Isolation of Chemical Constituents from Leaves of Ginkgo Biloba for Achieving Standardization

“Isolation of chemical constituents from leaves of Ginkgo biloba for achieving standardization”

SYNOPSIS FOR M.PHARM DISSERTATION

SUBMITTED TO RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA

CHANDRA DEV NALLURU I M. PHARM DEPARTMENT OF PHARMACOGNOSY PES COLLEGE OF PHARMACY, BANGALORE – 560050 KARNATAKA. (2011 – 2013)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BANGALORE. ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECTS FOR P.G. DISSERTATION

1 1. Name of the candidate and address(in CHANDRA DEV NALLURU, block letters) S/O N.PAPAIAH CHOWDARY, 54-14/8-7/C,FLAT NO.104, SRIKARA RESIDENCY, BHARATHI NAGAR MAIN ROAD, VIJAYAWADA,PIN-520007 KRISHNA (DIST),AP. PES COLLEGE OF PHARMACY 2. Name of the institution 50 FEET ROAD, HANUMANTHANAGAR, BANGALORE - 560050

3. Course of study and subject MASTER OF PHARMACY IN PHARMACOGNOSY

4. Date of the admission 03.09.2011

5. Title of the topic:

“ISOLATION OF CHEMICAL CONSTITUENTS FROM LEAVES OF GINKGO BILOBA FOR ACHIEVING STANDARDIZATION”

2 6. BRIEF RESUME OF THE INTENDED WORK 6.1 NEED FOR THE STUDY : Plants have been a major source of therapeutic agents for alleviation or cure of human diseases since time immemorial. These traditional systems of medicine continue to cater into the medicinal needs of the about 80% world population. With time being many traditional systems immerge and come into existence in various parts of the globe. The popular among them are Ayurveda, Siddha, Traditional Chinese medicine, Yoruba medicine, Kampo, Unani, and Homeopathy etc. These systems of medicine are heavily dependent upon medicinal plants which are used as herbal medicines.1 A herbal medicine is a medicinal preparation made from a plant and can include the fresh or

dried herb or seeds, chopped or powdered or an advanced form of the herb usually maid via extraction by a solvent such as water or an organic solvent (e.g., methanol, ethanol). Such advanced herbal preparations, often processed in a way that establishes relatively fixed parameters, are often called standardized extracts.2 Ginkgo biloba is gaining recognition for improving memory loss, in the treatment of Alzheimer’s disease ,antioxidant activity and scavenging for free radicals etc. In this work, we recognize the importance of using established modern scientific methods and criteria to characterize the compounds from Ginkgo biloba by column chromatographic fractions with the help of major peaks/spots corresponding to TLC/HPTLC. PLANT PROFILE: English Name3 : Maidenhair Tree Ginkgo biloba:4 Kingdom: Plantae Subkingdom: Tracheobionta Superdivision: Spermatophyta Division: Ginkgophyta Class: Ginkgoopsida Order: Ginkgoales Family: Ginkgoaceae Genus: Ginkgo L. Species: Ginkgo biloba L. Habitate5: The trees are ornamental and considered sacred in China and Japan.Plantations for the production of medicinal materials are common in China, Japan, Korea and France. The ginkgo tree thrives in full sun and average soil. It is hardy and tolerant of pollution,which makes it

3 valuable for planting along city streets. Morphology Description3: Deciduous,resinous,dioecious tree,to 60 m or more tall.Branches stiff,with both elongated and spur shoots.Leaves alternately clustered,fan shaped,cut in middle,dichotomously veined,long petioled.The female reproductive structures consisting of 2 ovules,rarely more on a long peduncle,usually only 1 maturing.Seeds plum like in appearance,yellowish,drupe-like,to 2.5 cm long,long peduncled,outer coat fleshy,inner coat stony. Chemical Constituents6: Ginkgo contains many chemical constituents. Most of them fall into two categories Flavnoids and Terpenoids or Terpene lactoids. The type of flavonoid in Ginkgo is called Ginkgolide. Ginkgolides are very unique compounds exclusive to Ginkgo and are broken down into separate ginkgolides A, B, C, J, and M. Each Ginkgolide has a different degree of potency. Ginkgolide B is considered the most active. Other flavnoids present are quercitin ( a stronger antioxidant than vitamin E), and kaempferol. Terpene lactones are the active constituents that give ginkgo a bitter strong flavor and helps increase blood circulation. Other constituents present are Amino Acid-6-hydrozykynurenic acid, Dimeric flavones (bilobetin, ginkgetin, isoginkgetin, scieadopitysin), Proanthocyanidins, ginkgolic acid, ascorbic acid, carotenoids, and Bilobalide.The nuts contain essential oil, fatty acid, tannin, and resin. USES5:  Ginkgo used in improving memory loss, vertigo, tinitus and fatigue.  Useful in the treatment of Alzheimer’s disease, helping to improve cognitive function.  Ginkgo flavanoids help protect vascular walls through antioxidant activity,scavenging for free radicals and reducing capillary fragility.  Antibacterial and antifungal,PAF-receptor binding antagonistic.Used in asthma,sputum and cough,leucorrhoea3.

6.2 Literature : Polymeric proanthocyanidins were eluted using Sephadex LH-20 gel chromatography of an extract from Gingko biloba leaves. A purified proanthocyanidin polymer accounted for 86.6% of the total proanthocyanidins, and for 37.7% of the total antioxidant activity of this leaf extract.

4 Structure elucidation was done by subjecting the polymer to acidic depolymerization in the presence of phloroglucinol. The structures of the resulting flavan-3-ols and phloroglucinol adducts were determined on the basis of 1D- and reverse 2D-NMR spectra of their peracetylated derivatives, MALDI-TOF-MS and CD-spectroscopy7. Commercial preparations of Ginkgo biloba are very complex mixtures prepared from raw leaf extracts. H1 NMR-based metabolomics and hyphenation of high-performance liquid chromatography, photo-diode array detection, mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy (HPLC-PDA-MS-SPE-NMR) were used for investigation of 16 commercially available preparations of Ginkgo biloba. The standardized extracts originated from Denmark, Italy, Sweden, and United Kingdom, and the results show that H1 NMR spectra allow simultaneous assessment of the content as well as identity of flavonoid glycosides and TTLs based on a very simple sample-preparation procedure consisting of extraction, evaporation and reconstitution in acetone. Automated integration of H1 NMR spectral segments provides relative distribution plots of TTLs based on their H-12 resonances. The present study shows that (1)H NMR-based metabolomics is an attractive method for non- selective and comprehensive analysis of Ginkgo extracts8. The G. biloba 11S seed storage protein ginnacin was purified by sequential anion-exchange and gel-filtration chromatography. A crystallization screen was performed and well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained. There are six protomers in an asymmetric unit9. The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds showed homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 A resolution and from SeMet-rGnk2 at 2.79 A resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P2(1)3, with unit-cell parameters a = b = c = 143.2 A10. An interlaboratory study was conducted for evaluation of a method to determine the flavonol aglycones quercetin, kaempferol, and isorhamnetin in Ginkgo biloba products. The method calculates total glycosides based on these aglycones formed after acid hydrolysis. Twelve matrixes were chosen for study by 12 collaborating laboratories. Test materials included crude

5 leaf material, standardized dry powder extract, single and multiple entity finished products, ethanol and glycerol tinctures, and National Institute of Standards and Technology (NIST) standard reference materials (SRMs). Results from 11 laboratories were used for the final calculations. Eight of the 12 matrixes evaluated produced acceptable results for total flavonol glycosides; repeatability relative standard deviations (RSDr); and reproducibility relative standard deviations (RSDR). Four additional matrixes, consisting of 3 tablets and 1 soft gel product (Ginkgold, Ginkoba, Ginkogen, and GinkgoPhytosome, respectively), showed greater total flavonol glycosides, RSDr values , and RSDR values. This method is recommended for determination of total flavonol glycosides calculated from quercetin, kaempferol, and isorhamnetin in dry powder extracts, crude leaf material, liquid extracts, and a select finished product11. New assay based on high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) was developed for the measurement of the terpenoid lactones in ginkgo products such as leaf powder and extracts. Initially, the MS/MS fragmentation pathways of ginkgolides were investigated to identify abundant fragment ions that might be useful for the sensitive and selective detection of ginkgolides and bilobalide during LC/MS/MS. This LC/MS/MS assay facilitated the rapid quantitative analysis of ginkgolides A, B, C and J and bilobalide in ginkgo dietary supplements with excellent recovery, reproducibity, accuracy and sensitivity12. A long-chain betulaprenol-type polyprenol mixture was isolated from the leaves of Ginkgo biloba as acetate. The structure was determined by mass spectroscopy, H1-NMR spectroscopy and C13-N.M.R spectroscopy. The mixture contained polyprenols-14-22, predominantly polyprenols-17, -18 and -19, and consisted of the dimethylallyl terminal unit (omega-terminal), two trans-isoprene residues, a sequence of 11-19 cis-isoprene residues and a terminal hydroxylated isoprene unit (alpha-terminal) aligned in order13. H1 NMR profiles (fingerprints) of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, quercetin, kaempferol, isorhamnetin, isoquercetin, and rutin in DMSO-d6 were obtained through the examination of 1D H1 NMR and 2D H1,H1-COSY data, in combination with H1 iterative full spin analysis (HiFSA). The computational analysis of discrete spin systems allowed a detailed characterization of all the H1 NMR signals in terms of chemical shifts (δ(H) ) and spin-spin coupling constants (J(HH) ), regardless of signal overlap and higher order

6 coupling effects.Precise H1 NMR assignments of the sugar moieties of isoquercetin and rutin are reported for the first time14. Terpene lactones such as bilobalide, ginkgolides A, B, C, and J are major bioactive compounds of Ginkgo biloba L. Purification of these compounds is tedious due to their similar chemical nature. For the purpose of developing an effective and efficient method for both analytical and preparative separation of terpene lactones in G. biloba, an innovative enhanced high-speed countercurrent chromatography (HSCCC) method was established. Taking advantage of quantitative H1 NMR (qHNMR) methodology, partition coefficients (K) of individual terpene lactones were calculated directly from crude G. biloba leaf extract, using their H-12 signals as distinguishing feature15. A fingerprint analysis method was developed for Ginkgo biloba leaves and was successfully used for quality evaluation of related health foods by HPLC with electrospray ionization MS. Fifteen samples of G. biloba leaves, which were collected from 15 different locations ,were analyzed and identified. By both peak analysis and similarity analysis of the fingerprint chromatograms, variation of constituents was easily observed in the leaves from different sources. The fingerprint-based strategy of the developed method provided improved quality control of G. biloba leaves and products16. The methanolic extract of Ginkgo biloba L. (Ginkgoaceae) was investigated as an inhibitor of pancreatic lipase (PL) in an attempt to explain its hypolipidaemic activity. In vitro assay of G. biloba leaves extract revealed a substantial PL inhibition activity (IC(50) = 16.5 µg/mL). Terpene trilactones, including ginkgolides and bilobalide, were found to fit within the binding pocket of PL via several attractive interactions with key amino acids. Experimentally, ginkgolides A, B, and bilobalide were found to inhibit PL significantly17. A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method based on isocratic elution has been developed and validated for the simultaneous quantitation of flavonols (myricetin, quercetin, kaempferol, and isorhamnetin), flavones (luteolin and apigenin), and phenolic acids (chlorogenic, caffeic, ellagic, and rosmarinic acids) as important constituents in fruits, vegetables, and medicinal plants. Analysis was achieved on a C- 18 column at ambient temperature. The wavelengths used for the detection of flavonols, flavones, and phenolic acids were 370, 350, and 325 nm, respectively. The plants with the highest flavonoids were found to be Rosa damascena , Solidago virgaurea , Ginkgo biloba , and

7 Camellia sinensis.18 G. biloba medications are used to improve memory, to treat neuronal disorders such as tinnitus or intermittent claudication, and to improve brain metabolism and peripheral blood flow. The most well-known ones are flavonoids and terpene lactones, but they also include allergenic and toxic compounds such as ginkgotoxin 19. Determinations made by employing capillary gas chromatography technique with FID detection were preceded by derivatization using BSTFA with TMCS addition at 1200c. Cholesterol was used as an internal standard. Validation of the method shows no interferences with concurrent constituents; average resolution (R), controlled for peaks of cholesterol and ginkgolide A. In the temperature program used (from 500c to 3000c) the analyte retention times range from 11.2 min. (bilobalide) to 13.8 min. (ginkgolide C) and are of high repeatability of relative values (RRT): RSD = 0.05% / 0.07% for ginkgolides20. Using the higher sensitivity of the MS detector, three trace constituents were found along with ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC) and bilobalide (BD). Two compounds (tR 57.8 and 56.7 min) were identified as 1, 10-dihydroxy-3, 14- didehydroginkgolide and 10-hydroxy-3, 14-didehydroginkgolide, named as ginkgolide K and ginkgolide L, respectively, according to the characteristics of their UV, MS, IR and NMR spectra. It lead to the discovery of ginkgolide compounds containing double bond21. A novel straight forward approach to selective separation for flavonoid compounds was reported. The solid phase material was prepared by copolymerization using allyl-bromide- modified chitosan as macromonomer, and ethylene glycol dimethacrylate as cross-linker. The material was evaluated by chromatographic analysis; it exhibited high selectivity separation for quercetin and its structural analogues using different mobile phases. The material could directly trap a specific class of compounds including quercetin and kaempferol from the hydrolyzate of Ginkgo biloba extract. This demonstrated the possibility of direct extraction of certain constituents from herbs22. An Normal Phase-HPLC method both with diode-array (DAD) and electrochemical detection (ED) was developed and validated for the determination of quercetin and kaempferol, the principal active constituents in phytopharmaceuticals of Ginkgo biloba. Calculated retention of the two flavonoids was contrasted with experimental values in five different reversed phase columns for methanol-water, acetonitrile-water, THF-water and dioxane-hexane binary mixtures

8 as mobile phases. The capacity factor k, selectivity alpha and asymmetry factor F were evaluated and compared in DAD-RP-HPLC, DAD-NP-HPLC, ED-RP-HPLC and ED-NP-HPLC. The electrochemical method based on differential pulse voltammetry (DPV) with a C-PVC electrode resolved the quercetin and kaempferol peaks and exhibited a two orders higher sensitivity in comparison with a carbon fiber electrode23.

6.3 OBJECTIVES OF THE STUDY:

1. Collection and authentication of leaves of Ginkgo biloba.

2. Extraction of plant material by Alcohol. 3. Development of TLC and HPTLC profiles. 4. Fractionation and isolation of Markers by column chromatography. 5. Characterization of isolated compounds by U.V, I.R, N.M.R, Mass. 6. Estimation of markers by HPLC/HPTLC 7.MATERIAL AND METHODS 7.1 SOURCE OF DATA: Journals searched on RGUHS-Digital library, NCSI-IISc, Text books of Pharmacognosy and Botany and Library of Natural Remedies Pvt. Ltd. Place of work: PES College of Pharmacy,Bengaluru-560050 Natural Remedies Pvt. Ltd., Bengaluru- 560 100 WEBSITES : WWW.PUBMED.COM, WWW.GOOGLE.COM, WWW.IJP- ONLINE.COM WWW.SCIENCEDIRECT.COM , WWW.HELINIT.COM 7.2 METHOD OF COLLECTION OF DATA : COLLECTION OF PLANT : The leaves of Ginkgo biloba will be collected from Natural Remedies Pvt Ltd, Bengaluru. EXTRACTION : After size reduction the leaves will be subjected for extraction by using alcohol. DEVELOPMENT OF TLC AND HPTLC :

9 After preliminary extraction the alcoholic extract will be subjected to TLC and HPTLC studies.

ISOLATION OF MARKERS : The alcoholic extract will be subjected by column chromatography for the fractionation and isolation of chemical constituents. PURITY DETERMINATION AND CHARECTERIZATION: The isolated markers will be characterized by suitable methods such as TLC, HPLC, GC, M.P followed by spectroscopic methods. QUANTIFICATION AND STANDARDISATION: The extracts will be quantified with respect to the isolated markers and standardized by using suitable analytical methods such as HPLC, HPTLC. 7.3 DOSE THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS? -NO- 7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3? -NOT APPLICABLE-

Bibilography : 1. Pawar VMPS. Medicinal and Aromatic plants; 2006. 2. Bekker MHJ M-VJ. Anxiety Disorders: Sex differences in prevalence, degree and background, but gender – Natural treatment. Gender Medicine; 2007. 3. ND Prajapati,SS Purohit,Arun K. Sharma,Tarun kumar, A Hand book of Medicinal Plants pg-252 and 253. 4. http://plants.usda.gov/java/profile?symbol=GIBI2(UnitedStatesDepartmentof Agriculture,Natural Resources Conservation Service) cited on 25/06/2012. 5. www.sussex.ac.uk/efm/documents/gbiloba.pdf cited on 25/06/2012. 6. http://www.herballegacy.com/Nelson_ginkgo_Chemical.html cited on 25/06/2012. 7. Qaadan F,Nahrstedt A,Schmidt M,Mansoor K. Polyphenols from Ginkgo biloba. Scientia Pharmaceutica. 2010;78(4):897-907.

10 8. Agnolet S, Jaroszewski JW, Verpoorte R, Staerk D. H NMR-based metabolomics combined with HPLC-PDA-MS-SPE-NMR for investigation of standardized Ginkgo biloba preparations. Metabolomics.2010 ;6(2):292-302. 9. Jin T, Chen YW, Howard A, Zhang YZ. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin. Acta crystallographica.Section F,Structural Biology and Crysatllization Communications. 2008 ;64(7):641-4. 10. Miyakawa T, Sawano Y, Miyazono K, Hatano K, Tanokura M. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Acta crystallographica.Section F,Structural Biology and Crysatllization Communications. 2007;63(9):737-9. 11. Gray D, LeVanseler K, Meide P, Waysek EH. Evaluation of a method to determine flavonol aglycones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography: collaborative study. Journal of AOAC International. 2007;90(1):43-53. 12. Sun Y, Li W, Fitzloff JF, van Breemen RB. Liquid chromatography/electrospray tandem mass spectrometry of terpenoid lactones in Ginkgo biloba. Journal of Mass Spectrometry. 2005;40(3):373-9. 13. Ibata K, Mizuno M, Takigawa T, Tanaka Y. Long-chain betulaprenol-type polyprenols from the leaves of Ginkgo biloba. The Biochemical Journal. 1983;213(2):305-11. 14. Napolitano JG, Lankin DC, Chen SN, Pauli GF. Complete H1 NMR spectral analysis of ten chemical markers of Ginkgo biloba. Magnetic Resonance in Chemistry. available

online 22 June 2012 . doi: 10.1002/mrc.3829. 15. Qiu F, Friesen JB, McAlpine JB, Pauli GF. Design of countercurrent separation of Ginkgo bilobaterpene lactones by nuclear magnetic resonance. Journal of Chromatography. 2012;1242:26-34.

16. Song J, Fang G, Zhang Y, Deng Q, Wang S. Fingerprint analysis of Ginkgo biloba leaves and related health foods by high-performance liquid chromatography/electrospray ionization-mass spectrometry. Journal of AOAC

11 International. 2010;93(6):1798-805. 17. Bustanji Y, Al-Masri IM, Mohammad M, Hudaib M, Tawaha K, Tarazi H, Alkhatib HS. Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba. Journal of enzyme inhibition and medicinal chemistry. 2011(4):453-9. 18. Haghi G, Hatami A. Simultaneous Quantification of Flavonoids and Phenolic Acids in Plant Materials by a Newly Developed Isocratic High-Performance Liquid Chromatography Approach. Journal of Agricultural and Food chemistry. 2010. 58 (20), 10812–6. 19. Leistner E, Drewke C. Ginkgo biloba and ginkgotoxin. Journal of natural products. 2010 ;73(1):86-92. 20. Krzek J, Czekaj JS, Rzeszutko W, Ekiert RJ. Validation of capillary gas chromatographic method for determination of bilobalide and ginkgolides A, B, C in Ginkgo biloba dry and liquid extracts. Acta Poloniae Pharmaceutica. 2007;64(4):303-10. 21. Wang Y, Sheng LS, Lou FC. Analysis and structure identification of trace constituent in the total ginkgolide by using LC/DAD/ESI/MS. Yao Xue Xue Bao. 2001;36(8):606-8. 22. Xia YQ, Guo TY, Zhao HL, Song MD, Zhang BH, Zhang BL. A novel solid phase for selective separation of flavonoid compounds. Journal of separation science. 2007 ; 30(9):1300-6. 23. Aguilar-Sánchez R, Ahuatl-García F, Dávila-Jiménez MM, Elizalde-González MP, Guevara-Villa MR. Chromatographic and electrochemical determination of quercetin and kaempferol in phytopharmaceuticals. Journal of pharmaceutical and biomedical analysis. 2005;38(2):239-49.

12 08. NAME OF THE CANDIDATE CHANDRA DEV NALLURU

09. SIGNATURE OF THE CANDIDATE (CHANDRA DEV NALLURU)

10. REMARKS OF THE GUIDE

Prof. Dr K. LAKSHMAN, 10.1 NAME AND DESIGNATION OF THE PROFESSOR&HOD, GUIDE DEPARTMENT OF PHARMACOGNOSY PES COLLEGE OF PHARMACY, BANGALORE-50.

10.2 SIGNATURE

10.3 CO-GUIDE (IF ANY) Not Applicable

. 10.4 SIGNATURE Not Applicable

11. HEAD OF THE DEPARTMENT Prof. Dr. K. Lakshman

REMARKS OF THE HEAD:

11.1 SIGNATURE

12. REMARKS OF THE PRINCIPAL

SIGNATURE Prof. Dr. S.MOHAN