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High performance liquid chromatographic method

for quantification of berberine in Tinospora
cordifolia
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V. Sivakumar et al. / Journal of Pharmacy Research 2011,4(10),3649-3651

Research Article
ISSN: 0974-6943

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High performance liquid chromatographic method for

quantification of berberine in Tinospora cordifolia
V. Sivakumar 1* and M.S. Dhana Rajan 2
Research Scholar, Bharathiar University, Coimbatore, Tamilnadu. India.
2
Principal, Jaya College of Arts and Science, Thiruninravur. - 602 024. Tamilnadu. India.
1

Received on: 19-06-2011; Revised on: 08-07-2011; Accepted on:01-10-2011

ABSTRACT
A sensitive, simple and accurate HPLC method has been developed for the quantification of berberine, a isoquinoline alkaloid in dry stem of Tinospora cordifolia
(wild) Miers. ex Hook.f. and Thoms. Chromatographic analysis was performed using pet- ether, methanol, aqueous and chloroform extracts, on using a solvent
system, comprising of Acetonitril: water 60:40 (V/V) as mobile phase at a flow rate of 0.5ml/min. And 265nm gives good separation of berberine at Rt 5.15min.
The maximum of berberine content was observed in methanol extract when compared to other test samples. The proposed HPLC method is rapid and accurate for
quantitative monitoring of berberine in Tinospora cordifolia.
Keywords: Tinospora cordifolia, berberine, HPLC.
INTRODUCTION
Herbal medicines have being used by billions of people around the world for
thousands of years. Herbal medicines play very important role in health care
of 80% population of the world especially developing countries. As per the
traditional medicines programme of the world health organization (WHO)
nearly 80 % of the world population used phyto-products due to low toxicity
and known pharmacological activity. [1] Herbal medicine is very popular in
different system of medicines like Indian system of medicine, Chinese system
of the medicine, unani naturopathy, oestropathy and homeopathy. [2] In recent
years, interest on plant based drugs has increased considerably. Annual growth
rate between 5-15% for trade of plant based drugs and raw materials is indicative of growing demand for herbal drugs. But the quality control and quality
assurance still remains a challenge because of the high variability of chemical
components involved. The major problem of quality assurance of herbal medicine has been solved to a great extent with the help of chromatographic and
spectral fingerprints analysis. [3]
Tinospora cordifolia is one such plant which is widely used in indigenous
system of medicine.[4] It is a large, glabrous, succulent, deciduous climbing
shrub belonging to the family menispermaceae.[5] It is distributed throughout
tropical India subcontinent, Sri Lanka and china, ascending to an altitude of
1200m. The stem of Tinospora cordifolia is rather succulent with long filiform fleshy aerial roots from the branches. The bark is creamy white to grey,
deeply left rosette like lenticels. The leaves are membranous and cordate. The
flowers are small and yellow or greenish yellow. [6]
Tinospora cordifolia known as Amrita (Guduchi) in Sanskrit, Shindilkodi in
Tamil, it is a widely used in folk and ayurvedic systems of medicine. The term
Amrita is attributed to its ability to impart youthfulness, vitality and longevity to the consumer. [7] The large numbers of compounds have been isolated
from the aerial parts, stem and roots of Tinospora cordifolia . Guduchi is
widely used in Ayurvedic system of medicine Rasayanas to the immune
system and the body resistance against infections. In modern medicine Tinospora
cordifolia used for the treatment of general weakness, fever, dyspepsia, dysentery, gonorrhoea, urinary diseases, viral hepatitis and anaemia more recently,
the immunomodulatory properties, antineoplastic activities and hypoglycemic activitie have been reported. [8,9]
HPLC is a popular method for the analysis of herbal medicines because it is
easy to learn and use and is not limited by the volatility or stability of the
sample compounds. HPLC method has emerged as an efficient tool for phy-

*Corresponding author.
V.Sivakumar,
Assistant professor,
Department of Pharmacy,
Sri Lakshmi Narasimha College of Pharmacy,
Palluru-517 132. Andhra Pradesh. India
Tel.: + 91E-mail:sivakumarv2k1@gmail.com

tochemical evaluation of herbal drugs. No HPLC method has been developed

in literature for the quantification of berberine from Tinospora cordifolia stem
power petroleum-ether, methanol, aqueous and chloroform extracts comparative analysis hence, an HPLC method has been devised in the present research
work, which may be used as an alternative method for quantification and
standardization of the Tinospora cordifolia using berberine as marker, the
proposed HPLC method is simple, precise and sensitive.
MATERIALS AND METHODS
Plant materials
The stem plant material of Tinospora cordifolia was collected fresh from
Vellore District area in Tamilnadu. The plant stem was authenticated by the
Herbarium of Botany Directorate in National Institute of Herbal Science,
Plant Anatomy Research Center, Chennai. A voucher specimen (No: TC08)
was deposited in the Center.
Preparation of extract from Tinospora cordifolia
The dried powdered stem of Tinospora cordifolia was allowed to pass through
ss sieve (20 mesh). It was extracted and then to exhaustion (soxhlet) with
various solvents like petroleum ether, methanol, aqueous and chloroform.
The solvents were removed under vacuum to get solid mass and use further
analysis. [10]
Preparation of standard solution
A stock solution of standard berberine was prepared in 5ml volumetric flask by
dissolving 1.7mg of accurate weighed berberine standard in about 3ml of solvent (acetonitril: water) 60:40 followed by sonication for 5min. and finally
making the volume up to mark with solvent.
Preparation of sample solution
Stock solution of samples was prepared by transferring 3.8mg of accurately
weighed; pet-ether extract in 1ml solvent (acetonitril: water) 60:40, 2.8mg of
methanol extract in 1ml solvent, 2.7mg of aqueous extract in 1ml solvent and
2.0mg of chloroform extract of Tinospora cordifolia stem power in the volumetric flasks and then sonicated for 15min. at room temperature. The content of the flask were filtered thought whattman filter paper No.41. The
filtrate was collected and use further analysis.
Chemical materials
Acetonitrile, methanol, chloroform, petroleum ether, were used of AR grade
(S.D. Fine chemicals, Baroda) and standard berberine from sigma Alrich, Bangalore.
Chromatographic conditions.[11,12]
Injection volume - 20l
Flow rate 0.5ml/min.
Mobile phase Acetonitril: water (60:40)
Detection wave length 265 nm
Mode isocratic
Retention time (Rt) berberine 5.15min.

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V. Sivakumar et al. / Journal of Pharmacy Research 2011,4(10),3649-3651

Figure 1. HPLC chromatogram of standard berberine.Berberine peak

at the Rt 5.15min. detected at a wavelength of 265nm.

Figure 2. HPLC chromatogram of petroleum-ether extract of Tinospora

cordifolia peak at the Rt 5.15min. correspond to berberine detected at a
wavelength of 265nm.

Figure 4. HPLC chromatogram of aqueous extract of Tinospora cordifolia

peak at the Rt 5.15min. correspond to berberine detected at a wavelength
of 265nm.

Figure 5. HPLC chromatogram of chloroform extract of Tinospora

cordifolia peak at the Rt 5.15min. correspond to berberine detected at a
wavelength of 265nm.
RESULT AND DISCUSSION
The present method was conducted to identifying and quantifying the berberine from Tinospora cordifolia medicinal plants stem various different fractions. Berberine peaks from solutions of various extract like pet- ether; methanol, aqueous and chloroform were identified by comparing their Rt values with
these obtained by chromatography of the standard under the same conditions.
The peaks of Rt 5.15min. was observed in the chromatograms obtained from
fractions like pet- ether, methanol, aqueous and chloroform, the chromatograms of standards and test samples are shown in Fig 1 to 5 respectively. The
berberine content of fractions of sample I (pet- ether), sample II (methanol), sample III (aqueous), sample IV (chloroform) was 0.002% (W/W),
0.223% (W/W), 0.150% (W/W), 0.020% (W/W) respectively. The more
amount of berberine was present in methanol extract when compared to other
fractions.
CONCLUSION
The HPLC technique is quite, simple, accurate, precise, reproducible and sensitive. This method can be used for routing analysis of berberine in cured drugs
and prepared formulations and also its used for standardization and quality
control of herbal products of traditional medicine containing Tinospora
cordifolia as an ingredient can be explored.

Figure 3. HPLC chromatogram of methanol extract of Tinospora cordifolia

peak at the Rt 5.15min. correspond to berberine detected at a wavelength
of 265nm.

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Source of support: Nil, Conflict of interest: None Declared

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