HPLC-UV analysis of antioxidants in Citrus sinensis stem and root extracts.
Summary: The present study was conducted to determine the antioxidant and anti-bacterial activities; estimate total phenolic, polyphenolic and pigments contents in citrus sinensis. Methods employed for the determination of antibacterial activity were an agar well diffusion method and determination of minimum inhibitory concentration and minimum bactericidal concentration. The total phenolic contents and polyphenol were determined by Follin Ciocalteu reagent method.
The pigments like chlorophyll a, chlorophyll b, [beta]-carotene and lycopene were estimated by using UV/visible spectrophotometer. The extract was subjected to HPLC analysis for antioxidants concentration. The zone of inhibition of the root extract was 1.87, 1.56, 1.50 and 1.90 cm whereas for the stem extract it was 1.69, 1.62, 1.20, 1.59 cm against Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis and Bacillus cereus respectively. The phenolic and polyphenolic contents were expressed as mg GAE/g and were found to be 5.14, 5.17 and 5.19, 5.12 in stem and root respectively. Chlorogenic acid, quercetin, morin, rutin, phloroglucinol and hydroxy benzoic acid were detected in stem at retention time of 6.36, 10.26, 12.80, 22.77, 35.53 and 36.23 respectively. While chlorogenic acid, quercetin, rutin, mandalic acid and hydroxy benzoic acid were detected in root extract at retention time of 6.58, 10.44, 22.63, 30.57 and 36.04 respectively.
Keywords: Citrus sinensis; High performance liquid chromatography; Polyphenols; Antioxidants; Chlorophyll.
Medicinal plants have been used as herbal remedies throughout human history to prevent and cure diseases . WHO defined medicinal plant as a plant which in one or more of its parts, contains substances that can be used for therapeutic purposes or which are substrates for the synthesis of useful drugs .
The generation of large amount of free radicals, particularly reactive oxygen species and their high activity plays an important role in the progression of a great number of pathological disturbances like inflammation, atherosclerosis, stroke, heart disease, diabetes mellitus, multiple sclerosis, cancer, Parkinson's disease, Alzheimer's disease, etc. [3-6].
Chlorophyll content is one of the important component of photosynthetic activity. It is of particular importance to precision agriculture and is an indicator of photosynthetic activity .
Rutaceae is the family of flowering plants belonging to the order Sapindales. It is valuable as a source of edible fruit and as ornamentals. It includes about 160 genera and 1,700 species distributed throughout the world, especially in warm temperate and tropical regions. The flowers are generally perfect (containing both male and female reproductive organs in the same flower) or sometimes unisexual. They are arranged in inflorescences, which facilitates pollination by insects such as small flies and bees . Citrus sinensis is the botanical name of the commonly cultivated sweet orange or simply orange. It is a member of the Rutaceae family and belongs to the genus citrus that is a large genus of flowering plants .
In the present study, the anti-bacterial activity, estimating total phenolic content, measuring polyphenol, finding pigments that include chlorophyll a, chlorophyll b, [beta]-carotene and lycopene, the antioxidant activity and determining the phenolic compound through High performance liquid chromatography (HPLC) of Citrus sinensis were performed.
Plant sample collection
Citrus sinensis plant was collected from Chakdara, lower Dir, KPK, Pakistan. The dried plant were then powdered by grinding with the help of mechanical grinder. The powered plant was dissolved in water and kept for one week with constant shaking at different time intervals. After one week the mixtures were filtered through filter paper. From filtered solution the solvent (water) was removed under reduced pressure in a rotatory evaporator and the extract was obtained in semisolid form. It was then kept in open atmosphere to evaporate the remaining solvent and was thus converted into solid form. The crude extracts were kept in a refrigerator at a temperature of 4AdegC.
Amoxicillin was used as a standard drug for the antimicrobial activity. Two Gram positive [Bacillus cereus (ATCC8035), Enterococcus faecalis (ATCC29212)] and two Gram negative [Klebsiella pneumonia (ATCC13833), Escherichia coli (ATCC25922)] bacterial strains were used in this study. For the preparation of standard antibiotic solution, powders of the antibiotic amoxicillin (purity 100%) were accurately weighed and dissolved in sterile distilled water (0.03g/15ml). The standard solutions were stored at - 20AdegC. The growth media nutrient agar and the petri dishes were sterilized at 121AdegC for 3 hours in autoclave. Then four petri dishes were taken and the agar nutrient solution was transferred to each petri dish. Holes were made by cork borer in each agar plate at a proper distance from one another and standard antibiotic and crude extracts of Citrus sinensis were introduced in the holes by using the micropipette.
Now each agar plate was inoculated with different bacterial strains with the help of cotton swab. After 24 hours of incubation period at 37AdegC in incubator the antimicrobial activities were determined from the diameter of the inhibition zone formed by the extracts of Citrus sinensis around the holes. By measuring the zone diameter of inhibition created by Citrus sinensis extracts were compared with the inhibition zone created by standard antibiotic (amoxicillin). The Minimum Inhibitory Concentration was found by preparing five different concentration solution of extracts was prepared ranging from 0.02, 0.04, 0.06, 0.08 and 0.1 mg/ml. Now nutrient broth solution was prepared and 9 ml of broth solution was taken in five test-tube. After that add 1, 1 ml from each of the five extract solution to the test-tube were added and bacterial strain was added to these five test tube.
The test tubes were placed in incubator for 24 hours and after this minimum inhibitory concentration against selected bacteria were determined. The minimum bactericidal concentration value was determined for same test tubes after 3 days incubation time through changes in the turbidity.
Determination of free radical scavenging activity
About 0.04 gm of DPPH was accurately weighed with the help of digital balance and dissolve in distilled methanol to get the required concentration solution (0.04 gm/100ml). Then 1.00 gm of extract was accurately weighed and dissolved separately in distilled methanol to get the required solution in 20.0 ml volume. Then this solution was stored as Citrus sinensis stock solutions. One solution of 5.0 ml pure distilled methanol + 0.0 ml Citrus sinensis extracts solutions were used as blank. This solution was used as control solution. After this five dilute solutions of the extracts of Citrus sinensis were prepared. Then 1 ml of stock solution of DPPH was added to the control solution and diluted solution of extracts. Then all these solutions were kept in dark place for 30 minutes. After this their absorbance at 517 nm were noted through spectrophotometer and percent free radical scavenging activity (%RSA) was calculated by the below given formula;
Total phenolic acid content
The total phenolic contents in Citrus sinensis were estimated by Folin Ciocalteu reagent method using Gallic acid as standard phenolic compound. 1.0 gm of plant extract was added into a flask containing 9.0 ml of distilled water. Then 1.0 ml of Follin Ciocalteu reagent was added and the mixture was mixed thoroughly. After 5.0 minute of incubations, 10.0 ml of 7% Na2CO3 were added. Then the mixture was diluted to 25.0 ml with the addition of 4.0 ml of distilled water. The mixture was then incubated at room temperature for 90 minute. Finally the absorbance was measured using UV/Visible spectrophotometer at 750 nm. The total phenolic acid content was expressed as mg GAE/g sample.
Total Polyphenol Assay
Total polyphenol was measured using Follin Ciocalteu method. About 1.0 g extracts of stem and root were mixed with 2 ml of Follin Ciocalteu reagent and 20 ml of H2O, and incubated at room temperature for 3 minute. Then 10 ml of 20 % of Na2CO3 was added to the mixture. Then total polyphenol was determined after 1 hour of incubation at room temperature .The absorbance of the resulting mixture was measured at 765 nm .Quantification was done with respect to the standard curve of Gallic acid. The result was expressed as GAE, mg /g of dry weight.
Determination of Pigments Contents
Pigments such as Chlorophyll a, Chlorophyll b, [beta]-carotene and Lycopene were determined through spectrophotometer at 453, 505, 645 and 663 nm respectively. 1 gm of each sample was mixed with acetone-hexane (4:6) and then placed on shaker for 1 hour. After shaking, the absorbance at particular wavelength as mention above was noted and the results were expressed as mg/100 ml of the sample.
Determination of phenolic contents through HPLC
For the preparation of extracts, 10 mL of methanol and water in 1:1 were mixed with 1 gram of the powdered stem and root samples. These mixtures were then heated on hot plat at 70 AdegC for 1 hour. After cooling, the samples were filtered with Whatman filter paper. Then these filtrates were centrifuged at 6000 rpm for 15 minutes. After centrifugation it was filtered again through syringe filters into HPLC vials. The vials containing the samples were labelled with proper code. The determinations of phenolic compounds were carried out by means of Agilent 1260 HPLC system. The separation was achieved via Agilent Zorbax Eclipse C18 column. The identification was performed by using retention times, available standards and UV spectra. Quantification of the identified compounds was on the basis of percent peaks values.
HPLC chromatogram of Citrus sinensis is shown in the Fig. 3. Only polyphenolic compound were targeted.
Antimicrobial activities of the methanolic extracts of Citrus sinesis
The aqueous extract of stem and root of Citrus sinensis was tested for its antimicrobial potentials and their values are shown in Table-1 while graphically in Fig. 1 and 2 for stem and root aqueous extracts respectively. The MIC and MBC values stem and root extracts of Citrus sinensis against Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis and Bacillus cereus are given in Table-2.
Table-1: Antibacterial activity (zone of inhibition in cm) of aqueous extracts of stem and root extracts of citrus sinensis
Bacteria###Stem extract Standard antibiotic Root extract
Table-2: MIC and MBC (mg/ml) of Citrus sinensis stem and root extracts against selected bacteria
The free radical scavenging activities of the aqueous extract of Citrus sinensis have been shown in Table-3 for both stem and root.
Table-3: Free radical scavenging activities of the aqueous extracts of citrus sinensis.
Concentration###%RSA of stem###%RSA of root
Total phenolic acid content
The total phenolic contents of Citrus sinensis were estimated by Follin Ciocalteu reagent method using Gallic acid as standard phenolic compound. The total phenolic acid content was expressed as mg GAE/g sample and were 5.14 and 5.19 in stem and root respectively that were found by formula as given below:
Total polyphenols were measured using Follin Ciocalteu method. Quantification was done with respect to the standard curve of Gallic acid. The result was expressed as GAE, mg /g of dry weight and was found to be 5.17 and 5.12 in stem and root extracts respectively using formula 2.
Determination of Pigments Contents
The pigment contents were determined by the method devised by Nagata and Yamashita . The equation devised by them is given as follow, and the results have been shown in Table-4.
Table-1: Concentration of different pigments contents.
###(mg/100ml) in roots###(mg/100ml) in stem
Determination of phenolic contents through HPLC
The identification of the phenolic compounds was carried out by comparing with the standard curve of the phenolic compounds shown Fig. 3. The HPLC chromatogram of the compounds present in root aqueous extract of Citrus sinensis has been shown in Fig. 4. While comparing with the standard curve of the phenolic compounds chlorogenic acid, quercetin, rutin, mandalic acid and hydro benzoic acid were detected. These compounds were eluted from the column with retention time of 6.58, 10.44, 22.63, 30.57 and 36.04 respectively. Using single point calibration formula the concentration of these compound were calculated and were found to be 9.53, 0.03, 0.02, 3.04x10-3 and 1.71 u/g respectively. The following formula was used for calculation of antioxidants concentration in samples.
Where CX = Concentration of unknown, AS = Peak area of standard, Ax = Peak area of unknown, Cs = Concentration of standard
The HPLC chromatogram of compounds that were identified in stem of Citrus sinensis have been shown in Fig. 5. By comparing this chromatogram with the standard curve of the phenolic compounds they were identified as chlorogenic acid, quercetin, morin, rutin, phloroglucinol and hydro benzoic acid these compound with retention time of 6.36, 10.26, 12.80, 22.77, 35.53 and 36.23 respectively. Using single point calibration formula the concentration of these compound were calculated and were found to be 20.94, 0.09, 247480.58, 0.01, 2.70 and 0.69 u/g respectively.
As new drug-resistant bacterial strains has emerged, herbal drugs are being looked as very important source for discovery of new agents for treating various ailments related to bacterial infections. In this study of antibacterial potentials of aqueous extract of Citrus sinensis roots and stem have been determined. Aqueous extract of Citrus sinensis stem and roots were screened against four bacterial strains. The result obtained in study showed that the tested sample of Citrus sinensis plant extracts have potential antibacterial activities against gram positive and gram negative bacterial strains. Comparatively root extract showed good results than the stem extract (Table-1). The values minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) have been shown in Table-2. This study correlates with the study of Uchechi et al.  in which he studied antibacterial activity of aqueous and ethanolic extract of Citrus sinensis.
Oxidative stress causes serious cell damage leading to a variety of human diseases. Phenolic compounds such as flavonoids may contribute directly to anti-oxidative action through various mechanisms like radical scavenging by H-donation and prevention of chain initiation by donating electrons. In present study, DPPH radical scavenging assay was used to find out the potential of Citrus sinensis stem and roots as a candidate for neutraceutical and to determine its pharmacological significance as an antioxidant. Comparatively stem extract was more potent than root. Also with increase in concentration there was an increase in the free radical scavenging activity for both stem and root extract (Table-3). This study correlates with the study of Cillard, and Cillard , in which they investigated the antioxidant and anti-inflammatory potentials of the ethanolic extract of Citrus sinensis, Linn.
Total phenolic acid and total polyphenol contents
The total phenolic and polyphenolic content were determined by Follin Ciocalteu reagent method at 750 and 765 nm respectively and were expressed as mg GAE/g (Table-4). Quantification was done with respect to the standard curve of Gallic acid. The total phenolic contents were found to be 5.14 and 5.19 whereas the polyphenolic contents were found to be 5.17 and 5.12 in stem and root extracts respectively. This study correlates with the study of Cillard, and Cillard  in which the author have determined the total phenolic acid contents of the ethanolic extract of Citrus sinensis, linn. (sweet orange) stem-bark.
The concentration of different pigments such as Chlorophyll a, Chlorophyll b, [beta]-carotene and Lycopene were determined with the help of spectrophotometer in both stem and root aqueous extracts of Citrus sinensis at 453,505,645 and 663 nm respectively. The pigment contents were calculated through formulae 3 to 6 and results have been presented in Table-4. This study correlates with the study of Gonclaves et al. , in which the authors have determined different pigments at three different temperatures.
Phenolic compound detected through HPLC
The HPLC chromatogram of Citrus sinensis root extract showed a total of five polyphenolic compounds; chlorogenic acid, quercetin, rutin, mandalic acid and hydroxy benzoic acid that were identified through comparison with standard chromatogram of eight standard antioxidants (Fig. 3). While in HPLC chromatogram of Citrus sinensis stem a total of six polyphenolic compounds; chlorogenic acid, quercetin, morin, rutin, phloroglucinol and hydroxy benzoic acid, were detected. These compounds have been shown with their respective peak position and retention time in Fig. 4 and 5. This study correlates with the study of Soares et al.  in which they investigated the Citrus sinensis through HPLC by a method developed for quantifying hesperitin and rutin levels.
In this study, the antibacterial and antioxidant activity of methanolic extracts of Citrus sinensis were determined. The highest antibacterial activity was recorded against Escherichia coli. The zone of inhibition of the root extract were; 1.87, 1.56, 1.50 and 1.90 cm whereas for the stem extract it were; 1.69, 1.62, 1.20, 1.59 cm against Escherichia coli, Klebsiella pneumonia, Enterococcus faecalis and Bacillus cereus respectively. The total phenolic contents and polyphenols were determined by Folin Ciocalteu reagent method, the results were expressed as mg GAE/g and were found to be 5.14, 5.17 in stem and 5.19, 5.12 in root extracts respectively. The pigments like chlorophyll a, chlorophyll b, [beta]-carotene and lycopene were also determined.
The extract was subjected to HPLC analysis and chlorogenic acid, quercetin, morin, rutin, phloroglucinol and hydro benzoic acid were detected in stem extract at retention time of 6.36, 10.26, 12.80, 22.77, 35.53 and 36.23 respectively. While chlorogenic acid, quercetin, rutin, mandalic acid and hydro benzoic acid were detected in root extract at retention time of 6.58, 10.44, 22.63, 30.57 and 36.04 respectively. It was concluded that Citrus sinensis can be used in medicines as it has antibacterial, antioxidant potential and contains a number of bioactive compounds. The present study will open a new research area to be investigated in future.
We are thankful to Higher Education Commission of Pakistan for providing financial support [Project No: 20-2515].
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|Title Annotation:||high performance liquid chromatography, ultraviolet|
|Author:||Zahoor, Muhammad; Shah, Abdul Bari; Gul, Sana; Amin, Sania|
|Publication:||Journal of the Chemical Society of Pakistan|
|Article Type:||Technical report|
|Date:||Jun 30, 2018|
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