Effect of Host Trees on Berberine Content in Stem of Tinospora cordifolia Climber: A Preliminary Study

 

Wazbir Hussain Barlaskar1, Neeta Raj Sharma1*, Gaurav Kumar1, Sanjeev Kataria2

1Lovely Professional University, Phagwara, Punjab, India

2Herbal Health Research Consortium, Amritsar, Punjab, India

*Corresponding Author E-mail: neeta.raj@lpu.co.in

 

ABSTRACT:

Present study was aimed to analyze berberine concentration in Tinospora cordifolia stems collected from Ficus, Azadirachta, Syzygium, Populus and Eucalyptus. High Performance Liquid Chromatography equipped with UV detector was used for analytical study of berberine from the stem of Tinospora cordifolia. Methanol (100%) as a diluent was used for sample extraction. Using potassium dihydrogen phosphate and acetonitrile as a mobile phase in a ratio of 65 to 35 respectively and Zorbax XDB C-18, 250x 4.6mm 5-micron column, berberine was separated at 254nm wavelength. Maximum concentration of berberine was detected in the stem of Tinospora cordifolia climbed on Ficus (0.087%) followed by Azadirachta (0.084%). The least concentration of berberine was quantified in Eucalyptus (0.011%) on dried wt. basis.

 

KEYWORDS:  Berberine, Tinospora cordifolia, HPLC chromatograms

 

 


INTRODUCTION:

Tinospora cordifolia (family: Menispermaceace) is succulent, glabrous, broadly scattering, climber with extensively elongated twining branches. It is named as Guduchi (Sanskrit), Gulancha (English), and Giloya (Hindi). It spreads all through the tropical region of India up to 1200 m above the sea level. Tinospora is very common and well known plant in Auyrvedic system, owing to its multiple usage evidenced with this drug in identification of its capacity to impart vitality, and longevity to the consumer.1 It is broadly used in traditional and Ayurvedic systems of medicine for its general tonic, anticancer2, antiulcer3, antipyretic4, antihepatitis5, immunomodulatory6, antioxidant7, hypoglycaemic8, antineoplastic, cardiotonic, antibacterial, antimicrobial, antiinflammatory, antiarthritic9 analgesic and diuretic properties.10

 

It is a popular reliance among the Ayurvedic professionals that Satva from Guduchi, climbing on Neem tree (Azadirachta indica) is more bitter and effective and is said to impart the medicinal worth of Neem also which is one of the hosts of this shrub. Antioxidant activity of Tinospora cordifolia grown on Ficus religiosa showed maximum activity (41.73 μM Fe (II)/g) when compared with various tree species. 11 It has already been established by existing literature that plants affect other plants in their nearby vicinity through allelochemicals. Berberine is found to be a universal marker in Tinospora cordifolia. Moreover, berberine is an isoquinoline alkaloid 12possesses therapeutic applications.10 Therefore this study investigates the effect of various hosts on the concentration of berberine in the stem of T. cordifolia. The present study included the stem of T. cordifolia climbed on wall to compare efficacy of various hosts in concentrating berberine. This study can be very helpful in selecting high phytochemical containing strains of T. cordifolia and hence with high therapeutic efficacy for other medicinal uses.

 

 

MATERIALS AND METHODS:

Instrument:

High performance liquid chromatography (Agilent Technologies 1260 infinity) equipped with UV detector was used to perform HPLC analysis of berberine in stem samples of T. Cordifolia from Ficus, Azadirachta, Syzygium, Populus and Eucalyptus and also the stem climbed on wall.

 

Mobile phase and diluent:

Two different mobile phases (Mobile Phase-A: B) were used in present study.

 

Mobile phase-A :

1.36 grams of Potassium dihydrogen phosphate (KH2PO4) was added into 1 litre capacity volumetric flask containing about 700 ml HPLC Grade water which was followed by adding 0.5 ml orthophosphoric acid with continuous mixing and made up the volume upto 1 litre. Further, mobile phase was filtered through 0.45µ nylon filter and sonicated for 5 minutes.

 

Mobile phase-B:

HPLC grade Acetonitrile (100%) was used as another mobile phase.

 

Mobile phase Composition:

Both the mobile phases were mixed properly in ratio of 65 to 35 i.e., Mobile phase-A: Mobile Phase-B (65:35).

 

Diluent:

100% Methanol was used as diluent.

 

Standard and Sample preparation:

Standard preparation:

Accurately weighed Berberine (5.0 mg) was dissolved in methanol (50 ml) by sonication. The volume was adjusted upto 100 ml mark with methanol. Standard berberine solution thus prepared and filtered using 0.22µ syringe filter.

 

Sample Preparation:

Sample preparation was done referring the method used by Srinivasan et al. 200813. Further, 2.0 gram of sample was taken into 50 ml. capacity volumetric flask and 30 ml. of methanol was added followed by sonication for 30 minutes and volume was made up to 50 ml. by adding methanol. Sample solution was filtered through 0.22µ syringe filter.

 

Chromatographic conditions:

Following Chromatographic conditions were used in present investigation.

Column: Zorbax XDB C-18, 250x 4.6mm 5-micron

Wavelength: 254 nm

Injection volume: 20 microliter

Mode: Isocratic

Column Temperature: 25°C

Runtime: 30 minute

 

RESULT AND DISCUSSION:

It is a time-honoured faith in Ayurveda that extract obtained from Tinospora species climber on Neem (Azadirachta indica) tree is bitter and also more effective owing to the medicinal potential of Neem also.14 Radical scavenging and immunomodulatory potential of T. cordifoila collected from four different trees (viz A. indica, A. leucophloea, B. monosperma, P. juliflora) was studied by Narkhede et al. 2014, and reported that Tinospora cordifoila grown on Azadirachta indica showed highest potential except humoral immune response.14 Such studies caught attention of author to conduct preliminary study on comparative HPLC analysis of berberine, one of the potential bioactive compounds of Tinospora cordifolia grown on different trees namely Ficus, Azadirachta, Syzygium, Populus and Eucalyptus species and also climbed on wall, to evaluate whether there is any effect of host trees in concentrating the bioactive constituent in this climber.

 

HPLC analysis was done for Berberine standard, followed by samples obtained (on dried weight basis) from Tinospora cordifolia collected from Ficus, Azadirachta, Syzygium, Populus and Eucalyptus tree species. In HPLC analysis peak of berberine was detected at 254nm at retention time of 3.4 min (Figure 1). Further, Tinospora cordifolia raw samples on dry weight basis were analyzed and loss on drying was also recorded. HPLC analysis results revealed that Ficus showed (Table 1) highest concentration of beriberin (0.087% dry wt. basis: Figure: 7) which was found very close to Azadirachta (0.084%; Figure: 6) with a marginal difference and further followed by Syzygium (0.027%; Figure: 5), Populus (0.017%; Figure: 4), Tinospora cordifolia grown on wall (0.018%; Figure: 2) and Eucalyptus (0.011%; Figure:3) respectively. Ficus and Azadirachta species indicated better berberine content in stems of Tinospora cordifolia as compared to other tree species investigated in present study which is also in agreement of earlier study conducted by Baighet al. (2014)11, on highest antioxidant activity (1.73 μM Fe (II)/g) of Tinospora cordifolia climber on Ficus, when compared with Eucalyptus hybrid and Roystonea regia. No pertinent research reports are available on the effect of host plant on enhanced concentration of berberine, as one of the active constituents of this climber and also on efficiency of Tinospora cordifolia grown on various host. However, Comparative immunomoduatroy potential of Tinospora cordifolia, Tinospora sinensis and neem-guduchi (Tinospora cordifolia grown on Neem) was studied, and reported that neem-guduchi has more efficiency for immunomodulation which supports the claim of prehistoric Ayurvedic literature. Considering available literature and present work, an extensive in vivo study is required to establish the fact on efficacy of active principles also obtained from Tinospora cordifolia grown on various host trees. The study in this direction will be helpful in exploring the molecular level studies on the factors responsible for selective efficacy of active principles and molecular interactions.


 

Table1: HPLC analysis of Berberine in Tinospora cordifolia collected from different trees

RS. No.

Sample name

Area (mAu)

Weight

% Assay (Dried basis)

1.

Standard

2065025

5.46mg in 100 ml methanol

 

2.

Wall

233949

2047.81mg in methanol

0.018

3.

Eucalyptus

146173

2057.31mg in methanol

0.011

4.

Populus

247288

2138.23mg in 50 ml methanol

0.017

5.

Syzygium

395483

2028.22 mg in methanol

0.027

6.

Azadirachta

870909

2029.85mg in methanol

0.084

7.

Ficus

964049

2025.39 mg in methanol

0.087

 

 

Figure 1. HPLC analysis of Berberine standard

 

Figure 2. HPLC analysis of Berberine from Tinospora cordifolia grown on wall

 

Figure 3. HPLC analysis of Berberine from Tinospora cordifolia climbed on Eucalyptus sp.

 

Figure 4. HPLC analysis of Berberine from Tinospora cordifolia climbed on Populusspecies

 

Figure 5. HPLC analysis of Berberine from Tinospora cordifolia climbed on Syzygium species

 

Figure 6. HPLC analysis of Berberine from Tinospora cordifolia climbed on Azadirachta species

 

Figure 7. High Performance Liquid Chromatogram of Berberine from Tinospora cordifolia climbed on Ficus species

 


 

CONCLUSION:

It is hence concluded by present preliminary study, that the higher concentration of berberine in the stems of Tinospora cordifolia grown on Ficus and Azadirachta species could beat tributed to the allelopathic effect of host on the climber which was noted positive in making Tinospora cordifolia comparatively more  effective  than  other  tree  species  studied.  An extensive study is recommended to identify therapeutically active Tinospora sp. grown on various supports/trees.

 

ACKNOWLEDGEMENTS:

Authors wish to thank management of Lovely Professional University for providing necessary facilities to carry out this study.

 

CONFLICT OF INTEREST STATEMENT:

Authors do not have any conflict of interest.

 

REFERENCES:

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11.  Baigh MA, Verma KC, Kaur S, Sachdeva A and Sharma NR. Antioxidant potential of Tinospora cordifolia climber from Eucalyptus hybrid, Ficus religiosa and Roystonearegia. International Research Journal of Biological Sciences. 3 (5); 2014:5-9.

12.  Budavari S. The Merck Index. Merck and Co Inc, Whitehouse Station, NJ. 2001.

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14.  Narkhede AN, Jagtap SD, Kasote DM, Kulkarni OP and Harsulkar AM. Comparative immunomodulation potential of Tinospora cordifolia (Willd.) Miers ex Hook. F., Tinospora sinensis (Lour.) Merrill and Tinospora cordifolia growing on Azadirachta indica A. Juss. Indian Journal of Experimental Biology. 52 (8); 2014:808-813.

 

 

 

 

 

 

Received on 06.01.2017             Modified on 16.01.2017

Accepted on 20.02.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(3): 818-822.

DOI: 10.5958/0974-360X.2017.00154.8