Characterization of secondary metabolites in different parts of Ocimum gratissimum L. by in vitro antioxidant activity and high-performance liquid chromatography–diode-array detector analysis
Sharmistha Ganguly1, Jyoti Kumar1, Tapan Seal2
1 University Department of Botany, Ranchi University, Jharkhand, India
2 Department of Plant Chemistry, Botanical Survey of India, Howrah, West Bengal, India
|Date of Submission||23-Dec-2020|
|Date of Decision||21-Jan-2021|
|Date of Acceptance||23-Feb-2021|
|Date of Web Publication||12-Jul-2021|
Department of Plant Chemistry, Botanical Survey of India, Howrah, West Bengal
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Ocimum gratissimum L. (OG) has medicinal importance, especially in the treatment of epilepsy, fever, diarrhea, mental illness, etc., Objectives: The present study focused on evaluation of the total phenolic content, total flavonoid content, and reducing and antioxidizing activities from methanolic extract of different plant parts, namely leaves, stem, roots, and flowers of OG. Materials and Methods: The quantification of phenolic and polyphenolic compounds was assessed by high-performance liquid chromatography (HPLC). Results: The results revealed that the root extract showed the highest phenolic content (323.93 ± 2.062 μg/mg) and reducing property (150.57 ± 1.76 μg/mg), whereas the highest flavonoid content was found in leaves 72.09 ± 1.269 μg/mg. The highest radical scavenging activity (68.12% ± 0.527%) was observed for the root extract in 2, 2-diphenyl-1-picrylhydrazyl method. Similar significant results were observed by 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) method as well for all investigated extracts. HPLC–diode-array detector analysis was done to find the secondary metabolite composition of the methanol extracts which lead to the characterization of different flavonoids and phenolic acids in OG extracts. Conclusion: These results showed that this species can be a source of phenolic compounds and antioxidants, thereby suggesting it for consumption as a source of natural food antioxidant to help hinder against various detrimental effects of reactive species-induced ailments.
Keywords: Antioxidants, flavonoids, high-performance liquid chromatography, Ocimum gratissimum L., phenolic compounds
|How to cite this article:|
Ganguly S, Kumar J, Seal T. Characterization of secondary metabolites in different parts of Ocimum gratissimum L. by in vitro antioxidant activity and high-performance liquid chromatography–diode-array detector analysis. Phcog Mag 2021;17:209-15
|How to cite this URL:|
Ganguly S, Kumar J, Seal T. Characterization of secondary metabolites in different parts of Ocimum gratissimum L. by in vitro antioxidant activity and high-performance liquid chromatography–diode-array detector analysis. Phcog Mag [serial online] 2021 [cited 2021 Sep 17];17:209-15. Available from: http://www.phcog.com/text.asp?2021/17/74/209/321256
The present study aimed to investigate the antioxidant activities of different plant parts viz. leaves, stem, roots and flowers of Ocimum gratissimum. The antioxidant properties were evaluated by using DPPH and ABTS radical scavenging activity, reducing power and estimation of total phenolic and flavonoid content. The quantification of phenolic acids and flavonoids were carried out by the high performance liquid chromatography (HPLC). The result of this investigation showed that the methanol extracts of roots of the plant showed higher antioxidant activity as compared to the other parts. The HPLC analysis also showed the presence of phenolic acids and flavonoids in various amounts in these parts which can conclude them as potent natural antioxidant.
Abbreviations used: OG: Ocimum gratissimum L.; HPLC: High-performance liquid chromatography; DPPH: 2, 2-diphenyl-1-picrylhydrazyl; ABTS: 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); HPLC-DAD: High-performance liquid chromatography–diode-array detector; ROS: Reactive oxygen species; BHT: Butylated hydroxytoluene; UV-VIS: Ultraviolet-visible; TPC: Total phenolic content; GAE: Gallic acid equivalent; DE: Dry extract of the concentrate; TFC: Total flavonoid content; RE: Rutin equivalent; AAE: Ascorbic acid equivalent; ANOVA: Univariate analysis of variance; Ac: Absorbance of control; At: Absorbance of test; USP: U. S: Pharmacopeia; ICH: International conference on Harmonization; IC: Inhibitory Concentration; DPM: Dry plant material; HIV: Human immunodeficiency infection; μg/mg: microgram per milligram; cm: centimeter; g: gram; mg: milligram; ml: milliliter; μg/ml: microgram per milliliter; nm: nanometer; μm: micrometer; μl: microliter; min: minutes; ml/min: milliliter per minute; mg/g: microgram per gram.
| Introduction|| |
Oxidation is an inevitable process in all living organisms. Free and nonfree radical species are simultaneously included in reactive oxygen species (ROS) production, thereby leading to hazardous and dreadful effects in the form of fatal diseases such as diabetes, atherosclerosis, ischemic cardiac disease, inflammation, suppression of immunity, diseases with neurodegeneration, cancer, and so on. These free radicals are produced as by-products during the formation of energy by the cell in the form of superoxide and hydroxyl radicals, etc., These ROS are beneficial in limited concentration but have detrimental effects on the cell when its concentration rises, thereby leading to oxidative stress. The living cell counteracts these mechanisms by producing antioxidants either naturally or in the form of food supplements. Several researches support the fact that flavonoids and phenolic compounds are potential antioxidants. The antioxidants scavenge the free radicals and compensate the damage caused by ROS. In present days, medicinal plants are on highlights for being the natural sources of powerful antioxidants. Researchers are focusing on commercial development of these medicinal plants to enhance the health industries. Several evidences also claim that dietary phytometabolites such as flavonoids, phenols, and tannins have been used against various diseases and even in maintenance of health since ages.,
Ocimum gratissimum L. (OG), Lamiaceae, is a native plant to the African continent but is inhabited in India, Bangladesh, China, Thailand, Haiti, Egypt, and Indonesia as well. This plant is mentioned in several books of traditional medicines, especially Ayurveda and Unani medicinal practices. The prominent therapeutic potentialities of the plant have been customarily utilized in the treatment of epilepsy, fever, diarrhea, mental illness, upper respiratory tract infections, cough, and conjunctivitis.
Meanwhile, researchers have searched several ways through which the medicinally important plant paves a pathway toward sustenance and solution of deadly diseases like cancer by cell reinforcement. Various studies have proved that the naturally occurring secondary metabolites in plants show a defensive mechanism against the genotoxicity which is caused during oxidative stress. Evaluating the reducing activities and antioxidizing potentialities of OG and estimation of phenolic and polyphenolic compounds by high-performance liquid chromatography (HPLC) from its different plant parts, namely leaves, stem, roots, and flowers was the main aim of this investigation. The novelty of our study was to establish a comparative evaluation by estimating the phenolic compounds, flavonoid, and antioxidant activities of different parts of OG and quantifying the active phenolic and polyphenolic compounds present in the different parts through HPLC analysis and plant could be a potential source of natural antioxidants which corroborate its efficiency in traditional medicine and might be exploited on an industrial level as food additive and herbal drug formulations.
| Materials and Methods|| |
The fresh plant material was collected from the locality of Hooghly and was sent to Central National Herbarium, Botanical Survey of India, Kolkata, for authentication. The voucher specimen (RU/BOT/JK/SG/001) was deposited in University Herbarium of Department of Botany, Ranchi University, Ranchi, for preservation. Each plant part was rinsed thoroughly with tap water. Then, samples were cut into small pieces and air-dried at room temperature.
Chemicals and equipments
To investigate the antioxidant activities, the phenolic and polyphenolic standards such as gallic acid, protocatechuic acid, catechin, rutin, gentisic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid, chlorogenic acid, p-hydroxy benzoic acid, ellagic acid, myricetin, quercetin, naringin, apigenin, and kaempferol along with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1, 1-diphenyl–2–picrylhydrazyl (DPPH) were procured from Sigma Chemical Co. (St. Louis, MO, USA). The other chemicals such as Folin–Ciocalteus's phenol reagent, potassium ferricyanide, potassium per sulfate, aluminum chloride, ferric chloride, sodium carbonate, sodium dihydrogen phosphate, and trifluoroacetic acid and the HPLC grade solvents such as acetonitrile, methanol, and water were obtained from Merck (Germany). Reversed-phase HPLC analysis was achieved using Dionex Ultimate 3000 liquid chromatography coupled with a diode-array detector with 5 cm flow cell and gradient elution of mobile phase system, using Chromeleon system manager as data processor. The separation was achieved by a reversed-phase Acclaim C18 column (5 μ particle size, 250 mm × 4.6 mm).
Extraction of plant materials
The plant parts, namely leaves, stem, roots, and flowers were dried, pulverized, and were kept in a container which was airtight to analyze the phytochemicals. The plant material (1 g) was taken in 20 ml of 70% aqueous ethanol and agitation on magnetic stirrer for 2 h. The normal room temperature was maintained overnight. The extracts were filtered and undergone evaporation to dryness. The leftover dry extract (DE) was scrapped and weighed. The DE (25 mg) was dissolved in a minimum concentration of methanol and the volume was adjusted up to 25 ml. The extract was kept in a volumetric flask (25 ml) for further analysis.
Estimation of total phenolic content
The Folin–Ciocalteus's methodology was followed to evaluate the total phenolic content (TPC) in various concentrations (100, 200, 400, 800, and 1000 μg/ml) from different plant parts. With 0.5 ml of tenfold diluted Folin–Ciocalteu's reagent, 4.5 ml of distilled water and 0.5 ml of the extract were added and mixed. The mixture was incubated for an hour by maintaining the room temperature and then absorbance was taken at 765 nm. The experiment was performed in triplicates and was communicated as gallic acid equivalent (GAE) in μg/mg DE of the plant materials.
Estimation of total flavonoid content
To 2 ml of extracts in different concentrations (100, 200, 400, 800, and 1000 μg/ml), 2 ml of 2% AlCl3 in ethanol was added followed by incubation in the dark by maintaining the room temperature for an hour. Then, absorbance was measured at 420 nm. The total flavonoid content was calculated and expressed as rutin equivalent (RE) in μg/mg of DE.
Estimation of reducing power
The reducing power was evaluated following the protocol given by Datta et al. 2019. The reducing activity was evaluated and expressed as ascorbic acid equivalent (AAE) in μg/mg of DE.
Estimation of antioxidant activities
The antioxidizing potentialities were determined from plant extracts of different parts using DPPH. Besides, ABTS radical cation was also used for analyzing the free radical scavenging property. The potentiality of DPPH and ABTS radical scavenger was deliberated, using the following equation:
DPPH/ABTS scavenge (%) = ([Ac– At]/Ac) × 100
Where Ac signifies the absorbance in the absence of sample extract, i.e., DPPH/ABTS solution and At signifies the absorbance with sample extracts in different concentrations.
Estimation of phenolic acids and polyphenolic compounds by high-performance liquid chromatography
The HPLC analysis for estimation of phenolic acids and polyphenolic compounds was performed following the protocol by Datta et al. 2019 which was validated by standard USP and ICH guidelines. The methanol extract of OG (20 mg) was mixed in 20 ml mobile phase solvent (methanol: 0.5% acetic acid in water: 1:9) and before injecting into the HPLC system, the filtration of the test solution was done through 0.45 μm membrane filter. Methanol (solvent A) was being used as a mobile solvent phase and 0.5% aqueous acetic acid solution (solvent B). The column temperature was maintained at 25°C thermostatically and the injection volume was kept at 20 μl. The gradient elusion was prepared in different percentages, namely solvent A (10%) and solvent B (90%) with a flow rate in the first 27 min being 1 ml/min to 0.7 ml/min. The solvent A changed from 10% to 40% and flow rate was reduced to 0.7 ml/min in 50 min and for 40% solvent A and 60% solvent B, the flow rate was 0.7 ml/min at first for 2 min and afterward flow rate was decreased from 0.7 to 0.3 ml/min in 65 min, and from 40% to 44% solvent A, the flow rate was 0.3–0.7 ml/min in 70 min. From 44% solvent A, the flow rate was 0.7–1 ml/min for 10 min duration then swapped from 44% to 58% with a flow rate of 1 ml/min for 5 min followed by 58%–70% solvent A in 98 min with a consistent flow rate of 1 ml/min. Before the injection of another sample, the flexible stage was resumed to the initial condition (solvent A: solvent B: 10:90) at 101 min and was allowed to run for the next 4 min. The total running time for the complete estimation of each sample was 105 min. The retention time and spiking with standards under comparable conditions were the recognition point for compound identification. The measurement of integrated peak area was carried out to quantify the phenolic acids and flavonoids. The quantification of phenolic acids and polyphenolic compounds was calculated by using the calibration curve and plotting peak area against respective standard sample concentration.
All the experiments were carried out using triplicate samples and the results were presented as mean ± standard error by using the Statistical Package for the Social Sciences (SPSS variant 7.5) (SPSS Inc, International Business Machines (IBM), USA). Tukey's test at 95% confidence level and statistical significance was followed, thereby accepting at P < 0.05 level. The linear regression graphs were used to calculate the IC50 values.
| Results and Discussion|| |
The TPC, flavonoid content, reducing activity, and antioxidizing activity from different parts of the plant of OG are exhibited in [Table 1].
|Table 1: Antioxidant activities of different plant parts of Ocimum gratissimum L.|
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Total phenolic content
The TPC was communicated as GAE which was expressed in μg/mg dry plant material (DPM), as shown in [Table 1] and [Figure 1]. The highest phenolic content was noticed in root extract (323.93 ± 2.062 μg/mg GAE). The lowest phenolic content was recorded in leaf extract (182.40 ± 1.26 μg/mg GAE). The phenolic compounds are the most vital secondary metabolites in the plant which are responsible for the antioxidizing activity by inactivation of lipid free radicals and prevention of hydroperoxide conversion to oxyradicals., This is the reason that the evaluation of TPC serves as a promising parameter to estimate the antioxidant potential. HPLC can detect the different components present in different amounts in the extract. In previous researches, the phenolic contents of leaves were reported to have the highest amount. However, the present study exhibits a striking difference in the results.
|Figure 1: Graphical representation showing total phenolic content, total flavonoid content, and total reducing activity from different plant parts, namely leaves, stem, roots, and flowers of Ocimum gratissimum L.|
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Total flavonoid content
Flavonoids are also an extensive group of natural metabolites and are prominent for their health-promoting benefits through cell signaling., In many studies, it has been proven to have several pharmacological and biological activities which also include anti-inflammatory, anti-carcinogenic, anti-thrombotic, anti-allergic, hepatoprotective, and even free radical scavenging activities. The highest amount of flavonoid was recorded in leaves (72.09 ± 1.269 μg/mg RE) and the lowest in stem (25.45 ± 0.286 μg/mg RE) which is represented in [Table 1] and [Figure 1]. The result showed a good congruence with the earlier studies where leaf extracts were reported to have the highest flavonoid content.,,
Total reducing power
The reducing activity is expressed in μg AAE/mg of DPM, as shown in [Table 1] and [Figure 1]. The highest reducing activity was recorded in root extract (150.57 ± 1.76 μg/mg AAE) and the lowest was recorded in leaf extract (76.21 ± 1.004 μg/mg AAE). The reducing potential of the extracts actually measures its ability to donate electrons. The promising results show that the root extract of the plant has ability to donate electrons and hence can also have the ability to scavenge free radical.
Free radical scavenging of DPPH and ABTS is communicated in percentage of inhibition (%) of DE of plant materials, as shown in [Table 1] and [Figure 2]. DPPH radical scavenging was recorded in root extract (68.12% ± 0.527%) immediately followed by stem extract (67.09% ± 0.732%). In ABTS radical scavenging, the stem extract exhibited highest inhibition (93.01 ± 0.079%) and lowest inhibition showed by the leaves extract (57.40 ± 0.079%).
|Figure 2: Graphical representation showing comparative antioxidant activity by 2, 2- diphenyl-1-picrylhydrazyl and 2, 2'- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) method|
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The methodology followed to evaluate the antioxidant activity is dependent on the reaction condition and substrate, therefore, all the methods do not exhibit similar value, but a similar pattern is noticed. The antioxidant activity was determined by using DPPH and ABTS. The results showed a very nominal difference that is why can be considered almost similar. Due to the presence of double bond in the C-ring and arrangement of hydroxyl group, flavonoids possess higher antioxidant activity. An eminent free radical scavenging activity is exhibited either due to the presence of ortho group (quercetin), hydroxyl group in meta position (kaempferol), or compounds having a double bond in the C-ring structure, between C2 and C3 hydroxyl group. It has also been previously reported that the molecular weight of phenolic compound is directly proportional to the free radical scavenging activity, depending on the number of aromatic rings and hydroxyl groups being substituted. The present study showed that in both DPPH and ABTS assays, the root and the stem extract showed almost similar results and hence both can be considered to have good antioxidizing activity.
Identification and quantification of phenolic acids and polyphenolic compounds in Ocimum gratissimum by high-performance liquid chromatography
In the analysis, the phenolic acids such as gallic acid, gentisic acid, chlorogenic acid, protocatechuic acid, caffeic acid, p-hydroxyl benzoic acid, vanillic acid, p-coumaric acid, syringic acid, sinapic acid, ferulic acid, ellagic acid, and salicylic acid and the flavonoids such as catechin, naringin, rutin, myricetin, quercetin, naringenin, apigenin, and kaempferol were detected which were being conveyed as μg/mg DPM and are represented in [Table 2]. All the compounds under investigation have responded at 280 nm and separated successfully. Besides, the identified compounds which were recorded by the absorption spectra [Figure 3], [Figure 4], [Figure 5], [Figure 6] are also comparable for all plant extracts as well as standard substances. Caffeic acid and salicylic acid were reported only in leaf and root extracts. p-hydroxy benzoic acid and quercetin were absent only in root extract. Rutin was found to be absent in only stem extract. The leaf extract of the plant contains highest amount of p-hydroxy benzoic acid (0.053 ± 0.001 μg/mg DPM), caffeic acid (0.184 ± 0.001 μg/mg DPM), myricetin (0.415 ± 0.001 μg/mg DPM), quercetin (0.399 ± 0.001 μg/mg DPM), and kaempferol (0.172 ± 0.001 μg/mg DPM). The stem extract was found to show highest amount of gentisic acid (0.352 ± 0.001 μg/mg DPM), chlorogenic acid (0.342 ± 0.001 μg/mg DPM), vanillic acid (1.155 ± 0.001 μg/mg DPM), syringic acid (0.556 ± 0.001 μg/mg DPM), and naringenin (2.454 ± 0.001 μg/mg DPM).
|Table 2: Quantitative estimation of phenolic acids and flavonoids of different plant parts of Ocimum gratissimum by high-performance liquid chromatography|
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|Figure 3: High-performance liquid chromatography chromatogram of leaf extract of Ocimum gratissimum L.|
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|Figure 4: High-performance liquid chromatography chromatogram of stem extract of Ocimum gratissimum L.|
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|Figure 5: High-performance liquid chromatography chromatogram of root extract of Ocimum gratissimum L.|
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|Figure 6: High-performance liquid chromatography chromatogram of flower extract of Ocimum gratissimum L.|
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The highest amount of gallic acid was recorded in the root extract of the plant (0.645 ± 0.001 μg/mg DPM), protocatechuic acid (1.464 ± 0.001 μg/mg DPM), gentisic acid (0.628 ± 0.001 μg/mg DPM), catechin (0.726 ± 0.001 μg/mg DPM), p-coumaric acid (5.080 ± 0.001 μg/mg DPM), ferulic acid (2.712 ± 0.001 μg/mg DPM), sinapic acid (0.827 ± 0.001 μg/mg DPM), salicylic acid (0.376 ± 0.001 μg/mg DPM), naringin (7.242 ± 0.001 μg/mg DPM), rutin (1.280 ± 0.001 μg/mg DPM), and ellagic acid (36.288 ± 0.001 μg/mg DPM).
The HPLC analysis detected the presence of gallic acid which is generally found in plants either in the form of esters or in the free states which goes about as a pioneering anticancerous agent. Some normal vegetables too have some amount of gallic acids which is comparable to the amount of gallic acid in methanol extract present in all parts of the plant, such as chili pepper (3.33 mg/g), lemon (2.03 mg/g), spinach (1.82 mg/g), onion bulb (1.55 mg/g), cabbage (0.49 mg/g), and so on., The presence of protocatechuic acid in the plant can be concluded to be helpful in treating different diseases such as neurodegenerative and hepatic disorders The occurrence of p-hydroxybenzoic acid in the plant also justifies its usage againt liver ailments such as cirrhosis of the liver and liver malignancy, The usage consumption of this plant containing chlorogenic acid may be useful in decreasing the glucose levels, thereby serving as an antidiabetic agent. The plant extracts were found to contain catechin which demonstrates that this plant may be added to cell reinforcement and restorative properties. The occurrence of vanillic acid in the plant which was investigated may get related to having hepatoprotective activities. The presence of caffeic acid helps increasing the production of collagen, premature aging, and antimicrobial and antioxidant activity. The noticeable amount of syringic acid in the plant extract is remarkable for its antimalignancy, hostile to proliferative, narcotic, decongestant, and hepatoprotective activities. p-coumaric acid has been measured in OG which demonstrates its defensive capacity to reduce the formation of carcinogenic nitrosamines in the stomach. The ferulic acid which is one of the significant phenolics is exceptionally found in this plant promptly brings down the cholesterol level in serum and builds sperm reasonability The presence of sinapic acid was also recognized in the plant which has antimicrobial and anticancerous. Rutin is a polyphenolic compound with glycosidic linkage having natural impacts having antidiabetic effects and anticancerous properties. A sufficient amount of rutin in this plant puts forward their potentiality as helpful operators and justifies the legends' application. An immense amount of ellagic acid was recognized in the plant under examination and utilization of it would be valuable for antimutagenic, antimicrobial, and cell reinforcement properties and inhibitors of human immunodeficiency infection. The plant parts also contain a good measure of myricetin and can be used as an antidiabetic agent. An extensive level of quercetin recognized in the plant under examination can be utilized for anticancerous, antihistamine, and also anti-inflammatory activities. The presence of apigenin might be helpful against cardiovascular diseases, neurological disorders, and mutagenesis. The prominent presence of kaempferol can be utilized to prevent the lipid proteins of low-density being oxidized, thereby indicating a potential defensive role in atherosclerosis, cardiovascular maladies, malignancy, and so on.
| Conclusion|| |
According to the above findings, it can be concluded that the plant OG can be exploited as a natural resource of antioxidants. The antioxidant and nutraceutical potentialities of this plant can promote its selection in such a group of plants which are used as nutritional supplements in food industry as well as in development of antioxidant-based drugs by pharmaceutical industries.
The authors would like to acknowledge Dr. A. A. Mao, Director, Botanical Survey of India, Kolkata, for providing all the laboratory facilities.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]