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Advances in Natural and Applied Sciences, 3(1): 88-94, 2009

ISSN 1995-0772
2009, American Eurasian Network for Scientific Information
This is a refereed journal and all articles are professionally screened and reviewed

ORIGINAL ARTICLE
Nutritional Composition, Antioxidant Ability and Flavonoid Content of Tinospora
crispa stem
1

Zulkhairi Amom, 1Hasnah Bahari, 1Sakinah Isemaail, 1Nur Amalina Ismail, 2Zamree
Md Shah, 2Mohd Shahidan Arsyad.
1

Department of Human Anatomy, Division of Physiology, Faculty of Medicine and Health Sciences, Universiti
Putra Malaysia, 43400 Serdang Selangor, Malaysia.
2
Herbal Technology Center, Forest Research Institute of Malaysia, Kepong, 52109 Kuala Lumpur, Malaysia.
Zulkhairi Amom, Hasnah Bahari, Sakinah Isemaail, Nur Amalina Ismail, Zamree Md Shah, Mohd
Shahidan Arsyad: Nutritional Composition, Antioxidant Ability and Flavonoid Content of Tinospora
crispa stem: Adv. in Nat. Appl. Sci., 3(1): 88-94, 2009
ABSTRACT
Although the plant Tinospora crispa was claimed by the traditional healer to be beneficial on human health
and able to combat degenerative diseases, scientific data on its health claims and the antioxidative properties
is scant. The present study was aimed to assess the nutritional composition and mineral content of the stem
of Tinospora crispa. The antioxidant ability, flavonoid and total phenolic content of the aqueous crude extract
of the stem were also investigated. Results from the proximate analysis revealed that T. crispa contains high
moisture content (77.9%), followed by carbohydrate content (19.4%). Protein, fat, ash and fiber are found at
low percentage. Calcium and potassium are the most abundant element measured. Other trace elements such
as magnesium, silicon, phosphorus and chlorine are very low. The antioxidant ability of the extract was
determined based on DPPH, TBA and FRAP assay. The data demonstrated that T. crispa extract possess high
antioxidative properties and the activity is comparable to the established antioxidants, such as BHT and vitamin
C. The flavonoids detected were catechin (1.58g/l), luteolin (0.85g/l), morin (1.44g/l), and rutin
(1.38g/l), which may be collectively responsible for the high antioxidant activity. The total phenolic content
is 0.29+0.01 mg (GAE)/100g of fresh sample. Findings from this study suggested that T. crispa could be a
valuable source of nutrients and natural antioxidant.
Key words: Tinospora crispa, nutritional composition, antioxidant, flavonoid
Introduction
Antioxidant compounds play an important role as a health-protecting factor. Antioxidants are substance
that significantly prevents or delays the oxidation of other molecules by inhibiting the initiation or propagation
of oxidizing chain reactions (Halliwell & Gutteridge, 1990). Free radicals are produced in normal and
pathological cell metabolism considering that oxidation is essential to most living organisms for the production
of energy to fuel biological processes. Oxygen-centered free radicals and other reactive oxygen species (ROS)
have been associated with the beginning of many diseases and degenerative processes in ageing (Halliwell,
1994). Almost all organisms are well protected against free radical damage by oxidative enzymes such as
superoxide dismutase and catalase or chemical compounds such as -tocopherol, vitamin C (ascorbic acid),
carotenoids, polyphenol compounds and glutathione (Betancur-Ancona et al., 2004). However, these systems
are frequently insufficient to totally prevent the damage, especially under the conditions of severe oxidative
stress, resulting in diseases and accelerated ageing (Ames et al., 1993).
Corresponding Author: Zulkhairi Hj Amom, PhD., Department of Human Anatomy, Division of Physiology, Faculty of
Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia.
Ph/Fax: 603-89472362
Email: zakirukm@hotmail.com; fradical@medic.upm.edu.my

Adv. in Nat. Appl. Sci., 3(1): 88-94, 2009

89

Natural products with antioxidant activity may be used to help the human body to reduce oxidative
damage. Many herbs, fruits and vegetables have been investigated for their antioxidant activities in the last
years (Dimitrios, 2006). Dietary sources have been recognized as safe and effective antioxidants in terms of
their efficiency and non-toxicity (Block et al., 1992, Tsai et al., 2005). The interest on the role of natural
antioxidants as a tool to prevent aging and degenerative diseases process is growing (Slater, 1991) due to the
toxicity of synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene
(BHT) at fairly high doses, which limits their therapeutic usage (Altmann et al., 1986, McCormick et al.,
1986).
Tinospora crispa, is an indigenous climber plant that commonly grows wild in Asean countries including
Malaysia. Known by various local names like akar patawali and akar seruntum (Noor & Ashcroft, 1989),
an infusion of the stems is consumed as vermifuge and decoction of the whole plant is used to treat cholera
and diabetes among the Malay community.
Its stem has been used by traditional folklore for various
therapeutic purposes such as treatment for diabetes, hypertension, stimulation of appetite and protection from
mosquito bites. Sometimes it was also used as an anti-parasitic agent in both man and domestic animals (Noor
et al., 1989, Kongsaktrakoon et al., 1994, Pathak et al., 1995). Despite its long usage among the traditional
folklore, scientific data supporting its health claims and the biological assessment of this plant is not thoroughly
studied. On the other hand, natural substances have been extensively studied especially during this decade as
an alternative treatment or a preventive measure. This has led our interest to explore the biological properties
of T. crispa in terms on its nutritional composition, the mineral and flavonoid content in the stem of the plant
as well as its antioxidant ability in vitro.
Materials and methods
Plant materials
Samples of fresh stems of T. crispa were collected from Universiti Putra Malaysia (UPM) after being
identified and confirmed by a plant taxonomist. A voucher specimen was deposited in the Institute of
Bioscience, UPM with voucher number as SK015.
Proximate analyses
Moisture, ash, crude fat, crude fiber and protein content of the fresh stem of T. crispa were determined
according to standard methods described by the Association of Official Analytical Chemists (AOAC, 1996).
Moisture content
The oven method was used to determine the moisture content. The percentage of dry matter was measured
using moisture balances. The moisture content was calculated using the formula:
Moisture (%) = weight of sample before drying weight of sample after drying x 100
))))))))))))))))))))))))))))))))))))))))))))))))))))))

Weight of sample before drying

Ash content
The oven method was used to determine the ash content. Two grams of the sample were added to a preweighed crucible and weighed. Then, the sample were placed in a furnace at 550C for 4 h, cooled in
desiccators and reweighed. The ash content was determined by the equation as follows:
% ash = weight of ash/weight of sample x 100
Fat content
For determination of fat content, the Soxhlet method was used. One hundred and fifty milliliters of
petroleum ether was poured over 5 g of T. crispa extracts in an extraction thimble. The thimble was placed
in a pre-weighed beaker covering anti-bumping cotton and placed in the Soxhlet for 8 h, after which the beaker
was dried in an oven, cooled and reweighed. The fat content of each sample was calculated by the equation
as follows:

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Adv. in Nat. Appl. Sci., 3(1): 88-94, 2009

% crude fat =

(weight of dried beaker + fat) ( weight of dried beaker + granules)

100

))))))))))))))))))))))))))))))))))))))))))))))))))))))

Weight of sample

Protein content
Crude protein content was determined using the Kjeldahl method. Nitrogen content was calculated using
the following equation:
N (%) = 14.01 x (ml titrant for sample - ml titrant for blank) x molarity of acid

100

))))))))))))))))))))))))))))))))))))))))))))))))))))))

Weight of sample

The crude protein content was then calculated by the equation as follows:
Protein (%) = N x 6:25 (protein factor specific to sample)
Crude fiber
Crude fiber was determined using fat-free samples.
following equation:

Crude fiber content was determined using the

% crude fibre = (beaker + residue weight - fibrebag weight) (beaker + ash weight)

100

))))))))))))))))))))))))))))))))))))))))))))))))))))))

Sample weight

Mineral content
The mineral elements present in the fresh stem of T. crispa were determined through energy dispersive
X-ray (EDX) analysis using Scanning electron microscopy (SEM). The fresh sample of the stem was sent to
Microscopy Unit, Institute of Bioscience, UPM to be analyzed.
Preparation of aqueous crude extract
The stems of T. crispa were washed thoroughly in flow tap water, dried vigorously by C-fold towel and
cut into small pieces. The specimens were dried in oven at 40C for 48 hrs until completely dried and
pulverized. An aqueous crude extract was prepared at 10% (w/v) by soaking 100 g of the powdered stem in
1000 ml distilled water and incubated in shaking water bath at the temperature of 60C for 6 h. Once filtered,
the filtrates were freeze dried and kept in air tight container at -20oC until used.
Determination of antioxidant ability
The free radical scavenging activity of T. crispa aqueous extract was determined using 1,1-diphenyl-2picrylhydrazyl (DPPH) assay. The Ferric Reducing Antioxidant Power (FRAP) assay and the Thiobarbituric
Acid (TBA) test were governed to evaluate the plants antioxidative properties. In all assessments, vitamin
C and Butylated Hydroxytoluene (BHT) were used as the standard.
1,1-diphenyl-2-picrylhydrazyl (DPPH ) assay
The scavenging activity of DPPH free radicals was determined according to the method recommended by
Lu and Yeap (2000) with minor modification. 0.45mM of DPPH was prepared by added 17.74mg of DPPH
into 100ml of absolute ethanol. 1ml of the 0.45mM DPPH was added into 0.5ml of the samples (T.crispa
extract, vitamin C, BHT and control). 0.5ml of the samples was prepared by added respective 5mg of the
crude extract, vitamin C and BHT into one ml of absolute ethanol. The mixture was kept in dark at room
temperature for 30 minutes. The absorbance of the free radical scavenging activity was measured by
spectrophotometer at 517nm. The percentage of inhibition of the sample against DPHH radicals was calculated
from the following equation:

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Adv. in Nat. Appl. Sci., 3(1): 88-94, 2009

% Inhibition =

[Absorbance of control Absorbance of test sample]

x 100

)))))))))))))))))))))))))))))))))))))))

Absorbance of control

Ferric Reducing Antioxidant Power (FRAP) assay

The ferric reducing antioxidant power (FRAP) assay gives an indication of the reducing ability of the plant
extract. This method was adapted from Benzie and Strain (1996). Ferric reducing antioxidant power reagent
was freshly prepared by mixing together 10mM 2, 4, 6-tripyridyl triazine (TPTZ) and 20mM ferric chloride
in 0.25M acetate buffer, pH 3.6. 100L of test sample was added to 300L of distilled water followed by 3mL
of FRAP reagent. The absorbance was read at 593 nm after 4 min incubation at room temperature against a
blank. The standard curve was constructed using ferrous sulphate (0.21mmol/L). Antioxidant activity could
be determined from the standard curve of ferrous sulphate using its measured absorbance. The results were
expressed in millimolar per liter. Antioxidant activity of the sample was compared with standard BHT and
vitamin C.
Thiobarbituric acid (TBA) test
The TBA test of T. crispa extract was determined using the method of Ottolenghi (1959) with slight
modification. Two (2) milliliters of 20% trichloroacetic acid and 2 ml of 0.67% 2-thiobarbituric acid were
added to 1 ml of sample solution. The mixture was placed in a boiling water bath and after cooling was
centrifuged at 3000 rpm for 20 min. Absorbance of supernatant was measured at 552 nm. The percentage of
inhibition was calculated from the following equation:
% Inhibition =

[Absorbance of control Absorbance of test sample]

x 100

))))))))))))))))))))))))))))))))))))))))

Absorbance of control
Determination of flavonoid content

Flavonoid standard such as catechin, luteolin, morin and rutin were dissolved in methanol to a
concentration of 1 mg/ml and stored protected from light at 20oC. Samples of T. crispa was dissolved in the
mobile phase of methanol-acetonitrile-water (40:15:45, v/v/v) with 1.0% acetic acid, filtered, and injected
directly. Chromatographic analysis was carried out by HIQ SIL C18V reverse-phase column ( 4.6 mm x 250
mm) packed with 5m diameter particles, and the flow rate was set at 1.0 ml/min.
Determination of total phenolic content
The total phenolic content of the extract was determined using the Folin-Ciocalteu assay following the
procedure described by Velioglu et al. (1998) with slight modification. An aliquot (200l) of the extract was
mixed with 1.5 ml Folin-Ciocalteu reagent (diluted 10-fold with distilled water) and left to stand at room
temperature. Following 5 min, 1.5ml of sodium bicarbonate (0.6M) was added to the reaction and left at room
temperature. Following 90 min, the absorbance was read at 725nm. Total phenolic content of the sample was
calculated as gallic acid equivalent (GAE) from the standard curve of gallic acid standard solution (20, 40, 60,
80, 100mg/L) and expressed as gallic acid equivalent (GAE) in mg per 100g of fresh sample.
Statistical analysis
Statistical analysis was performed using computer generated One-way ANOVA version 13.0. In all cases,
p < 0.05 was considered significant. All values are expressed as means S.D.
Results and discussion
Data on proximate and mineral analyses of T. crispa stem are shown in Table 1 and Table 2 respectively.
The results revealed that T. crispa possess high water content (77.9 %) in its stem. The stem contains high
carbohydrate which is 19.4%. Other substances like protein, fat, ash and total dietary fiber are found to be in
low concentration. Carbon is found at the highest percentage, followed by oxygen, while calcium and
potassium is the most abundant mineral measured. Other trace elements such as magnesium, silicon,
phosphorus and chlorine are available at a very low proportion.

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Adv. in Nat. Appl. Sci., 3(1): 88-94, 2009

Table 1: Proximate (protein, fat, carbohydrate, ash, moisture and total dietary fiber) content of T. crispa stem.
Percentage (%) by weight (w/w)
Protein
1.20
Fat
0.43
Carbohydrate
19.4
Ash
1.10
Moisture
77.9
Total Dietary Fiber
0.65
Table 2: Mineral content of T. crispa stem.
Carbon (C)
Oxygen (O)
Magnesium (Mg)
Silicon (Si)
Phosphorus (P)
Chlorine (Cl)
Potassium (K)
Calcium (Ca)
Values are expressed as mean SD.

Percentage (%) by weight (w/w)

52.26 1.48
33.77 2.11
0.79 0.11
0.27 0.03
1.21 0.09
0.85 0.07
4.42 0.58
6.17 0.57

Prior studies confirmed that chemical substances in plants including protein, carbohydrate, vitamin and
fiber also contribute to the antioxidant capacity (Betancur-Ancona et al., 2004). The plant proteins present in
the extract of grass pea seeds and soluble proteins of legume seeds contain compounds of strong antioxidant
activity, e.g. isoflavones, which are effective peroxyl radical scavengers (Patel et al., 2001). The amount of
ash measured in T. crispa extract is low compared to other herbs examined by Maisuthisakul et al. (2007).
There are inversely correlated between ash and antioxidant properties whereby ash contains minerals and heavy
metals (including iron) which can act as pro-oxidants (Maisuthisakul et al., 2007). Low ash content indicates
that T. crispa contains low pro-oxidant substances.
The DPPH assay was utilized to evaluate the antioxidant ability to scavenge free radicals. As shown
in Table 3, the scavenging activities of T. crispa extract, vitamin C and BHT on DPPH radicals were
compared. The test demonstrated the percentage of inhibition of up to 86% in the T. crispa extract. The free
radical scavenging effect was assessed by the discoloration of the DPPH solution (Sanchez et al., 2001). The
DPPH is a stable lipophilic radical (Scalzo, 2008) that can readily undergo scavenging by antioxidant (Lu &
Yeap, 2001). Antioxidant reacts with DPPH due to its hydrogen donating ability (Chen & Ho, 1994). The
interaction results in either the transfer of an electron or a hydrogen atom to DPPH, which neutralizes the free
radical (McCune & Johns, 2007). The result from this study proved that T. crispa extract is as effective as
established antioxidants like BHT and vitamin C in scavenging DPPH free radical.
Table 3: The antioxidant ability of T.crispa stems aqueous extract as measured by DPPH (scavenging activity), TBA (inhibition
percentage) and FRAP (FRAP value) assay.
Sample
Scavenging activity (%)
Inhibition percentage (%)
FRAP Value (mmol/l)
Vitamin C
96.360.90
73.25.14
1.050.00
BHT
96.510.95
75.86.08
1.030.03
T.crispa stems extract
86.510.07
39.25.14
0.890.07
Values are expressed as mean SD.

FRAP assay on the extract produced a FRAP value of 0.890.07 mmol/L. Antioxidant activity in plant
extract can be measured via the FRAP assay (Benzie & Strain, 1996) by looking at the reduction of the ferric
2,4,6-tripyridyl-s-triazin complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ). A blue colored FeIItripyridyltriazine compound is formed from colorless oxidized FeIII by the action of electron donating
antioxidants in plant samples. The result demonstrated that the FRAP value of T. crispa is comparable to
vitamin C and BHT, with difference of 0.16 0.06 mmol/L and 0.14 0.05 mmol/L respectively. This proves
that the extract possess high level of antioxidant properties.
T. crispa extract showed a significant difference of 39.202.97% in antioxidant activity compared to
controls via the TBA method. This is a colorimetric technique widely used to measure the extent of lipid
oxidation in samples (Ren et. al., 2008). The peroxidation of polyunsaturated fatty acid (PUFA) will yield
lower molecular compounds such as malondialdehyde (MDA) (Ledwozyw et al., 1986).
The flavonoids detected in T. crispa (Table 4) are catechin (1.58g/mg of sample), luteolin (0.85g/ mg
of sample), morin (1.44g/ mg of sample), and rutin (1.38g/ mg of sample). Catechin could prevent
cardiovascular disease (Du & Lou, 2008) by protecting LDL from oxidative damage through its free radical
quenching and metal chelating abilities (Rice-Evans et al.,, 1996). Luteolin has strong scavenging properties
for superoxide radicals (Cai et al.,, 1997) and poses as a potent physical quencher of singlet oxygen (Babenko
& Shakhova, 2006).

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Table 4: Flavonoid content of T.crispa stem extract.
Catechin
Luteolin
Morin
Rutin
Values are expressed as mean SD.

Flavonoid content (g/mg of sample)

1.58
0.85
1.44
1.38

Morin has certain biological activities, including antioxidant properties (Dai et al., 2006) and a modulatory
effect on lipoxygenase and cyclooxygenase activities in the arachidonic cascade (Babenko & Shakhova, 2006).
Rutin also has antioxidant properties and has been reported as essential to increase the strength of capillaries
and regulate their permeability (Rice-Evans et al.,, 1996).
Flavonoids belong to a polyphenol class and are powerful antioxidants (Rice-Evans et al.,, 1996) as well
as potent inhibitors of LDL oxidation via several mechanisms, including scavenging of free radicals and
chelation of transition metal ions (Maisuthisakul et al., 2007). Flavonoid intake decreases LDL and hastens
removal of cholesterol from peripheral tissue to the liver for catabolism and excretion (Tsai et al., 2005).
Flavonoids help liver cells to be more efficient in removing LDL from blood by increasing the LDL receptor
densities in liver and by binding to apolipoprotein B (Valero, 2005). Flavonoids found in the extract may be
responsible for the prevention of atherosclerotic plaque formation and improvement in the lipid profile (Yen
et al. 2002; Rice-Evans et al.,, 1996).
The total phenolic content for the stem aqueous extract was measured using the Folin-Ciocalteu assay. The
result produced the value of 0.29+0.01 mg (GAE)/100g of fresh sample. Phenolic and polyphenolic compounds
constitute the main class of natural antioxidants present in plants, foods, and beverages. More recently, several
researchers have shown a correlation between total phenolic content and the antioxidant activity of plant
materials (Turkoglu, 2007).
The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals
and oxygen species are present in biological systems from a wide variety of sources. These free radicals may
oxidize nucleic acids, proteins, lipids or DNA and can initiate degenerative disease. Antioxidant compounds
like phenolic acids and polyphenols will scavenge free radicals such as peroxide, hydroperoxide or lipid
peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (Halliwell & Gutteridge,
1999).
Conclusion
Through this study, we observed that T. crispa contains important nutrients, minerals and flavonoids that
are useful for human health. The results proved that T. crispa aqueous extract possess antioxidant activity
comparable with the established antioxidants, like BHT and vitamin C. The antioxidant activity of T. crispa
extract might be attributed to its effective hydrogen-donating ability and effective as scavenger of hydrogen
peroxide and free radicals. The results suggested that T. crispa is a valuable source of natural antioxidant and
can be potentially developed as herbal supplementary in combating free-radical mediated diseases.
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