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Antioxidant and Antimicrobial Activity of Cissus quadrangularis L.

Article in Journal of Medicinal Food · February 2003

DOI: 10.1089/109662003322233495 · Source: PubMed

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JOURNAL OF MEDICINAL FOOD
Volume 6, Number 2, 2003
© Mary Ann Liebert, Inc.

Antioxidant and Antimicrobial Activity of

Cissus quadrangularis L.

K.N. CHIDAMBARA MURTHY, M.Pharm, A. VANITHA, M.Sc.,

M. MAHADEVA SWAMY, Ph.D., and G.A. RAVISHANKAR, Ph.D.

ABSTRACT

Extracts of Cissus quadrangularis L. were tested for antioxidant activity by b-carotene linoleic
acid model and also by 1,1-diphenyl-2-picrylhydrazyl model. The ethyl acetate fraction of
both fresh and dry stem extracts at a concentration of 100 ppm showed 64.8% antioxidant ac-
tivity in the b-carotene linoleic acid system and 61.6% in the 1,1-diphenyl-2-picrylhydrazyl
system. This fraction showed the presence of sterols, vitamin C, and tannins as phytocon-
stituents. The antioxidant activity of methanol extract and aqueous extract were comparatively
less significant than that of ethyl acetate extract, and n-hexane extract showed the least ac-
tivity. The ethyl acetate extract and methanol extract of both fresh and dry stems further ex-
hibited antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis,
Bacillus cereus, Staphylococcus aureus, and Streptococcus species. The results of the study
have implications in the use of C. quadrangularis as an antibacterial agent and more so as an
antioxidant in several applications requiring these properties.

INTRODUCTION tamin C, carotene, anabolic steroid substances,

and columin.3 This plant has been used as an

C ISSUS QUADRANGULARIS L. belongs to family

Vitaceae and is an indigenous medicinal
plant of India1; it is known as asthisanghara in
anthelmintic, antidyspeptic, digestive tonic,
analgesic, and treatment for scurvy and
asthma.1,4 The alcoholic extract of the plant has
Sanskrit, meaning “which will strengthen the been shown to facilitate healing of fractured
bones.” C. quadrangularis is a tendril-climbing bone in rats.5 Singh et al.6 reported analgesic
shrub with stout, fleshy quadrangular stems activity of C. quadrangularis against Heffner’s
that is found throughout India and Ceylon (Fig. tail clip and Edely’s hot plate–induced analge-
1). The leaf portion constitutes only 5–8% of the sia. Two unsymmetrical tetracycline triter-
aerial plant parts; the fleshy, green stem is the penoids have been isolated from this plant:
major portion. The tendril shoots and young onocer-7-ene-3a,21-b-diol and onocer-7-ene-
leaves are used in various food preparations. 3b,21a-diol.7–9
The juice of the plant is said to be beneficial in This paper is the first to report antioxidant
scurvy.2 The plant contains high amounts of vi- and antimicrobial activity of C. quadrangularis.

Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570 013, India.

99
100 MURTHY ET AL.

40°C for 2 days (dry stems). The dried mater-

ial was powdered to pass a size 60 mesh, and
the powder was subjected to extraction for 8–10
hours, similar to that for fresh stems, using the
Soxhlet extractor. The individual extracts were
then pooled separately and concentrated under
vacuum at 40°C to get the dry mass of ex-
tractables. The solvents used for extraction were
n-hexane, ethyl acetate, methanol, and water.

Phytochemical test
The dry mass of extractables from fresh stem
extracts and from dry extracts were made up
as 1% solutions in the respective solvents and
subjected to phytochemical screening10 for
sterols, tannins, alkaloids, carbohydrates, and
ascorbic acid.
FIG. 1. Cissus quadrangularis stem.
Experimental procedure
MATERIALS AND METHODS Extracts of fresh and dry C. quadrangularis
stems were subjected to antioxidant assays us-
Materials ing a 1,1-diphenyl-2-picrylhydrazyl (DPPH)
model system and a b-carotene linoleic acid
All the solvents and chemicals used in the ex-
model system (B-CLAMS).
periment were of analytical or high-performance
liquid chromatography grade and were obtained
DPPH radical scanning assay. Different concen-
from Merck, Mumbai, India. Ultraviolet and vis-
trations (50, 100, 500, and 1,000 mg/ml [ppm])
ible spectra measurements were carried out with
of C. quadrangularis extractables and butylated
the use of a Shimadzu UV160A spectropho-
hydroxyanisole (BHA) were taken in test tubes,
tometer (Shimadzu Corp., Kyoto, Japan).
and the volume was adjusted to 500 ml by the
For antimicrobial assays, six bacterial cultures
addition of methanol. Then 5 ml of a 0.1 mM
were chosen: Bacillus subtilis, Bacillus cereus,
methanolic solution of DPPH was added, and
Staphylococcus aureus, Pseudomonas aeruginosa,
the tubes were shaken vigorously. They were
Escherichia coli, and Streptococcus species. These
then allowed to stand at 27 6 1°C for 20 min-
pure strains were obtained from the Depart-
utes.11 The control was prepared without any
ment of Food Microbiology of Central Food
extract, and methanol was used for baseline
Technological Research Institute, Mysore, In-
corrections in absorbance (OD) of samples
dia. In vitro antibacterial activity was deter-
measured at 517 nm. Radical scavenging activ-
mined by using nutrient broth agar.

Extraction TABLE 1. PERCENTAGE YIELD OF EXTRACTABLES IN

VARIOUS SOLVENTS
C. quadrangularis plants were collected from
the Mysore region and compared with herbar- % w/w Yield
ium specimens for authentication. The stems
were cleaned and divided into two batches. Extraction medium Fresh stem tissue Dry stem tissue
One batch (fresh stems) was directly subjected n-Hexane 0.66 3.6
to successive extractions using a Soxhlet ex- Ethyl acetate 1.69 4.5
tractor (Tensil Glass Works, Bangalore, India). Methanol 1.53 5.2
Water 2.24 5.9
The other batch was dried under vacuum at
ANTIOXIDANT ACTIVITY OF CISSUS QUADRANGULARIS 101

FIG. 2. Antioxidant activity of dry stem extractables of C. quadrangularis by DPPH (concentrations in ppm). *, sig-
nificant compared with BHA.

ity was expressed as % scavenging activity and oxyethylene sorbitan monopalmitate) were
was calculated by the following formula: mixed in 0.5 ml of chloroform. Chloroform was
removed at 40°C under vacuum using a rotary
% Radical scavenging activity 5 (Control evaporator. The resulting mixture was imme-
OD 2 Sample OD/ Control OD) 3 100 diately diluted with 10 ml of triple-distilled wa-
ter and mixed well for 1–2 minutes. The emul-
B-CLAMS antioxidant assay. The antioxidant ac- sion was further made up to 50 ml with
tivity of C. quadrangularis extractables was eval- oxygenated water. Aliquots (4 ml) of this emul-
uated by the B-CLAMS12 with a slight modifi- sion were transferred into test tubes containing
cation. First, 0.2 mg of b-carotene, 20 mg of 0.2 ml of C. quadrangularis test samples in
linoleic acid, and 200 mg of Tween-40 (poly- ethanol; BHA was used for comparative pur-

FIG. 3. Antioxidant activity of fresh stem extractables of C. quadrangularis DPPH (concentrations of extractables in
ppm). *, significant compared with BHA.
102 MURTHY ET AL.

FIG. 4. Antioxidant activity of dry stem extractables of C. quadrangularis B-CLAMS model (concentration of ex-
tractables in ppm). *, significant compared with BHA.

poses. A control containing 0.2 ml ethanol the color of b-carotene disappeared in the con-
without test sample and 4 ml of the emulsion trol reaction (t 5 180 minutes). A mixture pre-
was prepared. The tubes were placed in a wa- pared as described but without b-carotene
ter bath maintained at 50°C. Absorbance of served as a blank. All determinations were car-
each sample at 470 nm was measured at zero ried out in triplicate. Dose–response relation-
time (t 5 0). Measurement of absorbance was ships of antioxidant activity for C. quadrangu-
then continued, at 15–minute intervals, until laris extracts were determined at different

FIG. 5. Antioxidant activity of fresh stem extractables of C. quadrangularis B-CLAMS model (concentrations of ex-
tractables in ppm). *, significant compared with BHA.
ANTIOXIDANT ACTIVITY OF CISSUS QUADRANGULARIS 103

TABLE 2. PHYTOCHEMICALS PRESENT IN VARIOUS EXTRACTS

Fresh stem extracts Dry stem extracts

Phytochemical n-Hexane EtOAc MeOH H2 O n-Hexane EtOAc MeOH H2 O

Sterols 1 1 2 2 1 1 2 2
Polyphenols 2 1 1 2 2 1 1 1
Ascorbic acid 2 1 1 1 2 1 1 1
Carbohydrates 2 2 2 1 2 2 2 2
Alkaloids 2 2 2 2 2 2 2 2

EtOAc, ethyl acetate; MeOH, methanol; 1, present; 2, absent.

concentrations. The antioxidant activity (AA) the well to the inner margin of the surround-
of the extracts was evaluated in terms of bleach- ing bacterial growth.15 Tetracycline (50 mg) was
ing of the b-carotene, using the following for- used as standard for comparative purpose.
mula13:

AA 5 100[1 2 (A0 2 At)/(A00 2 A0t )] RESULTS AND DISCUSSION

where A0 and A00 are the absorbance values The yield of extractables obtained from C.
measured at zero time of the incubation for test quadrangularis using various solvents was ana-
sample and control, respectively, and At and lyzed (Table 1). Among the solvents used, wa-
A0t are the absorbance values measured in the ter yielded more extractables of both dry and
test sample and control, respectively, after in- fresh stems, followed by methanol and ethyl
cubation for 180 minutes. acetate. The yield of extractables from n-hexane
was minimal and constituted only lipid por-
Antimicrobial activity. Antimicrobial activity of tions. The free radical scavenging potential of
different extractables of C. quadrangularis was C. quadrangularis extractables at various con-
done by the agar well diffusion method. 14 Agar centrations was tested by the DPPH model
spread plates were prepared with 100 ml of 24- (Figs. 2 and 3). Antioxidants react with DPPH,
hour incubated suspension cultures of bacteria which is a stable free radical and converts to
inocula. A known concentration (1.0 mg/ml) of 1,1-diphenyl,2-picrylhydrazine. The degree of
ethyl acetate, methanol, and aqueous extracts discoloration indicated the scavenging prop-
was dissolved in the respective solvent, 50 ml erty of the extract. At 100 ppm, n-hexane, ethyl
of which was loaded into the wells. The same acetate, methanol, and water extracts of fresh
amount of solvent alone served as a control. Af- stem showed 14.3%, 66.2%, 19.3%, and 15.9%
ter 24–72 hours of incubation at 37 6 1°C, the antioxidant activity, respectively; the respec-
inhibition zone was measured from the edge of tive values for dry stem extracts were 12.3%,

TABLE 3. A NTIMICROBIAL A CTIVITY OF VARIOUS EXTRACTS OF CISSUS QUADRANGULARIS

L. STEM (DIAMETER OF INHIBITIO N ZONE IN MILLIMETER S)

Fresh stem Dry stem

Organism Standard EtOAc MeOH Water EtOAc MeOH Water

Bacillus subtilis 26 22 19 — 14 13 —
Bacillus cereus 36 15 13 09 16 15 11
Staphylococcus aureus 24 16 15 — 12 14 —
Pseudomonas aeruginosa 08 07 06 — 10 11 —
Streptococcus species 30 16 — — 20 — —
Escherichia coli 20 — — — — — —

EtOAc, ethyl acetate; MeOH, methanol; —, no activity.

104 MURTHY ET AL.

62.5%, 18.7%, and 39.6%. The antioxidant ac- studied (Table 3). Among the three extracts,
tivity of ethyl acetate extract was significantly the order of activity was ethyl acetate .
comparable to BHA (Figs. 2 and 3). The activ- methanol . aqueous extract. Aqueous extract
ity of extracts is attributed to their hydrogen showed little inhibitory activity. The Gram-
donating ability.16 It is known that free radicals positive bacteria (B. subtilis, B. cereus, Staphy-
cause auto-oxidation of unsaturated lipids in lococcus aureus, and Streptococcus species)
foods.17 Antioxidants are believed to intercept were highly susceptible, whereas Gram-neg-
the free radical chain of oxidation and to do- ative bacteria (P. aeruginosa and E. coli) ex-
nate hydrogen from the phenolic hydroxyl hibited high resistance to the plant extract.
groups, thereby forming a stable end product The results of this study have implications
that does not initiate or propagate further oxi- for the use of C. quadrangularis as an antibacte-
dation of lipids.11 rial agent and more so as an antioxidant in sev-
The antioxidant activity of C. quadrangularis eral applications requiring these properties.
extracts was also measured at various concen-
trations by bleaching of b-carotene and com-
parison with BHA (Figs. 3 and 4). C. quadran- ACKNOWLEDGMENT
gularis extracts in different solvents exhibited
varying degrees of antioxidant activity. The K.N.C. Murthy wishes to thank CSIR, Gov-
mechanism of bleaching of b-carotene is a free ernment of India, for the financial assistance.
radical–mediated phenomenon resulting from
the hydroperoxides formed from linoleic acid.
b-Carotene in this model undergoes rapid dis- REFERENCES
coloration in the absence of antioxidants. The
linoleic acid free radical, formed on abstraction 1. Nadakarni AK: Cissus quadrangularis. In: Indian Mate-
of a hydrogen atom from its diallylic methyl- ria Medica, 3rd Ed. Popular Book Depot, Bombay, In-
dia, 1954, pp. 1284–1286.
ene group, attacks the highly unsaturated b- 2. The Wealth of India: Vol. 2. Raw Materials, first supple-
carotene molecules. As b-carotene loses its ment. National Institute of Science and Communica-
double bonds by oxidation, the compound tion, CSIR, New Delhi, 1950, pp. 184–185.
loses its chromophore and characteristic orange 3. Deka DK, Lahon LC, Saikai J, Mukhit A: Effect of Cis-
color, which can be monitored spectrophoto- sus quadrangularis in accelerating healing process of
experimentally fractured radius and ulna of dog: A
metrically. The presence of different extracts
preliminary study. Indian J Pharmacol 1994;26:44–45.
can hinder the extent of b-carotene bleaching 4. Chopra NN, Chopra IC, Handa KL, Kapur LD. Ciss-
by neutralizing the linoleate free radical and sus quadranglularis. In: Indigenous Drugs of India. U. D.
other free radicals formed in the system. 18 The Dhar, Calcutta, 1958, pp. 669–670.
activity of ethyl acetate extracts of both dry and 5. Udupa KN, Prasad G: Biochemical calcium 45 stud-
fresh stems was significant when compared ies on effect of Cissus quadrangularis in fracture repair.
Indian J Med Res 1964;52:480–487.
with that of BHA (Figs. 4 and 5). The fresh stem 6. Singh SP, Mishra N, Dixit KS, Singh N, Kohli RP: An
extracts showed 49.1%, 53.6%, 67.1%, and experimental study of analgesic activity of Cissus
82.3% scavenging activity, respectively, at con- quadrangularis. Indian J Pharmacol 1984;7984:162–163.
centrations of 50, 100, 500, and 1,000 ppm, 7. Bhutani KK, Kapoor R, Atal CK: Two unsymmetrical
whereas the values for the dry stem extracts tetra cyclic triterpenoids from Cissus quadrangularis.
Phytochemistry 1984;23:407–410.
were 55.1%, 63.1%, 70.9%, and 78.7%, respec-
8. Anonymous: The Wealth of India: Vol. 1. Raw Materi-
tively. Further, the activity of methanolic ex- als, first supplement. National Institute of Science and
tract was significant and that of water and n- Communication, CSIR, New Delhi, 2000, pp. 263–265.
hexane were nonsignificant in both fresh and 9. Gupta MM, Verma RK: Unsymmetric tetracyclic
dry extract, compared with the activity of ethyl triterpenoid from Cissus quadrangularis. Phytochem-
acetate. istry 1990;29:336–337.
10. Kokate CK, Purohit CK, Gokhale SB: Phytochemical
The phytochemical contents of the various tests. In: Pharmacognosy. Nirali Prakashan, Pune, In-
extracts are shown in Table 2. dia, 1996, pp. 510–512.
Throughout the study, solvent did not 11. Singh RP, Chidambara Murthy KN, Jayaprakash GK:
show any inhibitory effect on the bacteria Studies on antioxidant activity of pomegranate peel
ANTIOXIDANT ACTIVITY OF CISSUS QUADRANGULARIS 105

and seed extracts using in vitro models. J Agric Food (Aruoma OI, Halliwell B, eds.). Taylor and Francis,
Chem 2002;50:86–89. London, 1991, pp. 17–35.
12. Jayaprakash GK, Singh RP, Sakkaraiah KK: Antioxidant 18. Chen HM, Muramoto K, Yamauchi F, Nokihara K:
activity of grape seed (Vitis vinifera) extracts on perox- Antioxidant activity of designed peptides based on
idation models in vitro. Food Chem 2001;73:285–290. the antioxidative peptides isolated from digest of
13. Hidalgo ME, Fernandez E, Quilhot W, Lissi E: An- a soyabean protein. J Agric Food Chem 1996;44:
tioxidant activity of depsides and depsidones. Phyto- 2619–2623.
chemistry 1994;37:1585–1587.
14. Schillinger U, Lucke FK: Antibacterial activity of Lac-
tobacillus sake isolated from meat. Appl Environ Micro-
Address reprint requests to:
biol 1989;55:1901–1906.
15. Selvi M, Selviraj R, Chidambaram A: Screening for an- Dr. G.A. Ravishankar
tibacterial activity of macro algae. Seaweed Res Util Scientist and Head
2001;23:59–63. Plant Cell Biotechnology Department
16. Shimada K, Fujikawa K, Yahara K, Nakamura T: An- Central Food Technological Research Institute
tioxidative properties of xanthan on the autoxida- Cheluvamba Mansion, Mysore 570 013
tion of soybean oil in cyclodextrin emulsion. J Agric
Food Chem 1992;40:945–948.
India
17. Kaur H, Perkins J: Free radicals and food additives.
In: The Free Radical Chemistry of Food Additives E-mail: pcbt@cscftri.ren.nic.in
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