! 12