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ANTIOXIDANT POTENTIAL OF THREE MEDICINAL PLANTS:
SIDA RETUSA Linn., URENA LOBATA Linn. AND
TRIUMFETTA RHOMBOIDEA Jacq.
6.1. Introduction
Reactive oxygen species (ROS) such as superoxide (O;), hydroxyl
radicals(0H ) and hydrogen peroxide (H2O2-) are considered to be important
factors in the etiology of several pathological conditions such as cardiovascular
diseases, diabetes. inflammation, cancer (Hemnani & Parihar, 1998; Halliwell &
Gutteridge, 1985). aging and neurodegenerative diseases (Beal, 1995; Thomas &
Kalyanaraman, 1997). ROS are degraded to non- reactive forms by enzymatic and
non- enzymatic antioxidant defense mechanisms.
Free radicals react with almost every known biological molecule in their
vicinity and damage protein, cause break down of DNA strands and initiate
peroxidation of various molecules. The hydroxyl radical is most reactive of all
and may be considered the ultimate damaging species whenever superoxide is
formed. ROS are implicated in carcinogenesis induced mutation and tumor
promotion. Antioxidants act as a masor defense against radical - mediated toxicity
by protecting the damages caused by free radicals. Inhibition of free radical
generation can serve as a facile system for identifying cancer preventive agents
(Troll et al.. 1994)
The traditional Indian medicine and the use of plant drugs against various
diseases receive considerable attention nowadays. Antioxidative components of
natural origin have attracted special interest because they can protect the human
body from free radicals (Osawa et a / , 1990) which are likely to be involved in the
pathophysiology of many diseases such as cancer (Babu et al., 2001). Many
antioxidative components of plant origin have protective activity against cancer
(Anto et a l . 1995). Free radical intermediates such as superoxide anion and
hydroxyl radicals are produced in living systems by various sources such as
ionization of water, X- rays and inflammatory phagocytes. These activated
oxygen species induce DNA strand breaks and chromosomal aberrations in
mammalian cells (C'erutii, 199 1).
It has been reported that the 'rasayanas' are rejuvenators and nutritional
supplements and possess strong antioxidant activity (Sharma et al., 1992). It has
been re-emphasized by the report of Scartezzini & Speroni (2000). A vast number
of literatures documenting the in vitro antioxidant property of polyphenol are
available. Polyphenol acts both as primary as well as secondary antioxidant, by
sequestration of metallic ion and by scavenging active oxygen species
(Morel et al., 1994). Flavones and isoflavones are hormones like phenolic
phytoestrogens of' dietary origin which influence intracellular enzymes, protein
synthesis, growth factor action. malignant cell proliferation, angiogenesis and
possessing major role as cancer protective compounds through several
mechanisms such as antioxidant effect (Formica & Regelson, 1995; Keli et aL,
1996; Meishiang et a l . 1997).
In order to contribute further to the knowledge of Indian traditional plants,
in the present study three medicinal plants were screened to determine their
antioxidant activity in vitro. Sida retusa Lhn. belonging to the family Malvaceae
is known a Kurumthotti in Malayalam and Bala in Sanskrit. The useful parts are
root system, aerial parts and seed. Of these the root system mainly used in
Ayurvedic formulations like Kshirabala prescribed for the treatment of
rheumatism. Acording to Shastri (1968) S. retusa is one of the ingredient of
famous Ayurvedic formulations such as Aravinda Asava, Bala Arishta. Kumary
Asava, Amrita Pvasha Ghrita, Agastya Rasayana and Chavana Prasha Lehya.
The plant is used in the treatment of tuberculosis, rheumatism and in combination
with other drugs and as an antidote to snake venom. Leaves and roots are useful in
urinary complaints. fever. heart disease. burning sensation, piles and all kind of
inflammation. Root is also used in treatment of leucorrhoea and rheumatism
( Nadkami, 1976; Krthikar & Basu, 1981).
Urena lohara Linn. is considered as a substitute of S. retusa, belonging to
the Malvaceae fsmily is known as Uram in Malayalam and Vanabenda in
Sanskrit. The root system of this plant is used as an external application for
lumbago and rheumatism (Nadkami. 1976). Triumfetta rhomboidea Jacq. is
considered as an adulterant of S. retusa is coming under the family Teliaceae.
Fruit, flowers and leaves are used in medicine. Bark and fresh leaves are used for
diarrhoea. Flowers rubbed with sugar and water are given in gonorrhoea to stop
the burning caused by urine (Nadkami. 1976).
Lipid peroxidation was measured by the method of Ohkawa et al. (1979)
by measuring the colour of thiobarbituric acid reactive substance (TBARS)
formed at the end of the reaction. Hydroxyl radical scavenging activity was
measured by studying the competition between deoxyribose and the test
compound by the method adopted by Elizabeth & Rao (1990). Superoxide radical
scavenging activity was determined by the NBT reduction method of Mc Cord &
Fridovich (1 969).
6.2.1. Review of literature
Sida species are indiscriminately used for the treatment as antituberculosis,
stomachics, nervous, urinary and cardiac diseases (Chopra et a/., 1958 &
Attygalle, 197 1 ) S. acuta Burm.f. and S. rhomboidea Roxb. are cultivated for the
production of fiber as a substihlte for jute. Extract of S. retusa showed sedative
effect as it decreased alertness, wakefulness and reactivity in mice (Thanam &
Kumari, 197 1 ) It has antirheumatic and antipyretic properties (Iyer & Kolammal,
1993). The various kinds of biological activities viz. antimicrobial and depression
of blood pressure in cats and dogs (Chopra, 1930), immunostimulant (Ghosal
et a[., 1988) antitumour. anti HIV, hepatoprotective and may serve for
hypothyroidism (Kotoky & Das, 2000), abortifacient, antipyretic, adaptogenic
hypoglycaemic (Aiyer & Koldmmal. 1962; Alam et al. 1991; Franzolti et al.,
2000) and antimalarial (Karou et a / , 2003) have generated interest in the
chemistry of this genus. which resulted in isolation and characterization of various
classes of chemical constituents from different species.
Ihe methanolic extract of roots of S. cordijolia Linn. checked growth of
dental plaque ~ i t h IC' 50 of 10-30 pg Iml. (Namba, et a/ . , 1985). The shoots
with black pepper are useful in excessive menstrual flow (Borthakur, 1992). A
familiar indigenous formulation, Shalparnydi churna, consisting of S. cordijolia.
as an ingredient has been maluated pharmacologically showed positive response
to irritable bowl syndrome (Sharma & Mishra, 1993). Oil from the plant is
beneficial for human skin (Pal, 1994).
Alcoholic extract of S. rhombifolia Linn. was found to be effective
antibacterial activity (tioyal & Rani, 1988 a; Alam et al., 1991; Muanza et al.,
1994). The leaf exhibited antitumour and anti H N activities as it was found
cytotoxic to 60 human cell lines (Muanza et. al. , 1995). Shylaraj (1998) observed
the quantification of ephedrine in in vitro culture of Sida species. It has anti-
inflammatory activity (Venkatesh el al.. 1999 & Franzolti et a/., 2000). Sankar
et at. (2000) studied the quantification of ephedrine in in-vitro cultures of Sida
species. A recent review by Khare et a/. (2002) on chemistry and pharmacology
of genus Sida revealed the medicinal properties of this 'wonderful herb'.
Mazumder et a/ (2001) studied the antibacterial activity of U. lobata.
Tariq (1985) studied the anti-inflammatory effect of Grewia populnifolia.
Cytotoxic activity of G trl~aefolia was observed by Badami et al. (2003).
Alam et a1 (1 99 1) obsewed the anti-inflammatory and antipyretic activities of
Triumfetta rotuntifolia. Pradhan et a1 (2003) studied the antiulcer activity of
roots of T. rhomboidea Jacq.
A vast number of literatures documenting the antioxidant property of
plants is listed below. (Jose & Kuttan . 1995; Anto et aL, 1995; Anto et al., 1996;
Anto eta/ . , 1998: Joy et al.. 1999; Shylesh & Padikkala, 1999; Martinez, et al.,
2000; Padma. et a/., 2000; Babu et al., 2001; Venkatesh, 2001; Ajaikumar, et al.,
2002; Aquino. et al.. 2002; Babu et al., 2002; Choudhary & Kale, 2002;
Ichikawa & Konishi, 2002; I'ieroni et al., 2002; Vayalil, 2002; Valentao, et al,,
2002; Auddy, et a/., 2003; Babu et a l , 2003; Bagul, et al., 2003; Gorinstein, et al.,
2003; Mary. et a/.. 2003; Soni. et a/., 2003; Velazquez et al., 2003;
Kamalakkannan. et al., 2003; Sabu & Kuttan, 2004.
Mushrooms also have antioxidant property. Some of the medicinally
important mushrooms which have antioxidant property are Pleurotus florida
(Jose et al., 2002) and Phellinus rimosus (Ajith & Janardhanan, 2002).
6.2.2. Chemical constituents of S. rhombifolia ssp. retusa
Root I aerial part consists of alkaloids, P-phenethylamine, N- methyl-P-
phenethylamine. ephedrine. Pseudo ephedrine, vasicinol, vasicinone, vasicine,
choline betainc. S-(+)-Nb5-methyl tryptophan methyl ester, hypaphorine methyl
ester and hypaphorine (Leslie et al., 1980, Prakash et al., 1981). Seed oil I leaves
contains fatty acids, sterculic acid , malval acid, linoleic acid, myristic acid,
palmitic acid, stearic acid, oleic acid and an unidentified fatty acid (Morghis et al.,
1976 & Bhatt et ul , 1983)
Leaves contain Aminoacids: Lysine, histidine, arginine, asparagines,
glutamine, alanine, valine. phenylalanine. leucine, aspartic acid, glutamic acid
glycine, serine. threonine, tyrosine and proline (Bhatt et al., 1983;
Lakshminarayanan et a1 1987). Aerial part consists of n-alkane (CIS CT5) long
chain alcohols, C',, C,? (Goyal & Rani, 1989). Whole plant I aerial part consists of
sterols: 0-sitosterol. cholesterol. 24-methylene cholesterol, campesterol,
22-dehydro campesterol and stigmasterol (Kumari, 1984), 8- stigmasterol,
spinasterol. 22-dihydrospinasterol (Goyal & Rani, 1988 b, Goyal & Rani, 1989).
Sankar et al. (2003) studied four species of Sida viz. S. rhombifolia ssp. retusa,
S.acuta, S. rhombifolia ssp. rhombifolia and S. cordifolia for their in vitro
secondary metabolite productivity at callus and cell suspension stages.
6.3. Materials and methods
6.3.1. Plant materials
The root system of' the S. retusa, U. lobata and T. rhomboidea were
collected from Kolenchery during the month of June and July. The roots were
dried under shade and powdered. The powder was extracted with methanol, 20%
methanol and pure water using Soxhlet apparatus. These extracts were used for
testing antioxidant property. Methanolic fractions, 20 % methanolic fractions and
pure water fraction of S. retusu. U. lobata and T. rhomboidea were used for initial
screening. The methanolic fractions were found to be effective and were used for
further study.
6.3.2. Animals
Male Wistar rats ( 1 50-200g) supplied by the small animal breeding station
of Kerala Agricultural University, Mannuthy, India were used. The animals were
maintained under standard environmental conditions. The liver of the rat was used
for preparing liver homogenate.
6.3.3. Preparation of liver homogenate
25% (ulv) liver homogenate in 30 mM KC1 was prepared. The tissue was
well ground in a cold mortar and pestle or homogenizer. Homogenate was kept for -4
15 minutes under cold condition and collected the clear homogenate from the top
for studies
6.3.4. Superoxide radical scavenging activity
Superoxide radical scavenging activity was determined by the Nitroblue
tetrazolium (NBT) reduction method of Mc Cord and Fridovich (1969). The
reaction mixture contained EDTA (0.1 M) containing 0.0015 % NaCN, riboflavin
(0.12 mM). NBT (1.5mM) and various concentrations of the extract (200-1000
pg), and phosphate buffer (pH 7.8) in a final volume of 3 ml. The tubes were
uniformly illuminated under an incandescent lamp for 15 minutes and the optical
density was measured at 530 nm before and after illumination. The percentage of
inhibition of' superoxide generation was evaluated by comparing the absorbance
values of the control and experimental tubes. A known antioxidant, curcumin
(1 - 100 p g) was used as reference.
6.3.5. Inhibition of lipid peroxide formation.
6.3.5.1. Induction by ~ e " / ascorbate system
The peroxide formation was measured by the method of Ohkawa et al.
(1 979) by measuring the colour of thiobarbituric acid reactive substance (TBARS)
formed at the end of the reaction. Malondialdehyde (MDA), which is formed, as
the end product in lipid peroxidation will react with thiobarbituric acid (TBA) to
give TBARS, which is pink ill colour, measured at 530 nm. The reaction mixture
contained rat liver homogenate (0.1 ml, 25% (wlv) in Tris-HC1 buffer
(20 mM, pH 7.0), KC1 150mM, ferrous ammonium sulphate (0.8 mM), ascorbic
acid (0.3 mM) and various concentrations of the drug (400-2000 pg) in a final
volume of 0.5 ml, was incubated for I hour at 3 7 ' ~ (Bishayee &
Balasubramanian. 1971 ).
The incubated reaction mixture (0.4 ml) was treated with 0.2 ml of 8 %
sodium dodecyl sulphate (SDS), thiobarbituric acid (1.5 ml, 8 %) and acetic acid
(1.5 ml, 20%, pH 3.5). The total volume was then made up to 4 ml by adding
distilled water and kept in a water bath at 1 0 0 " ~ for 1 hour. After cooling, 1 ml of
distilled water and 5 ml of a mixture of n-butanol-pyridine (15:l vlv) were added
and shaken vigorously and centrifuged at 4000 rpm for 10 minutes. The
absorbance of the organic layer was measured at 560 nm after centrifugation. The
percentage inhibition of lipid peroxide formation was determined by comparing
the results of the drug- treated and untreated samples. Curcumin (1 - 100 vg) was
used as reference
6.3.6. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was measured by studying the
competition between deoxyribose and the extract for hydroxyl radicals generated
from the ~ e ~ ' i a s c o r b a t e l ~ , ~ l ' A M ~ O ~ system. The hydroxyl radicals attack
deoxyribose, which eventually results in TBARS formation (Elizabeth & Rao,
1990). The reaction mixture contained deoxyribose (2.8 mM), FeCI3 (0.1 mM),
EDTA (0.1 mM). H,O>(lmM), ascorbate (0.1 mM), KH2P04-KOH buffer
(20mM, pH 7.4) and various concentrations of the drug (400-2000 pg) in a final
volume of I ml The reaction mixture was incubated for 1 hour at 3 7 ' ~ .
Deoxyribose degradation was measured as TBARS by the method of Ohkawa
et a/. (1979) and percentage inhibition was calculated. Curcumin (1-100 pg) was
used as reference.
6.3.7. Statistical Analysis
Method of Finney was adopted ( 1 971) to find 50% inhibition concentration
(IC 50) of plant extracts.
6.4. Observations
The methanolic fractions were found to be effective in S. retusa, U. lobata
and T. rhomboidea. So the methanolic fractions were selected for further study.
6.4.1. Superoxide radical scavenging activity
The methanolic extracts of the root of S. retusa, U, lobata and
7: rhomboidea were found to scavenge the superoxide radical generated by
photoreduction of riboflavin. The conccntration needed for 50% inhibition of
scavenging of superoxide was found to be 71.29 pg/ml, 470.60 pg/ml, 336.65
pglml and 6.3 pglml for S. retusa. 11. lobatn, T. rhomboidea and Curcumin
respectively (Table 27)
The methanolic extract of S. retusa root showed 40 % inhibition when
43.24 pdml of the extract was used and 60 % inhibition when 112.61 pg/ml of
the extract was used. The results are shown in Table 28.
Methanolic extract of U. lobata showed 40% inhibition when 405.15
pg/ml of the root extract was used. However, 539.69 pg/ml of the root extract of
U. lobata showed 60% inhibition, 234.76 pg/ml extract of T rhomboidea
inhibited 40 % reaction while 468.10 pglml extract of 7: rhomboidea extract
inhibited 60 %reaction (Table 29,30).
6.4.2. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity was calculated by hydroxyl radicals
generated by the ~ e " l a s c o r b a t e l ~ ~ T ~ 1 ~ ~ system was also found inhibited by
S. retusa, ti. lobata and 1: rhomboidea methanolic root extract. The
concentration of root extract needed for 50% inhibition was 1763.22 pg/ml,
1627.35 pgiml and 1346.03 pdml in S retusa, U, lobata and T. rhomboidea
respectively and that of Curcumin was 2.7 pg/ml (Table 27).
40% inhibition was obtained by 1178.09 pg Iml, 1074.71 pglml and
920.04 pg/ml root extract of S. retusa, CI. lobata and T rhomboidea respectively
(Table 3 1,32, 33)
6.4.3. Inhibition of lipid peroxidation
The generation of lipid peroxides by ~e~~ lasco rba t e in rat liver homogenate
was found inhibited by the addition of the root extract of S. retusa, 0. lobata and
T. rhomborderr fhe concentration of extract needed for 50% inhibition was
1130.24 pglml. 1 109.24 pglml and 1004.22 pglml in S. retusa, U. lobata and
T. rhomboidea respectively and that of Curcumin was 8.9 pg (Table 27).
40% inhibition was obtained by 713.55 pg/ml, 502.34 pg/ml and 565.19
pg/ml root extract of S. retusa, U. lobata and T rhomboidea respectively. 60%
inhibition was obtained by 172 1.16 &ml, 1792.59 pg/ml and 1698.61 pg/ml root
extract of S. retusa. 11. lobata and T rhomboidea respectively (Table 34,35,36).
6.5. Discussion
Oxygen derived free radicals such as the superoxide anion and hydroxyl
radicals are cytotoxic and promote tissue injury (Salim, 1987). Free radicals are
produced endogenously as a by-product of aerobic metabolism. They have
considerable chemical reactivity with biological molecules. Oxygen in high
concentrations can damage the brain, lungs and other organs. Inadequate dietary
intakes of antioxidant micronutrients, such as flavinoids and phenolic compounds,
which prevent free radical-mediated damage in vivo, contribute to disorders such
as atherosclerosis, cancer. ulcer and inflammatory diseases in living organisms
(Halliwell & Gutteridge. 1989). 95% of the oxygen taken in by the aerobic
organisms is fully reduced to I-1202 during the process of mitochondria1
respiration, a small percentage ( 4 % ) of the oxygen consumed is converted to
semi reduced species i.e. superoxide anion radical (OZ-), hydrogen peroxide
(H202-) and hydroxyl radical (OH-) (Kehrer & Smith, 1994).
Some disorders in which free radicals are implicated are Alzheimer's,
arthritis, Hemorrhoids. Parkinson's, Rheumatism, Heart attack, AIDS, Cataract,
Stroke, Cancer. Stress, Varicose veins, Heart diseases, Immune system disorders
and a long list of degenerative diseases, including aging (Hemnani & Parihar,
1998; Tiwari, 200 1; Pillai & Pillai, 2002). Over production of superoxide radical
takes place in various chronic inflammatory cases, induced by drug, toxin, stress,
tissue injury and heavy exercise. Hydroxyl radical is involved in inflammatory
processes. The inflammation is mainly caused by the generation of free radicals.
Hence the administration of .antioxidants may have a protective role in these
conditions. The tindings of present study give emphasis to this factor 71.29
pg/ml extract of S retusa root provide 50% inhibition of superoxide radical
(Table 28). U. lohota the substitute of S. retusa provide 50% inhibition of
superoxide radical scavenging at a concentration of 470.60pg/ml (Table 29).
While its adulterant, T rhomhoidea has IC 50 (50% inhibition concentration) of
336.65pgml (Table 30) in the case of superoxide scavenging activity (Text
fig. 34). Even though T. rlzomboidea is an adulterant of S. retusa, it has its own
medicinal property
The antioxidants are also known as free radical scavengers. For 50%
inhibition of reaction (hydroxyl scavenging activity) 1763.22 pglml. root extract
of Sida retusa is needed ('Table 31). However, 1627.35 pg/ml. root extract of
Urena labata Linn. is required for IC 50 (Table 32). 1346.03 pg/ml. root extract
of Triumfetta rhomboidea Jacq, is required for 50% scavenging of hydroxyl
radical(Tab1e 33).
Free radicals react with the cell membrane lipid and cause peroxidation of
polyunsaturated fatty acids and cause generation of further free radicals. In lipid
peroxidation, the 1C 50 is achieved by 1130.24 pglml root extract of S. retusa
(Table 34), 1109.24 pgrnl. root extract of U. lobata (Table 35) and 1004.22
pg/ml root extract of T. rhomboidea (Table 36).
The antioxidant property of the three taxa studied revealed that the
original, substitute and adulterant have medicinal properties and it varies from
taxa to taxa. 'The substitute U. lobata has its own medicinal properties.
Phytosterols, alkaloids and fatty acids are present in S. retusa Linn. (Khare et al.,
2002). This may be responsible for its antioxidant property. Such chemical
constituents present in adulterant may also have medicinal properties. The
chemical constituent responsible for antioxidant property is present in all the three
plants in varying concentrations. This study validates the traditional use of
S. retusa for the treatment of rheumatism.
Table 27. Effect of the methanolic extract of plant materials on oxygen derived free radical generation
Test material I C 50 (pg/ml) Superoxide hydroxyl Lipid peroxidation radical radical
Sida r e t ~ s u 71.29113.61 1763.221211.43 1130.24i112.08
Urenn loburc~ 470.60-1: 17.17 1627.35+182.46 1109.241141.39
Triumfettu 330.65143.1 8 1346.03*116.76 1004.22*125.48 rhomboideu
Curcumin* 6.310.06 2.7.tO.07 8.910.05 - Values are mean * SE (n=4)
* Reference - Mary et a/. , 2003
Table 28. Data of superoxide radical scavenging of Sida retusa
~
~ ~ Table of Percentiles 7-.--.-- ~
Standard 95.0% Fiducial C1
Error Lower Upper 0.06937 0.095 16 0.001 184 0.5 168
4~ ~
0.006691 1.2148
Table 29. Data of superoxide radical scavenging of Urena lobata
1 Table of Percentiles 1 1 Standard 95.00% Fiducial C1 I
Percent Error Lower Upper
12.726 34.8123 83.9817 15.0955 53.3178 1 1 1.9056
3 -,,;>,,-. 16.4406 68.4921 132.5136
- 17.3234 81.875 149.5147
Table 30. Data of superoxide radical scavenging of Triumfetta rhomboidea
r-- - -
- -- .- Table of Percentiles
Standard 95.00% Fiducial C1 I
Table 32. Data of hydroxyl radical scavenging of Urena lobata
1 Table of Percentiles I 1 Standard 95.00% Fiducial C1 I
Error Lower Upper
5.4894 / 0.1781 1 23.4404
Table 33. Data of hydroxyl radical scavenging of Triumfetta rhomboidea
~ --
-- --- Table of Percentiles
Standard 95.00% Fiducial CI -- -~~
Percent I Percentile Lower 7--
Upper 1 I 0.502 25.4133 -.f - - .
2 1 - - -.L!!L402 1.8541 48.866 27.3647 3.9963 7 1.8002
4 1 23.3869 6.9094 94.4975 __i -
52.4039 28.5706 10.587 117.0902
33.4888 15.0294 139.6569
___C _ 38.1541 20.241 1 162.2508
8 1 90.0337 42.577 26.2295 184.9115 ___+ - -
9 i 111.9719 46.7661 33.004 207.67
Table 34. Data of lipid peroxidation inhibition of Sida retusa
- - - --- - - Table of Percentiles
Standard 95.00% Fiducial C1
Percent Percentile I - - ' 1. --- L-JRO~ Lower upper
Table 35. Data of lipid peroxidation inhibition of Urena lobata
-
Table of Percentiles
Standard 95.00% Fiduci
-.
Table 36. Data of lipid peroxidation inhibition of Triumfetta rhon~boide~
~~. ~~ - - ~- ~ -- -
--
1 2 3 Name of tam
1. Supemxi& radical 2. Hydroxyl radical 3. Lipid peroxidation
Tex Figure 34. Bar chart showing IC 50 (wml) of S.retwa U lobato a d I: rhomboidea
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