Quantitative Estimation of Total Phenols, Flavonoids and Antioxidant Potential of Cayratia auriculata (In-vitro study)

 

Nagaraju Kancherla1, Anusha. D2*, Chitra. K3, Ravi Babu. K4

1Department of Pharmacology, GSL Medical College and General Hospital,

Rajamahendravaram, Andhra Pradesh, 533296, (Ph.D) SRIHER- Chennai, India.

2Department of Pharmacology, Sri Ramachandra Institute of Higher Education and Research,

Porur, Chennai - 600116, India.

3Department of Pharmaceutical Chemistry, Sri Ramachandra Institute of Higher Education and Research,

Porur, Chennai - 600116, India.

4Department of Pharmacology, GSL Medical College and General Hospital,

Rajamahendravaram, Andhra Pradesh – 533296, India.

*Corresponding Author E-mail: drdanusha@gmail.com

 

ABSTRACT:

The current study was aimed for screening of preliminary phytochemical constituents, estimation of total phenols, flavonoids and evaluation of antioxidant properties of various extracts of Cayratia auriculata (in vitro). Shade dried powder of Cayratia auriculata was exposed to Soxhlet extraction technique with expanding order of extremity of solvents, for example, hexane >chloroform,> ethyl acetate> methanol. Subjective phytochemical screening was finished by standard methods. Total phenolic content was determined by the Folin-Ciocalteau reagent technique using gallic acid as reference standard. Total flavonoid content was determined by the colorimetric technique, utilizing quercetin as standard. In vitro, antioxidant capacity was assessed by utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Phytochemical investigation suggested that the existence of different phytochemical constituents like alkaloids, flavonoids, phenolic mixtures and tannins, proteins, sugars, glycosides, steroids, triterpenoids, and saponins in the all extracts of Cayratia auriculata in varying degree. The methanolic extract showed higher total phenolic 383.99±1.11 mg GAE/g dry extract and total flavonoid content was 64.11±8.89)mg QE/g dry extract when contrasted with different extracts. Methanolic extract demonstrated most elevated Antioxidant activity 96.46±1.97µg/ml when contrasted with different extracts. Among the four extracts of studied plant methanol extract presented the potent free radical scavenging effect with its significantly lower IC50 value (80.66 μg/ml) in comparison with ethyl acetate, chloroform and hexane extracts. The discoveries of the current examination reinforce the expected ability of Cayratia auriculata as a decent option for the examination of novel antioxidant agents for treating physiological disorders.

 

KEYWORDS: Cayratia auriculata, Methanolic extract, Phytochemical, Antioxidant, DPPH Free Radical.

 

 


INTRODUCTION:

Taking into account World Health Organization, customary medication is characterized as numerous wellbeing usage, viewpoints, and information and thinks merging mineral, home-grown, and additionally animal-based medicaments, manual methods, otherworldly treatments, and activities used in a blend of uniquely to keep up prosperity just as to analyze, treat, or forestall illness1. Above 35,000 plant species are being used in a few human societies worldwide for restorative devotions2. Crude medications are by and large the dried pieces of therapeutic plants (entire plants, leaves, blossoms, seeds, bark roots, stem wood, and, and so on) that structure the significant crude materials for the creation of conventional cures in a few frameworks of meds like Homeopathy, Siddha, Ayurvedic, Unani and so on Cayratia auriculata has a place with local of south Indian species. Cayratia auriculata (Roxb.) bet is an equivalent of Cyphostemma auriculatum (Roxb). It is a climber all around filled in dry evergreen to dry deciduous woods and very much circulated in Andhra Pradesh, Tamilnadu, Maharashtra, Madhya Pradesh, Orissa,  Gujarat, Goa, Karnataka, Kerala, Bihar, West Bengal, Bangladesh, Myanmar, and Rajasthan3,4,5,6. Ethnomedicinal study on Cayaratia auriculata shows that this plant is perhaps the most ordinarily utilized in old stories medication, in cardiovascular problems, as a blood purifier, in intestinal worm diseases, wound boil, ear infection as tonic, dog bites, purulent injuries rheumatism, in injury recuperating, as astringent, hydrocele, tumors, cold and hack. In veterinary medication, it is utilized to animal's diarrhoea and blood dysentery7,8. Phytochemical investigations of Vitaceae family species recommend the presence of tannins, alkaloids, saponins, flavonoids, steroids, terpenoids, and stilbenoids9. While considerable literature survey doesn't report the antioxidant properties of Cayratia auriculata, the current examination endeavoured to break down fundamental phytoconstituents and to assess the quantitative determination of total phenolic as well as flavonoid content and followed by antioxidant potential in the different crude extracts of Cayratia auriculata (in-vitro).

 

MATERIALS AND METHODS:

Collection and Authentication of plant:

Collection of Cayratia auriculata plant material was done in August 2018 from the Araku valley in Eastern Ghats of Visakhapatnam, Andhra Pradesh, India. The taxonomic identification of Cayratia auriculata was affirmed and confirmed by Dr.S.B. Padal, professor, department of botany, Andhra University (AU), Visakhapatnam-530 003, Andhra Pradesh, India and Plant herbarium was arranged and kept at herbarium of botany department (Herbarium no.AU-B.D.H–22228). Undesirable residue molecule from new plant material was taken out by washing under running tap water and allowed to shade dry for 14 days. The shade-dried plant material mixed to a fine powder, preserved in airtight containers at 4°C in a refrigerator until use.

 

Preparation of extracts from Cayratia auriculata:

Around 30g of dried powder of Cayratia auriculata was exposed to Successive dissolvable extractions by utilizing a Soxhlet contraption following the standard technique10. The powder was pressed into a cover and removal procedure completed till the dissolvable in the extractor siphon tube turned dreary by utilizing four solvents with expanding request of their extremity, for example, hexane, chloroform, ethyl acetate, and methanol, successively. Concentrate filtration was completed with filter paper, Whatman No. 1 (42), and separated extracts were concentrated by rotary vacuum evaporator and drying with the assistance of a water bath. Semi-solid concentrates were preserved at 4°C until further utilization. The extracts were assessed for the following investigations viz. phytochemical examination, determination of the  total phenolic as well as total flavonoid content and also antioxidant potential (In-vitro). The percentage (%) of extraction obtained was estimated11,12.

 

                                       Wt.of the crude extract obtained (g)

Yield of extract (%) = –––––––––––––––––––––––––––––––– × 100

                               Wt.  of crude  powder utilised for extraction(g)

 

Preliminary Phytochemical analysis:

The preliminary phytochemical investigation was performed with freshly prepared four extracts of Cayratia auriculata by using standard protocols for the existence of different phytoconstituents viz. flavonoids, proteins, alkaloids, tannins, glycosides, anthraquinones, terpenoids, carbohydrates, coumarins, phlobatannins, saponins, and steroids by utilizing standard conventions13,14.

 

Quantitative estimation of total phenolic content (Folin-Ciocalteu Assay):

Estimation of TFC (total phenolic content): The TFC of all four crude extracts of Cayratia auriculata was assessed with the Folin-Ciocalteu reagent (combination of phosphomolybdate & phosphotungstate) with certain adjustments15. The concentration of (1mg/1 ml) solutions was set up by adding 10gm of powdered extracts of Cayratia auriculata to 10ml respective solvents this was taken as the stock solution, various concentrations 50, 100, 150, 200, 250, 300, 350, 400 450 and 500μg/ml with this stock solution was made following similar protocol for standard (Gallic acid). Briefly, Folin Ciocalteu reagent at a volume of (1.0ml) was put to these concentrations and shacked well. Permitted to allow for 300 sec undisturbed, followed by addition of 4 ml sodium carbonate 20% (w/v) solution of was done. Waited for 30 min, blue color was obtained. The optical density of color created was recorded at 765 nm in the ultraviolet (UV) - Visible spectrophotometer. Three replicates were taken for every concentration standard and test sample and the mean value of OD was obtained and plotted against the corresponding concentration to compute extrapolation of a calibration curve, which resembled Beer's Law. From the average OD values, content of total phenols levels were calculated in relation with gallic acid (standard) equivalents (50-1000 μg/ml),  Y = 0.0018x – 0.0572 ; R2 = 0.9916).The total phenolic content  of tested extracts was communicated as mg GAE/g dried plant extract (mg/g gallic acid equivalent .

 

Determination of total flavonoid content:

The content of total flavonoid of Cayratia auriculata plant extracts was estimated with the (AlCl₃) aluminum chloride spectrophotometric assay by using quercetin as a reference standard16. In short, to 1ml of the test sample or standard (quercetin) at a concentration of (1mg/ml) addition of 4ml of distilled water was done. Later equal volume of (0.3ml) of 5% (NaNO3) sodium nitrite and 10% aluminum chloride (AlCl₃) solutions were added and subjected for incubation at 370C for 5 minutes followed by the addition of 2ml of 1M NaOH (sodium hydroxide) was done, by adding remaining distilled water the final test reaction mixture made to 10ml. The reaction mixture made into homogenous by shaker (test tube) and the absorbance of the chromophore formed was estimated at 510nm in a triple manner, utilizing a UV-Visible spectrophotometer and the Blank solution taken as the test mixture with no aluminum chloride solution. The content of the Total flavonoid of each extract was communicated as mg QE/g dried plant extract (mg/g quercetin equivalent).

 

Estimation of antioxidant potential by DPPH (2, 2 -Diphenyl-1-picrylhydrazyl) assay:

Assessment free radical reduction potential of various crude extracts of Cayratia auriculata carried out by using (DPPH) 2, 2-diphenyl-1-picrylhydrazyl which is a stable free radical, concerning free radical-reducing ability or the capability of hydrogen donating property by the protocol followed by Braca et al. with minor alterations17. Various concentrations from 50 to 1000 μg/ml of crude extracts, standard (ascorbic acid) and (0.004%) of DPPH solutions were prepared in methanol. Equivalent volume (1 ml) DPPH solutions (0.004%) (of freshly prepared) in methanol was added to different concentrations of methanol, ethyl acetate, chloroform, and hexane crude extracts of Cayratia auriculata and standard (ascorbic acid) separately. The mixture was made homogenous by vigorous shaking and allowed for incubation (15- 30minutes) in the darkroom at 370C and the (OD) optical density recorded at 517nm by UV-Visible Spectrophotometer.  The Standard selected for this experiment was vitamin C and methanol used as methanol. DPPH in methanol without extract/ standard was kept for control. The scavenging efficiency of the tested extracts or standard was determined by the level of capability of decolourisation of DPPH- deep purple color to varying shades of yellow color. Lowering optical density (OD) of the reaction mixture reveals the peak potential antioxidant capability of tested compounds. The all the values of the present analysis was taken three times and their mean was used for the final data. The percentage (%) scavenging effect of DPPH was expressed with the formula: DPPH scavenging capacity (%) = [(Abs(c) –Abs (t))/ Abs(c)] ×100. In this (Abs(c)) is the control sample absorbance, and (Abs (t)) is the test sample or standard absorbance. The IC50 value represents the concentration, at which inhibition 50% of the DPPH free radical by the antioxidants (test sample or standard) was also calculated in addition to the percentage of inhibition.

 

RESULTS AND DISCUSSION:

Preliminary Phytochemical analysis:

In the course of the most recent couple of years, ethno-medication plants have assumed a pivotal part in the anticipation and therapy of various sicknesses, there are different natural and new restorative mixtures accomplished from plant origin18.  Many types of the Vitaceae family show famous antioxidant and anticancer activities19. In the current investigation, 30 g of Cayratia auriculata powder gave a complete yield of various quantities of extracts with four solvents (Table 1). The phytochemical investigation uncovered the presence of rich auxiliary metabolites incorporated in the plant that flavonoids, proteins, alkaloids, tannins, glycosides, anthraquinones, terpenoids carbohydrates, coumarins, phlobatannins, saponins, and steroids (Table 2). It is recognized that different polyphenolic compounds, similar to phenolic acids, flavonoids, and tannins, which have unbelievable antioxidant and anticancer exercises, are bountiful in plant materials. A few examinations uncovered the positive relationship of the high dietary admission of common antioxidants with decreased malignant growth mortality and coronary illness just as with life expectanc20. Several dietary polyphenolic components acquired from the plant-derived extracts or plants demonstrated correspondingly high antioxidant and cancer prevention agents. Diet having antioxidant-rich vegetables and fruits surprisingly diminishes the danger of numerous malignant growth infections showing that antioxidants could be fundamental specialists for the concealment of various physiological disorders21.


 

Table 1: Detailed data of extraction of plant material of Cayratia auriculata

Name of the plant and part used

Cayratia auriculata, whole plant

Weight during collection(g)

2575 g

Weight after shade drying (g)

183 g

Percentage(%) of water in plant material

92.89 %

Weight used for extraction

30 g

The volume of solvent used for extraction

300 ml

Type of extract

Weight of bottle

used for evaporation (g)

Weight of bottle with

extract after evaporation (g)

Weight of the

final extract (g)

(%) Percentage of

yield extract (w/w)

Hexane extract

54.8

58.9

4.1

13.33%

Chloroform extract

54.8

59.3

4.5

15.00%

Ethyl acetate extract

54.8

59.7

4.9

16.33%

Methanol extract

54.8

60

5.2

17.33%


Table 2: Results of Qualitative Preliminary Phytochemical Analysis of the Hexane, Chloroform, Ethyl Acetate, and Methanol  

Extracts of Cayratia auriculata: (-) = Absence, (+) = Traces, (++) = Moderate, (+++) = Abundant of Phytoconstituents

Phytoconstituents

Hexane Extract

Chloroform Extract

Ethyl acetate Extract

Methanol Extract

Alkaloids

+

+

+

+

Anthraquinones

+

+

Carbohydrates

+

+

+

+

Coumarins

+

Flavonoids

+

++

++

+++

Glycosides

+

+

Proteins

+

+

+

Phlobatannins

+

+

+

+

Saponins

+

+

+

++

Steroids

+

Tannins

+

+

++

+++

Terpenoids

+

+

++

++

 

Quantitative estimation of total phenolic content (Folin-Ciocalteu Assay):

In the current examination, the total level of phenols of Cayratia auriculata crude extracts was estimated by FCR (Folin–Ciocalteu reagent) method. Based on the results obtained from the present study it is confirmed that all extracts of Cayratia auriculata possessing considerable levels of phenols, among the other extracts methanol extract exhibited a high level of phenolic content. In methanol extract, total phenolic content is 383.99±1.11mg GAE/g dry extract, followed by ethyl acetate extract (300.66±1.66)mg/g GAE/g dry extract, chloroform (229.55±1.67)mg/g GAE/g dry extract, hexane extract (150.66±1.66)mg/g GAE/g dry extract (Figure:1a &1b). Generally many medicinal plants having high content antioxidants like phenols and polyphenols. Several literature surveys proved that there is a good relationship between phenolic content and their antioxidant properties22. The level of phenolic content in plant extracts is significantly responsible for their antioxidant properties. It is well known that redox properties exhibited by the phenolic compounds are accountable for their antioxidant activity, which plays an important role in decomposing peroxides and neutralizing free radicals23. Based on the strong interactions of phenols with lipid peroxy radical (LOO.), hydroxyl radical (HO.), superoxide anion radical (O2-) which are highly reactive oxygen free radicals, it is indicated that antioxidant potentials of plant extract directly proportional to their phenolics content24,25,26. The antioxidant activity of phytochemicals is responsible for the significant decrease in mortality rates and the occurrence of numerous human disorders27. The result strongly provides evidence that some of the pharmacological properties of this plant are could be due to the presence of an abundant amount of valuable phenolic compounds. The hydroxyl groups present phenols and polyphenols are responsible for free radical scavenging and antioxidant properties, depending on this phenomenon for rapid evaluation of antioxidant and free radical scavenging properties crude extracts, the content of total phenols used as a standard reference28,29.

 

 

Figure 1a: Standard curve of Gallic acid (GA), absorbance against concentration. y = 0.0018x – 0.0557; R2 = 0.9916 in the estimation phenolic content model.

 

 

Figure 1b: Graphical representation of total phenol content (mg GAE/g DE) of methanol, ethyl acetate, chloroform, and total hexane extracts of Cayratia auriculata in total phenolic content determination model. Mg = milligram; g = gram;

DE = Dried Extract.

 

Determination of total flavonoids content:

The total flavonoid content for methanol, ethyl acetate, chloroform, and hexane crude extract was calculated with the aluminum chloride spectrophotometric method by taking quercetin as standard. Formation of stable complexes by Aluminium chloride due to its interaction with the functional groups such as C-5 or C-3 hydroxide (OH) group and C-4 keto(C=O)groups of flavonoids. Flavonoids are a group of phytoconstituents well known as plant pigments having comparable substance structures. Flavonoids are accounted for that showing many pharmacological and biological properties, for example, antibacterial, antidiabetic, anticancer, and anti-inflammatory activities30. They are responsible for modifying the pathways of cell signalling and are responsible for the production of numerous metabolic important enzymes like lipooxygenase, iNOS, cycloxygenase, and are having high potent scavenging of free radical and antioxidants properties. Numerous C-glycoflavonoids were produced artificially and /or obtained naturally from plant extracts; their structure-activity relationship was determined31. From the mean absorbance values, content of total flavonoid levels were calculated in relation to quercetin (standard) equivalents (50-1000μg/ml), Y = 0.0009x+0.0303; R2 = 0.9943.Total flavonoid content in methanol extract is (64.11±8.89) mg QE/g dry extract, followed by ethyl acetate extract (31.88±3.33)mg QE/g dry extract, chloroform (19.66±6.66)mg QE/g dry extract, hexane extract (6.33±2.22)mg QE/g dry extract (Figure: 2a and 2b). Because of the outcomes revealed by the present study, it is confirmed that all extracts of Cayratia auriculata possessing considerable levels of flavonoids, among the other extracts methanol extract exhibited a high level of flavonoid content.

 

 

Figure 2a: Standard curve of Quercetin (QE), absorbance Concentration y = 0.0009x + 0.0303;  R2 = 0.9943. In total flavonoid content determination model.

 

 

Figure 2b: Graphical representation of total flavonoid content against (TFC) (mg QE/gDE) of various extracts of Cayratia Auriculata in total   flavonoid content determination model. QE=Quercetin Equivalent; mg=milligram; g= gram;  

 

Estimation of antioxidant potential by DPPH (2, 2 -Diphenyl-1-picrylhydrazyl) assay:

Free radicals produced in the body are normally scavenged by natural endogenous antioxidant systems which in turn further prevent their harmful effects. These antioxidant systems are greatly playing a crucial role in reducing the harmful effects on nucleic acids (DNA), carbohydrates, lipids and enzymes by neutralizing them. The basic reason for cellular damage by oxidative stress is mainly due to inadequate levels of reactive oxygen species such as( peroxides, hydroxyl radical) and antioxidant systems(glutathione peroxidase)32,33. Such type of cellular damage is typically linked to aging and also many neurodegenerative disorders such as Alzheimer's disease, Amyotrophic lateral sclerosis, Multiple sclerosis, and Parkinson's disease34,35. All four extracts of Cayratia auriculata exhibited considerable levels of antioxidant activity while methanol extract showed high antioxidant activity when compared to other extracts indicated by the outcomes of the present study. Generally, potent antioxidant potential is confirmed by a highest (%) percentage of scavenging effect of free radical and lower Inhibitory Concentration (IC50) values. Extracts of methanol, ethyl acetate, chloroform, and hexane with six different concentrations used in the present study (50,100, 200, 400, 800, and 1000μg/mL), at a concentration of 1000 μg/ml, highest scavenging potential of free radical activity exhibited by methanol extract i.e. (96.46±1.97µg/ml), while ascorbic acid (reference standard) at that same concentration exhibited (97.18±1.12µg/ml), which were close to each other. However, the extracts showed free radical scavenging effect in decreasing order as follows: MECA(96.46±1.97µg/ml)>EECA(94.38±3.12µg/ml)>CECA (92.13±1.69µg/ml) > HECA (90.44±2.80µg/ml). Among the four extracts of studied plant methanol extract presented the potent scavenging effect of free radicals having significantly lower inhibitory concentration (IC50) values in comparison with ethyl acetate, chloroform, and hexane extracts, being 80.66 μg/ml, 117.86μg/ml, 151.54μg/ml, and 169.96μg/ml, respectively. On the other side, reference standard (ascorbic acid) exhibited the IC50 value was 62.95mg/ml (Figure: 3a and 3b).

 

 

Figure 3(a): Graphical representation of (%) Percentage of Inhibition of various concentrations of Ascorbic acid of Cayratia auriculata in model.

 

Figure 3(b): Graphical representation of Inhibitory Concentration (IC50) of various concentrations of (standard)  extracts Ascorbic acid (standard) and extracts of Cayratia auriculata DPPH Assay in DPPH Assay model.

 

By using excel sheets (2007) graphs were generated inhibitory concentration (IC50) values of ascorbic acid and methanol extracts (Figure: 4 & 5). The above results proved that all the extracts of Cayratia auriculata had a noticeable amount of free radical scavenging activity among them methanol extract was considered a good free radical scavenging activity when compared to other extracts.

 

CONCLUSION:

Considerable levels of existence of phytochemical compounds such as phenolic acids, flavonoids, tannins, and other compounds revealed by the phytochemical analysis provide strong evidence that Cayratia auriculata is one of the good sources for valuable phytoconstituents as like other species in the vitaceae family. Strong antioxidant properties are exhibited by plant-derived polyphenolic compounds and flavonoids, which are predominantly extracted by methanol extracts and also other solvents to a considerable level. Aluminum chloride assay and FCR assay were performed to determine the level of phenolic and flavonoid content in the present study plant extracts, significant outcomes were revealed for phenolic as well as flavonoid content, later in vitro antioxidant property was analyzed by DPPH assay, the strong antioxidant activity observed by methanol in comparison  to others tested compounds including the standard antioxidant (ascorbic acid)  and the values are close to standard in a dose-dependent manner. From the above results, the present study undoubtedly reveals that Cayratia auriculata is a potent phytochemical source and also possessing efficient antioxidant potential. Further studies are recommended to categorize and isolation of bioactive compounds offering their antioxidant property of the crude extracts of Cayratia auriculata which in turn helps to evaluate the novel drugs from this medicinally valuable species. 

 

ACKNOWLEDGEMENT:

We thank the GSL Educational society and “Whizbang Bioresearch” laboratories for their invaluable support to carry out the present study.

 

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Received on 28.06.2021            Modified on 02.10.2021

Accepted on 07.12.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(10):4570-4576.

DOI: 10.52711/0974-360X.2022.00767