Synopsis for m. Pharm dissertation submitted to




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Isolation of chemical constituents from aerial parts of Hypericum perforatum (St. John’s wort) towards achieving standardization”

SYNOPSIS FOR

M.PHARM DISSERTATION
SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA



BY
K VISHNU PRIYA

I M. PHARM

DEPARTMENT OF PHARMACOGNOSY

PES COLLEGE OF PHARMACY, BANGALORE – 560050

KARNATAKA.

(2012 – 2014)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE.

ANNEXURE-II


PROFORMA FOR REGISTRATION OF SUBJECTS FOR P.G. DISSERTATION





1.

Name of the candidate and address(in block letters)

K VISHNU PRIYA,


D/0 K RANGA SWAMY,

20/207 C-17,

VIJAY NAGAR COLONY (S),

GUNTAKAL. PIN-515801

ANANTAPUR(DIST),AP.



2.

Name of the institution

PES COLLEGE OF PHARMACY


50 FEET ROAD, HANUMANTHANAGAR, BANGALORE - 560050


3.



Course of study and subject

MASTER OF PHARMACY IN

PHARMACOGNOSY






4.

Date of the admission



12.07.2012

5.

Title of the topic:

“ISOLATION OF CHEMICAL CONSTITUENTS FROM AERIAL PARTS OF HYPERICUM PERFORATUM ( St. JOHN’S WORT) TOWARDS ACHIEVING STANDARDIZATION”



6.


BRIEF RESUME OF THE INTENDED WORK

6.1 NEED FOR THE STUDY :

In recent years, plant derived products are increasingly being sought out as medicinal products, nutraceuticals and cosmetics and are available in health food shops and pharmacies over the counter as selfmedication or also as drugs prescribed in the non-allopathic systems1,2. Herbal medicines widely used in health-care in both developed and developing countries are complex chemical mixtures prepared from plants and are limited in their effectiveness because they are poorly absorbed when taken orally3.

According to an estimate of the World Health Organization (WHO), about 80% of the world population still uses herbs and other traditional medicines for their primary health care needs4. Herbal formulations have reached widespread acceptability as therapeutic agents for diabetics, arthritics, liver diseases, cough remedies, memory enhancers and adoptogens5. As per WHO definition, there are three kinds of herbal medicines: raw plant material, processed plant material and medicinal herbal products. Herbal drugs are finished labeled products that contain active ingredients such as aerial or underground parts of plant or other plant material or combination thereof, whether in the crude state or as plant preparations.

The use of herbal medicines has increased remarkably in line with the global trend of people returning to natural therapies6. Herbal medicine products are dietary supplements that people take to improve their health and are sold as tablets, capsules, powders, teas, extracts and fresh or dried plants7. Herbals are traditionally considered harmless and increasingly being consumed by people without prescription. However, some can cause health problems, some are not effective and some may interact with other drugs. Standardization of herbal formulations is essential in order to assess the quality of drugs, based on the concentration of their active principles8.

Quality evaluation of herbal preparation is a fundamental requirement of industry and other organization dealing with ayurvedic and herbal products. The growing use of botanicals (drug and other products derived from plants) by the public is forcing moves to assess the health claims of these agents and to develop standards of quality and manufacture. It is evident that the herbal industry needs to follow strict guidelines and such regulations are necessary. Herbal drugs regulations in India as well as an overview of regulatory status of herbal medicine in USA, China, Australia, Brazil, Canada and Germany has been reported9. According to WHO guidelines, an herbal product needs to be standardized with respect to safety before releasing it into the market.

However scientific data on their efficacy, pharmacological properties and action mechanism as well as on their chemical constituents have so far been lacking. Hypericum perforatum is used as anti-depressant, anti-inflammatory, anti-spasmodic, stimulates bile flow. In this work we recognize the importance of using established modern scientific methods and criteria to characterize the compounds from Hypericum perforatum by column chromatographic fractions with the help of major peeks/spots corresponding to TLC/HPTLC.



PLANT PROFILE:10

Synonyms: Tipton’s weed, rosin rose

English Name : St. John’s wort

Hypericum perforatum

Kingdom : Plantae

Order : Malpighiales

Family : Hypericaceae

Genus : Hypericum

Species : H.perforatum


Habitate: Hypericum perforatum is native to Britain and Europe. It grows wild throughout much of the world. In India, found in Himalayas in moist and shady places.

Morphology Description: 11

Hypericum perforatum is a yellow flowering stoloniferous perennial herb with extensive, creeping rhizomes. Its stems are erect, branched in the upper section, and can grow to 1m high. It has opposing stalkless , narrow, oblong leaves that are 12 mm long to slightly larger. The leaves are yellow-green in colour , exhibit translucent dots when held up to the light giving them a perforated appearance. Its flowers measure up to 2.5 cm across, has five petals and coloured bright yellow, and appear in broad cymes at the ends of the upper branches. Sepals are pointed. And there are many stamens which are united at the base into three bundles. The pollen grains are ellipsoidal.

Chemical Constituents12

Naphthodianthrones - is the principle component for a class of chemical constituents called quinones, which contain two of the most studied chemicals thought to be responsible for the anti-depressive action of St. John’s Wort: hypericin and pseudohypericin.

Flavanoids, a large group of biologically active molecules that fall under the class of polyphenols. The flavanoids present in St. John’s Wort include proanthocyanidin, hyperin, biflavone, amentoflavone and quercetin among others. Flavanoids are found in the highest concentrations in the hypericum flowers.  

Other chemical compounds that have been identified include terpenoids, which contain essential or volatile oils, xanthones and coumarins.  Many of these compounds, found primarily in the leaves and flowers.

Tannins, a subgroup of the large phenol class.  These tannins are responsible for much of the medicinal action relating to superficial wound or injury care. 

USES13

˃ Mild to moderate depressive states

˃ Antianxiety / Antistress

˃ Restlessness and irritability

˃ Antiviral and antibacterial

6.2 Literature :

Naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3, II8-biapigenin) and phenolic acids (chlorogenic acid, 3-Ocoumaroylquinic acid).were characterized from Hypericum perforatum by LC/DAD/SPE/NMR and LC/UV/MS14

Two sterols were isolated from Hypericum perforatum. The isolated sterols were identified as β-sitosterol and ergosterol by means of UV-,NMR-spectroscopy, mass spectrometry.15

By high-speed countercurrent chromatography furohyperforin, was isolated from Hypericum perforatum L. using a solvent system composed of hexane-methanol-water (2:1.75:0.25, v/ v/ v.) and its structure was elucidated on the basis of HPLC-MS-MS, 1HNMR, and 13CNMR analysis.16

The petroleum ether extracts yielded phloroglucinol compound hyperforin with HPLC purity over 98%. The ethyl acetate extracts yielded naphthodianthrone compound hypericin with HPLC purity about 95%. The n-butanol extract yielded two flavones, rutin and hyperoside, with HPLC purity > 95%. The structures of isolated compounds were identified by ESI-MS, 1HNMR and 13CNMR.17

3, 8’- bisapigenin was isolated from Hypericum perforatum for the substantiation of new approaches to the standardization of raw material. And the total flavonoids and anthracene derivatives were determined by the use of spectrophotometry methods.18

Six known flavonoids (quercitrin, hyperoside, avicularin, rutin, quercetin, and kaempferol) and a new flavonol glycoside named 6''-O-acetyl quercetin 3-O-β-D-alloside were isolated from the aerial parts of Hypericum perforatum. The structures were determined based on spectroscopic methods.19

Five compounds. from Hypericum perforatum L. were isolated and identified as quercetin, avicularin, quercitrin, isoquercitrin and hyperin by chromatography methods.20

Detailed procedure for the isolation of naphthodianthrones from St. John's Wort by an accelerated extraction and separation of marker compounds was reported by preparative high-performance liquid. Chromatography (HPLC) with photodiode array detector .21

Two new xanthone derivatives, 1-hydroxy-5,6,7-trimethoxyxanthone and 3-O-methylpaxanthone (I), were isolated from callus of Hypericum perforatum with the known paxanthone, cadensin G, 1-hydroxy-6,7-dimethoxyxanthone, 1,3,6,7-tetrahydroxyxanthone, and 1,3,5,6-tetrahydroxyxanthone and vanillic acid, β-sitosterol, and stigmasterol. The structures of the new compounds were established by spectroscopic methods. 22



The free and esterified triterpene alcoholics and sterols α -Amyrin, taraxasterol, ψ-taraxasterol, lupeol, β-amyrin, taraxerol, isobauerenol, stigmasterol, campesterol, 28-isofucosterol and cholesterol were isolated from Hypericum perforatum and their components were identified by GC and GC/MS.23

Hypericin and hyperforin were isolated by modified Soxhlet extraction with 80% ethanol and detected by HPLC with UV-VIS detection at 592 and 292 nm in less than 4 min of retention time. 24

Hypericin and pseudohypericin were isolated from Hypericum perforatum hydro-alcoholic extracts and are monitored by LC-MS and NMR analyses.25

Hyperforin from Hypericum perforatum was standardized by HPTLC method and was used for quantification. The lowest detectable limit of Hyperforin was found up to 100 ng and seperated from other constituents.26

From the upper phase solvent system of ethyl acetate-methanol-water (5:2:5, v/ v/ v) yields two hydrophobic compounds. Including 28.4 mg of hyperforin with a HPLC purity of 97.28% and 32.7 mg of adhyperforin with a HPLC purity of 97.81% were isolated. From the lower phase of ethylacetate-methanol-n-butanol-water (5:2:2.5:12, v/ v/ v/ v) three hydrophilic compounds. Of 12.7 mg of 3,4,5-O-tricaffeoylquinic acid with a HPLC purity of 98.82%, 15.2 mg of 1,3,5-O-tricaffeoylquinic acid with a HPLC purity of 99.46% and 42.5 mg of 3-O-caffeoylquinic acid with a HPLC purity of 96.90%, were obtained in a one step extraction-separation process..)And the chemical structures of all 5 compounds. above mentioned were identified by UV, MS and NMR.27

Two prenylated phloroglucinol derivatives. And 15 known compounds were isolated from the aerial parts of Hypericum perforatum. Their structures were determined on the basis of spectroscopic methods. 28

From Hypericum perforatum, biflavonoids were isolated by column chromatography on Sephadex LH 20 and preparative TLC. The structure of the main compound was elucidated by UV, 1H and 13C NMR spectroscopy and MS and identified as I3,II8-biapigenin (I)29

Nine compounds isolated and their structures were elucidated as D-Mannitol (1), 1, 2- benzenedicarboxylic acid bis(1-methylpropyl) ester (2), (7E, 6R,9S)-9-hydroxy-4,7-megastigmadien-3-one (3), (6S,9R)- roseoside (4) , 2,6-dimethoxy-4-hydroquinone-l-O- β -D-glucopyranoside (5), 2,6-di-methoxy-4-hydroxybenzyl alcohol 1 -O- β -D-glucopyranoside (6), syringate 4-O- β -glucopyranoside (7), hypericin (8), skyrin-6-0- β -Dglucopyranoside (9) , (R)-2,3-dihydroxypropyl 3,4-dihydroxy-benzoate (10). At a concentration of 2 micromol x L(-1), compound 8 inhibited recombinant human PTP1B with inhibitory rate of 96.4% and IC50 of 2.5 micromol x L(-1).30

Five compounds, 3-epiursolic acid, myriaboric acid, 1,6-dihydroxy-4-methoxyxanthone, 1,7-dihydroxyxanthone and 2,5- dimethyl-7-hydroxychromone, were isolated from the aerial parts of Hypericum perforatum.31

Sixty-six compounds were identified in the essential oils obtained by hydro distillation of aerial parts of Hypericum perforatum with germacrene D (13.7%), α-pinene (5.1%), (E)-caryophyllene (4.7%), n-dodecanol (4.5%), caryophyllene oxide (4.2%), bicyclogermacrene (3.8%), and spathulenol (3.4%) as the main constituents by GC-MS.32

Amentoflavone was isolated as a minor compound from Hypericum perforatum and its structure was elucidated by NMR.33

6.3 OBJECTIVES OF THE STUDY

1. Extraction of plant material by Alcohol.

2. Development of TLC and HPTLC profiles.

3. Fractionation and isolation of compounds by column chromatography.

4. Characterization of isolated compounds by U.V, I.R, N.M.R, Mass spectroscopy.

5. Estimation of compounds 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- 560 050.

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 aerial parts of plant Hypericum perforatum will be collected from Natural Remedies Pvt Ltd, Bengaluru.



EXTRACTION :

After size reduction the plant will be subjected for extraction by using alcohol.



DEVELOPMENT OF TLC AND HPTLC :

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, and GC, M.P, Rf values 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. Gautam V, Raman RMV, Ashish K. Exporting Indian healthcare (Export potential of Ayurveda and Siddha products and services) Road beyond boundaries (The case of selected Indian healthcare systems).Gautam V, Raman RMV, Ashish K. (eds.) Export-Import Bank of India; Mumbai: 2003; p 4–54.

  2. Sagar Bhanu PS, Zafar R, Panwar R. Herbal drug standardization. The Indian Pharmacist 2005; 4(35):19-22.

  3. Amit J, Sunil C, Vimal K, Anupam P. Phytosomes: A revolution in herbal drugs. The Pharma Review 2007; 11- 13.

  4. http://www.who.int/research/en. Cited on 7/1/12.

  5. Patel PM, Patel NM, Goyal RK. Quality control of herbal products. The Indian Pharmacist 2006; 5(45):26-30.

  6. Vaidya ADB, Devasagayam TPA. Current status of herbal drugs in India: An overview. J Clin Biochem 2007; 41(1):1–11.

  7. http://www.morethanvitamins.co.uk/herbal-remedies-c-24.html. Cited on 7/1/12.

  8. Yadav NP, Dixit VK. Recent approaches in herbal drug standardization. Int J Integr Biol 2008; 2:195-203.

  9. Sachan V, Kohli Y, Gautam R. Regulatory issues for herbal products – a review.

Pharmainfo.net;http://www.pharmainfo.net/justvishal/publications/regulatoryissues-herbal-products-review. Cited on 7/1/12.

  1. Prajapati ND, Purohit SS, Sharma AK., Kumar T, Hand book of Medicinal Plants- A

Complete source book, Agrobios (India) 2003, 283.

11. Kiritikar KR, Basu BD. Text book of Indian Medicinal Plants, Vol-1:255.

12. http://www.herballegacy.com/Nelson_Chemical.html. Cited on 8/1/12.

13. Dr V. Rajpal, Testing and extraction methods of medicinal herbs. Text book of

Standardization of botanicals.Vol-1:155.

14. Tatsis EC, Boeren S, Exarchou V, Troganis AN, Vervoort J, Gerothanassis IP,

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR

And/or LC/MS. Phytochemistry. 2007; 68(3):383-93.

15. Pravdivtseva OE, Kurkin VA. Sterols of Hypericum perforatum L. herbs. Khimiya

Rastitel’nogo Syr'ya. 2011; 4(2):333-4.

16. Li Yan, Cao X. Isolation and characterization of furohyperforin from Hypericum perforatum l. by high-speed countercurrent chromatography. Journal of Liquid Chromatography & Related Technologies. 2012; 35(18): 2558-66.

17. Cao X, Wang Q, Li Y, Bai G, Ren H, Xu C, et al. Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography. Journal of Chromatography, B: Analytical Technologies in the Biomedical and Life Sciences.2011; 879(7-8):480-8.

18. Pravdivtseva OE, Kurkin VA. Studies om the substantiation of new approaches to the standardization of raw material and preparations of Hypericum perforatum L. Khimiya Rastitel’nogo Syr'ya. 2008; 1:81-6.

19. Lu Y, Zhang Z, Shi G, Meng J, Tan R. New antifungal flavonol glycoside from Hypericum perforatum. Acta Botanica Sinica. 2002; 44(6):743-5.

20. Zhou J, Hu Y, Xiao M, Zhu Y. Flavonoids from Hypericum perforatum L. Guangzhou Zhongyiyao Daxue Xuebao. 2006; 23(5):416-8.

21. Puri S, Handa G, Kalsotra AK, Gupta VK, Shawl AS, Suri OP, et al. Preparative High- Performance Liquid Chromatographic Separation of Naphthodianthrones from St. John's Wort. Journal of Chromatographic Science. 2006; 44(4):177-80.

22. Ferrari F, Pasqua G, Monacelli B, Cimino P, Botta B. Xanthones from calli of Hypericum perforatum subsp. perforatum. Natural Product Research. 2005; 19(2):171-6.

23. Ganeva Y, Chanev C, Dentchev T, Vitanova D. Triterpenoids and sterols from Hypericum perforatum. Dokladi na Bulgarskata Akademiya na Naukite. 2003;56(4):37-40

24. Pavlik M, Vacek J, Klejdus B, Kuban V. High-performance liquid chromatographic study of the effect of sucrose and polyethylene glycol on production of hypericin and hyperforin by Hypericum perforatum L. Chemicke Listy. 2007; 101(7):556-62.

25. Karioti A, Vincieri FF, Bilia AR, Rapid and efficient purification of naphthodianthrones from St. John's wort extract by using liquid-liquid extraction and SEC. Journal of Separation Science. 2009; 32(9):1374-82.

26. Tewari N, Menghani H, Hamarapurkar P, Suthar A, Chauhan VS, Developing a new high performance thin layer chromatography method for quantitative estimation of Hyperforin in Hypericum perforatum. Pharmacognosy Magazine. 2008; 4(16):304-8.

27. Zhang Y, Liu C, Yu M, Zhang Z, Qi Y, Wang J, et al. Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L. Journal of Chromatography A. 2011;1218(20):2827-34.

28. Hashida C, Tanaka N, Kashiwada Y, Ogawa M, Takaishi Y. Prenylated phloroglucinol derivatives from Hypericum perforatum var. angustifolium. Chemical & Pharmaceutical Bulletin. 2008; 56(8):1164-7.

29. Berghoefer R, Hoelzl J. Biflavonoids in Hypericum perforatum. Planta Medica. 1987; 53(2):216-7.

30. Ma J, Yang J, Ji T, WangAiguo;, Su Y. Chemical constituents from Hypericum perforatum. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China journal of Chinese materia medica. 2012; 37(16):2408-12.

31. Yin Z-Q, Wang Y, Ye W-C, Zhao S-X. Chemical constituents of Hypericum perforatum (St. John's wort) growing in China. Biochemical Systematics and Ecology. 2004; 32(5):521-3.

32. Sharopov, Farukh S, Gulmurodov, Isomiddin S, Setzer, William N. Essential oil composition of Hypericum perforatum L. Journal of Chemical and Pharmaceutical Research. 2010;2(6):284-90
33. Berghoefer R, Hoelzl J. Biflavonoids from Hypericum perforatum L. Part 2. Isolation of I3',

II8-biapigenin (amentoflavone) from Hypericum perforatum. Planta Medica 1989; 55(1):91.







08.

NAME OF THE CANDIDATE

K VISHNU PRIYA


09.

SIGNATURE OF THE CANDIDATE

(K VISHNU PRIYA)



10.

REMARKS OF THE GUIDE




10.1


NAME AND DESIGNATION OF THE GUIDE



G. MURUGANANTHAN,

ASST.PROFESSOR

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




11.1


SIGNATURE



12.

REMARKS OF THE PRINCIPAL


SIGNATURE

Prof. Dr. S.MOHAN




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