Proforma for registration of subjects for

“DEVELOPMENT OF STANDARDIZATION PROFILE FOR THE ISOLATED CONSTITUENTS OF TINOSPORA CORDIFOLIA”

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  Nature always stands as a golden mark to exemplify the outstanding phenomena of symbiosis. Medicinal plants are small but important part of biological heritage of this earth. Herbal medicine has been enjoying renaissance among the customers throughout the world. In many cases, if they bridge the gap between the availability and demand for modern medicine, these herbal medicines can play a vital role in the health care.
  

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  According to WHO guidelines for methodologies on research and evaluation of traditional medicines, the first step in assuring quality, safety and efficacy of traditional medicines is its correct identification. However, one of the impediments in acceptance of the herbal formulations is the lack of standardization and quality control profiles.
  

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  It is observed that, quality control of crude drugs and herbal formulations is of paramount importance in justifying their acceptability in modern system of medicine. Due to its complex nature and inherent variability of chemical constituents of plant based drugs, it is difficult to establish quality control parameters or profiles for herbal material. Hence, to solve the major problem faced by herbal drug industry, Standardization of herbal drugs and plant materials and their formulation is the need of the day.
  

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  Tinospora cordifolia (Menispermaceae), known as Amrita (guduchi) is one such plant which is widely used in indigenous system of medicine. The term Amrita meaning divine nectar is attributed to this drug in recognition of this capacity to impart youthfulness, vitality and longevity to the consumer. It is a large, glabrous climber with succulent, corky grooved stems, which has continued to draw the attention of researchers all over the world for over 50 years. The estimated annual demand of this species used in preparation of crude herbal drugs in Indian system of medicine is 10000 tonnes.
  

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  The research work done on Tinospora cordifolia ranges from experimental to clinical studies and from phytochemistry to bioefficacy in diverse areas. Extensive literature survey revealed that good amount of reports available on isolation of many constituents from Tinospora cordifolia.
  

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  The plant has been well researched for its therapeutic actions like Immunomodulatory, Antineoplastic, Cardioprotective, Allergic rhinitis, Antioxidant, Hepatoprotective, Anti cancer, Antiulcer, Antipyretic, Antihepatitic, Hypoglycaemic, Antibacterial, Antimicrobial, Antileishmanial, Antiinflammatory, Antiarthritic, Analgesic, Diuretic properties and many others.
  

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  But, uniformity of quality is promoted only by the use of standards by which the quality of extracts may be assessed. Upon compiling the available literature on research work done on Tinospora cordifolia mainly on isolation of constituents and their therapeutic utility till recent, from the early beginning, and considering the acclaimed medicinal values of Tinospora cordifolia as a potential source of therapeutic aids, the present work
  

is planned to fill the niche areas which has to be systematically filled in research works related to Tinospora cordifolia.

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  In support of ongoing research works, the gaps (niche) that the present work is intended to fill are:
  

1. 
   Clearly defined chromatographic profiles with identified constituents are not available in the literature.
   

2. 
   Phytochemical reference substances (corresponding to the isolated or reported constituents) on par with industrial demand are still not available.
   

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  All these loopwholes creates the utmost need to develop standardization profiles for the isolated constituents of Tinospora cordifolia. Thus the present work is planned to give a bird view regarding standardization of isolated components from Tinospora cordifolia.
  

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  Cordioside, a clerodane furano diterpene glucoside was isolated from the stems of the plant Tinospora cordifolia and its structural determination was done. In this study, the plant material (5gm) was extracted with 70% EtOH at room temp., The extract was evaporated to dryness in vacuo and the resulting residue was suspended in H20 and extracted successively with CHC13 and n-BuOH. The n-BuOH extract was freed from solvent and partially purified by MPLC using mixtures of CHC13 and MeOH as eluting solvents. Careful flash chromatography of the CHCI3-MeOH (9: 1) eluate allowed the isolation of Cordioside. The structure of cordioside isolated was characterized on the basis of NMR spectroscopy.⁽¹⁾
  

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  Tinosponone and tinocordioside were isolated from Tinospora cordifolia in which the plant material (5 kg) was extracted with 70% aq. EtOH at room temp. The EtOH was evaporated and the remaining extract washed with petrol and then extracted with n- BuOH. The n-BuOH extract was freed from solvent and subjected to repeated flash chromatography over silica gel (230-400 mesh. Merck) to yield tinosponone (0.120 g) and tinocordioside (0.064 g) whose structures were established by spectroscopic studies and chemical correlation.⁽²⁾
  

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  Two new norditerpene furan glycosides (cordifoliside D and cordifoliside E) were isolated, as their tetraacetates, from the polar butanol extract of Tinospora cordifolia stems. Fresh stems were subjected to cold extraction with MeOH, through percolation. Isolation of compounds were carried out following the experimental and chromatographic procedure. Thus, a combination of exhaustive radial and preparative TLC resulted in the isolation of cordifoliside D and E as well as Palmatosides C and F, ecdysterone, makisterone A and N-trans-feruloyl tyramine as their respective acetates. The structural elucidations and relative configurations are based on high-resolution 1D and 2D NMR spectroscopy.⁽³⁾
  

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  A new daucane-type sesquiterpene glucoside, tinocordifolioside was isolated from the stems of Tinospora cordifolia. The powdered stem (5 kg) was extracted with 70% aq. EtOH at room temp. After removal of the EtOH by evaporation the remaining extract was washed with petrol and then extracted with n-BuOH. The n-BuOH extract was freed from solvent and on repeated flash chromatography over silica gel (230-400 mesh, Merck) with EtOAc-MeOH (49: 1) yielded a fraction. Fraction 1 was found to be a mixture. This fraction was collected, concentrated, dried and stirred with Ac₂0 and pyridine at room temperature for 16 hr. The solvent was then removed in vacuo. Careful flash chromatography using hexane-EtOAc (7:3) allowed the isolation of compound Tinocordifolioside tetraacetate (35 mg) whose structure was established by detailed spectroscopic studies.⁽⁴⁾
  

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  A new daucane-type sesquiterpene, tinocordifolin, has been isolated from the stem of Tinospora cordifolia. The powdered stem of T. cordifolia (500 g) was soxhlet extracted successively with hexane and EtOAc. The concentrated EtOAc extract was subjected to column chromatography over silica gel eluting with hexane-EtOAc (17:3). This fraction on repeated chromatography using hexane-EtOAc (9:1) yielded compound Tinocordifolin (15 mg). Further elution of the column with EtOAc gave a mixture, which on acetylation with Ac₂O in pyridine, gave compound N-trans-feruloyl tyramine diacetate. Isolation of compound Tinocordifolioside from the n-BuOH extract was
  

carried out. Careful flash chromatography using CHCl₃ allowed the isolation of

compound 2 and further elution with CHCl3-MeOH (99:1) gave 4-hydroxy-3-methoxy benzoic acid. Enzymatic hydrolysis of tinocordifolioside with b-glucosidase by the usual procedure yielded an aglycone found to be identical with tinocordifolin (TLC, co-TLC). Tinocordifolin structure was then established by detailed spectroscopic studies.⁽⁵⁾

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  Polysaccharide from Tinospora cordifolia was isolated, purified, hydrolysed, trimethylsilylated and then subjected to GC/MS studies. The following types of linkages were noticed: Terminal/glucose, 4/xylose, 4/glucose, 4,6/glucose and 2,3,4,6/glucose. and the polysaccharide composition was estimated as follows: glucose 98.0%, arabinose 0.5%, rhamnose 0.2%, xylose 0.8%, mannose 0.2% and galactose 0.3%.⁽⁶⁾
  

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  Four new clerodane furano diterpene glucosides (Amritosides A, B, C, and D) were isolated as their acetates from Tinospora cordifolia stems. The stem powder (5gm) was extracted with 70% aq. EtOH at room temperature. After removal of EtOH by evaporation, the remaining extract was washed with petrol and CHCl3 and then extracted with n-BuOH. The n-BuOH extract later subjected for MPLC (Silica gel 230-400 mesh) with CHCl3: MeOH (9:1 and 8:2) giving fractions F1 and F2. These fractions were collected, concentrated, dried and stirred separately with Ac₂O and pyridine at room temperature for 16 hr. The solvent was then removed in vacuo afforded Fr. 1Ac and Fr. 2Ac respectively. Careful flash chromatography of Fr. 1Ac using hexane : EtOAc (7:3) isolated Amritoside C pentaacetate (15mg) and Amritoside D tetraacetate (11mg). Careful flash chromatography of Fr. 2Ac using hexane : EtOAc (1:4) isolated Amritoside A pentaacetate (19mg) and Amritoside B pentaacetate (10mg).⁽⁷⁾
  

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  A simple and reproducible high performance thin layer chromatography method for the determination of tinosporaside in Tinospora cordifolia was developed and was described. This method involves separation of compounds by TLC on pre-coated silica gel 60F 254 plates with a solvent system of toluene: acetone: water (5:15:1) and scanned using densitometric scanner in UV reflectance photomode at 220 nm. The linearity was observed in the range of 0.5 to 8 mg. The tinosporaside content of 0.40% w/w was
  

observed in test sample. The average percentage recovery value of 99.24±0.49 was

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  To understand and correlate the medicinal uses of Tinospora cordifolia (Willd.) Hook. F. & Thomson (Menispermaceae), Ocimum sanctum L. (Lamiaceae), Moringa oleifera Lam. (Moringaceae) and Phyllanthus niruri L. (Euphorbiaceae), trace element studies on the aqueous extract of these medicinal plants have been carried out using particle-induced X-ray emission technique. A 2-MeV proton beam was used to identify and characterize major and minor elements namely Cl, K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, Br, and Sr in them. Results have revealed that these elements are present in varying concentrations in the selected plants. Notable results include very high concentrations of Cl, K, and Ca in all the leaf samples, appreciable levels of Mn in all plants, high Zn content in T. cordifolia, and the aqueous extract of Moringa leaves compared to others and relative higher concentrations of Cr in all the plants.⁽⁹⁾
  

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  A high performance liquid chromatographic method for the estimation of berberine in the stem of Tinospora cordifolia (Willd.) Miers. ex Hook.f. and Thoms. and Tinospora sinensis (Lour.) Merill was described. The dried stems of Tinospora cordifolia and Tinospora sinensis were defatted with pet. Ether (60-80⁰). The marc was dried and further extracted with methanol. The concentration of berberine in methanol extract was determined using a C-18 reverse phase column with a mobile phase of acetonitrile:water (10:90 v/v) at a flow rate of 0.6 ml/min and with UV detectable at 266 nm. TLC and HPTLC comparison of both the species revealed significant variation in the chemical constitution of the two species.⁽¹⁰⁾
  

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  Although many constituents have been extracted and isolated from Tinospora cordifolia, the procedure available for isolation or for standardization of this drug is not meeting the industrial requirements. Hence the main objective of this study is to develop standard method for isolation and to develop standardization profile for isolated constituents of Tinospora cordifolia.
  

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  To record the chromatographic profiles of different extracts of Tinospora cordifolia.
  

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  To target and isolate the major peaks (HPLC) / spots (HPTLC) present in the chromatographic profiles.
  

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  To characterize various constituents by spectral studies.
  

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  To establish standard profiles for phytochemical reference substances of Tinospora cordifolia.
  

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  To estimate isolated constituents by HPLC or/and HPTLC.
  

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  Thus to confirm or/and extend the previous findings of Tinospora cordifolia.
  

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  Electronic data (Internet).
  
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  Published research papers.
  
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  Review articles from journals.
  
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  Library of IISC, and Govt. College of pharmacy.
  
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  Indian herbal pharmacopoeia revised new edition 2002
  
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  Library of Natural Remedies, Bangalore.
  

1. 
   Collection of the plant material and authentication.
   
2. 
   Drying and storage of plant material.
   
3. 
   Successive extraction of constituents using various solvents of increasing polarity.
   
4. 
   Isolation and detection of various constituents by spectral and chromatographic studies.
   
5. 
   Development of Chromatographic profiles (TLC, HPTLC) for isolated constituents.
   
6. 
   Subjecting the isolated constituents for spectral studies like NMR, Mass for their characterization.
   
7. 
   Establishing the standardization profiles for markers.
   
8. 
   Quantitatively study the isolated constituents from various extracts by HPLC or/and HPTLC.
   

1. 
   Versha Wazir, Rakesh Maurya, Randhir SK. Cordioside, a clerodane furano diterpene glucoside from Tinospora cordifolia. Phytochemistry 1995; 38 (2):447-49
   

2. 
   Rakesh Maurya, Versha Wazir, Anjulika Tyagi, Randhir SK. Clerodane diterpenoids from Tinospora cordifolia. Phytochemistry 1995; 38( 3): 659-61
   

3. 
   Gangan VD, Pradhan P, Sipahimalani AT, Banerji A. Norditerpene furan glycosides from Tinospora cordifolia. Phytochemistry 1995; 39 (5): 1139-42
   

4. 
   Rakesh Maurya, Kanaya LD, Sukhdev SH. A sesquiterpene glucoside from Tinospora cordifolia. Phytochemistry 1997; 44(4): 749-50.
   

5. 
   Rakesh Maurya, Sukhdev SH. Tinocordifolin, a sesquiterpene from Tinospora cordifolia. Phytochemistry 1998; 49(5): 1343-45.
   

6. 
   Jahfar M, Azadi P. Glycosyl composition of polysaccharide from Tinospora cordifolia. Acta Pharm 2003; 53 (1): 65-9
   

7. 
   Rakesh Maurya, Lila RM, Prasoon Gupta, Mishra PK, Geetu Singh, Yadav PP. Amritosides A, B, C and D: Clerodane furano diterpene glucosides from Tinospora cordifolia. Phytochemistry 2004; 65: 2051-55.
   

8. 
   Puratchimani V, Jha S. HPTLC Standardisation of Tinospora cordifolia using Tinosporaside. Indian Journal of Pharmaceutical Sciences 2007; 69 (4): 576-78.
   

9. 
   Gowrishankar P. et., al., Trace Element Studies on Tinospora cordifolia (Menispermaceae), Ocimum sanctum (Lamiaceae), Moringa oleifera (Moringaceae) and Phyllanthus niruri (Euphorbiaceae). Biological Trace Element Research, 10.1007/s12011-009-8439-1
   

10. 
    Srinivasan GV, Unnikrishnan KP, Remashree AB, Balachandran I. HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis. Indian Journal of Pharmaceutical Sciences 2008; 70 (1): 96-99
    

1. 
   Guide:
   

2. 
   Signature
   

3. 
   Co-guide
   

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   Signature
   

5. 
   Head of the department
   

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   Signature
   

DEPT. OF PHARMACOGNOSY,

GOVT. COLLEGE OF PHARMACY,

# 2, P. KALINGA RAO ROAD,

SUBBAIAH CIRCLE,

BANGALORE-560027.

GOVT COLLEGE OF PHARMACY,