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International Journal of Pharmaceutical Research & Allied Sciences, 2019, 8(4):121-129

ISSN : 2277-3657
Research Article CODEN(USA) : IJPRPM

Anti-hemolytic Activity and Antioxidant Studies of Caralluma quadrangula:

Laboratory of Phytomedicine and Therapeutics, Prince Fahd Bin Sultan Research Chair, Department of
Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, Kingdom of Saudi
Arabia

*Email: sh.ghulam @ ut.edu.sa

ABSTRACT

Background: Since ancient times, plants and its derivatives have been used in traditional medicine to cure
human diseases. In the past few decades, the research on medicinal plants has gained significant attention due
to the medicinal potential of certain phytochemicals against cancer and metabolic disorders. The present study
has examined the alcoholic extract of Caralluma quadrangula (Ca qu) for its quantitative and qualitative
composition and its anti-oxidant as well as anti-hemolytic properties. The findings have potential implications
for plausible intervention in reactive oxygen species (ROS) mediated pathologies. Materials and Methods: An
80 % aqueous-methanol extract of areal parts of Ca qu was prepared. It was subjected to qualitative and
quantitative phytochemical analysis. Anti-oxidant potential was determined by inhibition of 2,2-Diphenyl-1-
pycrylhydrazyl (DPPH) and 2,2’ -Azino-bis-3- ethylbenzthiazoline-6-sulfonic acid (ABTS) radicals; while, anti-
hemolytic activity was determined by the ability of the extract to protect human RBCs from oxidative insult.
Results: The extract showed abundance of polyphenolic and flavonoid compounds at concentrations of 8.6 GAE
% w/w and 0.90 mg QE % w/w, respectively. Tannins, alkaloids and saponins were present at the
concentration of 8.50 mg TAE % w/w, 2.8 mg % w/w and 20.07 mg % w/w, respectively. Qualitative HPLC
column chromatography indicated the presence of rutin in the extract. In an increasing concentration range
from 31.25 to 2000 μg/ml the extract provided significant protection to RBCs from membrane damage induced
by ROS. In the DPPH and ABTS inhibition assays, the extract showed a dose-dependent inhibition of the
radicals in the concentration range of 50 -1000 μg/ml and 10-250 μg/ml, respectively. Conclusion: The hydro-
alcoholic extract of Ca qu contains several classes of important phytochemicals with known therapeutic
significance. The extract possesses significant anti-oxidant and anti-hemolytic potential as demonstrated in
standard assays. The findings can be exploited for advanced studies on pharmacological premises for
intervention in different diseases that are associated with an imbalanced production of ROS/free radicals in
cells including certain anemic disorders and cancers. The formulations derived from the plant are expected to
possess therapeutic advantage as nutraceuticals or as adjuvants with standard treatment regimen.

Key words: Caralluma quadrangula, anti-oxidant, anti-hemolytic, nutraceutical.

INTRODUCTION

Throughout human civilizations, plants and their derivatives have been used in traditional medicine for the
therapeutic management of several diseases and thus have affected human health for centuries. The medicinal
Showket Hussain Bhat et al. Int.J. Pharm. Res. Allied Sci., 2019, 8(4):121-129

plant Ca qu is found in Saudi Arabia and the neighboring countries in Arabian Gulf peninsula [1, 2]. In these
regions, it is used as a component of traditional medicine in treatment of metabolic disorders including diabetes
[3]. The genus caramulla is particularly known to be rich in pregnane glycosides with medicinal properties
particularly for the control of obesity and hyperglycemia. In a more recent study, the rats fed with the HF diet
and russelioside B (25 mg/kg; pregnane glycoside from caralluma) resulted in diminishing body weight gain as
compared to the control group fed with HF diet alone. Enriching the diet with doubled russelioside B dose led to
further reduction in body weight gain as compared to control group. The anti-obesity affect was partly attributed
to its anti-inflammatory and adipokine modulating activities, in addition to its favorable effect on energy
expenditure [4]. When administered at a dose of 50 mg/kg in streptozotocin induced diabetic rats, russelioside B
was shown to regulate few key carbohydrate metabolizing enzymes, thereby improving the fasting serum
glucose level, glycated hemoglobin, serum insulin level and lipid profile [5]. In a biologically guided
fractionation approach, few isolated pregnane glycosides from Ca qu have also been shown to be cytotoxic
against MFC7 breast cancer cell lines [6]. Moreover fractionated organic and aqueous extracts form Ca qu have
also been demonstrated to possess antimicrobial properties [7].
Extensive studies have implicated the role of ROS in the pathogenesis of human diseases and disorders
including cancers and few blood disorders like hemolytic anemia [8]. It important to note that the
phytochemicals in herbal extracts, especially polyphenols including flavonoids, tannins, proanthocyanids,
catechins have been shown to scavenge and neutralize ROS in deranged cells preventing the damage or at least
partly restoring the normal cellular functions [9]. Studies have shown that nutraceuticals derived from plants are
highly beneficial in human health [10] and may serve to reverse certain pathologies. Interestingly, from the last
decade the genus caralluma has been studied for developing nutraceuticals given its medicinal properties. For
example, in a double blind placebo controlled clinical trial, the extracts from Caramulla fimbriata have been
shown to play a favorable role in curbing central obesity (significant weight loss), the key component of
metabolic syndrome [11]. Furthermore, C. fimbriata extract has received Generally Recognized as a Safe
(GRAS) status (FDA) for use as a nutraceutical to combat obesity, a well-known and serious public health
concern across many developed and developing nations. In fact, an extract of C. fimbriata (Slimaluna(®),
Gencor Nutrients, Anaheim, CA, USA) is used as an anti-obesity agent and appetite suppressor [12].
The aim of the current investigation was to study the basic phytochemical content and to evaluate antioxidant
and anti-hemolytic potential of the extract obtained from the areal parts of Ca qu. As depicted in Figure 1, we
hypothesize that the extract has the potential to be developed as a nutraceutical product that can be used to
intervene in certain human diseases for which ROS have been associated as a causative mechanism; for example
cancers and hemolytic anemia.

Figure 1.The scheme to hypothesize that phyto-constituents of Caralluma quadrangula extract are bio-effective
antioxidants. It can be developed into a nutraceutical product to impede certain reactive oxygen species
associated disorders.

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MATERIALS AND METHODS

DPPH, ABTS, anhydrous potassium dihydrogen orthophosphate, orthophosphoric acid, kampferol, gallic acid,
rutin, lutiolin and quercitin were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All solvents used
were analytical and HPLC grade.
Plant collection and extraction: Ca qu was collected from its natural desert habitats from the Northwestern
region of Saudi Arabia around the city of Tabuk. The plant was authenticated by an expert taxonomist by
comparing the collected sample with the Ca qu specimen (RH 3254) preserved in the herbarium at the
Department of Biology, College of Science, University of Tabuk. The Areal parts were dried under shade for
few days. The dried plant material was crushed into powdered form with a wooden mortar and pestle and further
ground to fine dry powder using a blender. 100 g of dry powder was then soaked in 1 L of 80% methanol in a
conical flask placed in a shaking water bath at 40°C for 24 hr. The mixture was then filtered through double-
layered, clean cheese cloth and then again through double-layered Whatman paper (Sigma-Aldrich Co., St.
Louis, MO, USA). The procedure was repeated twice and the collective filtrate was then concentrated under
reduced pressure at 35°C using a Buchi Rotavapor R-210 (Flawil, Switzerland). The extract was dried in a
vacuum freeze dryer. The residual material was found to weigh 12.25 g (representing a yield of 12.25 %) and
stored at -35 C for further use.
Phytochemical determination (quantitative): Several major classes of phytochemicals were determined by
authenticated procedures which included phenolics, flavonoids, tannins, alkaloids, and saponins. The
quantitative determination of the total phenolic and flavonoid contents were determined using gallic acid and
quercitin as reference standards respectively [13,14]. Tannins were determined using tannic acid as a reference
standard [15]. Alkaloids and saponins were determined by gravimetric estimation as described by Harborne et
al. [16].
Phytochemical determination (qualitative): HPLC was used for the qualitative determination of flavonoids
for which a standard flavonoid mixture containing kampferol, rutin, lutiolin and quercitin was used. Shimadzu
LC 2010 CHT HPLC was used for the chromatographic separation equipped with autosampler and a UV
detector detector (370 nm). The chromatographic separation was achieved on a Purosphere, C18, 5 µ (250 x 4.6
mm) reverse phase analytical column. Mobile phase A was HPLC grade water: buffer (0.136 g of anhydrous
potassium dihydrogen orthophosphate/L, pH adjusted to 2.8 with orthophosphoric acid). It was filtered by
passing through 0.45 µm filter and further degassed by using bath sonicator. The mobile phase B was HPLC
grade acetonitrile. Injection volume was 10 μL and oven temp was set at 30oC. The mobile phase was pumped at
1.5 ml/min at room temperature. The gradient program was as follows: 0-6 min B was 5%, 6-10 min B was
25%, 10-25 min B was 65%, 25-30 min B was 90%; 30-35 min B was ramped to 5% and followed by
equilibration of column till another 5 minutes.
Antioxidant activity by scavenging of DPPH radicals: The DPPH radical-scavenging activity was carried out
according to the Blois method [17]. DPPH (0.3 mM) was added to each sample. After incubation for 30 min in
the dark at room temperature, the absorbance was measured at 518 nm using a microplate reader. Vitamin C was
used as a positive control. The free radical-scavenging capacity was expressed by IC50.
Antioxidant activity by scavenging of ABTS radicals: The ABTS assay was adopted to determine the ability
of the Ca qu extract to neutralize the ABTS radical cation and compared to that of vitamin C [18]. The radical
cation was prepared by mixing 7 mM ABTS with 2.45 mM potassium persulfate (1:1 v/v). The mixture was
incubated for 24 h until completion of the reaction to attain a stable absorbance. PBS was mixed with ABTS
radical solution to achieve an absorbance of 0.7 (±0.02) at 732 nm. The experiment was executed with diluted
ABTS radical solution mixed with samples, and the measurements were taken at 734 nm after 30 min. The
decrease in absorbance reflected the antioxidant capacity of the samples and was expressed by IC50.
Anti-hemolytic assay (Isolation of RBCs and protection again H2O2 induced hemolysis): Human Blood was
obtained by venipuncture in EDTA tubes and RBCs were isolated and stored according to Nabavi et al. [19].
Different concentrations of the each extracts (0.5 mL) were added to erythrocyte suspension (4%, 2 mL) and the
volume was made up to 5 mL with saline buffer. Reaction mixtures were incubated for 5 min at room
temperature and then 0.5 mL of H2O2 solution in saline buffer was added to induce hemolysis. After incubation
(240 min) at room temperature, the reaction mixture was centrifuged at 250g for 10 min and the extent of
hemolysis was determined by measuring the absorbance at 540 nm corresponding to hemoglobin liberation [20].
Statistical analysis: The experiments were performed in three different sets, with each set in triplicate. The data
are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using an

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analysis of variance (ANOVA), followed by an F-test using SPSS version 11.5 (SPSS, Inc., Chicago, IL).
Values of P that were ≤0.05 were considered significant. For HPLC analysis LC solutions software was used.

RESULTS

Phytochemical composition (quantitative) of the extract show the presence of varied phytochemical
classes: The results as tabulated in Table 1, showed that the extract contained total phenolics at a concentration
of 8.60 ± 0.76 mg GAE % w/w, total flavonoids were 0.90 ± 0.01 mg QE % w/w, total tannin content at 8.50
±0.66 mg TAE % w/w, alkaloids at 2.80 ±0.15 mg % w/w and saponins 20.07 mg %w/w. The phytochemical
contents of extract displayed an array of bioactive metabolites which have been well implicated to benefit and
intervene in certain metabolic diseases.
Table 1. The phytochemicals determined in the hydro-methanolic extract of Caralluma quadrangular. Most
determined phytochemical classes are effective antioxidants and are capable of limiting reactive oxygen species
associated pathologies.
Phytochemical Class Quantity
TP (mg GAE % w/w) 08.60 ±0.76
TF (mg QE % w/w) 00.90 ±0.01
Saponins (mg %w/w) 20.07 ±1.85
Tannins mg TAE % w/w 08.50 ±0.66
Alkaloids (mg %w/w) 02.80 ±0.15
TP = Total polyphenols; TF = Total flavonoids; GAE = Gallic acid equivalent; QE =
equivalent; TAE = Tannic acid equivalent; Values are mean ±
Quercitin;
SD of three replicates.

Qualitative phytochemical analysis display the presence of flavonoid rutin: As shown in Figure 2, the
extract chromatogram displayed a peak at RT 9.000, strongly indicative of the presence of rutin in the extract.
Rutin has effective pharmacological properties that include anti-carcinogenic, antioxidant, cardio-protective,
vaso-protective, neuro-protective and cyto-protective activities [21].

Figure 2. HPLC chromatogram of the hydro-alcoholic extract of Caralluma quadrangula. The peak
corresponding to the retention time of 9.00 min is highly indicative of the presence of rutin. Rutin is a strong
anti-oxidant and possess varied pharmacological properties as mentioned.

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DPPH radical scavenging: As shown in Figure 3, the inhibition of DDPH radicals was dose- dependent in
relation to concentration of the extract, and more than 85% inhibition could be seen at a concentration of 1000
μg/ml extract in the reaction mixture. The unpaired electron on DPPH confers a strong absorbance at 517 nm,
giving the radical a purple color. With the exposure to antioxidants, it is reduced resulting in decreased
absorbance due to the formation of yellow colored anti-radical diphenylpicryl hydrazine.

Figure 3. DPPH inhibition (%) by the hydro-alcoholic extract of Caralluma quadrangular.The values reported
are the mean ± SEM of three independent experiments. A dose-dependent inhibition of DPPH radicals can be
seen as the concentration of the extract was increased in the given range.

Inhibition of ABTS radicals: As can be observed from Figure 4, the final extract concentration in the reaction
mixture in the range of 10-250 μg/ml had a dose dependent effect on the scavenging of ABTS radicals. ABTS is
a well-studied substrate for peroxidases and is often utilized to examine the antioxidant properties of plant
derived compounds.

Figure 4. The radical scavenging capacity of the aqueous-alcoholic Caralluma quadrangula extract expressed
by its ability to inhibit ABTS•+ radicals. The values reported are the mean ±SEM of three independent
experiments. A dose-dependent inhibition of ABTS radicals can be seen as the concentration of the extract was
increased in the given range.

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The extract alleviates the hydrogen peroxide-induced RBC membrane damage (Anti-hemolytic): The
results of the anti-hemolytic assay are shown in Figure 5. A clear dose- dependent relation among the extract
dose and potential to inhibit RBC lysis can be observed. An inhibition of near 90% RBC lysis was observed at
the highest dose of 2000 μg/ml of the extract in the reaction mixture. The phytconstuitents of the extract
scavenge hydrogen peroxide and peroxide radicals generated, protecting the RBC from membrane damage
induced by oxidative insult, which otherwise will lead to hemoglobin leakage into the reaction mixture.

Figure 5. Anti-hemolytic assay showing the effect of increasing concentrations of the Caralluma quadrangula
extract on inhibition of RBC lysis induced by hydrogen peroxide. As the concentration of the extract was
increased, the lysis of RBCs was decreased in the given range. This can be attributed to scavenging of hydrogen
peroxide which results in decreasing the oxidation of polyunsaturated fatty acids in RBC membrane, and
therefore of cell lysis.

DISCUSSION

Aqueous-alcoholic solvents are excellent combinations to extract polyphenols including flavonoids such as
flavonols [22, 23]. The aqueous methanolic extract of Ca qu showed rich contents of different phytochemicals
as presented in Table 1. These plant-derived secondary metabolites possess spectra of pharmacological
properties attributable to their ability to interfere with multiple signaling cascades that are critical to
heterogeneous metabolic diseases [24]. Few previous studies have also demonstrated the antioxidant potential of
genus Caralluma and richness in bioactive metabolites [25-27]. As observed in the HPLC chromatogram
(Figure 2), the peak eluting at 9.000 min is highly indicative of the presence of rutin in the extract. Rutin which
is mainly present in glycosidic form in plants is a potent antioxidant and has potential therapeutic implications
in several chronic diseases such as different cancers and neurodegenerative disorders [28, 29]. The low toxicity
associated with rutin and its derivatives in mammalian cells along with potent attenuation of ROS in human
cells makes it a potential candidate for use as nutraceutical as well [30]. The two anti-oxidant assays resulted in
an IC50 of 323.19 μg/ml and 69.68 μg/ml for DPPH and ABTS radicals, respectively. As in Figure 3, our results
demonstrate significant scavenging activities of DPPH radicals. DPPH is a stable free radical that can donate
hydrogen when reacts with antioxidant compounds and is reduced to diphenyl picrylhydrazine. It is therefore
presumable that in our experimental system at least rutin and other unidentified anti-oxidative polyphenols
contribute to attenuate the DPPH radicals as well as the peroxide radicals generated in RBC hemolysis model.
Our study has specifically used the experimental model of RBC hemolysis to evaluate the scavenging capability
of Ca qu. The phenols and tannins in the extract could easily donate electrons to H2O2, thereby neutralizing it
into water. As shown in Figure 5, with increasing dose of the extract the hemoglobin leakage that reflects RBC
membrane damage is reduced significantly. A similar reflection of a dose dependent scavenging effect of ABTS
radicals can be seen in the antioxidant assay as shown in Figure 4. The enolic groups on the aromatic ring of
polyphenols/flavonoids are excellent electron donors and thereby easily get oxidized. The presence of alkaloids
in the extract (Table 1) possibly has an additive effect on this scavenging activity. In fact the imine group(s) in

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plant alkaloids like capsaicin, protoberberines, jatrorrhizine and piperine is/are also excellent nucleophile(s) and
readily donate electron to highly unstable ROS thereby neutralizing their effects. Such scavenging mechanisms
have been well studied and reviewed by others [31]. Peroxidation of cellular membranes induced by ROS is a
major event in oxidative damage in living systems [32, 33]. It is believed that due the presence of high content
of polyunsaturated fatty acids in erythrocyte cell membrane, these serve as an acceptable model to study the
lipid peroxidation. ROS generated by hydrogen peroxide/or radicals generated directly in cellular metabolism
leads to the peroxidation of cell membrane lipids causing membrane damage and the consequent hemoglobin
leakage. In our study, the inhibition of hemolysis reached 90% after treatment at 2000 µg/mL extract, likely
reflecting a cumulative scavenging antioxidant effect of constituent polyphenols/flavonoids and alkaloids and is
in agreement with other studies [34]. Several diseases and disorders are a result of excessive production of ROS
including cancers and can aggravate others like hemolytic anemia. Antioxidant molecules derived from plants
are effective in neutralizing ROS, thereby compromising their deleterious effects in the cells. Given Ca qu plant
is edible and has been shown to be non-toxic towards mammalian cells, it would be of interest to investigate its
potential as nutraceutical to counter ROS related pathologies. Interestingly, one nutraceutical product from
genus Caralluma is already FDA approved, and used for appetite and weight loss to control obesity [12]. The
scavenging ability of ROS by the Ca qu extract and its anti-hemolytic activity, as demonstrated in our study are
reflective of its potential to attenuate ROS burden in diseased cells such as cancers and certain blood disorders.
Several such studies in literature provide evidence for the protective effects of plant extracts and their
derivatives against ROS mediated damage of red blood cells [35, 36]. It is recognized that the natural products
reflect the richest source for new drugs and/or pharmaceutical leads due to their enormous structural diversity
and pleiotropic action mechanism.

CONCLUSION

The hydro-alcoholic extract of Ca qu is rich in antioxidant phytochemicals and have possible therapeutic
implication in ROS associated impairments that include chronic diseases such as cancer and certain blood
disorders. We provide experimental evidences that the extract has the potential to be exploited as nutraceutical
or as an adjuvant with standard therapies. Further studies are worthwhile to examine the extract for its capacity
towards the development of nutraceutical formulations to contest such pathologies.

ACKNOWLEDGEMENTS

We acknowledge the kind support provided by the Deanship of Scientific Research, University of Tabuk,
Saudi Arabia (S-1436-0115). We also acknowledge Natural Remedies pvt Ltd, Bangalore, India for supporting
few techniques.

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