RP-HPLC ESTIMATION OF QUERCETIN AND SALICYLIC ACID IN CAPSICUM

ANNUUM AND THEIR CORRELATION WITH IN-VITRO ANTI-

INFLAMMATORY ACTIVITY

MOHAMMED SHARIQ IQBAL1*, ZAHRA IQBAL2

1Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus,

INDIA 226028

2Department of Botany, University of Lucknow, Uttar Pradesh-226007 India

*Corresponding author; e-Mail: msiqbal@lko.amity.edu, shariq0903@gmail.com

Abstract

In most of the quercetin QE and salicylic acid SA important properties, its ability to modulate

inflammation is significant. However in Capsicum annuum CA, QE and SA anti-

inflammatory activity was not correlated and elucidated. In the present study QE and SA in

CA was estimated and its in-vitro anti-inflammatory activity (pilot study) was determined.

Estimation of QE and SA in CA was done using RP-HPLC. In-vitro anti-inflammatory

activity of methanolic extract of CA, QE and SA was compared and correlation was

established. The results were found to be significant with percentage yield of methanolic

extract of CA, to be (1.9 ±0.2 %). Using RP-HPLC, QE was found to be 0.0223±0.0012%

while SA was found to be 0.0598±0.0029%. A comparative in-vitro anti- inflammatory study

showed noteworthy results. Primarily the inhibition of albumin denaturation IAD and

antiproteinase activity APA was studied and was found maximum in QE with

98.230±1.589% and 59.906±1.529% respectively. Heat induced haemolysis HIH of

erythrocytes showed maximum in SA (71.830±2.838%). Whereas hypotonicity induced

haemolysis HPIH showed significant activity with QE (76.770±3.475%). When lipoxygenase

LOX and cyclooxygenase COX inhibition was estimated it was found maximum in QE with

56.930±4.069% and 61.660±3.135% respectively. When correlation was established a strong

positive correlation of 0.9 was observed, thereby postulating the role of QE and SA to be the

active phytochemical of CA accountable for its anti-inflammatory activity. Therefore further

research is desirable to fully elucidate the phytochemicals accountable for its anti-

inflammatory activity to develop better herbal drug formulations.

Keywords: Anti-inflammatory, Capsicum annuum, quercetin, RP-HPLC, salicylic acid.

INTRODUCTION

Fruits and vegetables are known to be natural source of biologically active compounds

(Wesolowska et al 2011, Bae et al 2012, Ha et al 2007). Flavonoids, vitamins C and

E, anthocyanins, dietary fiber, phenolics, and carotenoids are the chief phyto-

compounds found in fruits and vegetables (García-Closas et al 2007; Iqbal et al 2017).

Phytochemicals, in particular like antioxidant and radical-scavenging activities attributes

lower risk of developing several chronic diseases (Ha et al 2007). As a result of naturally

occurring antioxidants, which commonly exist in numerous combinations,

investigators are now looking forward for its improved and synergistic effects (Srinivas et al

2009, Conforti et al 2007).

CA is one of the vegetable in which a range of antioxidants are found. CA belongs to the

family Solanaceae, and genus Capsicum, that is comprised of about 200 varieties (Oboh et al

2011, Menichini et al 2009). CA is consumed globally and their significance

has enlarged step by step to put them among the foremost consumed spice crops around

the world (Ciz et al 2010). Primarily it is consumed as food and also used as

additives in food trade. They even have a major role in ancient drugs. It is noted that in

Indian, American, and Chinese ancient therapeutic applications CA is extensively used. Some

of the therapeutic applications are  inflammatory disease, abdomen aches, rheumatism, skin

rashes and dog/snake bites (Aggarwal et al 2008, Hwang et al 2009). The medicinal value of

this plant is due to is various phytochemicals found in the form of flavonoids (QE), phenolics

(SA) and other antioxidants of various therapeutic importances (Shaimaa et al 2016).

In most of the QE’s important properties, its ability to modulate inflammation is noteworthy.

Since, we tend to currently perceive that inflammation is involved in almost every chronic

human disease; therefore having an inflammatory modulator like QE is just like an

armamentarium. Min et al (2007) reported that QE precludes the formation of inflammatory

cytokines by impeding the effects of cellular mediator (NF-kappaB), related with both

chronic inflammation and cancer. QE inhibit the inflammation producing enzymes like COX

and LOX, that leads to a decline in inflammatory mediator’s prostaglandins and leukotrienes

(Warren et al 2009). Therapeutic mediators that block the COX enzymes are frequently used

in the treatment of agonizing situations such as arthritis, and LOX inhibitors are used in the

treatment of asthma. QE exerts anti-inflammatory and anti-allergic effects by preventing the

formation of histamine from basophils and mast cells. QE ability to preclude allergic effects

has an incredible implication in treatment and deterrence of bronchitis and asthma (Chaabi et

al 2007). Several studies have shown that pre-treatment of QE or its compounds on animals

have affectedly reduced the reactions to chemicals which trigger asthma attacks (Jiang et al

2007; Jung et al 2007; Rogerio et al 2007). In a study by Moon et al (2008), comparison on

inhaled QE with prescribed asthma drugs in guinea-pigs was done.

SA is another phytochemical found in CA. Salicylates compounds are widely used for their,

antipyretic and anti-inflammatory properties (Insel, 1991). The most frequently recognized

and used salicylates are sodium salicyclates, SA (2-hydroxybenzoic acid) and aspirin

(acetylsalicylic acid). They are comprehensively used for the relief in inflammation,

headache, and blood thinner for cardiac diseases (Rainsford, 1984). The mechanism action of

salicylates is based on inhibition of prostaglandin and its derivatives synthesis that cause

inflammation, pain, rise in temperature and related diseases (Meade et al, 1993). Meanwhile,

there is advancement of new anti-inflammatory, analgesic and antipyretic compounds that

can compete with salicylates compounds.

The present study was aimed to estimate flavonoid and phenolic phytochemical (QE and SA

respectively) in CA plant by using RP-HPLC. Further it is to determine the in-vitro anti-

inflammatory effect (pilot study) of methanolic extract of CA and QE, SA (flavonoid and

phenolic phytochemicals of CA respectively). The correlation of QE and SA was also

determined in the study. The investigation would help pharmaceutical and neutraceutical

industry to develop anti-inflammatory drug from natural source.

MATERIALS AND METHODS

Plant sample of CA were obtained from herbal garden, Amity University Uttar Pradesh,

Lucknow campus. The sample was thoroughly washed with tap water followed by distilled water. For drying washed tissues were dried on blotting paper and spread out at room temperature in shade. Shade dried materials were ground to fine powder with tissue blender. The extracts were prepared according to Khoddami et al (2013) with few modifications. Methanol as solvent was used to prepare extracts from the plant sample. The extract was

filtered through Whatman filter paper no. 1 and evaporated till dryness. The dried extract was

dissolved to 1mg/ml stock solution with methanol, filtered with 0.45 μm syringe filter for analysis. The stock solutions were diluted to different concentrations of 500 µg/ mL, 400 µg/ mL, 300 µg/ mL, 200 µg/ mL, and 100µg/ mL for further analysis. Standard drugs were used as control at a concentration of 100µg/mL.

RP-HPLC analysis of QE and SA

Reagents

Authentic standard of QE dihydrate extrapure (SRL Pvt. Ltd.) and SA (Hi-Media) was

purchased. HPLC-grade acetonitrile, methanol, water (MERCK) and phosphate buffer, acetic

acid were used for estimation analysis by RP-HPLC.

Instrumentation

RP-HPLC analysis was carried out using a UFLC Shimadzu corporation Kyoto, Japan

chromatograph Model with pump Model LC-20AD, and UV-Vis detector Model SPD-20A.

The C-18 column (250 × 4.6 mm) was made of stainless steel having matrix of 5 μm particle

diameter, (Shiseido, Japan). Data acquisition was performed using Lab Solution Lite software

(Shimadzu Corporation Kyoto, Japan).

RP-HPLC Conditions

RP-HPLC assays were carried out for QE and SA by the protocol of Lee et al (2002) and

Pushpa et al (2014) respectively with some modifications. QE was estimated by RP-HPLC

method in an isocratic system with a flow rate of 1.0 ml/min. The mobile phase was

acetonitrile and 1% v/v acetic acid (60%:40% v/v). The detection wavelength was at 362 nm,

with retention time of 7.47 minutes. SA was estimated by RP-HPLC method in an isocratic

system with a flow rate of 1.0 ml/min. The mobile phase was phosphate buffer (pH 3.0) and

methanol (80:20 v/v). The detection wavelength was at 230 nm, with retention time of 5.07

minutes. All determinations were performed at ambient temperature (25-300C). Before use,

the mobile phase was filtered through 0.22 μm disposable syringe (Millipore filters) followed by degassing by bath sonicator.

Stock standard solutions were prepared by dissolving 10 mg standards of QE and SA, in 10

mL mobile phase respectively. For calibration curve standard solution of (1mg/ml) was

diluted using mobile phase. The concentration ranges from 0.0005 to 0.2 mg/ mL was used.

Each standard solution was injected (20 μL) and data was analyzed, further chromatographed

under the specified conditions described previously. Each standard and unknown was run in

triplicate. Peak area was plotted against the corresponding concentrations to obtain the

calibration graph. There after a linear relationship was obtained.

IN-VITRO ASSESSMENT OF ANTI-INFLAMMATORY ACTIVITY

Inhibition of albumin denaturation

The anti-inflammatory activity (pilot study) was done according to the protocol of Rastogi et

al (2018) and Sakat et al (2010) with some modifications. The reaction mixture consists of

equal volume of test extracts of different concentrations (100 – 500 μg/ml) and 1% aqueous

solution of bovine albumin (Fraction V). The pH of the reaction mixture was adjusted using

small amount of 1N HCl. The sample extracts were incubated at 37˚C for 20 min and then

heated to 51˚C for 20 min. The absorbance was measured after cooling the samples at room

temperature. The turbidity formed was measured at 660nm using UV-Visible

Spectrophotometer (Model: Shimadzu UV-1800). The Percentage IAD was calculated as

follows:

Percentage inhibition = (Abs Control –Abs Sample) X 100/ Abs control

Antiproteinase activity

The activity was performed by the protocol of Sakat et al (2010) and Oyedepo et al (1995),

with some modifications. The reaction mixture (2 ml) containing 0.001% trypsin, 1 ml of

1mM Tris HCl buffer (pH 7.4) and 1 ml test sample of different concentrations (100 – 500

μg/ml). The mixture was incubated at 37˚C for 5 min and then 1 ml of 0.02% (w/v) casein

was added. The mixture was incubated for an additional 20 min at 37˚C. 2 ml of 2%

perchloric acid was added to arrest the reaction. Cloudy suspension was centrifuged and the

absorbance of the supernatant was read at 210 nm against buffer as blank. The percentage

APA was calculated.

Percentage inhibition = (Abs control –Abs sample) X 100/ Abs control

Membrane stabilization

Preparation of Red Blood cells (RBCs) suspension (Sakat et al 2010; Sadique et al 1989). The

blood was obtained from healthy human volunteer who had not taken any non-steroidal anti-

inflammatory drugs for 2 weeks prior to the experiment. The blood sample was centrifuged at

3000 rpm for 10min and was washed three times with equal volume of normal saline. The

amount of blood was measured and re-constituted as 10% v/v in normal saline suspension.

Heat induced haemolysis

The activity was done according to the protocol of Sakat et al (2010) and Shinde et al (1999)

with some modifications. The reaction mixture (2ml) comprised of 1 ml test sample at

different concentrations (100 - 500 μg/ml) and 10% RBCs suspension (1 ml). For control,

only saline was added to the test tube. Aspirin as a reference was used. The reaction mixture

was incubated in water bath for 30 minutes, at 56 ºC. The reaction was stopped by cooling the

reaction mixture under running tap water. The reaction mixture was then centrifuged at 2500

rpm for 5 min. The supernatant collected was used to take absorbance at 560 nm. The

experiment was performed in triplicates. The Percentage HIH was calculated as follows:

Percentage inhibition = (Abs control –Abs sample) X 100/ Abs control

Hypotonicity-induced haemolysis

HPIH was done according to the protocol of Azeem et al (2010) with some modifications.

Extracts of different concentration (100-500μg/ml), reference sample (Diclofenac sodium

100μg/ml), and control were individually mixed with phosphate buffer pH 7.0 (1ml),

hyposaline (2ml) and red blood cell suspension (0.5ml). The reaction mixtures were

incubated at 37˚C for 30minutes. It was then centrifuged at 3000rpm. The supernatant was

transferred and absorbance was taken at 560nm. The percentage HPIH was calculated by

assuming control as 100%.

Percentage protection = 100- (OD sample/OD control) x 100

Anti-lipoxygenase activity

LOX was studied with minor modifications, using linoleic acid as substrate and lipoxidase as

enzyme (Shinde et al 1999). Test samples were dissolved in 2M borate buffer pH 9.0

(0.25ml) and (0.25ml) lipoxidase enzyme solution (20,000U/ml). The reaction mixture was

incubated for 5 min at 25˚C. Then 0.6mM lenoleic acid solution (1.0ml) was added. The

reaction mixture was vertexed and absorbance was measured at 234nm. Indomethacin as a

reference was used. The percent inhibition was calculated from the following equation.

Percent inhibition= [{Abs control- Abs sample}/Abs control] x 100

Cyclooxygenase Activity

COX activity was measured by the protocol of Viji and Helen (2008) with some minor

modifications. The assay mixture consisted of Tris- HCl buffer, hemoglobin, glutathione and

enzyme. The assay was initiated by addition of arachidonic acid. It was then ended after 20

min of incubation at 37ºC by adding 0.2 ml of 10% tricarboxylic acid in 1N HCl. It was then

added tert-Butyl alcohol (0.2 ml) and heated in a boiling water bath for 20 min. The reaction

mixture was centrifuged at 1000 rpm for 5 min. The supernatant obtained was measured at

for COX activity at 632 nm. The percent inhibition was calculated from the following

equation.

Percent inhibition= [{Abs control- Abs sample}/Abs control] x 100

STATISTICAL ANALYSIS

Results are expressed as mean ± standard deviation. Correlation was established between methanolic extract of CA, QE and SA using Microsoft excel.

RESULT AND DISCUSSION

RP-HPLC estimation of QE and SA

The solubility of QE and SA were good in methanol, therefore methanol as a solvent was

considered. Percentage yield of methanolic extract of CA, were found to be (1.9 ±0.2 %).

Before quantitative analysis of QE and SA in methanolic extract of CA by RP-HPLC a linear

calibration curve of standards QE and SA was prepared. The concentration ranges from

0.0005 to 0.2 mg/ mL. The RP-HPLC conditions used are already explained in detail in the

method section. The mean coefficient of determination (R2) for QE was 0.999 whereas %

RSD was found to be 22.74 and for SA the mean coefficient of determination (R2) was 0.997

whereas % RSD was found to be 16.25 Linearity, precision, accuracy, selectivity and

robustness of data were taken care for the validation and reproducibility of the method.

RP-HPLC estimation of flavonoid and phenolic phytochemicals (QE and SA respectively) of

CA showed significant results (figure 1). QE was estimated with (0.0223±0.0012%) while

SA was found to be (0.0598±0.0029%). In a similar study by Shaimaa et al (2016),

phytochemical screening of CA and Capsicum frutescens was done. The results showed

significant content of flavonoids and phenolic compounds. It was found that sufficient

amount of QE and SA was present in the samples. The results were also in good agreement

with the report of Howard et at (2000) where phytochemical screening of flavonoids and

phenolic compounds of CA was done.

In-Vitro Assessment of Anti-Inflammatory Activity

A comparative in-vitro anti- inflammatory study of methanolic extract of CA, QE and SA

(Phytochemicals of CA) was studied and showed significant results. Primarily the IAD was

studied and was found maximum in QE (98.230±1.589%), while minimum in SA

(89.193±1.580%). The results are represented in figure 2 which shows significant variation in

in-vitro anti-inflammatory activity of methanolic extract of CA, QE and SA. In a study by

Sengupta and Sengupta (2003) reported the binding of QE with human serum albumin

(HSA), revealing no substantial perturbation in the secondary structure of HSA as a result

protein denaturation is inhibited. Aurica et al (2016) reported the effect of QE on thermal

denaturation of bovine serum albumin (BSA), thus inhibiting protein denaturation. When

APA was done it was found maximum in QE with (59.906±1.529%) and minimum was

observed in methanolic extract of CA with (7.530±1.600%). The results were in accordance

to the report of Robert et al (2001), where the inhibition of protein was reported for anti-

inflammatory activity. QE’s enzyme inhibitory action covers phospholipases that can

catalyse the discharge of arachidonic acid from phospholipids present in cell membranes.

Arachidonic acid plays a key role as a substrate for biomolecules like inflammatory

prostaglandins, thromboxane and leukotrienes (Parul and Deepak 2007).

HIH of erythrocytes showed significant results with maximum in SA (71.830±2.838%) and

minimum in QE (3.066±0.792%). Whereas HPIH showed significant activity with QE

(76.770±3.475%) while minimum in methanolic extract of CA (18.120±1.426%). The results

were in accordance to the report of Farhina et al (2015) where SA showed 62.12±0.26% in

heat induced conditions while 71.90±29% in inhibition of haemolysis of erythrocytes

(HRBC). It can be assumed from the experiential results, that the action of membrane

stabilization and erythrocyte lysis inhibitory property of CA may be the probable mechanism

of its anti-inflammatory activity.

When lypoxigenase inhibition was estimated it was found maximum in QE (56.930±4.069%),

while minimum was recorded in methanolic extract of CA (14.343±1.796%). COX inhibitory

assay showed maximum in QE (61.660±3.135%) and minimum in methanolic extract of CA

(15.163±1.267%). The results postulate the role of QE in LOX and COX inhibition, resulting

in its potent anti-inflammatory activity. Inhibition of enzymes (LOX and COX) by QE, which

catalyses and alter arachidonic acid to its metabolites (Middleton and Drzewiecki 1985;

Yoshimoto et al 1983; Della et al 1988). Thus, decreasing the levels of these metabolites as

well as histamine levels, leads in sustaining the normal level of body tissue and its structures,

thereby, showing potent anti-inflammatory activity.

When correlation was established between methanolic extract of CA, QE and SA in all the

anti-inflammatory activities, a strong positive correlation of 0.9 was observed, thereby

postulating the role of QE and SA (flavonoid and phenolic phytochemicals respectively) to be

the active phytochemical of CA for its anti-inflammatory activity. Results are in table 1. In a

similar study by Aline et al (2012), the flavonoids and phenolic components of Capsicum

baccatum showed significant anti-inflammatory activity and the correlation was established

between the two factors was also found noteworthy. Therefore, QE and SA found in CA are

the active phytochemicals responsible for anti-inflammatory activity.

CONCLUSION

The results exhibited methanolic extract of CA, QE and SA to a potent anti-inflammatory

agent. QE and SA being the active phytochemicals found in CA significant anti-inflammatory

activity. Strong positive correlation between methanolic extract of CA, QE and SA confirms

the anti-inflammatory property of flavonoid and phenolic phytochemicals of CA. Further

work needs to be undertaken to fully elucidate the mode of mechanism of the phytochemicals

responsible for anti-inflammatory action. The study would help to develop improved herbal

drug formulations in pharmaceutical and neutraceutical industry.

COMPLIANCE WITH ETHICAL STANDARDS:

Funding: No funding was provided for the study.

Conflict of Interest:  Authors worked for this manuscript declares that they have no conflict

of interest.

Ethical approval: All procedures performed in studies involving human participants were in

accordance with the ethical standards of the institutional and/or national research committee

and with the 1964 Helsinki declaration and its later amendments or comparable ethical

standards.

Informed consent: Informed consent was obtained from all individual participants included

in the study.

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Figure 1. Chromatogram of standard and plant extract of Quercetin (362nm) and salicylic acid (230nm) with retention time of 7.47 mins and 5.07 mins respectively. Data analyzed and acquired using Lab Solution Lite software

Figure 2. Comparison based on graphical representation of in-vitro anti-inflammatory activity of methanolic extract of Capsicum annuum, quercetin and salicylic acid, showing significant variation. The results are expressed in ± S.D where N=3.

Table 1. Correlation matrix between methanolic extract of Capsicum annuum (MeOH CA), quercetin (QE) and salicylic acid (SA)

Inhibition of protein (albumin) denaturation Antiproteinase activity

MeOH CA QE SAMeOH CA 1

QE 0.908440475 1SA 0.95342902 0.961393 1

MeOH CA QE SAMeOH CA 1

QE 0.966305 1SA 0.965972 0.996826843 1

Heat induced haemolysis Hypotonicity induced haemolysis

MeOH CA QE SAMeOH CA 1

QE 0.923532 1SA 0.988789 0.875726728 1

MeOH CA QE SAMeOH CA 1

QE 0.960559 1SA 0.989565 0.973660399 1

Lypoxigenase inhibition Cyclooxygenase Inhibition

MeOH CA QE SAMeOH CA 1

QE 0.99738 1SA 0.987683 0.99476808 1

MeOH CA QE SAMeOH CA 1

QE 0.987012 1SA 0.988282 0.999822906 1